Identification of the genome-wide TPS gene family of Cinnamomum camphora var. linaloolifera and functional validation of linalool

Identification of the genome-wide TPS gene family of Cinnamomum camphora var. linaloolifera and functional validation of linalool | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Identification of the genome-wide TPS gene family of Cinnamomum camphora var. linaloolifera and functional validation of linalool Wei-Hong Sun, Wen-Hui Li, Zhuang Zhao, Yu Lv, Shuang-Quan Zou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6432060/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Cinnamomum camphora var. linaloolifera is known for its richness in linalool, which is an acyclic monoterpene widely used in the fragrance and flavour industries. However, limited information is available regarding the genome-wide identification and characterization of the key genes for linalool synthesis in C. camphora var. linaloolifera . Results Here, based on the whole genome data of Nan’an 1, a variety of C. camphora var. linaloolifera , a total of 46 CcTPS genes were identified and were classified into five subfamilies, among which TPS-b (28 members) and TPS-g (four members) clustered closely together. Functional annotation results indicated that all members of the TPS-b and TPS-g groups were associated with the synthesis of monoterpenes. Multiple sequence alignment analysis results showed that the RRX8W domain, which is mainly involved in cyclic isomerization, is relatively conserved in TPS-b and completely lost in TPS-g, suggesting that genes in the TPS-g subfamily are involved in the biosynthesis of acyclic monoterpenes. Expression analysis revealed that the expression levels of CcTPS14 , CcTPS15 , CcTPS16 and CcTPS32 from TPS-g subfamily were higher in stems and leaves than in fruits. Further results of gene function validation confirmed that these four genes all produced linalool at levels higher than 30%. Conclusion our research found that some of the ancient members of TPS-b resulted from the loss of the RRX8W structural domain responsible for cyclic isomerization, resulting in the TPS-g subfamily, which is responsible for the biosynthesis of the acyclic monoterpene linalool. Theresults provide a basis for further exploration of linalool biosynthesis and accumulation. Linalool biosynthesis Terpene synthase (TPS) gene family Cinnamomum camphora var. linaloolifera RRX8W domain evolution Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Terpenoids are the largest class of natural products produced by plants and play an important role in plant pollination, defence, and information exchange [ 1 ]. All terpenoids are derived from a limited number of acyclic prenyl pyrophosphate precursors and are categorized according to the number of condensed five-carbon isoprenoid units into hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, triterpenes, and polyterpenes [ 2 ]. Terpene synthase genes (TPS) are key enzymes that catalyse these precursors and have two conserved structural domains, the RRX8W arginine domain at the N-terminus and the DDXXD aspartate domain at the C-terminus [ 3 ]. The TPS gene family contains seven subfamilies: TPS-c is the ancestral branch, TPS-d is unique to gymnosperms and encodes monoterpene, sesquiterpene, and diterpene synthases, while TPS-a, TPS-b, and TPS-g are angiosperm-specific and encode monoterpene, sesquiterpene, and diterpene synthases, respectively [ 4 ]. TPS-e/f is found in both angiosperms and gymnosperms and encodes copalyl diphosphate and ent-kaurene, which are intermediates in the synthesis of the hormone gibberellin [ 5 – 7 ]. TPS-h is only found in Selaginella moellendorffii [ 8 ]. Enabled by the increasing availability of genomic resources, the TPS family has been systematically id entified in various plant species; for example, a large expansion of the TPS gene family was found in the genomes of the terpenoid-rich Lauraceae family, especially a significant expansion of TPS-b and TPS-a [ 4 , 9 – 12 ]. Linalool is an acyclic monoterpene alcohol with a pleasant aroma that is widely utilized in the fragrance and flavour industries [ 13 , 14 ]. The chemical structure of natural linalool exists as two enantiomers, (S)- and (R)-linalool, which have different odours and biological properties [ 15 ]. It is recognized that the odour of natural linalool is far superior to that of synthetic linalool and that certain drugs can only use natural linalool as a starting material [ 16 – 18 ]. Natural linalool is more economically valuable in the flavour market due to its "natural" label, which is in line with emerging consumer preferences [ 16 ]. However, the low production of linalool products from natural plant tissues results in a shortage of natural linalool in the market [ 16 , 19 ]. C. camphora is rich in terpenoids and is the major source of linalool, especially camphora var. linaloolifera , 90% of which is extracted from fresh branches [ 20 ]. In recent years, researchers have explored the molecular mechanism of terpenoid biosynthesis of major components of C. camphora essential oil, but they have not focused on linalool biosynthesis based on genome data [ 12 , 21 , 22 ]. Recently, our research group reported a high-quality chromosome-level reference genome of Nan’an 1, a new variety of C. camphora var. linaloolifera with high linalool content [ 20 ]. Here, this study performed a comprehensive and systematic genome-wide analysis of the TPS gene family in the Nan’an 1 genome to identify the key genes involved in linalool synthesis and to further validate the function of the TPS gene in linalool synthesis. Materials and methods Plant material Nan’an 1, a new variety of C. camphora var. linaloolifera , was grown and planted in the Banlin State-owned Forest Farm in Anxi, Fujian Province (117°57′ E, 24°55′ N), by Xiamen Peony Fragrance Industry Co., Ltd. Healthy reproductive and nutritional organs with excellent development and no disease or insect damage were randomly sampled in four different directions (east, south, west, and north) and stored in liquid nitrogen for transcriptome sequencing and qRT‒PCR verification. Three relevant biological replicates were generated for each sample. Identification of the TPS gene family To identify the TPS genes in the Nan’an 1 genome, the Hidden Markov Model (HMM) profile of the Terpene_synth domain (PF01397) and the Terpene_synth_c domain (PF03936), downloaded from the Pfam database ( http://pfam.xfam.org , Pfam 3.0), were used to search the Nan’an 1 genome. All of the proteins with E-values lower than 10 − 3 were selected [ 23 ]. Using Arabidopsis TPS genes as queries, the predicted Nan’an 1 TPS genes ( CcTPSs ) were checked by BLASTP searches ( https://blast.ncbi.nlm.nih.gov/Blast . cgi). Then, the predicted TPS models detected were examined manually. Candidate CcTPS members were deredundantly confirmed using NCBI-CDD ( https://www.ncbi.nlm.nih.gov/cdd ) and Pfam ( http://pfam.xfam.org/ ) for conserved structural domains. Finally, 46 TPS genes ( CcTPS ) were identified in the Nan’an 1 genome. ProtParam ( https://web.ExPASy.org/protparam ) was used to calculate the physicochemical properties[ 24 ]. Secondary structural analysis was performed by using SOPMA ( https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html ). Plant-mPLoc ( http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/ ) [ 25 ] and SignalP v4.1 ( http://www.cbs.dtu.dk/services/SignalP-4.1/ ) [ 26 ] were used for subcellular localization and signal peptide prediction, respectively, and the TPS gene function prediction was used for website ( http://www.nipgr.ac.in/terzyme.html ) [ 27 ]. Analysis of chromosomal localization and multiple sequence alignment The CcTPS genes were subjected to multiple sequence alignment using the MUSCLE program, and conserved structural domains were displayed using Genedoc. Chromosome location analysis was visualized using MapChart software [ 28 ]. BLASTP was used for all CcTPS proteins for comparison, and MCScanX was used for duplication events [ 29 ]. KaKs2 Calculator 2.0 was used to calculate the duplicate gene kaks values and gene divergence times [ 30 ]. Analysis of the correlations, gene structure, and conserved motifs The CcTPS genes were analysed for correlations using the R package corrplot. The maximum value for motif prediction was set to 15 when the CcTPS genes were predicted at the MEME website ( https://meme-suite.org/meme/ ) [ 31 ]. GSDS ( http://gsds.gao-lab.org/index.php ) was used to display the structural information of the CcTPS genes [ 32 ]. Analysis of cis-acting elements and phylogenetic trees MUSCLE software was used to align the sequences of the TPS proteins from Nan’an 1 ( C. camphora var. linaloolifera ) and six other plants with known genomes ( Selaginella moellendorffii , Abies grandis , Oryza sativa , Cinnamomum kanehirae , Populus trichocarpa , A. thaliana ) [ 33 ]. IQ-TREE software was used to construct an ML tree with the JTT model, and the bootstrap value was 1000 replicates [ 34 ]. The phylogenetic tree was polished using Evoview ( https://evolgenius.info//evolview-v2/ ) [ 35 ]. PlantCARE ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) [ 36 ] was used to extract the 2000 bp upstream of CcTPS promoters for cis -acting element analysis. Transcriptome analysis of the CcTPS gene family High-quality RNA was extracted from different tissues of Nan’an 1, including young leaves, mature leaves, stems, young fruits (green fruits), mature fruits (red fruits), and later ripening fruits (purple fruits), for transcriptome sequencing. The clean transcriptome sequencing data were assembled using Trinityc [ 37 ] (min_kmer_cov set to 2, other parameters default), with the Nan’an 1 genome as the reference genome. RSEM [ 38 ] and DESeq2 were used to obtain FPKM values and to conduct gene differential expression analysis. The FPKM values of CcTPS s in different tissues were visualized by using TBtools software [ 39 ]. qRT‒PCR evaluation A total plant RNA kit (Polysaccharide Polyphenol Plant Total RNA Extraction Kit, developed by Hangzhou Bo Ri Technology Co., Ltd.) was used to extract RNA from stems, young leaves, mature leaves, and mature fruits of Nan’an 1, and the quality RNA was detected by electrophoresis. The qualified RNA was used as a template to construct the RT reaction solution according to the TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix instructions. Twenty microlitres of the whole reaction system was utilized, inactivated at 85°C for 5 seconds to produce cDNA, and then kept at -20°C. For qRT‒PCR, the following reaction parameters were chosen: 94°C for 30 s; 94°C for 5 s; 60°C for 30 s; 40 cycles; 72°C for 10 s; 95°C for 15 s; 60°C for 1 min; and 95°C for 1 s. The relative expression of each target gene in the samples was then determined using CcEF1a as the internal reference gene (Supplementary Table 1). Each reaction technique included three biological replicates, and each biological replicate included three technical replications. Prokaryotic expression of CcTPS14, CcTPS15, CcTPS16 and CcTPS32 in E. coli and detection by GC‒MS Prokaryotic expression of CcTPS14, CcTPS15, CcTPS16 and CcTPS32 in E. coli and detection by GC‒MS The ORFs of CcTPS14 , CcTPS15 , CcTPS16 and CcTPS32 were isolated and ligated into the pET32a vector BglII XhoI sites. The recombinant plasmids and the absence plasmids were all transformed into E. coli BL21 (DE3) pLysS cells (TransGen, China). Single colonies containing the recombinant plasmid were selected into 3 mL of LB liquid medium (amp resistant) and cultivated in 10 µl tubes at 37℃ with shaking until the concentration of the bacterial solution reached OD600, approximately 0.6–0.8 h. Then, 0.1 mM IPTG was added to the recombinant CcTPS14 , CcTPS15 , CcTPS16 and CcTPS32 enzymes, and the culture was continued at 16°C with shaking for 16 hours to induce protein expression. The cultivated bacteria were collected by centrifugation at 5000 rpm for 5 min and resuspended in 40 mL of precooled GST equilibrium solution. Ultrasonically crush the bacteria with parameters set to 200 W, 2.5 s, 5 s pause, and 80 cycles. After centrifugation at 10,000 rpm at 4℃ for 5 min, the supernatant and the precipitate were collected. A portion of the supernatant and the precipitate were subjected to SDS‒PAGE, the remaining supernatant was placed in a 50 ml centrifuge tube, and the precipitate was set aside at 4℃. Results Identification of CcTPS gene family members and analysis of their physicochemical properties A total of 46 CcTPS genes were identified in the Nan’an genome and named CcTPS1 to CcTPS46 based on their location on the chromosome (Table 1 ). Physicochemical property analysis showed that the protein length of the 46 CcTPSs varied from 306 ( CcTPS8 ) to 2597 aa ( CcTPS30 ), the relative molecular masses ranged from 35.00 kDa ( CcTPS8 ) to 301.08 kDa ( CcTPS30 ), isoelectric points (pI) ranged from 4.49 ( CcTPS17 ) to 7.81 ( CcTPS39 ), and only six CcTPSs of the PI were greater than seven. Secondary structure prediction revealed an alpha helix between 54.78% ( CcTPS10 ) and 78.88% ( CcTPS14 ), beta turns between 2.15% ( CcTPS28 ) and 7.04% ( CcTPS10 ), irregular curls between 15.32% ( CcTPS14 ) and 34.47% ( CcTPS1 ), and extended chains between 1.58% ( CcTPS33 ) and 13.18% ( CcTPS10 ). Except for CcTPS4 , CcTPS9 , and CcTP26 , the remaining CcTPSs were marked in chloroplasts rather than in the cytoplasm. The prediction result of the signal peptide is that all CcTPS genes have no signal peptide. Table 1 Localization, physicochemical properties, and secondary structure of CcTPS genes. Gene name Gene ID Length MW(kDa) pI Subcellular localization Alpha helix (%) Extended strand (%) Beta turn (%) Random coil (%) Signal CcTPS1 Maker00011790 850 96.35 7.51 Chloroplast 55.41 6.24 3.88 34.47 NO CcTPS2 Maker00028622 435 50.73 5.66 Chloroplast 66.67 4.6 2.53 26.21 NO CcTPS3 Maker00012602 528 60.48 5.87 Chloroplast 71.02 2.84 3.6 22.54 NO CcTPS4 Maker00012641 870 99.57 5.51 Chloroplast, Cytoplasm 68.97 4.94 4.25 21.84 NO CcTPS5 Maker00012720 553 63.89 5.66 Chloroplast 71.97 2.53 2.89 22.6 NO CcTPS6 Maker00010115 558 64.24 5.3 Chloroplast 69.89 3.05 2.51 24.55 NO CcTPS7 Maker00005406 681 77.84 5.21 Chloroplast 65.49 6.02 4.11 24.38 NO CcTPS8 Maker00018207 306 35.00 5.4 Chloroplast 61.11 2.61 2.94 33.33 NO CcTPS9 Maker00018214 897 103.50 6.26 Chloroplast, Cytoplasm 68.45 6.02 4.91 20.62 NO CcTPS10 Maker00005791 1108 126.23 7.31 Chloroplast 54.78 13.18 7.04 25 NO CcTPS11 Maker00020936 551 63.46 5.13 Chloroplast 67.88 4.17 3.63 24.32 NO CcTPS12 Maker00007365 1335 153.08 6.29 Chloroplast 58.05 9.81 5.24 26.89 NO CcTPS13 Maker00025120 741 85.24 5.74 Chloroplast 68.29 5.94 5.13 20.65 NO CcTPS14 Maker00024100 483 55.63 6.8 Chloroplast 78.88 2.48 3.31 15.32 NO CcTPS15 Maker00014813 814 93.36 6.57 Chloroplast 60.57 7.99 6.51 24.94 NO CcTPS16 Maker00014818 434 49.83 6.76 Chloroplast 70.28 5.53 3.69 20.51 NO CcTPS17 Maker00014809 496 56.78 4.49 Chloroplast 69.15 6.05 2.82 21.98 NO CcTPS18 Maker00001539 596 68.35 5.89 Chloroplast 61.07 8.89 5.2 24.83 NO CcTPS19 Maker00001514 431 49.58 4.75 Chloroplast 70.3 3.94 3.48 22.27 NO CcTPS20 Maker00001509 346 40.07 6.35 Chloroplast 74.57 2.31 3.47 19.65 NO CcTPS21 Maker00011988 1404 161.15 5.89 Chloroplast 57.34 9.4 6.48 26.78 NO CcTPS22 Maker00011978 546 63.18 5.02 Chloroplast 70.7 4.03 3.3 21.98 NO CcTPS23 Maker00023025 558 64.56 5.52 Chloroplast 68.28 3.94 3.41 24.37 NO CcTPS24 Maker00018905 1130 131.04 5.18 Chloroplast 64.96 6.46 4.51 24.07 NO CcTPS25 Maker00018899 1098 127.72 4.92 Chloroplast 70.13 4.64 3.37 21.86 NO CcTPS26 Maker00014805 475 54.66 5.18 Chloroplast, Cytoplasm 59.58 8.63 4.21 27.58 NO CcTPS27 Maker00014802 562 65.17 5.43 Chloroplast 67.79 4.45 3.02 24.73 NO CcTPS28 Maker00014789 465 53.94 7.26 Chloroplast 68.6 4.52 2.15 24.73 NO CcTPS29 Maker00014796 545 63.18 5.25 Chloroplast 66.06 4.95 2.39 26.61 NO CcTPS30 Maker00014782 2597 301.08 6.51 Chloroplast 60.8 9.66 6.66 22.87 NO CcTPS31 Maker00014804 484 56.00 7.44 Chloroplast 68.6 4.13 2.89 24.38 NO CcTPS32 Maker00015451 1074 123.80 5.29 Chloroplast 69.27 4.56 4.1 22.07 NO CcTPS33 Maker00015350 571 66.47 6.11 Chloroplast 67.6 1.58 2.98 27.85 NO CcTPS34 Maker00017669 1429 164.62 6.48 Chloroplast 59.27 7.21 5.32 28.2 NO CcTPS35 Maker00017748 398 45.55 6.34 Chloroplast 69.6 3.77 4.27 22.36 NO CcTPS36 Maker00015605 1122 130.50 6.81 Chloroplast 68.36 4.63 5.53 21.48 NO CcTPS37 Maker00015620 427 49.77 6.11 Chloroplast 74.47 3.28 3.28 18.97 NO CcTPS38 Maker00015608 737 84.95 6.39 Chloroplast 63.09 6.24 3.66 27 NO CcTPS39 Maker00018575 566 64.37 7.81 Chloroplast 61.13 6.01 3 29.86 NO CcTPS40 Maker00018262 540 62.49 6.79 Chloroplast 72.04 4.26 2.96 20.74 NO CcTPS41 Maker00014305 635 72.98 7.06 Chloroplast 64.72 5.83 2.99 26.46 NO CcTPS42 Maker00014318 443 51.37 6.43 Chloroplast 72.23 4.29 4.29 19.19 NO CcTPS43 Maker00021750 424 49.02 6.11 Chloroplast 72.64 4.01 3.3 20.05 NO CcTPS44 Maker00022851 444 51.26 5.96 Chloroplast 72.75 2.93 3.6 20.72 NO CcTPS45 Maker00021963 607 69.74 6.51 Chloroplast 65.57 3.79 3.29 27.35 NO CcTPS46 Maker00017397 448 51.02 5.99 Chloroplast 77.90 2.46 3.35 16.29 NO Phylogenetic analysis of the CcTPS gene family To reveal the evolutionary relationship of the CcTPS gene family, a phylogenetic tree (Fig. 1 ) was constructed based on the 257 TPS genes from Nan’an 1 (46 members), C. kanehirai (90 members), O. sativa (31 members), A. thaliana (33 members), P. trichocarpa (32 members), A. grandis (11 members) and S . moellendorffii (14 members). All TPSs were classified into seven subfamilies: TPS-a, TPS-b, TPS-c, TPS-d, TPS-e/f, and TPS-g, in which TPS-b underwent a significant expansion. Among them, 46 CcTPS genes were classified as TPS-a, TPS-b, TPS-c, TPS-e/f, TPS-g, and TPS-b. TPS-b had the most members with 28 members, followed by TPS-a with 12 members, TPS-g with four members, and TPS-e/f with one member each. In addition, functional enrichment suggested that all the members of TPS-a encode sesquiterpene synthase, TPS-b members encode monoterpene synthase, TPS-g encode acyclic monoterpenes, and TPS-c and TPS-e/f encode diterpene synthase (Supplementary Table 2). Multiple sequence alignment Multiple sequence alignment analysis (Fig. 2 ) suggested that most CcTPS have three conserved structural domains: RRX8W, RXR, and DDXXD. Among them, the DDXXD domain is the most conserved, with deletions or variants found only in the C-terminus of CcTPS10 and CcTPS8 in TPS-a and in the C-terminus of CcTPS1 in TPS-c. The function of this domain is to bind divalent magnesium ions or manganese ions for substrate cleavage. The RXR domain is second only to the DDXXD domain in conservation, with mutations observed in five genes (TPS-a: CcTPS24 , CcTPS10 ; TPS-b: CcTPS35 and CcTPS2 ; TPS-c: CcTPS1 ). This domain is currently only found in Orchidaceae and Mentha longifolia , and its function is unclear [ 40 , 41 ]. The RRX8W domain, which is involved in the synthesis of cyclic monoterpenes [ 42 ], shows relative conservation in TPS-a and TPS-b, whereas significant mutations or deletions have occurred in the N-terminus of the rest of the subfamilies, e.g., the RRX8W domain is lost in all members of TPS-g ( CcTPS14 , CcTPS15 , CcTPS16 , and CcTPS32 ). This implies that TPS-g members are involved in the synthesis of acyclic monoterpenes.s Gene structure and conserved motif analysis To investigate the diversity of motif components among CcTPS , the motif distribution in 46 CcTPS proteins was investigated using the online tool MEME program (Figs. 3 A, 3 B). A total of 15 conserved motifs were identified, of which 13 motifs appeared in the TPS-b subfamily and six motifs appeared in the TPS-c subfamily. Among these motifs, motifs 3, 4, and 13 were found to be more conserved and retained in most CcTPSs . Combined with the results of multiple sequence alignment analysis, motifs 3, 4, and 13 corresponding to DDXXD, RXR, and RRX8W were identified, respectively. It is important to note that most members of TPS-b possess the conserved motif 13 (RRX8W domain), which plays an essential role in the catalysis of monoterpene cyclization. Conversely, all members of TPS-g lack motif 13 (Fig. 3 B). Furthermore, motif 13 and motif 8 are the conserved domains of terpene synthase (PF01397) at the N-terminus, while motif 3 and motif 2 are the conserved domains of terpene synthase C (PF03936) (Fig. 3 B). Genes with more variable members of introns and exons have more complex structures [ 43 ]. Gene structure analysis showed that the number of exons in the CcTPS genes ranged from five to 35, and the number of introns ranged from four to 34 (Fig. 3 C). Most members of the subfamily contain the same number of introns and exons, but CcTPS30 in the TPS-b group has the highest number of exons and introns, and CcTPS40 has the lowest number of exons and introns. The untranslated regions (UTRs) interact with RNA-binding proteins involved in posttranscriptional regulation of genes [ 44 ]. Only CcTPS28 (TPS-a), CcTPS13 (TPS-b), CcTPS3 (TPS-g), and CcTPS32 (TPS-g) contain UTRs. Chromosomal localization and synteny analysis 46 CcTPS genes were unevenly distributed throughout seven chromosomes and one scaffold (Fig. 4 ). The largest number of CcTPS genes (13 members) was located on chromosome 10, followed by chromosome 3 (eight members) and chromosome 7 (seven members). In contrast, chromosomes 1, 2 and 12 displayed only one CcTPS gene. The synteny analysis suggested that the CcTPSs had seven segmental duplication gene pairs ( CcTPS32 and CcTPS14 ; CcTPS38 and CcTPS42 ; CcTPS38 and CcTPS43 ; CcTPS36 and CcTPS46 ; CcTPS38 and CcTPS18 ; CcTPS42 and CcTPS19 ; and CcTPS41 and CcTPS18 ) and one tandem duplication gene pair ( CcTPS27 and CcTPS28 ) (Fig. 4 ). Interestingly, the segmental duplication genes are from the TPS-b subfamily, and the tandem duplication genes are from the TPS-a subfamily. The K a/ K s ratio of these gene pairs was less than one, indicating that these genes underwent significant purifying selection during their evolution. The calculated divergence time of segmental duplication and tandem duplication gene pairs was approximately 3.69 Mya ~ 71.16 Mya (million years ago), of which six gene pairs (85%) experienced α events and one gene pair (15%) underwent β events (Table 2 ). These results indicate that the duplication events of the CcTPS genes are mainly caused by α events. Table 2 The differentiation time and gene duplication events of the CcTPS gene pairs. Duplicated gene pairs Duplicated gene pairs Ka Ks Ka/Ks Purify selcetion Duplicated type Time (MYA) Duplication event CcTPS38 CcTPS42 0.058870266 0.067280307 0.874999963 yes WGD 3.6967 Cc-α CcTPS38 CcTPS43 0.008641966 0.038505247 0.224436068 yes WGD 2.1157 Cc-α CcTPS36 CcTPS46 0.386682802 1.29511625 0.298569956 yes WGD 71.1602 Cc-β CcTPS38 CcTPS18 0.112363873 0.21134456 0.531662006 yes WGD 11.6123 Cc-α CcTPS42 CcTPS19 0.052975454 0.085371247 0.620530398 yes WGD 4.6907 Cc-α CcTPS41 CcTPS18 0.025626409 0.091066749 0.281402475 yes WGD 5.0037 Cc-α CcTPS27 CcTPS28 0.058640445 0.17472081 0.335623699 yes tandem ~ ~ To further infer the phylogenetic mechanisms of the CcTPSs , this study constructed a comparative collinear relationship of C. camphora var. linaloolifera (Nan’an 1) associated with five magnoliid plants ( Magnolia biondii, Piper nigrum, C. kanehirae, Chimonanthus salicifolius and Aristolochia fimbriata ) (Fig. 5 ). There were 14 TPS gene pairs in C. camphora var. linaloolifera and C. kanehirae , 15 in M . biondii , nine in C . salicifolius , four in P. nigrum and one in A. fimbriata (Supplementary Table 3). These orthologous gene pairs have a K a /K s ratio of less than one, indicating that the TPS genes of magnoliid plants underwent strong purifying selection during evolution. The study further calculated the divergence time of gene pairs between C. camphora var. linaloolifera and the other four magnoliid plants and found that the divergence time of C. camphora var. linaloolifera and C. kanehirae (2.56 Mya ~ 136.79 Mya) was later than that of C. camphora var. linaloolifera and other plants (the divergence time of C. camphora var. linaloolifera and M. biondii was 53 Mya ~ 158.44 Mya; that of P. nigrum was 57.81 ~ 209.27 Mya; that of C . salicifolius was 48.72 Mya ~ 139.23 Mya; and that of A . fimbriata was 189.03 Mya) (Supplementary Table 3). Expression correlation analysis among CcTPS members The Pearson product-moment correlation coefficient (pcc) value of CcTPS27 / CcTPS28 (TPS-a) of the tandem duplication gene pair was 0.89 (Fig. 6 ). Among the seven segmental duplication gene pairs, the expression profiles of five gene pairs were positively correlated ( CcTPS38 / CcTPS42 : 0.55; CcTPS38 / CcTPS43 : 0.02; CcTPS38 / CcTPS46 : 0.037; CcTPS38 / CcTPS18 : 0.036; CcTPS42 / CcTPS19 : 0.88), and the expression levels of one gene pair were negatively correlated ( CcTPS32 / CcTPS14 : -0.062). Since most of the segmental duplications in CcTPSs displayed positive correlations, it was likely that the segmental genes functioned in collaboration to work together. Analysis of promoter cis-acting elements To ascertain the potential biological functions of the CcTPS genes, cis -acting element analysis was performed. A total of 1,169 cis -acting regulatory elements were identified in the CcTPS gene promoter, and these cis -elements can be divided into three categories: plant growth and development, stress responsiveness, and hormone responsiveness (Figs. 7 A, B). The first type of cis -element related to plant growth and development mainly contained the following ten categories: AAGAA motif (16.03%) and GCN4 motif (6.13%) related to endosperm development; AT-rich element (3.30%), CCAAT box (10.37%), and MRE (11.32%) related to flowering regulation; CAT box (15.09%) related to meristem; RY element (3.83%) related to seed development; as-1 (17.92%) related to bud development; O2 site (10.37%) related to zein metabolism; and circadian (6.60%) related to circadian rhythm (Fig. 7 C). The second type of cis- element was related to stress response elements, such as MYC (48.02%), ARE (29.26%), STRE (35.62%), MBS (8.39%), LTR (7.88%), DRE1 (0.76%), WUN (5.34%), WRE3 (10.68%), and TC-rich repeats (2.03%) (Fig. 7 D). Among them, MYC responded to MeJA, MBS responded to drought induction, LTR responded to low temperature stress, WUN is related to injury response, and WRE3 and TC-rich repeats are related to plant defence. The last type of cis -element is mainly involved in phytohormone responses (Fig. 7 E). ABREs (12.54%) are associated with abscisic acid. The GARE motif (2.86%), P-box (5.37%) and TATC-box (2.86%) are related to gibberellin synthesis. The TCA element (5.01%) is related to growth. The TGACG motif (6.81%) and MYC motif (48.02%) are related to methyl jasmonate synthesis. EREs (6.98%) are related to ethylene. These results indicated that the TPS genes may be regulated by a variety of hormones and that they respond to multiple abiotic stresses. CcTPS gene expression patterns in different tissues To understand the expression patterns of TPS genes in different tissues of Nan’an 1, the study performed an RNA transcription analysis on young leaves (YL), mature leaves (ML), stems (St), young fruits (green fruit; fruit I), mature fruits (red fruit; fruit II), and late ripening fruits (purple fruit; fruit III) of Nan’an 1 (Supplementary Fig. 1; Supplementary Table 4). The expression results showed that except for CcTPS2 and CcTPS31 , the remaining CcTPS s were expressed in stems, young leaves, and mature leaves. The stems and young leaves contained 31 (70.45%) and 32 (72.72%) highly expressed CcTPS genes, respectively. A total of 21 (45.1%) CcTPS genes were expressed at three different fruit development stages. With fruit development, the expression of 14 CcTPS genes was downregulated, and the expression of four CcTPS genes was upregulated. Validation of the expression of key genes involved in linalool synthesis In addition, based on our gene structure and annotation analysis of the CcTPS genes described above, the study inferred that four genes of the TPS-g subfamily are key enzymes in the final step of linalool synthesis. The four TPS-g members of Nan’an 1 exhibited two distinct expression patterns (Fig. 8 A; Supplementary Table 4). Among these genes, CcTPS14 , CcTPS15 and CcTPS16 were highly expressed in stems and mature fruits and were also expressed in young leaves but not in old leaves. CcTPS32 was highly expressed in stems, old leaves, and young fruits but not in other tissues. Further validated CcTPS14 , CcTPS15 , CcTPS16 and CcTPS32 in the TPS-g subfamily in stems, leaves, and roots was achieved through qRT‒PCR (Fig. 8 B). The results showed that CcTPS14 , CcTPS15 , and CcTPS16 had the highest expression in the stem, followed by the young leaves, and CcTPS32 had the highest expression in the old leaves, followed by the stem. The qRT‒PCR results were consistent with the transcriptome expression results. Validation of functional genes for linalool synthesis After four linalool synthesis genes, CcTPS14 , CcTPS15 , CcTPS16 and CcTPS32 were cloned, the expression vectors were constructed, and the four genes were successfully ligated into the constructed expression vectors and fused with His tags by enzymatic digestion experiments (Fig. 9 A). To verify the catalytic activity of the four linalool synthesis genes and the products generated, the study adsorbed the proteins on a nickel column and then eluted the purified proteins by SDS‒PAGE. The results showed that the molecular weights of the soluble proteins of CcTPS14 , CcTPS15 , CcTPS16 and CcTPS32 were 56, 94, 60 and 58 kDa, respectively, which were consistent with the predictions by the software, implying that enzyme activity experiments could be performed (Fig. 9 B). Linalool is an acyclic monoterpene, so the study detected the activity of the recombinant protein by adding GPP, the substrate needed for monoterpene production, and detected the enzymatic activity products by GC‒MS (Fig. 9 C). CcTPS14 , CcTPS15 , CcTPS16 , and CcTPS32 were able to produce linalool at levels ranging from 30.14–36.60% (Supplementary Tables 5–8). Since one gene can catalyse the generation of multiple products from the same substrate, the study found that CcTPS14 also produced the monoterpene Geranial, and CcTPS15 , CcTPS16 and CcTPS32 produced monoterpenes such as Thujene and D-Limonene. Discussion C. camphora belongs to the family Lauraceae and is an aromatic tree with important economic and ecological value. According to the main volatile components of its leaf essential oils, C. camphora can be subdivided into linalool, borneol, camphor, cineole, and nerolidol types [ 21 , 45 , 46 ]. C. camphora var. linaloolifera belongs to the linalool type, is native to China and is the main source of natural linalool. Natural linalool is in short supply in the market due to its optical activity, its far superior odour to synthetic linalool, and the fact that certain drugs can only use natural linalool as a starting material [ 15 – 18 ]. Our group successfully cultivated a new cultivar of C. camphora var. linaloolifera , Nan'an1, whose branch and leaf essential oil rate is as high as 3.07%, and 90% of the essential oil is linalool, which is much higher than that of the other C. camphora var. linaloolifera (0.173%~2.668%) [ 47 ]. Therefore, Nan'an1 is the best material for studying natural linalool biosynthesis. Biosynthetic biology offers a more effective and reasonable solution for the production of natural chemicals [ 48 ]. Transcriptome sequencing analysis has become the premier means for researchers to explore the biosynthesis of secondary metabolites, but the technique may miss functional genes that are active at specific times and does not provide insight into their evolution and improve their production [ 49 ]. The whole-genome sequence provides access to comprehensive genetic resources and compensates for the shortcomings of the transcriptome. The combined mode of transcriptomics and genomics is currently a popular trend for elucidating the biosynthetic pathways of natural products. Based on the whole-genome sequence at the chromosome level of Nan'an 1 reported by our group [ 20 ], a total of 46 CcTPS genes were identified, much less than the number of TPS in other Lauraceae species. The reason for this is that the screening of TPS genes in this study was conditioned on the retention of structural domains containing both terpene_synthase (PF01397) and terpene_synthase_C (PF03936) domains, whereas genes with one of these domains were retained in other studies. This shows that the CcTPS genes this study identified are more accurate. Phylogenetic analysis classified the 46 CcTPSs into five subfamilies, including TPS-a (12 members), TPS-b (28 members), TPS-c (one member), TPS-e/f (one member), and TPS-g (four members). A significant expansion of the TPS-b subfamily occurred, consistent with results observed in other Lauraceae, which may favour the biosynthesis and accumulation of aromatic terpenes in plants of this family. Seven segment duplication gene pairs were identified in the CcTSP gene family of Nan’an 1, and all were from the TPS-b subfamily. Of these, six gene pairs were generated by α events, and one gene pair was generated by β events. These results suggested that segment duplication events and palaeopolyploidy events are major drivers of the expansion of the TPS-b branch in the Nan’an 1 CcTPS gene family. Linalool is an acyclic monoterpene [ 17 , 18 ]. Functional annotation results showed that all members of TPS-b and TPS-g were annotated as monoterpene synthases. In this study, it also found that the gene structures of TPS-b and TPS-g are similar and that TPS-g is a clade closely related to TPS-b. These findings are consistent with those of previous studies [ 50 , 51 ]. The domains of genes in the same subfamily are roughly similar, and in long-term evolution, the variation or loss of the domains of some genes may form a new subfamily [ 52 ]. The plant TPS genes contain the conserved domains RRX8W, RXR and DXXD [ 53 ], of which the function of the RRX8W domain primarily performs cyclic isomerization [ 54 ]. Interestingly, the RRX8W domain was completely lost in members of the TPS-g subfamily. Thus, the study speculate that the TPS-g group may be a subgroup of branches derived from the TPS-b branch that lost the RRX8W domain during evolution and inherited the function of linalool synthesis. In addition, AtTPS14 is a crucial gene for the production of linalool in A. thaliana [ 55 ]. The phylogenetic tree showed that AtTPS14 clustered with four genes ( CcTPS14 , CcTPS15 , CcTPS16 , and CcTPS32 ) of TPS-g, further implying that these genes are involved in linalool synthesis. The content of essential oils (approximately 95% of terpenoids) produced by fresh stems (3.07 ± 0.17%) and leaves (2.04 ± 0.02%) of ‘Nan’an 1’ is significantly higher than that produced by fresh fruits (0.85 ± 0.01%), and the content of linalool in essential oils is up to 90.43% [ 47 ]. Expression analysis showed that the four linalool synthesis genes were highly expressed mainly in stems and leaves, with CcTPS14 , CcTPS15 , and CcTPS16 having the highest expression in stems and CcTPS32 having the highest expression in mature leaves. Gene function validation showed that CcTPS14 , CcTPS15 , CcTPS16 and CcTPS32 were all able to produce linalool with more than 30% linalool content. Ultimately, this study demonstrated that four genes of the TPS-g subfamily play pivotal roles in linalool biosynthesis and accumulation in C. camphora var. linaloolifera , which provides a basis for an in-depth understanding of the molecular mechanisms of terpenoid formation in C. camphora . Conclusion In this work, it conducted a comprehensive and systematic genome-wide analysis of the TPS gene family in Nan’an 1, a new variety of C. camphora var. linaloolifera with high linalool content. A total of 46 TPS genes were identified, and their promoters contained numerous stress cis -acting elements and hormone cis -acting elements, suggesting that CcTPS s play an important role in plant defence and information exchange. The phylogenetic results showed that the CcTPSs were categorized into five subfamilies, of which the TPS-b subfamily was significantly expanded due to segment duplication events and palaeopolyploidy events. TPS-b and TPS-g clustered together, and their members were mainly involved in monoterpene synthesis. Notably, the RRX8W domain involved in cyclic isomerization is conserved in most members of TPS-b but is lost in all members of TPS-g. Domain variations such as deletions or mutations promote the creation of a new subfamily. Therefore, this study suggest that some members of the ancient TPS-b subfamily lost the RRX8W domain during evolution and may have formed the TPS-g subfamily, which is mainly involved in the synthesis of acyclic monoterpenes. The content of essential oil extracted from the fresh stems and leaves of ‘Nan 'an 1’ was much higher than that from the fresh fruit, and linalool was the main component in the essential oil. The results of expression analysis and qRT‒PCR showed that the four linalool synthesis genes ( CcTPS14 , CcTPS15 , CcTPS16 , and CcTPS32 ) from TPS-g were highly expressed in stems and leaves. In vitro enzyme activity verification proved that these four key genes can produce high levels of linalool. In conclusion, the study screened four key genes for linalool synthesis in the Nan’an 1 genome by biological information and gene functional validation. These novel results provide a basis for the subsequent functional validation of the linalool type of other Lauraceae plants and have significant biological significance for promoting the development of the linalool type plant industry and for high linalool breeding, providing a good molecular genetic foundation. Declarations Supplemental information Additional supporting information can be found online in the Supporting Information section at the end of this article. Ethics approval and consent to participate Nan'an 1 material were obained from the Banlin State-owned Forest Farm (117°57′ E, 24°55′ N) in Anxi, Fujian Province, and Professor Shuang-Quan zou ensured that we obtained permission for the collection the Nan'an 1 and formally identified the plant material used in the research. Consent for publication Not applicable. Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding The current study was supported by Fujian forestry science and technology plan project salt-tolerance mechanism of Cinnamomum camphor and its application in coastal mountain afforestation (2024FKJ25) and application demonstration of Cinnamomum camphor seed in coastal ecological landscape forest transformation (2024TG08). Author Contribution SZ: conceived & designed the research. WS, WL and YL: performed the bioinformatics analysis, data curation & writing; WH and ZZ: helped with collecting samples & field work. WS: writing-review & editing. All authors have read and agreed to the published version of the manuscript. Data Availability All raw transcriptome sequences described in this manuscript have been submitted to the BioProject/GSA, National Genomics Data Center (NGDC) PRJCA002001/CRA004220. References Pichersky E, Gershenzon J. The formation and function of plant volatiles: perfumes for pollinator attraction and defense. 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Furubayashi M, Ikezumi M, Kajiwara J, Iwasaki M, Fujii A, Li L, Saito K, Umeno D. A high-throughput colorimetric screening assay for terpene synthase activity based on substrate consumption. PLoS ONE. 2014;9:e93317. http://dx.doi.org/10.1371/journal.pone.0093317 . Aubourg S, Lecharny A, Bohlmann J. Genomic analysis of the terpenoid synthase (AtTPS) gene family of Arabidopsis thaliana . Mol Genet Genomics. 2002;267:730–45. http://dx.doi.org/10.1007/s00438-002-0709-y . Additional Declarations No competing interests reported. Supplementary Files Supplementary.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6432060","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":454308672,"identity":"584ae5f8-73a9-4533-8254-dac3a47eb666","order_by":0,"name":"Wei-Hong Sun","email":"","orcid":"","institution":"Zunyi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Wei-Hong","middleName":"","lastName":"Sun","suffix":""},{"id":454308673,"identity":"226c0666-2954-4568-b6c2-9902eb3dd978","order_by":1,"name":"Wen-Hui Li","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Wen-Hui","middleName":"","lastName":"Li","suffix":""},{"id":454308674,"identity":"75f7e869-0f0e-4738-9e3b-d20820a1174a","order_by":2,"name":"Zhuang Zhao","email":"","orcid":"","institution":"Zunyi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Zhuang","middleName":"","lastName":"Zhao","suffix":""},{"id":454308675,"identity":"0b3be441-8f8c-4eef-b3b8-a11855d2655f","order_by":3,"name":"Yu Lv","email":"","orcid":"","institution":"Zunyi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Lv","suffix":""},{"id":454308676,"identity":"691d2c13-1482-41b6-ade4-b6931a4a33cc","order_by":4,"name":"Shuang-Quan Zou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAr0lEQVRIiWNgGAWjYHACxgcVDHIghgHRWpgNzjAYk6aFTYI0LfLtPWYVB2oMEhvYm7dJMNTcIayFseeM2Y0Dx4BaeI6VSTAce0ZYC7NEjtntD2x/EhuADAnGhsOEtbABVRYc+Ae0Rf4NkVp4gFoYDrYBtUjwEKlFgudYscTBPgPjNp60YouEY0RokW9v3vjhwDcD2X72wxtvfKghQgscsIGIBBI0jIJRMApGwSjAAwB3TzYmbIw+TAAAAABJRU5ErkJggg==","orcid":"","institution":"Fujian Colleges and Universities Engineering Research Institute of Conservation and Utilization of Natural Bioresources, Fujian Agriculture and Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Shuang-Quan","middleName":"","lastName":"Zou","suffix":""}],"badges":[],"createdAt":"2025-04-12 04:23:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6432060/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6432060/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82506546,"identity":"fca223d1-9f9a-48ae-bf66-c03530ec58b5","added_by":"auto","created_at":"2025-05-12 09:41:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2691944,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of 257 TPS genes from \u003cem\u003eC. camphora\u003c/em\u003evar. l\u003cem\u003einaloolifera\u003c/em\u003e and six other species.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6432060/v1/20fc82e603f0362f956bea69.png"},{"id":82506551,"identity":"4efd5f3e-3033-414c-ac9e-b04a1ed729c8","added_by":"auto","created_at":"2025-05-12 09:41:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5905128,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple sequence alignment of conserved structural domains of \u003cem\u003eCcTPS\u003c/em\u003e genes.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6432060/v1/2cef4e1ca218e066b12f70fe.png"},{"id":82506547,"identity":"a5a583a5-f38c-424f-ac36-9c3118c40754","added_by":"auto","created_at":"2025-05-12 09:41:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7530789,"visible":true,"origin":"","legend":"\u003cp\u003eConserved motifs and gene structure of \u003cem\u003eCcTPS\u003c/em\u003e genes. (\u003cstrong\u003eA\u003c/strong\u003e) Phylogenetic tree of the \u003cem\u003eCcTPS\u003c/em\u003e genes. (\u003cstrong\u003eB\u003c/strong\u003e) Conserved motifs of the \u003cem\u003eCcTPS\u003c/em\u003e genes. (\u003cstrong\u003eC\u003c/strong\u003e) Gene structure of the \u003cem\u003eCcTPS\u003c/em\u003e genes.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6432060/v1/c752be1cbdea510b3b86fe16.png"},{"id":82506930,"identity":"273d1092-fab4-4f4b-b129-aeac27403a88","added_by":"auto","created_at":"2025-05-12 09:49:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":15888645,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal localization of the \u003cem\u003eCcTPS\u003c/em\u003e gene distribution. The purple shading of the image represents tandem duplication genes. Genes labelled in red, pink, and blue are segmental duplication genes.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6432060/v1/00a3257cf96e69a0cf466395.png"},{"id":82506931,"identity":"44c37f71-1613-4b42-a38d-5e45e722fd0f","added_by":"auto","created_at":"2025-05-12 09:49:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1266745,"visible":true,"origin":"","legend":"\u003cp\u003eSynteny analysis of \u003cem\u003eTPS\u003c/em\u003e genes in\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eC. camphora \u003c/em\u003evar.\u003cem\u003e linaloolifera \u003c/em\u003eand five magnoliid plants.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6432060/v1/659d8b45837337e9648a660d.png"},{"id":82507709,"identity":"156d937a-5743-491b-a8b0-cdacf56b6a1a","added_by":"auto","created_at":"2025-05-12 09:57:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":8332840,"visible":true,"origin":"","legend":"\u003cp\u003eExpression correlation analysis of \u003cem\u003eCsTPS\u003c/em\u003e genes.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6432060/v1/d6245e4b16e3312ed912a33f.png"},{"id":82506549,"identity":"666b158b-ce8b-4eea-a7b0-40875eef122d","added_by":"auto","created_at":"2025-05-12 09:41:22","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":195856,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of \u003cem\u003ecis\u003c/em\u003e-acting elements in the promoters of the \u003cem\u003eCcTPS \u003c/em\u003egenes. (\u003cstrong\u003eA\u003c/strong\u003e) Distribution of \u003cem\u003ecis\u003c/em\u003e-elements. (\u003cstrong\u003eB\u003c/strong\u003e) Histogram of \u003cem\u003ecis\u003c/em\u003e-elements per \u003cem\u003eCcTPS\u003c/em\u003e gene with functions in plant growth and development, stress responsiveness, or phytohormone responsiveness. (\u003cstrong\u003eC\u003c/strong\u003e) Pie chart of \u003cem\u003ecis\u003c/em\u003e-elements associated with function in plant growth and development. (\u003cstrong\u003eD\u003c/strong\u003e) Pie chart of \u003cem\u003ecis\u003c/em\u003e-elements associated with function in plant stress responsiveness. (\u003cstrong\u003eE\u003c/strong\u003e) Pie chart of \u003cem\u003ecis\u003c/em\u003e-elements associated with function in plant phytohormone responsiveness.\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6432060/v1/9cd5f9d1ef62bf8e3b30aeed.jpeg"},{"id":82506560,"identity":"2732344c-6539-4799-89e7-f0beed5e2b9e","added_by":"auto","created_at":"2025-05-12 09:41:23","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1579296,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiles of\u003cem\u003e \u003c/em\u003efour key genes involved in linalool synthesis. (\u003cstrong\u003eA\u003c/strong\u003e) Transcriptome expression data of four linalool synthesis genes in different tissues. (\u003cstrong\u003eB\u003c/strong\u003e) qRT‒PCR validation of selected genes in linalool synthesis. S, steam; YL, young leaves; OL, old leaves; Re, red fruit.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-6432060/v1/24a7121e192e547eec80bdac.png"},{"id":82506556,"identity":"6e653e9d-989a-4909-aa94-2e6a75c612c3","added_by":"auto","created_at":"2025-05-12 09:41:23","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":97864,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional characterizations of TPS genes contributing to the biosynthesis of linalool. (\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eExpression vectors for \u003cem\u003eCcTPS14\u003c/em\u003e, \u003cem\u003eCcTPS15\u003c/em\u003e, \u003cem\u003eCcTPS16\u003c/em\u003e, and \u003cem\u003eCcTPS32\u003c/em\u003e. (\u003cstrong\u003eB\u003c/strong\u003e) \u003cem\u003eCcTPS14\u003c/em\u003e, \u003cem\u003eCcTPS15\u003c/em\u003e, \u003cem\u003eCcTPS16\u003c/em\u003e, and \u003cem\u003eCcTPS32\u003c/em\u003eprotein expression purification and SDS‒PAGE detection. (\u003cstrong\u003eC\u003c/strong\u003e) \u003cem\u003eCcTPS14\u003c/em\u003e, \u003cem\u003eCcTPS15\u003c/em\u003e, \u003cem\u003eCcTPS16\u003c/em\u003e, and \u003cem\u003eCcTPS32 \u003c/em\u003eenzymatic activity product assays.\u003c/p\u003e","description":"","filename":"image9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6432060/v1/544bba0ceab184d0e4029ff0.jpeg"},{"id":84383191,"identity":"ed74e2df-3318-4299-9f1d-3809d482ccfd","added_by":"auto","created_at":"2025-06-11 09:32:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":42727221,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6432060/v1/54f96a3a-e7e7-47d2-890e-1fbc06c35573.pdf"},{"id":82506929,"identity":"a7844220-d3ac-4cc4-82d1-10e88eda6997","added_by":"auto","created_at":"2025-05-12 09:49:22","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":969673,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-6432060/v1/9275ffa471cb37a1df84e9bb.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification of the genome-wide TPS gene family of Cinnamomum camphora var. linaloolifera and functional validation of linalool","fulltext":[{"header":"Background","content":"\u003cp\u003eTerpenoids are the largest class of natural products produced by plants and play an important role in plant pollination, defence, and information exchange [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. All terpenoids are derived from a limited number of acyclic prenyl pyrophosphate precursors and are categorized according to the number of condensed five-carbon isoprenoid units into hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, triterpenes, and polyterpenes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Terpene synthase genes (TPS) are key enzymes that catalyse these precursors and have two conserved structural domains, the RRX8W arginine domain at the N-terminus and the DDXXD aspartate domain at the C-terminus [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The TPS gene family contains seven subfamilies: TPS-c is the ancestral branch, TPS-d is unique to gymnosperms and encodes monoterpene, sesquiterpene, and diterpene synthases, while TPS-a, TPS-b, and TPS-g are angiosperm-specific and encode monoterpene, sesquiterpene, and diterpene synthases, respectively [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. TPS-e/f is found in both angiosperms and gymnosperms and encodes copalyl diphosphate and ent-kaurene, which are intermediates in the synthesis of the hormone gibberellin [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. TPS-h is only found in \u003cem\u003eSelaginella moellendorffii\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Enabled by the increasing availability of genomic resources, the TPS family has been systematically id entified in various plant species; for example, a large expansion of the TPS gene family was found in the genomes of the terpenoid-rich Lauraceae family, especially a significant expansion of TPS-b and TPS-a [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLinalool is an acyclic monoterpene alcohol with a pleasant aroma that is widely utilized in the fragrance and flavour industries [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The chemical structure of natural linalool exists as two enantiomers, (S)- and (R)-linalool, which have different odours and biological properties [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. It is recognized that the odour of natural linalool is far superior to that of synthetic linalool and that certain drugs can only use natural linalool as a starting material [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Natural linalool is more economically valuable in the flavour market due to its \"natural\" label, which is in line with emerging consumer preferences [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, the low production of linalool products from natural plant tissues results in a shortage of natural linalool in the market [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. \u003cem\u003eC. camphora\u003c/em\u003e is rich in terpenoids and is the major source of linalool, especially \u003cem\u003ecamphora\u003c/em\u003e var. \u003cem\u003elinaloolifera\u003c/em\u003e, 90% of which is extracted from fresh branches [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In recent years, researchers have explored the molecular mechanism of terpenoid biosynthesis of major components of \u003cem\u003eC. camphora\u003c/em\u003e essential oil, but they have not focused on linalool biosynthesis based on genome data [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently, our research group reported a high-quality chromosome-level reference genome of Nan\u0026rsquo;an 1, a new variety of \u003cem\u003eC. camphora\u003c/em\u003e var. \u003cem\u003elinaloolifera\u003c/em\u003e with high linalool content [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Here, this study performed a comprehensive and systematic genome-wide analysis of the TPS gene family in the Nan\u0026rsquo;an 1 genome to identify the key genes involved in linalool synthesis and to further validate the function of the TPS gene in linalool synthesis.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material\u003c/h2\u003e \u003cp\u003eNan\u0026rsquo;an 1, a new variety of \u003cem\u003eC. camphora\u003c/em\u003e var. \u003cem\u003elinaloolifera\u003c/em\u003e, was grown and planted in the Banlin State-owned Forest Farm in Anxi, Fujian Province (117\u0026deg;57\u0026prime; E, 24\u0026deg;55\u0026prime; N), by Xiamen Peony Fragrance Industry Co., Ltd. Healthy reproductive and nutritional organs with excellent development and no disease or insect damage were randomly sampled in four different directions (east, south, west, and north) and stored in liquid nitrogen for transcriptome sequencing and qRT‒PCR verification. Three relevant biological replicates were generated for each sample.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIdentification of the TPS gene family\u003c/h3\u003e\n\u003cp\u003eTo identify the TPS genes in the Nan\u0026rsquo;an 1 genome, the Hidden Markov Model (HMM) profile of the Terpene_synth domain (PF01397) and the Terpene_synth_c domain (PF03936), downloaded from the Pfam database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.xfam.org\u003c/span\u003e\u003cspan address=\"http://pfam.xfam.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, Pfam 3.0), were used to search the Nan\u0026rsquo;an 1 genome. All of the proteins with E-values lower than 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e were selected [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Using \u003cem\u003eArabidopsis\u003c/em\u003e TPS genes as queries, the predicted Nan\u0026rsquo;an 1 TPS genes (\u003cem\u003eCcTPSs\u003c/em\u003e) were checked by BLASTP searches (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. cgi). Then, the predicted TPS models detected were examined manually. Candidate \u003cem\u003eCcTPS\u003c/em\u003e members were deredundantly confirmed using NCBI-CDD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/cdd\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/cdd\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Pfam (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.xfam.org/\u003c/span\u003e\u003cspan address=\"http://pfam.xfam.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for conserved structural domains. Finally, 46 TPS genes (\u003cem\u003eCcTPS\u003c/em\u003e) were identified in the Nan\u0026rsquo;an 1 genome.\u003c/p\u003e \u003cp\u003eProtParam (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.ExPASy.org/protparam\u003c/span\u003e\u003cspan address=\"https://web.ExPASy.org/protparam\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to calculate the physicochemical properties[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Secondary structural analysis was performed by using SOPMA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html\u003c/span\u003e\u003cspan address=\"https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Plant-mPLoc (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.csbio.sjtu.edu.cn/bioinf/plant-multi/\u003c/span\u003e\u003cspan address=\"http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and SignalP v4.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cbs.dtu.dk/services/SignalP-4.1/\u003c/span\u003e\u003cspan address=\"http://www.cbs.dtu.dk/services/SignalP-4.1/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] were used for subcellular localization and signal peptide prediction, respectively, and the TPS gene function prediction was used for website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.nipgr.ac.in/terzyme.html\u003c/span\u003e\u003cspan address=\"http://www.nipgr.ac.in/terzyme.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eAnalysis of chromosomal localization and multiple sequence alignment\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eCcTPS\u003c/em\u003e genes were subjected to multiple sequence alignment using the MUSCLE program, and conserved structural domains were displayed using Genedoc. Chromosome location analysis was visualized using MapChart software [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. BLASTP was used for all \u003cem\u003eCcTPS\u003c/em\u003e proteins for comparison, and MCScanX was used for duplication events [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. KaKs2 Calculator 2.0 was used to calculate the duplicate gene kaks values and gene divergence times [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eAnalysis of the correlations, gene structure, and conserved motifs\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eCcTPS\u003c/em\u003e genes were analysed for correlations using the R package corrplot. The maximum value for motif prediction was set to 15 when the \u003cem\u003eCcTPS\u003c/em\u003e genes were predicted at the MEME website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. GSDS (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds.gao-lab.org/index.php\u003c/span\u003e\u003cspan address=\"http://gsds.gao-lab.org/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to display the structural information of the \u003cem\u003eCcTPS\u003c/em\u003e genes [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eAnalysis of cis-acting elements and phylogenetic trees\u003c/h3\u003e\n\u003cp\u003eMUSCLE software was used to align the sequences of the TPS proteins from Nan\u0026rsquo;an 1 (\u003cem\u003eC. camphora\u003c/em\u003e var. \u003cem\u003elinaloolifera\u003c/em\u003e) and six other plants with known genomes (\u003cem\u003eSelaginella moellendorffii\u003c/em\u003e, \u003cem\u003eAbies grandis\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eCinnamomum kanehirae\u003c/em\u003e, \u003cem\u003ePopulus trichocarpa\u003c/em\u003e, \u003cem\u003eA. thaliana\u003c/em\u003e) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. IQ-TREE software was used to construct an ML tree with the JTT model, and the bootstrap value was 1000 replicates [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The phylogenetic tree was polished using Evoview (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://evolgenius.info//evolview-v2/\u003c/span\u003e\u003cspan address=\"https://evolgenius.info//evolview-v2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. PlantCARE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] was used to extract the 2000 bp upstream of \u003cem\u003eCcTPS\u003c/em\u003e promoters for \u003cem\u003ecis\u003c/em\u003e-acting element analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTranscriptome analysis of the\u003c/b\u003e \u003cb\u003eCcTPS\u003c/b\u003e \u003cb\u003egene family\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHigh-quality RNA was extracted from different tissues of Nan\u0026rsquo;an 1, including young leaves, mature leaves, stems, young fruits (green fruits), mature fruits (red fruits), and later ripening fruits (purple fruits), for transcriptome sequencing. The clean transcriptome sequencing data were assembled using Trinityc [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] (min_kmer_cov set to 2, other parameters default), with the Nan\u0026rsquo;an 1 genome as the reference genome. RSEM [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and DESeq2 were used to obtain FPKM values and to conduct gene differential expression analysis. The FPKM values of \u003cem\u003eCcTPS\u003c/em\u003es in different tissues were visualized by using TBtools software [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eqRT‒PCR evaluation\u003c/h2\u003e \u003cp\u003eA total plant RNA kit (Polysaccharide Polyphenol Plant Total RNA Extraction Kit, developed by Hangzhou Bo Ri Technology Co., Ltd.) was used to extract RNA from stems, young leaves, mature leaves, and mature fruits of Nan\u0026rsquo;an 1, and the quality RNA was detected by electrophoresis. The qualified RNA was used as a template to construct the RT reaction solution according to the TransScript\u0026reg; One-Step gDNA Removal and cDNA Synthesis SuperMix instructions. Twenty microlitres of the whole reaction system was utilized, inactivated at 85\u0026deg;C for 5 seconds to produce cDNA, and then kept at -20\u0026deg;C. For qRT‒PCR, the following reaction parameters were chosen: 94\u0026deg;C for 30 s; 94\u0026deg;C for 5 s; 60\u0026deg;C for 30 s; 40 cycles; 72\u0026deg;C for 10 s; 95\u0026deg;C for 15 s; 60\u0026deg;C for 1 min; and 95\u0026deg;C for 1 s. The relative expression of each target gene in the samples was then determined using \u003cem\u003eCcEF1a\u003c/em\u003e as the internal reference gene (Supplementary Table\u0026nbsp;1). Each reaction technique included three biological replicates, and each biological replicate included three technical replications.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProkaryotic expression of CcTPS14, CcTPS15, CcTPS16 and CcTPS32 in E. coli and detection by GC‒MS\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eProkaryotic expression of CcTPS14, CcTPS15, CcTPS16 and CcTPS32 in E. coli and detection by GC‒MS\u003c/div\u003e \u003cp\u003eThe ORFs of \u003cem\u003eCcTPS14\u003c/em\u003e, \u003cem\u003eCcTPS15\u003c/em\u003e, \u003cem\u003eCcTPS16\u003c/em\u003e and \u003cem\u003eCcTPS32\u003c/em\u003e were isolated and ligated into the pET32a vector BglII XhoI sites. The recombinant plasmids and the absence plasmids were all transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) pLysS cells (TransGen, China). Single colonies containing the recombinant plasmid were selected into 3 mL of LB liquid medium (amp resistant) and cultivated in 10 \u0026micro;l tubes at 37℃ with shaking until the concentration of the bacterial solution reached OD600, approximately 0.6\u0026ndash;0.8 h. Then, 0.1 mM IPTG was added to the recombinant \u003cem\u003eCcTPS14\u003c/em\u003e, \u003cem\u003eCcTPS15\u003c/em\u003e, \u003cem\u003eCcTPS16\u003c/em\u003e and \u003cem\u003eCcTPS32\u003c/em\u003e enzymes, and the culture was continued at 16\u0026deg;C with shaking for 16 hours to induce protein expression. The cultivated bacteria were collected by centrifugation at 5000 rpm for 5 min and resuspended in 40 mL of precooled GST equilibrium solution. Ultrasonically crush the bacteria with parameters set to 200 W, 2.5 s, 5 s pause, and 80 cycles. After centrifugation at 10,000 rpm at 4℃ for 5 min, the supernatant and the precipitate were collected. A portion of the supernatant and the precipitate were subjected to SDS‒PAGE, the remaining supernatant was placed in a 50 ml centrifuge tube, and the precipitate was set aside at 4℃.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of CcTPS gene family members and analysis of their physicochemical properties\u003c/h2\u003e \u003cp\u003eA total of 46 \u003cem\u003eCcTPS\u003c/em\u003e genes were identified in the Nan\u0026rsquo;an genome and named \u003cem\u003eCcTPS1\u003c/em\u003e to \u003cem\u003eCcTPS46\u003c/em\u003e based on their location on the chromosome (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Physicochemical property analysis showed that the protein length of the 46 \u003cem\u003eCcTPSs\u003c/em\u003e varied from 306 (\u003cem\u003eCcTPS8\u003c/em\u003e) to 2597 aa (\u003cem\u003eCcTPS30\u003c/em\u003e), the relative molecular masses ranged from 35.00 kDa (\u003cem\u003eCcTPS8\u003c/em\u003e) to 301.08 kDa (\u003cem\u003eCcTPS30\u003c/em\u003e), isoelectric points (pI) ranged from 4.49 (\u003cem\u003eCcTPS17\u003c/em\u003e) to 7.81 (\u003cem\u003eCcTPS39\u003c/em\u003e), and only six \u003cem\u003eCcTPSs\u003c/em\u003e of the PI were greater than seven. Secondary structure prediction revealed an alpha helix between 54.78% (\u003cem\u003eCcTPS10\u003c/em\u003e) and 78.88% (\u003cem\u003eCcTPS14\u003c/em\u003e), beta turns between 2.15% (\u003cem\u003eCcTPS28\u003c/em\u003e) and 7.04% (\u003cem\u003eCcTPS10\u003c/em\u003e), irregular curls between 15.32% (\u003cem\u003eCcTPS14\u003c/em\u003e) and 34.47% (\u003cem\u003eCcTPS1\u003c/em\u003e), and extended chains between 1.58% (\u003cem\u003eCcTPS33\u003c/em\u003e) and 13.18% (\u003cem\u003eCcTPS10\u003c/em\u003e). Except for \u003cem\u003eCcTPS4\u003c/em\u003e, \u003cem\u003eCcTPS9\u003c/em\u003e, and \u003cem\u003eCcTP26\u003c/em\u003e, the remaining \u003cem\u003eCcTPSs\u003c/em\u003e were marked in chloroplasts rather than in the cytoplasm. The prediction result of the signal peptide is that all \u003cem\u003eCcTPS\u003c/em\u003e genes have no signal peptide.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLocalization, physicochemical properties, and secondary structure of \u003cem\u003eCcTPS\u003c/em\u003e genes.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLength\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMW(kDa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003epI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSubcellular localization\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAlpha helix (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eExtended strand\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eBeta turn (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eRandom coil\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eSignal\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00011790\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e850\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e96.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e55.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e6.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e34.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00028622\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e435\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e66.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e26.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00012602\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e528\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e71.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e22.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00012641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e870\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast, Cytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e68.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e21.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS5\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00012720\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e553\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e63.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e71.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e22.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00010115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e558\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e64.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e69.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e24.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS7\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00005406\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e681\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e77.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e65.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e6.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e24.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS8\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00018207\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e306\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e35.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e61.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e33.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS9\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00018214\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e897\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e103.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast, Cytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e68.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e6.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e20.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS10\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00005791\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e126.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e54.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e13.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e7.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS11\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00020936\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e551\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e63.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e67.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e24.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS12\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00007365\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1335\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e153.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e58.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e9.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e26.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00025120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e741\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e85.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e68.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e5.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e20.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS14\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00024100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e483\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e55.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e78.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e15.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS15\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00014813\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e814\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e93.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e 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char=\".\" colname=\"c3\"\u003e \u003cp\u003e496\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e56.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e69.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e6.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e21.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e 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align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS19\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00001514\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e431\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e49.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e70.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e22.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS20\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00001509\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e346\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e 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\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e57.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e9.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e6.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e26.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS22\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00011978\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e546\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e63.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e70.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e21.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS23\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00023025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e558\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e64.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e68.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e24.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS24\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00018905\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e131.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e64.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e6.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e24.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS25\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00018899\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1098\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e127.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e70.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e21.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS26\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00014805\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e475\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e54.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast, Cytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e59.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e8.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e27.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS27\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00014802\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e562\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e65.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e67.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e24.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS28\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00014789\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e465\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e53.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e68.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e24.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS29\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00014796\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e545\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e63.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e66.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e26.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS30\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00014782\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2597\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e301.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e60.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e9.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e6.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e22.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS31\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00014804\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e484\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e56.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e68.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e24.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS32\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00015451\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1074\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e123.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e69.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e22.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS33\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00015350\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e571\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e66.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e67.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e27.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS34\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00017669\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1429\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e164.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e59.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e7.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e28.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS35\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00017748\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e398\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e69.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e22.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS36\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00015605\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1122\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e130.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e68.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e21.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS37\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00015620\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e427\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e49.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e74.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e18.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS38\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00015608\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e737\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e84.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e63.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e6.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS39\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00018575\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e566\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e64.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e61.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e6.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e29.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS40\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00018262\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e540\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e62.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e72.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e20.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS41\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00014305\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e635\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e72.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e64.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e5.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e26.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS42\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00014318\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e443\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e51.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e72.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e19.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS43\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00021750\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e424\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e49.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e72.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e20.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS44\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00022851\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e444\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e51.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e72.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e20.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS45\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00021963\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e607\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e69.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e65.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e27.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS46\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaker00017397\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e448\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e51.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e77.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e16.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis of the CcTPS gene family\u003c/h2\u003e \u003cp\u003eTo reveal the evolutionary relationship of the CcTPS gene family, a phylogenetic tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) was constructed based on the 257 TPS genes from Nan\u0026rsquo;an 1 (46 members), \u003cem\u003eC. kanehirai\u003c/em\u003e (90 members), \u003cem\u003eO. sativa\u003c/em\u003e (31 members), \u003cem\u003eA. thaliana\u003c/em\u003e (33 members), \u003cem\u003eP. trichocarpa\u003c/em\u003e (32 members), \u003cem\u003eA. grandis\u003c/em\u003e (11 members) and \u003cem\u003eS\u003c/em\u003e. \u003cem\u003emoellendorffii\u003c/em\u003e (14 members). All TPSs were classified into seven subfamilies: TPS-a, TPS-b, TPS-c, TPS-d, TPS-e/f, and TPS-g, in which TPS-b underwent a significant expansion. Among them, 46 \u003cem\u003eCcTPS\u003c/em\u003e genes were classified as TPS-a, TPS-b, TPS-c, TPS-e/f, TPS-g, and TPS-b. TPS-b had the most members with 28 members, followed by TPS-a with 12 members, TPS-g with four members, and TPS-e/f with one member each. In addition, functional enrichment suggested that all the members of TPS-a encode sesquiterpene synthase, TPS-b members encode monoterpene synthase, TPS-g encode acyclic monoterpenes, and TPS-c and TPS-e/f encode diterpene synthase (Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMultiple sequence alignment\u003c/h2\u003e \u003cp\u003eMultiple sequence alignment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) suggested that most \u003cem\u003eCcTPS\u003c/em\u003e have three conserved structural domains: RRX8W, RXR, and DDXXD. Among them, the DDXXD domain is the most conserved, with deletions or variants found only in the C-terminus of \u003cem\u003eCcTPS10\u003c/em\u003e and \u003cem\u003eCcTPS8\u003c/em\u003e in TPS-a and in the C-terminus of \u003cem\u003eCcTPS1\u003c/em\u003e in TPS-c. The function of this domain is to bind divalent magnesium ions or manganese ions for substrate cleavage. The RXR domain is second only to the DDXXD domain in conservation, with mutations observed in five genes (TPS-a: \u003cem\u003eCcTPS24\u003c/em\u003e, \u003cem\u003eCcTPS10\u003c/em\u003e; TPS-b: \u003cem\u003eCcTPS35\u003c/em\u003e and \u003cem\u003eCcTPS2\u003c/em\u003e; TPS-c: \u003cem\u003eCcTPS1\u003c/em\u003e). This domain is currently only found in Orchidaceae and \u003cem\u003eMentha longifolia\u003c/em\u003e, and its function is unclear [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The RRX8W domain, which is involved in the synthesis of cyclic monoterpenes [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], shows relative conservation in TPS-a and TPS-b, whereas significant mutations or deletions have occurred in the N-terminus of the rest of the subfamilies, e.g., the RRX8W domain is lost in all members of TPS-g (\u003cem\u003eCcTPS14\u003c/em\u003e, \u003cem\u003eCcTPS15\u003c/em\u003e, \u003cem\u003eCcTPS16\u003c/em\u003e, and \u003cem\u003eCcTPS32\u003c/em\u003e). This implies that TPS-g members are involved in the synthesis of acyclic monoterpenes.s\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGene structure and conserved motif analysis\u003c/h2\u003e \u003cp\u003eTo investigate the diversity of motif components among \u003cem\u003eCcTPS\u003c/em\u003e, the motif distribution in 46 \u003cem\u003eCcTPS\u003c/em\u003e proteins was investigated using the online tool MEME program (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). A total of 15 conserved motifs were identified, of which 13 motifs appeared in the TPS-b subfamily and six motifs appeared in the TPS-c subfamily. Among these motifs, motifs 3, 4, and 13 were found to be more conserved and retained in most \u003cem\u003eCcTPSs\u003c/em\u003e. Combined with the results of multiple sequence alignment analysis, motifs 3, 4, and 13 corresponding to DDXXD, RXR, and RRX8W were identified, respectively. It is important to note that most members of TPS-b possess the conserved motif 13 (RRX8W domain), which plays an essential role in the catalysis of monoterpene cyclization. Conversely, all members of TPS-g lack motif 13 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Furthermore, motif 13 and motif 8 are the conserved domains of terpene synthase (PF01397) at the N-terminus, while motif 3 and motif 2 are the conserved domains of terpene synthase C (PF03936) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGenes with more variable members of introns and exons have more complex structures [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Gene structure analysis showed that the number of exons in the \u003cem\u003eCcTPS\u003c/em\u003e genes ranged from five to 35, and the number of introns ranged from four to 34 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Most members of the subfamily contain the same number of introns and exons, but \u003cem\u003eCcTPS30\u003c/em\u003e in the TPS-b group has the highest number of exons and introns, and \u003cem\u003eCcTPS40\u003c/em\u003e has the lowest number of exons and introns. The untranslated regions (UTRs) interact with RNA-binding proteins involved in posttranscriptional regulation of genes [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Only \u003cem\u003eCcTPS28\u003c/em\u003e (TPS-a), \u003cem\u003eCcTPS13\u003c/em\u003e (TPS-b), \u003cem\u003eCcTPS3\u003c/em\u003e (TPS-g), and \u003cem\u003eCcTPS32\u003c/em\u003e (TPS-g) contain UTRs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eChromosomal localization and synteny analysis\u003c/h2\u003e \u003cp\u003e46 \u003cem\u003eCcTPS\u003c/em\u003e genes were unevenly distributed throughout seven chromosomes and one scaffold (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The largest number of \u003cem\u003eCcTPS\u003c/em\u003e genes (13 members) was located on chromosome 10, followed by chromosome 3 (eight members) and chromosome 7 (seven members). In contrast, chromosomes 1, 2 and 12 displayed only one \u003cem\u003eCcTPS\u003c/em\u003e gene. The synteny analysis suggested that the \u003cem\u003eCcTPSs\u003c/em\u003e had seven segmental duplication gene pairs (\u003cem\u003eCcTPS32\u003c/em\u003e and \u003cem\u003eCcTPS14\u003c/em\u003e; \u003cem\u003eCcTPS38\u003c/em\u003e and \u003cem\u003eCcTPS42\u003c/em\u003e; \u003cem\u003eCcTPS38\u003c/em\u003e and \u003cem\u003eCcTPS43\u003c/em\u003e; \u003cem\u003eCcTPS36\u003c/em\u003e and \u003cem\u003eCcTPS46\u003c/em\u003e; \u003cem\u003eCcTPS38\u003c/em\u003e and \u003cem\u003eCcTPS18\u003c/em\u003e; \u003cem\u003eCcTPS42\u003c/em\u003e and \u003cem\u003eCcTPS19\u003c/em\u003e; and \u003cem\u003eCcTPS41\u003c/em\u003e and \u003cem\u003eCcTPS18\u003c/em\u003e) and one tandem duplication gene pair (\u003cem\u003eCcTPS27\u003c/em\u003e and \u003cem\u003eCcTPS28\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Interestingly, the segmental duplication genes are from the TPS-b subfamily, and the tandem duplication genes are from the TPS-a subfamily. The \u003cem\u003eK\u003c/em\u003ea/\u003cem\u003eK\u003c/em\u003es ratio of these gene pairs was less than one, indicating that these genes underwent significant purifying selection during their evolution. The calculated divergence time of segmental duplication and tandem duplication gene pairs was approximately 3.69 Mya\u0026thinsp;~\u0026thinsp;71.16 Mya (million years ago), of which six gene pairs (85%) experienced α events and one gene pair (15%) underwent β events (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results indicate that the duplication events of the \u003cem\u003eCcTPS\u003c/em\u003e genes are mainly caused by α events.