Genome-wide identification of R2R3-MYB Family Genes and their Response to Stress in Dendrobium nobile

Genome-wide identification of R2R3-MYB Family Genes and their Response to Stress in Dendrobium nobile | 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 Genome-wide identification of R2R3-MYB Family Genes and their Response to Stress in Dendrobium nobile Liping Wu, Jizhou Fan, Xinglong Su, Weiyi Rao, Yingying Duan, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2749425/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 R2R3-MYB is one of the largest and most important gene families, participating in the regulation of plant growth and development and response to abiotic stresses. However, the function of R2R3-MYB genes in Dendrobium nobile remains largely unknown. Here, a comprehensive genome-wide analysis of D. nobile R2R3- DnMYB s was performed, investigating phylogenic relationships, gene structures, motif composition, chromosomal locations, collinearity analysis, and cis-acting elements. