Identification and Characterization of A Lanosterol synthase Gene from Sanghuangporus Baumii

Identification and Characterization of A Lanosterol synthase Gene from Sanghuangporus Baumii | 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 and Characterization of A Lanosterol synthase Gene from Sanghuangporus Baumii XuTong Wang, TingTing Sun, Jian Sun, Zengcai Liu, Li Zou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-203582/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 Lanosterol synthase (LS) is a key enzyme involved in the mevalonate pathway (MVA pathway) to produce lanosterol, which is a precursor for synthesizing Sanghuangporus baumii triterpenoids. To research the characteristics and construction of LS , LS ORF and promoter were cloned from S. baumii. A 2,445 bp S. baumii LS sequence was obtained by rapid amplification of cDNA ends (RACE) technology and recombinant PCR. S. baumii LS sequence includes a 5’-untranslated region (129 bp), a 3’-untranslated region (87 bp), and an open reading frame (2,229 bp) encoding a 734 amino acids. The molecular weight of LS is 84.99 kDa, and transcription start site of S. baumii LS promoter sequence ranged from 1 740 bp to 1790 bp. LS promoter contained 12 CAAT-boxes, 5 ABREs, 6 G-Boxes, 6 CGTCA-motifs, and so on. The S. baumii LS protein was expressed in E. coli BL21 (DE3) (84.99 kDa + 21.15 kDa tag protein). The transcription level of S. baumii LS was the highest on day 11 in mycelia (1.6-fold). Applied Biochemistry Biotechnology and Bioengineering Lanosterol synthase Promoter Sanghuangporus baumii Triterpenoids Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Sanghuangporus baumii , a traditional Chinese medicine, grows on the trunk of Syringa reticulat [ 1 ] . S . baumii used to belong to the genus of Inonotus or Phellinus and now belongs to the genus of Sanghuangporus [ 2 ] . Numerous studies have demonstrated that S . baumii possesses antitumor, antioxidant, and anti-inflammatory [ 3 ] . Besides, S . baumii contains many secondary metabolites, such as polysaccharides, flavonoids, and terpenes [ 1 ] . Triterpenoids in S . baumii are important pharmacological active substances, which have the anti-tumor, anti-inflammatory, anti-bacterial and antiviral effect [ 4 ] . Triterpenoids are complex mixtures, formed by six isoprene units and have a variety of structures. Because of the diversity of the triterpenoids structure, they have a wide range of pharmacological activities [ 5 ] . Triterpenoids are synthesized by the mevalonate (MVA) pathway and 1-deoxy-D-xylulose-5-phos-phate pathway (DXP) pathway [ 6 , 7 ] . Fungi triterpenoids in which are synthesized mainly through the MVA pathway (Fig. 1 ) [ 8 ] . Lanosterol is a common intermediate of triterpene and ergosterol biosynthesis [ 9 , 10 ] . The squalene synthase (SQS) catalyzes reaction from the isoprenoid pathway toward to sterol and triterpenoids biosynthesis, and lanosterol synthase (LS) catalyzes the cyclization of 2, 3-oxidosqualene to lanosterol [ 11 ] . The precursor of triterpenoids is lanosterol, but how lanosterol syntheses triterpenoids are still unknown [ 7 ] . Lanosterol synthase, a member of the OSC ((3S)-2, 3-oxidosqualene cyclase) family, is not only a key enzyme in cholesterol and steroid synthesis in animals but also in sterol and triterpenoids synthesis in plants and fungi. The enzyme activities are determined by a few key amino acids in the active site [ 5 ] . Therefore, amino acids structure and activities are of great importance for the study of enzyme function and catalytic mechanism. In Sacchar cerevisiae , a variety of cyclization products are produced by mutating lanosterol synthase His234, which means the lanosterol synthase is related to the deprotonation of cations on the four-ring structure [ 12 ] . The swiss-models of OSC were studied in herbal plants, which shows the OSC structure in Panax ginseng , Panax notoginseng , Taraxacum mongolicum , Cimicifuga racemosa , and Lotus corniculatus are stable. There are some variations in the random curl, most of them are distributed on the surface of the protein [ 13 ] . In Siraitia grosvenorii , homology modeling has been used to predict the 3D structure of OSC (Cycloartenol Synthase, CAS). The interaction between CAS and substrates are analyzed by molecular docking, which shows that Asp491, Cys492, Cys570, Tyr540, and His265 are the key catalytic sites in CAS [ 14 ] . At present, the LS gene has been cloned and examined in other organisms. For example, Ganoderma lucidum LS is cloned and transferred in an erg7 yeast strain lacking LS activity, which demonstrates that the cloned cDNA encodes a functional LS [ 15 ] . The ergosterol content in deficient mutant decreases to 42% than that of in wild strain after the Saccharomyces cerevisiae LS knockout cassette harboring the loxP-Marker-loxP element [ 16 ] . Poria cocos LS and promoter are cloned and then transformed into ERG7 hybrid diploid Saccharomyces cerevisiae strain YHR072W. The results show that the P . cocos LS gene mediates the formation of ergosterol in S. cerevisiae [ 17 ] . In S . baumii , acetyl-CoA acetyl transferase gene ( AACT ) [ 18 ] , 3-hydroxy-3-methylglutaryl-CoA synthase gene ( HMGS ) [ 19 ] , and squalene epoxidase gene ( SE ) [ 20 ] in MVA pathway have been cloned and expresses in E. coli , but S . baumi triterpenoids synthesis pathway is not fully understood. Therefore, it is highly important to analyze the characteristics of key genes at the beginning of the experiment. S . baumii LS and promoter was analyzed for the first time in this study. And we detected the transcription level of LS by real-time quantitative PCR further. Then, LS was constructed in pET-32a (+) and expressed in E. coli . Materials And Methods Strain and plasmid S. baumii was authenticated by visual observation and Internal Transcribed Spacer (ITS) identification. The pET-32a(+) vector was used as expression vectors. E. coli DH5α and BL21 (DE3) strains (Tiangen, Beijing, China) were purchased to expand reproduction and express recombinant vectors. RNA, cDNA and DNA extraction S. baumii mycelia were collected and washed by distilled water. Then ground to powder by using liquid nitrogen. Next, S. baumii RNA, cDNA, and DNA were extracted according to Wang's description [ 18 ] . Amplification of the full length of LS To obtain the full length of LS , the LS gene fragment should be cloned firstly. Primers ( LS -S, LS -A; Table 1) were designed according to S. baumii transcription data [ 21 ] . Using cDNA as a template, LS 3 'and 5' gene fragments were amplified [ 18 ] . The 3’ and 5’ RACE PCR amplification products were verified by AGE (agarose gel electrophoresis) and sequencing (Boshi, Harbin, China). Subsequently, A 468 bp LS 5’ fragment and a 2,011 bp 3’ fragment were obtained, and the 5’ and 3’ cDNA fragments were spliced using software, a 2,445 bp S. baumii LS sequence was obtained. Heterologous expression of LS in E. coli Trelief™ SoSoo Cloning Kit was used to structure the expression vector to express in E. coli . Primers ( LS-EcoR I, LS-Hind III; Table 1) (containing 20 bp homologous flanks, which complementary with the ends of the pET-32a(+) linearized vector) were designed for PCR amplification [ 18 ] . The PCR product with homologous flanks was purified using a MiniBEST DNA Fragment Purification Kit Ver.4.0 (TaKaRa). Then, the purified product and linearized vector were mixed according to the instructions. The recombinant vector was transferred to E. coli DH5α, and a single positive clone was inoculated in LB medium for extracting plasmid (pET- LS ). pET- LS and pET-32a (+) were transferred into the E. coli BL21 (DE3) competent cell. A single positive clone was selected and cultivated to OD600 = 0.5–0.8 in LB medium, respectively. Then, 1 mL bacteria solution from two kinds of vectors was fetched as controls. Whereafter, bacteria solution was induced using 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and incubated (shaking at 200 r/min, 28°C) [ 22 ] . Bacterial liquid and gel were treated according to Wang’s description [ 18 ] . At the end of running, the gel was stained with Coomassie Brilliant Blue Fast Staining solution (Solarbio). Amplification of the LS promoter LS promoter primers ( LS -P-f, LS -P-r; Table 1) were designed based on LS sequencing analysis and S. baumii genomic DNA. The PCR product was amplified and sequenced (Boshi, Harbin, China). Sequence analysis The LS ORF was obtained using ORF Finder ( https://ncbiinsights.ncbi.nlm.nih.gov/tag/orffinder/ ), and the LS sequence was compared using the NCBI database ( https://www.ncbi.nlm.nih.gov/ ). Phylogenetic trees were constructed using MEGA 6.0 with the neighbor-joining method, and LS sequences from other species were downloaded from NCBI. The transmembrane region was predicted by TMHMM Server v.2.0 ( https://www.hsls.pitt.edu/obrc/index.php?page=URL1164644151 ). The theoretical isoelectric point (pI), molecular weight (MW), amino acid composition and protein transmembrane structures were calculated by ExPASy ProtParam ( https://web.expasy.org/protparam/ ). The solubility of LS was analyzed by Protein-Sol ( http://protein-sol.manchester.ac.uk ). Subcellular localization was predicted through the POSRT II server ( https://psort.hgc.jp/cgi-bin/runpsort.pl ). LS sequences were aligned through ESPript ( http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi ). Domain architectures were analyzed by SMART ( http://smart.embl.de/smart/job_status.pl?jobid=601520980332911588497123qEJfXLcIpn ). The secondary structure of the LS was determined by the PredictantProtein tool ( https://www.predictprotein.org/ ). The three-dimensional structure of the LS was modeled by SWISS-MODEL ( https://www.swissmodel.expasy.org/interactive ). The three-dimensional structure model was evaluated by PROCHECK ( https://servicesn.mbi.ucla.edu/PROCHECK/ ). Prediction of signal peptide was performed using SignalP 4.0 software ( http://www.cbs.dtu.dk/services/SignalP/ ). The LS promoter was analyzed using PlantCARE ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) and the Berkeley Drosophila Genome Project tool ( http://www.fruitfly.org/seq_tools/promoter.html ). Transcription analysis of LS in the different development stage Total RNA from mycelia, primordia, and young fruiting bodies were extracted, and cDNA was produced using a PrimeScript RT Reagent Kit with gDNA Eraser (Takara). Quantitative real-time PCR (qRT-PCR) was performed on an Mx3000P Sequence Detection System (Agilent Technologies, California, USA). The reaction system was mixed as follow: 10 µL of SYBR GreenMaster Mix (Takara), 0.4 µL of LS -T-f (Table 1), 0.4 µL of LS -T-r (Table 1), 1 µL of cDNA from the different development stage and 8.2 µL of ddH 2 O. Each sample was analyzed in triplicate and repeated three times. Mycelia from day 9 served as a control sample, and β -tubulin was used as an internal reference for all qRT-PCR analyses. Relative transcription levels were calculated using the 2 − ΔΔCT method [ 23 ] . Variance (ANOVO) was used to analyze data, and P < 0.05 was considered statistically significant. Data analysis LS transcription levels data was compared with total triterpenoids content [ 18 ] . Line chart was drawn using Excel software. Results Sequence analysis of the LS sequence The S. baumii LS sequence we obtained includes a 129 bp 5’ UTR, an 87 bp 3’ UTR, and a 2,229 bp ORF encoding a 734 amino acids. Next, it was submitted to NCBI GenBank to get accession number (Genbank accession number: MT108416). S. baumii LS sequence shared 89% identity and 99% query cover with Inonotus obliquus (Genbank accession number: QEP49720.1). The 20 homologous LS sequences from NCBI were used for phylogenetic tree construction, which showed that S. baumii LS was most similar to I. obliquus , but more distantly related to Amanita thiersii LS and Amanita muscaria LS (Fig. 2 ). The molecular weight was 84.99 kDa and the theoretical isoelectric point was 5.87. Alanine (Ala) was the most common (8.5%), and valine (Val) was the least (5.3%) in the amino acid sequence. Instability index of protein was 48.38, which means LS was unstable [ 24 ] . LS did not exist transmembrane helix and signal peptide through TMHMM Server (v.2.0) and SignalP 4.0 Server analysis. And LS was a hydrophilic protein by ProtScale software analysis. Prediction by solubility and subcellular localization showed LS was an insoluble protein in the cytoplasmic. When compared LS sequences with other fungi (Fig. 3 ) and predicted conserved domains, they were relatively conservative. The SQ Hop_cyclase_N was located in the 76–362 amino acid region of S.baumii LS, and the SQ Hop_cyclase_C was located in the 382–716 amino acid region of S.baumii LS. The secondary structure of LS contained 46.36% α-helix, 2.56% β-sheet, and 51.08% random coil [ 25 ] . The three-dimensional structure of LS had 47.8% similarity with other LS by X-ray prediction [26−29] (Fig. 4 ). The three-dimensional structure model typically had 92% of residues in the allowed regions by PROCHECK soft, which suggested that the LS protein is theoretically reliable. Analysis of the LS promoter A 1,854 bp sequence of LS upstream was amplified and submitted in NCBI (Genbank accession number: MT108416). Promoter sequence was analyzed by software, which found sequence transcription start site ranged from 1,740 bp to 1,790 bp. LS promoter contained many acting elements (Table 2 ). Prokaryotic expression The prokaryotic expression of the pET- LS fusion protein was shown in a gel. Expected protein bands were consistent with software prediction (84.99 kDa + 21.15 kDa tag protein). The LS protease yield was correlated with induction time (Fig. 5 ). As shown in the figure, LS production was increasing with induction time. Relationship between LS transcription level and triterpenoids content The LS transcription level up-regulated 1.6-fold for the first time on day 11 in mycelia (Fig. 6 ), then decreased, and increased again to the primordial stage (1.5-fold). In the young fruit stage, the LS transcription level was reduced to a minimum (0.1-fold). The LS transcription level was highest on day 11 in mycelia and primordial stage, and it was significantly different from other stages. The change trend of LS transcription level was opposite to that of triterpenoids content after 11 days. LS transcription level was lowest, but triterpenoids content was higher in young fruiting bodies phase. Discussion At present, the increased yield of Sanghuangporus triterpenoids is mainly by optimizing the extraction method and changing the inducer. Inonotus obliquus is extracted by 5% (v/w) Viscozyme L, and the total triterpenoids are the highest (24.3 mg/g) [ 30 ] . Methyl jasmonate (MeJA) (150 mmol/L) can induce Inonotus baumii to enhance triterpenoids yield, which is 4.05-fold higher than that in water [ 31 ] . Although these methods can increase triterpenoids yield, they are insufficient for the triterpenoids production in factories. Therefore, improving the triterpene yield by molecular biotechnology is a popular research method. LS, a key enzyme in the MVA pathway, is a precursor of triterpenoid synthesis. A 2,229 bp S. baumii LS ORF sequence was obtained by PCR amplification and BLAST in NCBI. In Ganoderma lucidum , LS was found to contain a 2,181 bp ORF encoding a 726 amino acids [ 15 ] . In Saccharomyces cerevisiae , the LS gene coding region contains 2,901bp nucleotides [ 32 ] . In Poria cocos , a 2187 ORF was found out that it codes a 728 amino acid [ 17 ] . S. baumii LS ORF was longer than G . lucidum , S . cerevisiae , and P . cocos . In this study, we discovered S. baumii LS sequences and analyzed the molecular weight of S. baumii LS protein (84.99 kDa), and found the protein was unstable. According to signal peptide analysis, subcellular localization, and prediction of the transmembrane domain, LS is an insoluble protein in the cytoplasmic, which is consistent with the site of S. baumii MVA pathway. The site of transcription start in S. baumii LS promoter sequence ranged from 1 740 bp to 1790 bp. There are responsiveness acting elements contained by S. baumii LS promoter, which is similar to other species. In the past, AACT promoters in the S. baumii MVA pathway were cloned [ 18 ] . LS and AACT promoter all contained ABRE (cis-acting element involved in the abscisic acid responsiveness), CGTCA-motif (cis-acting regulatory element involved in the MeJA-responsiveness), LTR (cis-acting element involved in low-temperature responsiveness), TGACG-motif (cis-acting regulatory element involved in the MeJA-responsiveness), which showed triterpenoids synthesis may relay to abscisic acid, low-temperature, and MeJA. LS promoter contained more ARE (essential cis-acting regulatory element for the anaerobic induction), ATC-motif (part of a conserved DNA module involved in light responsiveness), Box 4 (part of a conserved DNA module involved in light responsiveness), GT1-motif (light-responsive element), MRE (MYB binding site involved in light responsiveness) and TGA-element (auxin-responsive element) than AACT promoter. LS promoter elements are related to anaerobic induction and light responsiveness, so the reaction to catalyzed LS may require anaerobic and light stimuli. In Poria cocos , the LS promoter region contains transcriptional sequesters associated with transcriptional regulation, including acid, light, methyl jasmine, etc [ 17 ] . In G . lucidum , the potential regulatory elements include the G-box/GT1-motif (light-responsive element), ABRE(cis-acting element involved in abscisic acid responsiveness), AuxRR-core (cis-acting regulatory element involved in auxin responsiveness), MBS (MYB binding site, involved in drought-inducibility), and Box-W1 (fungal elicitor responsive element). But no MeJA responsive element has been found in G . lucidum [ 15 ] . The S. baumii LS protein bands were consistent with software prediction (84.99 kDa + 21.15 kDa tag protein) and whose transcription level first up-regulated 1.6-fold on day 11 in mycelia, then decreased, and increased again in the primordial stage (1.5-fold). The S. baumii LS transcription level was the highest on day 11 in mycelia. In G . lucidum , gene transcription level is relatively low in the mycelia and then increased to the primordial level, which is also the highest level (about 8.39 fold compared with 10 d-old mycelia) [ 15 ] . This result is different from S. baumii LS expression, but they all have higher transcription level in the primordial stage. The variation trend of LS transcriptional level was opposite to that of triterpenoids content. This may be because the LS transcriptional level was inhibited with the accumulation of triterpenoids in S. baumii intracellular. This result is similar to zhang 's [ 33 ] research, and the triterpenoids content is the highest in the S. baumii mycelia. To sum up, S. baumii LS and promoter were cloned and analyzed for the first time. Subsequently, LS was constructed into the vector and expressed in E. coli BL21. The transcriptional level of LS was explored at different development stages. These studies help us to understand the LS as a key enzyme gene in the triterpenoids synthesis pathway. However, to understand the mechanism of triterpenoids synthesis and gene function better, it is necessary to study the overexpression and suppression expression of LS in S. baumii . Moreover, the transcriptional regulatory factors of LS gene upstream may also be the key factors controlling triterpenoids synthesis. Declarations Acknowledgements This research was supplied by the Fundamental Research Funds for the Central Universities, China (2572020AW18). Authors’ Contributions XuTong Wang and Li Zou designed the study. XuTong Wang and Jian Sun performed the experiments. XuTong Wang and Tingting Sun analyzed the results. XuTong Wang and Zengcai Liu wrote the manuscript. All of the authors read and approved the final manuscript. All relevant data are included in this paper. Materials are available upon reasonable request to the corresponding author. 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T., Zou L., Zhang L., Zhang J., Wang X. T. (2017) Methyl jasmonate induces triterpenoid biosynthesis in Inonotus baumii . Biotechnology & Biotechnological equipment 31 (2) , 312-317. Wang Q. H., Gao L. L., Liang H. C. (2015) 利用反义RNA技术抑制酿酒酵母羊毛甾醇合酶基因的表达 [Downregulation of lanosterol synthase gene expression by antisense RNA technology in Saccharomyces cerevisiae ]. Acta Pharmaceutica Sinica 50 (1) , 118-122. Chinese. Zhang L. F., Sun T. T., Zou L. (2015) 鲍姆纤孔菌总三萜的提取及其体外抗乳腺癌细胞MCF-7活性 [Extraction of total triterpenoids from Inonotus baumii and its inhibitory activity on breast cancer cells (MCF-7) in vitro]. Drug Evaluation Research 38 (5) , 497-502. Chinese. Tables Table 1 Primers used for PCR amplification Primers Sequences (5’- 3’) Descriptions LS -S CACCAAAGGGAGGACGGAGGAT For LS cDNA fragment sequence amplification. LS -A AGCTCTCTCCCCATCCTCCGTCCTC LS -F ATGTGGTCACCATTGGATGTCCCGG For full-length LS cDNA isolation. LS -R CTAGTGCAGATGTCCATTCCCATTTG LS - EcoR I CTGATATCGGATCC GAATTC ATGTGGTCACCATTGGATGTCC For pET- LS construction. LS - Hind III TCGAGTGCGGCCGC AAGCTT CTAGTGCAGATGTCCATTCCCA LS -P-f CCCGACTCTCGTTGCGAAGATACTA For LS promoter isolation LS -P-r ATTCCAGGTATTCCCAGACATGACG LS -T-f ACACAGTTCGCCCTTGAGAGCC For qRT-PCR analysis LS -T-r CATCTTCACGGCCCGTTCGATAGGT β-tubulin-f GCTGAATATCGTTCGTGCCC For qRT-PCR analysis β-tubulin-r ATCCGCCTTCCTCCTTACAGT Table 2 Cis-acting regulatory elements of the Promoter Type of cis-acting regulatory elements LS elements numbers AACT elements numbers Function sequence A-box 2 1 cis-acting regulatory element CCGTCC ABRE 5 6 cis-acting element involved in the abscisic acid responsiveness ACGTG ACA-motif 0 1 part of gapA in (gapA-CMA1) involved with light responsiveness AATCACAACCATA ARE 1 0 cis-acting regulatory element essential for the anaerobic induction AAACCA ATC-motif 1 0 part of a conserved DNA module involved in light responsiveness AGTAATCT Box 4 1 0 part of a conserved DNA module involved in light responsiveness ATTAAT CAAT-box 12 13 common cis-acting element in promoter and enhancer regions CCAAT CAT-box 0 1 cis-acting regulatory element related to meristem expression GCCACT CCAAT-box 1 2 MYBHv1 binding site CAACGG CGTCA-motif 6 5 cis-acting regulatory element involved in the MeJA-responsiveness CGTCA G-Box 4 9 cis-acting regulatory element involved in light responsiveness CACGTT/TACGTG GARE-motif 0 1 gibberellin-responsive element TCTGTTG GATA-motif 0 1 part of a light responsive element AAGGATAAGG GT1-motif 1 1 light responsive element GGTTAA GC-motif 0 1 enhancer-like element involved in anoxic specific inducibility CCCCCG LTR 1 3 cis-acting element involved in low-temperature responsiveness CCGAAA MSA-like 0 1 cis-acting element involved in cell cycle regulation (T/C)C(T/C)AACGG(T/C)(T/C)A P-box 0 2 gibberellin-responsive element CCTTTTG MBS 1 0 MYB binding site involved in drought-inducibility CAACTG MRE 1 0 MYB binding site involved in light responsiveness AACCTAA Sp1 2 2 light responsive element GGGCGG TCCC-motif 1 1 part of a light responsive element TCTCCCT TGA-element 2 0 auxin-responsive element AACGAC TGACG-motif 6 5 cis-acting regulatory element involved in the MeJA-responsiveness TGACG 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. 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Abbreviations: mevalonate (MVA), methylerythritol 4-phosphate (MEP), diphosphate isomerase (IDI), farnesyl-Diphosphate Synthase (FPS), squalene synthase (SQS), squalene epoxidase gene (SE), lanosterol synthase (LS).","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-203582/v1/5d10a510d07db2ef09ff0213.jpg"},{"id":6114532,"identity":"a561a598-9ddf-4e62-9974-44224fcb5f19","added_by":"auto","created_at":"2021-02-18 22:43:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":64520,"visible":true,"origin":"","legend":"Phylogenetic tree of LS proteins sequences of various species.","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-203582/v1/503e8b9a3939da0ceb669afd.jpg"},{"id":6114530,"identity":"32e548d7-c79b-4d65-b560-0afdf319ada2","added_by":"auto","created_at":"2021-02-18 22:43:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":426356,"visible":true,"origin":"","legend":"Alignment of the LS amino acid sequences from different species.\nSanghuangporus baumii (Sanghuangporus), Inonotus obliquus (Inonotus), Fomitiporia mediterranea (Fomitiporia), Phellinidium pouzarii (Phellinidium), Schizopora paradoxa (Schizopora).","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-203582/v1/c6e6c9c4ac104bf111c56adf.jpg"},{"id":6114872,"identity":"6a7d7abd-10e4-4910-bce1-6f37bd559008","added_by":"auto","created_at":"2021-02-18 22:46:15","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":57890,"visible":true,"origin":"","legend":"Three-dimensional structure schematic representation of S. baumii LS.","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-203582/v1/55646d6a0cd19ab43a00cc7f.jpg"},{"id":6114873,"identity":"aa23e584-e1cd-4a79-82e0-5dd3c254bef7","added_by":"auto","created_at":"2021-02-18 22:46:15","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":33769,"visible":true,"origin":"","legend":"Heterologous expression of S. baumii LS in E. coli via IPTG induction (1 mM) at 37°C for 0, 2, 4, 6, 8 and 10 h. Lane 1, protein markers (43-200 kDa); Lane 2 E. coli harbouring empty vector; Lane 3-8, E. coli harbouring the pET-LS construct induced by IPTG (1 mM) for 0, 2, 4, 6, 8, and 10 h.","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-203582/v1/636eaa7a2f022d829f829abe.jpg"},{"id":6114087,"identity":"f13293c3-2a4f-467a-bf4c-b8dfabba3daa","added_by":"auto","created_at":"2021-02-18 22:40:15","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":53940,"visible":true,"origin":"","legend":"LS transcriptional level and triterpenoids content in S. baumii in different development stages.","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-203582/v1/08fe84093db6e59a16eee4ed.jpg"},{"id":15671023,"identity":"afe0119b-65b9-4f8a-bee8-a58824855151","added_by":"auto","created_at":"2021-11-18 14:03:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":721760,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-203582/v1/9448551e-d6e4-4bb6-a3fd-85402ebb32ec.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eIdentification and Characterization of A Lanosterol synthase Gene from \u003cem\u003eSanghuangporus Baumii\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":" \u003cp\u003e \u003cem\u003eSanghuangporus baumii\u003c/em\u003e, a traditional Chinese medicine, grows on the trunk of \u003cem\u003eSyringa reticulat\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaumii\u003c/em\u003e used to belong to the genus of \u003cem\u003eInonotus\u003c/em\u003e or \u003cem\u003ePhellinus\u003c/em\u003e and now belongs to the genus of \u003cem\u003eSanghuangporus\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Numerous studies have demonstrated that \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaumii\u003c/em\u003e possesses antitumor, antioxidant, and anti-inflammatory \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Besides, \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaumii\u003c/em\u003e contains many secondary metabolites, such as polysaccharides, flavonoids, and terpenes \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Triterpenoids in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaumii\u003c/em\u003e are important pharmacological active substances, which have the anti-tumor, anti-inflammatory, anti-bacterial and antiviral effect \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTriterpenoids are complex mixtures, formed by six isoprene units and have a variety of structures. Because of the diversity of the triterpenoids structure, they have a wide range of pharmacological activities \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Triterpenoids are synthesized by the mevalonate (MVA) pathway and 1-deoxy-D-xylulose-5-phos-phate pathway (DXP) pathway \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Fungi triterpenoids in which are synthesized mainly through the MVA pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e1\u003c/span\u003e) \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLanosterol is a common intermediate of triterpene and ergosterol biosynthesis \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. The squalene synthase (SQS) catalyzes reaction from the isoprenoid pathway toward to sterol and triterpenoids biosynthesis, and lanosterol synthase (LS) catalyzes the cyclization of 2, 3-oxidosqualene to lanosterol \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. The precursor of triterpenoids is lanosterol, but how lanosterol syntheses triterpenoids are still unknown \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLanosterol synthase, a member of the OSC ((3S)-2, 3-oxidosqualene cyclase) family, is not only a key enzyme in cholesterol and steroid synthesis in animals but also in sterol and triterpenoids synthesis in plants and fungi. The enzyme activities are determined by a few key amino acids in the active site \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Therefore, amino acids structure and activities are of great importance for the study of enzyme function and catalytic mechanism. In \u003cem\u003eSacchar cerevisiae\u003c/em\u003e, a variety of cyclization products are produced by mutating lanosterol synthase His234, which means the lanosterol synthase is related to the deprotonation of cations on the four-ring structure \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. The swiss-models of OSC were studied in herbal plants, which shows the OSC structure in \u003cem\u003ePanax ginseng\u003c/em\u003e, \u003cem\u003ePanax notoginseng\u003c/em\u003e, \u003cem\u003eTaraxacum mongolicum\u003c/em\u003e, \u003cem\u003eCimicifuga racemosa\u003c/em\u003e, and \u003cem\u003eLotus corniculatus\u003c/em\u003e are stable. There are some variations in the random curl, most of them are distributed on the surface of the protein \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. In \u003cem\u003eSiraitia grosvenorii\u003c/em\u003e, homology modeling has been used to predict the 3D structure of OSC (Cycloartenol Synthase, CAS). The interaction between CAS and substrates are analyzed by molecular docking, which shows that Asp491, Cys492, Cys570, Tyr540, and His265 are the key catalytic sites in CAS \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAt present, the \u003cem\u003eLS\u003c/em\u003e gene has been cloned and examined in other organisms. For example, \u003cem\u003eGanoderma lucidum LS\u003c/em\u003e is cloned and transferred in an erg7 yeast strain lacking LS activity, which demonstrates that the cloned cDNA encodes a functional LS \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. The ergosterol content in deficient mutant decreases to 42% than that of in wild strain after the \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e LS knockout cassette harboring the loxP-Marker-loxP element \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003ePoria cocos LS\u003c/em\u003e and promoter are cloned and then transformed into ERG7 hybrid diploid \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e strain YHR072W. The results show that the \u003cem\u003eP\u003c/em\u003e. \u003cem\u003ecocos LS\u003c/em\u003e gene mediates the formation of ergosterol in \u003cem\u003eS. cerevisiae\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. In \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaumii\u003c/em\u003e, acetyl-CoA acetyl transferase gene (\u003cem\u003eAACT\u003c/em\u003e) \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, 3-hydroxy-3-methylglutaryl-CoA synthase gene (\u003cem\u003eHMGS\u003c/em\u003e) \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e, and squalene epoxidase gene (\u003cem\u003eSE\u003c/em\u003e) \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e in MVA pathway have been cloned and expresses in \u003cem\u003eE. coli\u003c/em\u003e, but \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaumi\u003c/em\u003e triterpenoids synthesis pathway is not fully understood. Therefore, it is highly important to analyze the characteristics of key genes at the beginning of the experiment.\u003c/p\u003e \u003cp\u003e \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaumii LS\u003c/em\u003e and promoter was analyzed for the first time in this study. And we detected the transcription level of \u003cem\u003eLS\u003c/em\u003e by real-time quantitative PCR further. Then, \u003cem\u003eLS\u003c/em\u003e was constructed in pET-32a (+) and expressed in \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e "},{"header":"Materials And Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrain and plasmid\u003c/h2\u003e \u003cp\u003e \u003cem\u003eS. baumii\u003c/em\u003e was authenticated by visual observation and Internal Transcribed Spacer (ITS) identification. The pET-32a(+) vector was used as expression vectors. \u003cem\u003eE. coli\u003c/em\u003e DH5α and BL21 (DE3) strains (Tiangen, Beijing, China) were purchased to expand reproduction and express recombinant vectors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRNA, cDNA and DNA extraction\u003c/h2\u003e \u003cp\u003e \u003cem\u003eS. baumii\u003c/em\u003e mycelia were collected and washed by distilled water. Then ground to powder by using liquid nitrogen. Next, \u003cem\u003eS. baumii\u003c/em\u003e RNA, cDNA, and DNA were extracted according to Wang's description \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAmplification of the full length of LS\u003c/h2\u003e \u003cp\u003eTo obtain the full length of \u003cem\u003eLS\u003c/em\u003e, the \u003cem\u003eLS\u003c/em\u003e gene fragment should be cloned firstly. Primers (\u003cem\u003eLS\u003c/em\u003e-S, \u003cem\u003eLS\u003c/em\u003e-A; Table\u0026nbsp;1) were designed according to \u003cem\u003eS. baumii\u003c/em\u003e transcription data \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Using cDNA as a template, \u003cem\u003eLS\u003c/em\u003e 3 'and 5' gene fragments were amplified \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. The 3\u0026rsquo; and 5\u0026rsquo; RACE PCR amplification products were verified by AGE (agarose gel electrophoresis) and sequencing (Boshi, Harbin, China). Subsequently, A 468 bp \u003cem\u003eLS\u003c/em\u003e 5\u0026rsquo; fragment and a 2,011 bp 3\u0026rsquo; fragment were obtained, and the 5\u0026rsquo; and 3\u0026rsquo; cDNA fragments were spliced using software, a 2,445 bp \u003cem\u003eS. baumii LS\u003c/em\u003e sequence was obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eHeterologous expression of LS in E. coli\u003c/h2\u003e \u003cp\u003eTrelief\u0026trade; SoSoo Cloning Kit was used to structure the expression vector to express in \u003cem\u003eE. coli\u003c/em\u003e. Primers (\u003cem\u003eLS-EcoR\u003c/em\u003eI, \u003cem\u003eLS-Hind\u003c/em\u003eIII; Table\u0026nbsp;1) (containing 20 bp homologous flanks, which complementary with the ends of the pET-32a(+) linearized vector) were designed for PCR amplification \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. The PCR product with homologous flanks was purified using a MiniBEST DNA Fragment Purification Kit Ver.4.0 (TaKaRa). Then, the purified product and linearized vector were mixed according to the instructions. The recombinant vector was transferred to \u003cem\u003eE. coli\u003c/em\u003e DH5α, and a single positive clone was inoculated in LB medium for extracting plasmid (pET-\u003cem\u003eLS\u003c/em\u003e).\u003c/p\u003e \u003cp\u003epET-\u003cem\u003eLS\u003c/em\u003e and pET-32a (+) were transferred into the \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) competent cell. A single positive clone was selected and cultivated to OD600\u0026thinsp;=\u0026thinsp;0.5\u0026ndash;0.8 in LB medium, respectively. Then, 1 mL bacteria solution from two kinds of vectors was fetched as controls. Whereafter, bacteria solution was induced using 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and incubated (shaking at 200 r/min, 28\u0026deg;C) \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBacterial liquid and gel were treated according to Wang\u0026rsquo;s description \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. At the end of running, the gel was stained with Coomassie Brilliant Blue Fast Staining solution (Solarbio).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAmplification of the LS promoter\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLS\u003c/em\u003e promoter primers (\u003cem\u003eLS\u003c/em\u003e-P-f, \u003cem\u003eLS\u003c/em\u003e-P-r; Table\u0026nbsp;1) were designed based on LS sequencing analysis and \u003cem\u003eS. baumii\u003c/em\u003e genomic DNA. The PCR product was amplified and sequenced (Boshi, Harbin, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSequence analysis\u003c/h2\u003e \u003cp\u003eThe LS ORF was obtained using ORF Finder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ncbiinsights.ncbi.nlm.nih.gov/tag/orffinder/\u003c/span\u003e\u003c/span\u003e), and the \u003cem\u003eLS\u003c/em\u003e sequence was compared using the NCBI database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003c/span\u003e). Phylogenetic trees were constructed using MEGA 6.0 with the neighbor-joining method, and LS sequences from other species were downloaded from NCBI.\u003c/p\u003e \u003cp\u003eThe transmembrane region was predicted by TMHMM Server v.2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.hsls.pitt.edu/obrc/index.php?page=URL1164644151\u003c/span\u003e\u003c/span\u003e). The theoretical isoelectric point (pI), molecular weight (MW), amino acid composition and protein transmembrane structures were calculated by ExPASy ProtParam (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003c/span\u003e). The solubility of LS was analyzed by Protein-Sol (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://protein-sol.manchester.ac.uk\u003c/span\u003e\u003c/span\u003e). Subcellular localization was predicted through the POSRT II server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://psort.hgc.jp/cgi-bin/runpsort.pl\u003c/span\u003e\u003c/span\u003e). LS sequences were aligned through ESPript (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi\u003c/span\u003e\u003c/span\u003e). Domain architectures were analyzed by SMART (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.embl.de/smart/job_status.pl?jobid=601520980332911588497123qEJfXLcIpn\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe secondary structure of the LS was determined by the PredictantProtein tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.predictprotein.org/\u003c/span\u003e\u003c/span\u003e). The three-dimensional structure of the LS was modeled by SWISS-MODEL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.swissmodel.expasy.org/interactive\u003c/span\u003e\u003c/span\u003e). The three-dimensional structure model was evaluated by PROCHECK (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://servicesn.mbi.ucla.edu/PROCHECK/\u003c/span\u003e\u003c/span\u003e). Prediction of signal peptide was performed using SignalP 4.0 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cbs.dtu.dk/services/SignalP/\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eLS\u003c/em\u003e promoter was analyzed using PlantCARE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003c/span\u003e) and the Berkeley Drosophila Genome Project tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.fruitfly.org/seq_tools/promoter.html\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eTranscription analysis of LS in the different development stage\u003c/h2\u003e \u003cp\u003eTotal RNA from mycelia, primordia, and young fruiting bodies were extracted, and cDNA was produced using a PrimeScript RT Reagent Kit with gDNA Eraser (Takara). Quantitative real-time PCR (qRT-PCR) was performed on an Mx3000P Sequence Detection System (Agilent Technologies, California, USA). The reaction system was mixed as follow: 10 \u0026micro;L of SYBR GreenMaster Mix (Takara), 0.4 \u0026micro;L of \u003cem\u003eLS\u003c/em\u003e-T-f (Table\u0026nbsp;1), 0.4 \u0026micro;L of \u003cem\u003eLS\u003c/em\u003e-T-r (Table\u0026nbsp;1), 1 \u0026micro;L of cDNA from the different development stage and 8.2 \u0026micro;L of ddH\u003csub\u003e2\u003c/sub\u003eO. Each sample was analyzed in triplicate and repeated three times. Mycelia from day 9 served as a control sample, and \u003cem\u003eβ\u003c/em\u003e-tubulin was used as an internal reference for all qRT-PCR analyses. Relative transcription levels were calculated using the 2\u003csup\u003e\u0026minus;\u0026thinsp;ΔΔCT\u003c/sup\u003e method\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Variance (ANOVO) was used to analyze data, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLS\u003c/em\u003e transcription levels data was compared with total triterpenoids content \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Line chart was drawn using Excel software.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSequence analysis of the LS sequence\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eS. baumii LS\u003c/em\u003e sequence we obtained includes a 129 bp 5\u0026rsquo; UTR, an 87 bp 3\u0026rsquo; UTR, and a 2,229 bp ORF encoding a 734 amino acids. Next, it was submitted to NCBI GenBank to get accession number (Genbank accession number: MT108416). \u003cem\u003eS. baumii\u003c/em\u003e LS sequence shared 89% identity and 99% query cover with \u003cem\u003eInonotus obliquus\u003c/em\u003e (Genbank accession number: QEP49720.1). The 20 homologous LS sequences from NCBI were used for phylogenetic tree construction, which showed that \u003cem\u003eS. baumii\u003c/em\u003e LS was most similar to \u003cem\u003eI. obliquus\u003c/em\u003e, but more distantly related to \u003cem\u003eAmanita thiersii LS\u003c/em\u003e and \u003cem\u003eAmanita muscaria LS\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The molecular weight was 84.99 kDa and the theoretical isoelectric point was 5.87. Alanine (Ala) was the most common (8.5%), and valine (Val) was the least (5.3%) in the amino acid sequence. Instability index of protein was 48.38, which means LS was unstable \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. LS did not exist transmembrane helix and signal peptide through TMHMM Server (v.2.0) and SignalP 4.0 Server analysis. And LS was a hydrophilic protein by ProtScale software analysis. Prediction by solubility and subcellular localization showed LS was an insoluble protein in the cytoplasmic. When compared LS sequences with other fungi (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and predicted conserved domains, they were relatively conservative. The SQ Hop_cyclase_N was located in the 76\u0026ndash;362 amino acid region of \u003cem\u003eS.baumii\u003c/em\u003e LS, and the SQ Hop_cyclase_C was located in the 382\u0026ndash;716 amino acid region of \u003cem\u003eS.baumii\u003c/em\u003e LS.\u003c/p\u003e \u003cp\u003eThe secondary structure of LS contained 46.36% α-helix, 2.56% β-sheet, and 51.08% random coil \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. The three-dimensional structure of LS had 47.8% similarity with other LS by X-ray prediction \u003csup\u003e[26\u0026minus;29]\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The three-dimensional structure model typically had 92% of residues in the allowed regions by PROCHECK soft, which suggested that the LS protein is theoretically reliable.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of the LS promoter\u003c/h2\u003e \u003cp\u003eA 1,854 bp sequence of \u003cem\u003eLS\u003c/em\u003e upstream was amplified and submitted in NCBI (Genbank accession number: MT108416). Promoter sequence was analyzed by software, which found sequence transcription start site ranged from 1,740 bp to 1,790 bp. \u003cem\u003eLS\u003c/em\u003e promoter contained many acting elements (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eProkaryotic expression\u003c/h2\u003e \u003cp\u003eThe prokaryotic expression of the pET-\u003cem\u003eLS\u003c/em\u003e fusion protein was shown in a gel. Expected protein bands were consistent with software prediction (84.99 kDa\u0026thinsp;+\u0026thinsp;21.15 kDa tag protein). The LS protease yield was correlated with induction time (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003e). As shown in the figure, LS production was increasing with induction time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRelationship between LS transcription level and triterpenoids content\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eLS\u003c/em\u003e transcription level up-regulated 1.6-fold for the first time on day 11 in mycelia (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), then decreased, and increased again to the primordial stage (1.5-fold). In the young fruit stage, the \u003cem\u003eLS\u003c/em\u003e transcription level was reduced to a minimum (0.1-fold). The \u003cem\u003eLS\u003c/em\u003e transcription level was highest on day 11 in mycelia and primordial stage, and it was significantly different from other stages.\u003c/p\u003e \u003cp\u003eThe change trend of \u003cem\u003eLS\u003c/em\u003e transcription level was opposite to that of triterpenoids content after 11 days. \u003cem\u003eLS\u003c/em\u003e transcription level was lowest, but triterpenoids content was higher in young fruiting bodies phase.\u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion","content":" \u003cp\u003eAt present, the increased yield of \u003cem\u003eSanghuangporus\u003c/em\u003e triterpenoids is mainly by optimizing the extraction method and changing the inducer. \u003cem\u003eInonotus obliquus\u003c/em\u003e is extracted by 5% (v/w) Viscozyme L, and the total triterpenoids are the highest (24.3 mg/g) \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Methyl jasmonate (MeJA) (150 mmol/L) can induce \u003cem\u003eInonotus baumii\u003c/em\u003e to enhance triterpenoids yield, which is 4.05-fold higher than that in water\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Although these methods can increase triterpenoids yield, they are insufficient for the triterpenoids production in factories. Therefore, improving the triterpene yield by molecular biotechnology is a popular research method.\u003c/p\u003e \u003cp\u003eLS, a key enzyme in the MVA pathway, is a precursor of triterpenoid synthesis. A 2,229 bp \u003cem\u003eS. baumii LS\u003c/em\u003e ORF sequence was obtained by PCR amplification and BLAST in NCBI. In \u003cem\u003eGanoderma lucidum\u003c/em\u003e, \u003cem\u003eLS\u003c/em\u003e was found to contain a 2,181 bp ORF encoding a 726 amino acids \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. In \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, the \u003cem\u003eLS\u003c/em\u003e gene coding region contains 2,901bp nucleotides \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. In \u003cem\u003ePoria cocos\u003c/em\u003e, a 2187 ORF was found out that it codes a 728 amino acid \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eS. baumii LS\u003c/em\u003e ORF was longer than \u003cem\u003eG\u003c/em\u003e. \u003cem\u003elucidum\u003c/em\u003e, \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecerevisiae\u003c/em\u003e, and \u003cem\u003eP\u003c/em\u003e. \u003cem\u003ecocos\u003c/em\u003e. In this study, we discovered \u003cem\u003eS. baumii LS\u003c/em\u003e sequences and analyzed the molecular weight of \u003cem\u003eS. baumii\u003c/em\u003e LS protein (84.99 kDa), and found the protein was unstable.\u003c/p\u003e \u003cp\u003eAccording to signal peptide analysis, subcellular localization, and prediction of the transmembrane domain, LS is an insoluble protein in the cytoplasmic, which is consistent with the site of \u003cem\u003eS. baumii\u003c/em\u003e MVA pathway.\u003c/p\u003e \u003cp\u003eThe site of transcription start in \u003cem\u003eS. baumii LS\u003c/em\u003e promoter sequence ranged from 1 740 bp to 1790 bp. There are responsiveness acting elements contained by \u003cem\u003eS. baumii LS\u003c/em\u003e promoter, which is similar to other species. In the past, \u003cem\u003eAACT\u003c/em\u003e promoters in the \u003cem\u003eS. baumii\u003c/em\u003e MVA pathway were cloned \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eLS\u003c/em\u003e and \u003cem\u003eAACT\u003c/em\u003e promoter all contained ABRE (cis-acting element involved in the abscisic acid responsiveness), CGTCA-motif (cis-acting regulatory element involved in the MeJA-responsiveness), LTR (cis-acting element involved in low-temperature responsiveness), TGACG-motif (cis-acting regulatory element involved in the MeJA-responsiveness), which showed triterpenoids synthesis may relay to abscisic acid, low-temperature, and MeJA. \u003cem\u003eLS\u003c/em\u003e promoter contained more ARE (essential cis-acting regulatory element for the anaerobic induction), ATC-motif (part of a conserved DNA module involved in light responsiveness), Box 4 (part of a conserved DNA module involved in light responsiveness), GT1-motif (light-responsive element), MRE (MYB binding site involved in light responsiveness) and TGA-element (auxin-responsive element) than \u003cem\u003eAACT\u003c/em\u003e promoter. \u003cem\u003eLS\u003c/em\u003e promoter elements are related to anaerobic induction and light responsiveness, so the reaction to catalyzed LS may require anaerobic and light stimuli.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003ePoria cocos\u003c/em\u003e, the LS promoter region contains transcriptional sequesters associated with transcriptional regulation, including acid, light, methyl jasmine, etc\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. In \u003cem\u003eG\u003c/em\u003e. \u003cem\u003elucidum\u003c/em\u003e, the potential regulatory elements include the G-box/GT1-motif (light-responsive element), ABRE(cis-acting element involved in abscisic acid responsiveness), AuxRR-core (cis-acting regulatory element involved in auxin responsiveness), MBS (MYB binding site, involved in drought-inducibility), and Box-W1 (fungal elicitor responsive element). But no MeJA responsive element has been found in \u003cem\u003eG\u003c/em\u003e. \u003cem\u003elucidum\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eS. baumii\u003c/em\u003e LS protein bands were consistent with software prediction (84.99 kDa\u0026thinsp;+\u0026thinsp;21.15 kDa tag protein) and whose transcription level first up-regulated 1.6-fold on day 11 in mycelia, then decreased, and increased again in the primordial stage (1.5-fold). The \u003cem\u003eS. baumii LS\u003c/em\u003e transcription level was the highest on day 11 in mycelia. In \u003cem\u003eG\u003c/em\u003e. \u003cem\u003elucidum\u003c/em\u003e, gene transcription level is relatively low in the mycelia and then increased to the primordial level, which is also the highest level (about 8.39 fold compared with 10 d-old mycelia) \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. This result is different from \u003cem\u003eS. baumii LS\u003c/em\u003e expression, but they all have higher transcription level in the primordial stage.\u003c/p\u003e \u003cp\u003eThe variation trend of \u003cem\u003eLS\u003c/em\u003e transcriptional level was opposite to that of triterpenoids content. This may be because the \u003cem\u003eLS\u003c/em\u003e transcriptional level was inhibited with the accumulation of triterpenoids in \u003cem\u003eS. baumii\u003c/em\u003e intracellular. This result is similar to zhang 's\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e research, and the triterpenoids content is the highest in the \u003cem\u003eS. baumii\u003c/em\u003e mycelia.\u003c/p\u003e \u003cp\u003eTo sum up, \u003cem\u003eS. baumii LS\u003c/em\u003e and promoter were cloned and analyzed for the first time. Subsequently, \u003cem\u003eLS\u003c/em\u003e was constructed into the vector and expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21. The transcriptional level of \u003cem\u003eLS\u003c/em\u003e was explored at different development stages. These studies help us to understand the \u003cem\u003eLS\u003c/em\u003e as a key enzyme gene in the triterpenoids synthesis pathway. However, to understand the mechanism of triterpenoids synthesis and gene function better, it is necessary to study the overexpression and suppression expression of \u003cem\u003eLS\u003c/em\u003e in \u003cem\u003eS. baumii\u003c/em\u003e. Moreover, the transcriptional regulatory factors of \u003cem\u003eLS\u003c/em\u003e gene upstream may also be the key factors controlling triterpenoids synthesis.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supplied by the Fundamental Research Funds for the Central Universities, China (2572020AW18).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e XuTong Wang and Li Zou designed the study. XuTong Wang and Jian Sun performed the experiments. XuTong Wang and Tingting Sun analyzed the results. XuTong Wang and Zengcai Liu wrote the manuscript. All of the authors read and approved the final manuscript. All relevant data are included in this paper. Materials are available upon reasonable request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e The authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman and Animal Rights and Informed Consent\u003c/strong\u003e The research performed did not involve any human or animal subjects. The research did not require informed consent.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eQi X., Zhang J., Chen Y. 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Chinese.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1 Primers used for PCR amplification\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003ePrimers\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"409\"\u003e\n\u003cp\u003eSequences (5\u0026rsquo;- 3\u0026rsquo;)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003eDescriptions\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e\u003cem\u003eLS\u003c/em\u003e-S\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"409\"\u003e\n\u003cp\u003eCACCAAAGGGAGGACGGAGGAT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003eFor \u003cem\u003eLS\u003c/em\u003e cDNA fragment sequence amplification.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e\u003cem\u003eLS\u003c/em\u003e-A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"409\"\u003e\n\u003cp\u003eAGCTCTCTCCCCATCCTCCGTCCTC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e\u003cem\u003eLS\u003c/em\u003e-F\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"409\"\u003e\n\u003cp\u003eATGTGGTCACCATTGGATGTCCCGG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003eFor full-length \u003cem\u003eLS\u003c/em\u003e cDNA isolation.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e\u003cem\u003eLS\u003c/em\u003e-R\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"409\"\u003e\n\u003cp\u003eCTAGTGCAGATGTCCATTCCCATTTG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e\u003cem\u003eLS\u003c/em\u003e-\u003cem\u003eEcoR\u003c/em\u003eI\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"409\"\u003e\n\u003cp\u003eCTGATATCGGATCC\u003cu\u003eGAATTC\u003c/u\u003eATGTGGTCACCATTGGATGTCC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003eFor pET-\u003cem\u003eLS \u003c/em\u003econstruction.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e\u003cem\u003eLS\u003c/em\u003e-\u003cem\u003eHind\u003c/em\u003eIII\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"409\"\u003e\n\u003cp\u003eTCGAGTGCGGCCGC\u003cu\u003eAAGCTT\u003c/u\u003eCTAGTGCAGATGTCCATTCCCA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e\u003cem\u003eLS\u003c/em\u003e-P-f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"409\"\u003e\n\u003cp\u003eCCCGACTCTCGTTGCGAAGATACTA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003eFor \u003cem\u003eLS\u003c/em\u003e promoter isolation\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e\u003cem\u003eLS\u003c/em\u003e-P-r\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"409\"\u003e\n\u003cp\u003eATTCCAGGTATTCCCAGACATGACG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e\u003cem\u003eLS\u003c/em\u003e-T-f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"409\"\u003e\n\u003cp\u003eACACAGTTCGCCCTTGAGAGCC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003eFor qRT-PCR analysis\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e\u003cem\u003eLS\u003c/em\u003e-T-r\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"409\"\u003e\n\u003cp\u003eCATCTTCACGGCCCGTTCGATAGGT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e\u0026beta;-tubulin-f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"409\"\u003e\n\u003cp\u003eGCTGAATATCGTTCGTGCCC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003eFor qRT-PCR analysis\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e\u0026beta;-tubulin-r\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"409\"\u003e\n\u003cp\u003eATCCGCCTTCCTCCTTACAGT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr /\u003eTable 2 Cis-acting regulatory elements of the Promoter\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eType of cis-acting regulatory elements\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003eLS elements numbers\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003eAACT elements numbers\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003eFunction\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003esequence\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eA-box\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003ecis-acting regulatory element\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003eCCGTCC\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eABRE\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003ecis-acting element involved in the abscisic acid responsiveness\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003eACGTG\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eACA-motif\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003epart of gapA in (gapA-CMA1) involved with light responsiveness\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003eAATCACAACCATA\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eARE\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003ecis-acting regulatory element essential for the anaerobic induction\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003eAAACCA\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eATC-motif\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003epart of a conserved DNA module involved in light responsiveness\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003eAGTAATCT\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eBox 4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003epart of a conserved DNA module involved in light responsiveness\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003eATTAAT\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eCAAT-box\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003ecommon cis-acting element in promoter and enhancer regions\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003eCCAAT\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eCAT-box\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003ecis-acting regulatory element related to meristem expression\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003eGCCACT\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eCCAAT-box\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003eMYBHv1 binding site\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003eCAACGG\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eCGTCA-motif\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003ecis-acting regulatory element involved in the MeJA-responsiveness\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003eCGTCA\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eG-Box\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003ecis-acting regulatory element involved in light responsiveness\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003eCACGTT/TACGTG\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eGARE-motif\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003egibberellin-responsive element\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003eTCTGTTG\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eGATA-motif\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003epart of a light responsive element\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003eAAGGATAAGG\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eGT1-motif\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003elight responsive element\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003eGGTTAA\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eGC-motif\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003eenhancer-like element involved in anoxic specific inducibility\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003eCCCCCG\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eLTR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003ecis-acting element involved in low-temperature responsiveness\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003eCCGAAA\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eMSA-like\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003ecis-acting element involved in cell cycle regulation\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003e(T/C)C(T/C)AACGG(T/C)(T/C)A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eP-box\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003egibberellin-responsive element\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003eCCTTTTG\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eMBS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003eMYB binding site involved in drought-inducibility\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003eCAACTG\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eMRE\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003eMYB binding site involved in light responsiveness\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003eAACCTAA\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eSp1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003elight responsive element\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"208\"\u003e\n\u003cp\u003eGGGCGG\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003eTCCC-motif\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"79\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003epart of a light responsive element\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd 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width=\"208\"\u003e\n\u003cp\u003eTGACG\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\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":"Lanosterol synthase, Promoter, Sanghuangporus baumii, Triterpenoids","lastPublishedDoi":"10.21203/rs.3.rs-203582/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-203582/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLanosterol synthase (LS) is a key enzyme involved in the mevalonate pathway (MVA pathway) to produce lanosterol, which is a precursor for synthesizing \u003cem\u003eSanghuangporus baumii \u003c/em\u003etriterpenoids. To research the characteristics and construction of \u003cem\u003eLS\u003c/em\u003e, \u003cem\u003eLS\u003c/em\u003e ORF and promoter were cloned from S. baumii. A 2,445 bp \u003cem\u003eS. baumii LS \u003c/em\u003esequence was obtained by rapid amplification of cDNA ends (RACE) technology and recombinant PCR. \u003cem\u003eS. baumii\u003c/em\u003e \u003cem\u003eLS\u003c/em\u003e sequence includes a 5’-untranslated region (129 bp), a 3’-untranslated region (87 bp), and an open reading frame (2,229 bp) encoding a 734 amino acids. The molecular weight of LS is 84.99 kDa, and transcription start site of \u003cem\u003eS. baumii LS\u003c/em\u003e promoter sequence ranged from 1 740 bp to 1790 bp.\u003cem\u003e LS\u003c/em\u003e promoter contained 12 CAAT-boxes, 5 ABREs, 6 G-Boxes, 6 CGTCA-motifs, and so on. The \u003cem\u003eS. baumii\u003c/em\u003e LS protein was expressed in E. coli BL21 (DE3) (84.99 kDa + 21.15 kDa tag protein). The transcription level of \u003cem\u003eS. baumii LS\u003c/em\u003e was the highest on day 11 in mycelia (1.6-fold).\u003c/p\u003e","manuscriptTitle":"Identification and Characterization of A Lanosterol synthase Gene from Sanghuangporus Baumii","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-18 22:40:13","doi":"10.21203/rs.3.rs-203582/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":"420f3f39-1d7e-46bc-8fa5-ce91b8291aca","owner":[],"postedDate":"February 18th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":2481313,"name":"Applied Biochemistry"},{"id":2481314,"name":"Biotechnology and Bioengineering"}],"tags":[],"updatedAt":"2021-02-18T22:43:14+00:00","versionOfRecord":[],"versionCreatedAt":"2021-02-18 22:40:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-203582","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-203582","identity":"rs-203582","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}