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe differentiation time and gene duplication events of the \u003cem\u003eCcTPS\u003c/em\u003e gene pairs.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDuplicated gene pairs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDuplicated gene pairs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKa/Ks\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePurify selcetion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDuplicated type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eTime (MYA)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDuplication event\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS38\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCcTPS42\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.058870266\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.067280307\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.874999963\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWGD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.6967\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCc-α\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS38\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCcTPS43\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.008641966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.038505247\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.224436068\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWGD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.1157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCc-α\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS36\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCcTPS46\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.386682802\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.29511625\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.298569956\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWGD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e71.1602\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCc-β\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS38\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCcTPS18\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.112363873\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.21134456\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.531662006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWGD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e11.6123\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCc-α\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS42\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCcTPS19\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.052975454\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.085371247\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.620530398\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWGD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.6907\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCc-α\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS41\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCcTPS18\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.025626409\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.091066749\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.281402475\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWGD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5.0037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCc-α\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcTPS27\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCcTPS28\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.058640445\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.17472081\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.335623699\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003etandem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e~\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e~\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo further infer the phylogenetic mechanisms of the \u003cem\u003eCcTPSs\u003c/em\u003e, this study constructed a comparative collinear relationship of \u003cem\u003eC. camphora var. linaloolifera\u003c/em\u003e (Nan\u0026rsquo;an 1) associated with five magnoliid plants (\u003cem\u003eMagnolia biondii, Piper nigrum, C. kanehirae, Chimonanthus salicifolius and Aristolochia fimbriata\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). There were 14 TPS gene pairs in \u003cem\u003eC. camphora var. linaloolifera\u003c/em\u003e and \u003cem\u003eC. kanehirae\u003c/em\u003e, 15 in \u003cem\u003eM\u003c/em\u003e. \u003cem\u003ebiondii\u003c/em\u003e, nine in \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esalicifolius\u003c/em\u003e, four in \u003cem\u003eP. nigrum\u003c/em\u003e and one in \u003cem\u003eA. fimbriata\u003c/em\u003e (Supplementary Table\u0026nbsp;3). These orthologous gene pairs have a \u003cem\u003eK\u003c/em\u003ea\u003cem\u003e/K\u003c/em\u003es ratio of less than one, indicating that the TPS genes of magnoliid plants underwent strong purifying selection during evolution. The study further calculated the divergence time of gene pairs between \u003cem\u003eC. camphora var. linaloolifera\u003c/em\u003e and the other four magnoliid plants and found that the divergence time of \u003cem\u003eC. camphora var. linaloolifera\u003c/em\u003e and \u003cem\u003eC. kanehirae\u003c/em\u003e (2.56 Mya\u0026thinsp;~\u0026thinsp;136.79 Mya) was later than that of \u003cem\u003eC. camphora var. linaloolifera\u003c/em\u003e and other plants (the divergence time of \u003cem\u003eC. camphora var. linaloolifera\u003c/em\u003e and \u003cem\u003eM. biondii\u003c/em\u003e was 53 Mya\u0026thinsp;~\u0026thinsp;158.44 Mya; \u003cem\u003ethat of P. nigrum\u003c/em\u003e was 57.81\u0026thinsp;~\u0026thinsp;209.27 Mya; that of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esalicifolius\u003c/em\u003e was 48.72 Mya\u0026thinsp;~\u0026thinsp;139.23 Mya; and that of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efimbriata\u003c/em\u003e was 189.03 Mya) (Supplementary Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eExpression correlation analysis among CcTPS members\u003c/h2\u003e \u003cp\u003eThe Pearson product-moment correlation coefficient (pcc) value of \u003cem\u003eCcTPS27\u003c/em\u003e/\u003cem\u003eCcTPS28\u003c/em\u003e (TPS-a) of the tandem duplication gene pair was 0.89 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Among the seven segmental duplication gene pairs, the expression profiles of five gene pairs were positively correlated (\u003cem\u003eCcTPS38\u003c/em\u003e/\u003cem\u003eCcTPS42\u003c/em\u003e: 0.55; \u003cem\u003eCcTPS38\u003c/em\u003e/\u003cem\u003eCcTPS43\u003c/em\u003e: 0.02; \u003cem\u003eCcTPS38\u003c/em\u003e/\u003cem\u003eCcTPS46\u003c/em\u003e: 0.037; \u003cem\u003eCcTPS38\u003c/em\u003e/\u003cem\u003eCcTPS18\u003c/em\u003e: 0.036; \u003cem\u003eCcTPS42\u003c/em\u003e/\u003cem\u003eCcTPS19\u003c/em\u003e: 0.88), and the expression levels of one gene pair were negatively correlated (\u003cem\u003eCcTPS32\u003c/em\u003e/\u003cem\u003eCcTPS14\u003c/em\u003e: -0.062). Since most of the segmental duplications in \u003cem\u003eCcTPSs\u003c/em\u003e displayed positive correlations, it was likely that the segmental genes functioned in collaboration to work together.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of promoter cis-acting elements\u003c/h2\u003e \u003cp\u003eTo ascertain the potential biological functions of the \u003cem\u003eCcTPS\u003c/em\u003e genes, \u003cem\u003ecis\u003c/em\u003e-acting element analysis was performed. A total of 1,169 \u003cem\u003ecis\u003c/em\u003e-acting regulatory elements were identified in the \u003cem\u003eCcTPS\u003c/em\u003e gene promoter, and these \u003cem\u003ecis\u003c/em\u003e-elements can be divided into three categories: plant growth and development, stress responsiveness, and hormone responsiveness (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). The first type of \u003cem\u003ecis\u003c/em\u003e-element related to plant growth and development mainly contained the following ten categories: AAGAA motif (16.03%) and GCN4 motif (6.13%) related to endosperm development; AT-rich element (3.30%), CCAAT box (10.37%), and MRE (11.32%) related to flowering regulation; CAT box (15.09%) related to meristem; RY element (3.83%) related to seed development; as-1 (17.92%) related to bud development; O2 site (10.37%) related to zein metabolism; and circadian (6.60%) related to circadian rhythm (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). The second type of \u003cem\u003ecis-\u003c/em\u003eelement was related to stress response elements, such as MYC (48.02%), ARE (29.26%), STRE (35.62%), MBS (8.39%), LTR (7.88%), DRE1 (0.76%), WUN (5.34%), WRE3 (10.68%), and TC-rich repeats (2.03%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Among them, MYC responded to MeJA, MBS responded to drought induction, LTR responded to low temperature stress, WUN is related to injury response, and WRE3 and TC-rich repeats are related to plant defence. The last type of \u003cem\u003ecis\u003c/em\u003e-element is mainly involved in phytohormone responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). ABREs (12.54%) are associated with abscisic acid. The GARE motif (2.86%), P-box (5.37%) and TATC-box (2.86%) are related to gibberellin synthesis. The TCA element (5.01%) is related to growth. The TGACG motif (6.81%) and MYC motif (48.02%) are related to methyl jasmonate synthesis. EREs (6.98%) are related to ethylene. These results indicated that the TPS genes may be regulated by a variety of hormones and that they respond to multiple abiotic stresses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCcTPS\u003c/b\u003e \u003cb\u003egene expression patterns in different tissues\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo understand the expression patterns of TPS genes in different tissues of Nan\u0026rsquo;an 1, the study performed an RNA transcription analysis on young leaves (YL), mature leaves (ML), stems (St), young fruits (green fruit; fruit I), mature fruits (red fruit; fruit II), and late ripening fruits (purple fruit; fruit III) of Nan\u0026rsquo;an 1 (Supplementary Fig.\u0026nbsp;1; Supplementary Table\u0026nbsp;4). The expression results showed that except for \u003cem\u003eCcTPS2\u003c/em\u003e and \u003cem\u003eCcTPS31\u003c/em\u003e, the remaining \u003cem\u003eCcTPS\u003c/em\u003es were expressed in stems, young leaves, and mature leaves. The stems and young leaves contained 31 (70.45%) and 32 (72.72%) highly expressed \u003cem\u003eCcTPS\u003c/em\u003e genes, respectively. A total of 21 (45.1%) \u003cem\u003eCcTPS\u003c/em\u003e genes were expressed at three different fruit development stages. With fruit development, the expression of 14 \u003cem\u003eCcTPS\u003c/em\u003e genes was downregulated, and the expression of four \u003cem\u003eCcTPS\u003c/em\u003e genes was upregulated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eValidation of the expression of key genes involved in linalool synthesis\u003c/h2\u003e \u003cp\u003eIn addition, based on our gene structure and annotation analysis of the \u003cem\u003eCcTPS\u003c/em\u003e genes described above, the study inferred that four genes of the TPS-g subfamily are key enzymes in the final step of linalool synthesis. The four TPS-g members of Nan\u0026rsquo;an 1 exhibited two distinct expression patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA; Supplementary Table\u0026nbsp;4). Among these genes, \u003cem\u003eCcTPS14\u003c/em\u003e, \u003cem\u003eCcTPS15\u003c/em\u003e and \u003cem\u003eCcTPS16\u003c/em\u003e were highly expressed in stems and mature fruits and were also expressed in young leaves but not in old leaves. \u003cem\u003eCcTPS32\u003c/em\u003e was highly expressed in stems, old leaves, and young fruits but not in other tissues. Further validated \u003cem\u003eCcTPS14\u003c/em\u003e, \u003cem\u003eCcTPS15\u003c/em\u003e, \u003cem\u003eCcTPS16\u003c/em\u003e and \u003cem\u003eCcTPS32\u003c/em\u003e in the TPS-g subfamily in stems, leaves, and roots was achieved through qRT‒PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). The results showed that \u003cem\u003eCcTPS14\u003c/em\u003e, \u003cem\u003eCcTPS15\u003c/em\u003e, and \u003cem\u003eCcTPS16\u003c/em\u003e had the highest expression in the stem, followed by the young leaves, and \u003cem\u003eCcTPS32\u003c/em\u003e had the highest expression in the old leaves, followed by the stem. The qRT‒PCR results were consistent with the transcriptome expression results.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eValidation of functional genes for linalool synthesis\u003c/h2\u003e \u003cp\u003eAfter four linalool synthesis genes, \u003cem\u003eCcTPS14\u003c/em\u003e, \u003cem\u003eCcTPS15\u003c/em\u003e, \u003cem\u003eCcTPS16\u003c/em\u003e and \u003cem\u003eCcTPS32\u003c/em\u003e were cloned, the expression vectors were constructed, and the four genes were successfully ligated into the constructed expression vectors and fused with His tags by enzymatic digestion experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). To verify the catalytic activity of the four linalool synthesis genes and the products generated, the study adsorbed the proteins on a nickel column and then eluted the purified proteins by SDS‒PAGE. The results showed that the molecular weights of the soluble proteins of \u003cem\u003eCcTPS14\u003c/em\u003e, \u003cem\u003eCcTPS15\u003c/em\u003e, \u003cem\u003eCcTPS16\u003c/em\u003e and \u003cem\u003eCcTPS32\u003c/em\u003e were 56, 94, 60 and 58 kDa, respectively, which were consistent with the predictions by the software, implying that enzyme activity experiments could be performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLinalool is an acyclic monoterpene, so the study detected the activity of the recombinant protein by adding GPP, the substrate needed for monoterpene production, and detected the enzymatic activity products by GC‒MS (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). \u003cem\u003eCcTPS14\u003c/em\u003e, \u003cem\u003eCcTPS15\u003c/em\u003e, \u003cem\u003eCcTPS16\u003c/em\u003e, and \u003cem\u003eCcTPS32\u003c/em\u003e were able to produce linalool at levels ranging from 30.14\u0026ndash;36.60% (Supplementary Tables\u0026nbsp;5\u0026ndash;8). Since one gene can catalyse the generation of multiple products from the same substrate, the study found that \u003cem\u003eCcTPS14\u003c/em\u003e also produced the monoterpene Geranial, and \u003cem\u003eCcTPS15\u003c/em\u003e, \u003cem\u003eCcTPS16\u003c/em\u003e and \u003cem\u003eCcTPS32\u003c/em\u003e produced monoterpenes such as Thujene and D-Limonene.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eC. camphora\u003c/em\u003e belongs to the family Lauraceae and is an aromatic tree with important economic and ecological value. According to the main volatile components of its leaf essential oils, \u003cem\u003eC. camphora\u003c/em\u003e can be subdivided into linalool, borneol, camphor, cineole, and nerolidol types [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. \u003cem\u003eC. camphora var. linaloolifera\u003c/em\u003e belongs to the linalool type, is native to China and is the main source of natural linalool. Natural linalool is in short supply in the market due to its optical activity, its far superior odour to synthetic linalool, and the fact that certain drugs can only use natural linalool as a starting material [\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Our group successfully cultivated a new cultivar of \u003cem\u003eC. camphora var. linaloolifera\u003c/em\u003e, Nan'an1, whose branch and leaf essential oil rate is as high as 3.07%, and 90% of the essential oil is linalool, which is much higher than that of the other \u003cem\u003eC. camphora var. linaloolifera\u003c/em\u003e (0.173%~2.668%) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Therefore, Nan'an1 is the best material for studying natural linalool biosynthesis.\u003c/p\u003e \u003cp\u003eBiosynthetic biology offers a more effective and reasonable solution for the production of natural chemicals [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Transcriptome sequencing analysis has become the premier means for researchers to explore the biosynthesis of secondary metabolites, but the technique may miss functional genes that are active at specific times and does not provide insight into their evolution and improve their production [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The whole-genome sequence provides access to comprehensive genetic resources and compensates for the shortcomings of the transcriptome. The combined mode of transcriptomics and genomics is currently a popular trend for elucidating the biosynthetic pathways of natural products. Based on the whole-genome sequence at the chromosome level of Nan'an 1 reported by our group [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], a total of 46 \u003cem\u003eCcTPS\u003c/em\u003e genes were identified, much less than the number of TPS in other Lauraceae species. The reason for this is that the screening of TPS genes in this study was conditioned on the retention of structural domains containing both terpene_synthase (PF01397) and terpene_synthase_C (PF03936) domains, whereas genes with one of these domains were retained in other studies. This shows that the \u003cem\u003eCcTPS\u003c/em\u003e genes this study identified are more accurate.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis classified the 46 \u003cem\u003eCcTPSs\u003c/em\u003e into five subfamilies, including TPS-a (12 members), TPS-b (28 members), TPS-c (one member), TPS-e/f (one member), and TPS-g (four members). A significant expansion of the TPS-b subfamily occurred, consistent with results observed in other Lauraceae, which may favour the biosynthesis and accumulation of aromatic terpenes in plants of this family. Seven segment duplication gene pairs were identified in the \u003cem\u003eCcTSP\u003c/em\u003e gene family of Nan\u0026rsquo;an 1, and all were from the TPS-b subfamily. Of these, six gene pairs were generated by α events, and one gene pair was generated by β events. These results suggested that segment duplication events and palaeopolyploidy events are major drivers of the expansion of the TPS-b branch in the Nan\u0026rsquo;an 1 CcTPS gene family.\u003c/p\u003e \u003cp\u003eLinalool is an acyclic monoterpene [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Functional annotation results showed that all members of TPS-b and TPS-g were annotated as monoterpene synthases. In this study, it also found that the gene structures of TPS-b and TPS-g are similar and that TPS-g is a clade closely related to TPS-b. These findings are consistent with those of previous studies [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The domains of genes in the same subfamily are roughly similar, and in long-term evolution, the variation or loss of the domains of some genes may form a new subfamily [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The plant TPS genes contain the conserved domains RRX8W, RXR and DXXD [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], of which the function of the RRX8W domain primarily performs cyclic isomerization [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Interestingly, the RRX8W domain was completely lost in members of the TPS-g subfamily. Thus, the study speculate that the TPS-g group may be a subgroup of branches derived from the TPS-b branch that lost the RRX8W domain during evolution and inherited the function of linalool synthesis.\u003c/p\u003e \u003cp\u003eIn addition, \u003cem\u003eAtTPS14\u003c/em\u003e is a crucial gene for the production of linalool in \u003cem\u003eA. thaliana\u003c/em\u003e [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The phylogenetic tree showed that \u003cem\u003eAtTPS14\u003c/em\u003e clustered with four genes (\u003cem\u003eCcTPS14\u003c/em\u003e, \u003cem\u003eCcTPS15\u003c/em\u003e, \u003cem\u003eCcTPS16\u003c/em\u003e, and \u003cem\u003eCcTPS32\u003c/em\u003e) of TPS-g, further implying that these genes are involved in linalool synthesis. The content of essential oils (approximately 95% of terpenoids) produced by fresh stems (3.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17%) and leaves (2.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02%) of \u0026lsquo;Nan\u0026rsquo;an 1\u0026rsquo; is significantly higher than that produced by fresh fruits (0.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%), and the content of linalool in essential oils is up to 90.43% [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Expression analysis showed that the four linalool synthesis genes were highly expressed mainly in stems and leaves, with \u003cem\u003eCcTPS14\u003c/em\u003e, \u003cem\u003eCcTPS15\u003c/em\u003e, and \u003cem\u003eCcTPS16\u003c/em\u003e having the highest expression in stems and \u003cem\u003eCcTPS32\u003c/em\u003e having the highest expression in mature leaves. Gene function validation showed that \u003cem\u003eCcTPS14\u003c/em\u003e, \u003cem\u003eCcTPS15\u003c/em\u003e, \u003cem\u003eCcTPS16\u003c/em\u003e and \u003cem\u003eCcTPS32\u003c/em\u003e were all able to produce linalool with more than 30% linalool content. Ultimately, this study demonstrated that four genes of the TPS-g subfamily play pivotal roles in linalool biosynthesis and accumulation in \u003cem\u003eC. camphora var. linaloolifera\u003c/em\u003e, which provides a basis for an in-depth understanding of the molecular mechanisms of terpenoid formation in \u003cem\u003eC. camphora\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this work, it conducted a comprehensive and systematic genome-wide analysis of the TPS gene family in Nan\u0026rsquo;an 1, a new variety of \u003cem\u003eC. camphora var. linaloolifera\u003c/em\u003e with high linalool content. A total of 46 TPS genes were identified, and their promoters contained numerous stress \u003cem\u003ecis\u003c/em\u003e-acting elements and hormone \u003cem\u003ecis\u003c/em\u003e-acting elements, suggesting that \u003cem\u003eCcTPS\u003c/em\u003es play an important role in plant defence and information exchange. The phylogenetic results showed that the \u003cem\u003eCcTPSs\u003c/em\u003e were categorized into five subfamilies, of which the TPS-b subfamily was significantly expanded due to segment duplication events and palaeopolyploidy events. TPS-b and TPS-g clustered together, and their members were mainly involved in monoterpene synthesis. Notably, the RRX8W domain involved in cyclic isomerization is conserved in most members of TPS-b but is lost in all members of TPS-g. Domain variations such as deletions or mutations promote the creation of a new subfamily. Therefore, this study suggest that some members of the ancient TPS-b subfamily lost the RRX8W domain during evolution and may have formed the TPS-g subfamily, which is mainly involved in the synthesis of acyclic monoterpenes. The content of essential oil extracted from the fresh stems and leaves of \u0026lsquo;Nan 'an 1\u0026rsquo; was much higher than that from the fresh fruit, and linalool was the main component in the essential oil. The results of expression analysis and qRT‒PCR showed that the four linalool synthesis genes (\u003cem\u003eCcTPS14\u003c/em\u003e, \u003cem\u003eCcTPS15\u003c/em\u003e, \u003cem\u003eCcTPS16\u003c/em\u003e, and \u003cem\u003eCcTPS32\u003c/em\u003e) from TPS-g were highly expressed in stems and leaves. In vitro enzyme activity verification proved that these four key genes can produce high levels of linalool. In conclusion, the study screened four key genes for linalool synthesis in the Nan\u0026rsquo;an 1 genome by biological information and gene functional validation. These novel results provide a basis for the subsequent functional validation of the linalool type of other Lauraceae plants and have significant biological significance for promoting the development of the linalool type plant industry and for high linalool breeding, providing a good molecular genetic foundation.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eSupplemental information\u003c/h2\u003e \u003cp\u003eAdditional supporting information can be found online in the Supporting Information section at the end of this article.\u003c/p\u003e \u003c/div\u003e\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e Nan'an 1 material were obained from the Banlin State-owned Forest Farm (117\u0026deg;57\u0026prime; E, 24\u0026deg;55\u0026prime; N) in Anxi, Fujian Province, and Professor Shuang-Quan zou ensured that we obtained permission for the collection the Nan'an 1 and formally identified the plant material used in the research.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe current study was supported by Fujian forestry science and technology plan project salt-tolerance mechanism of \u003cem\u003eCinnamomum camphor\u003c/em\u003e and its application in coastal mountain afforestation (2024FKJ25) and application demonstration of \u003cem\u003eCinnamomum camphor\u003c/em\u003e seed in coastal ecological landscape forest transformation (2024TG08).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSZ: conceived \u0026amp; designed the research. WS, WL and YL: performed the bioinformatics analysis, data curation \u0026amp; writing; WH and ZZ: helped with collecting samples \u0026amp; field work. WS: writing-review \u0026amp; editing. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll raw transcriptome sequences described in this manuscript have been submitted to the BioProject/GSA, National Genomics Data Center (NGDC) PRJCA002001/CRA004220.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePichersky E, Gershenzon J. The formation and function of plant volatiles: perfumes for pollinator attraction and defense. 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Mol Genet Genomics. 2002;267:730\u0026ndash;45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1007/s00438-002-0709-y\u003c/span\u003e\u003cspan address=\"10.1007/s00438-002-0709-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Linalool biosynthesis, Terpene synthase (TPS) gene family, Cinnamomum camphora var. linaloolifera, RRX8W domain evolution","lastPublishedDoi":"10.21203/rs.3.rs-6432060/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6432060/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003e\u003cem\u003eCinnamomum camphora\u003c/em\u003e var. \u003cem\u003elinaloolifera \u003c/em\u003eis known for its richness in linalool, which is an acyclic monoterpene widely used in the fragrance and flavour industries. However, limited information is available regarding the genome-wide identification and characterization of the key genes for linalool synthesis in \u003cem\u003eC. camphora var. linaloolifera\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e Here, based on the whole genome data of Nan’an 1, a variety of \u003cem\u003eC. camphora var. linaloolifera\u003c/em\u003e, a total of 46 \u003cem\u003eCcTPS\u003c/em\u003egenes were identified and were classified into five subfamilies, among which TPS-b (28 members) and TPS-g (four members) clustered closely together. Functional annotation results indicated that all members of the TPS-b and TPS-g groups were associated with the synthesis of monoterpenes. Multiple sequence alignment analysis results showed that the RRX8W domain, which is mainly involved in cyclic isomerization, is relatively conserved in TPS-b and completely lost in TPS-g, suggesting that genes in the TPS-g subfamily are involved in the biosynthesis of acyclic monoterpenes. Expression analysis revealed that the expression levels of \u003cem\u003eCcTPS14\u003c/em\u003e, \u003cem\u003eCcTPS15\u003c/em\u003e, \u003cem\u003eCcTPS16\u003c/em\u003eand \u003cem\u003eCcTPS32\u003c/em\u003e from TPS-g subfamily were higher in stems and leaves than in fruits. Further results of gene function validation confirmed that these four genes all produced linalool at levels higher than 30%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion \u003c/strong\u003eour research found that some of the ancient members of TPS-b resulted from the loss of the RRX8W structural domain responsible for cyclic isomerization, resulting in the TPS-g subfamily, which is responsible for the biosynthesis of the acyclic monoterpene linalool. Theresults provide a basis for further exploration of linalool biosynthesis and accumulation.\u003c/p\u003e","manuscriptTitle":"Identification of the genome-wide TPS gene family of Cinnamomum camphora var. linaloolifera and functional validation of linalool","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-12 09:41:17","doi":"10.21203/rs.3.rs-6432060/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f876fb68-aeb0-49f8-a9cc-7dc1e907f392","owner":[],"postedDate":"May 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-11T09:24:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-12 09:41:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6432060","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6432060","identity":"rs-6432060","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}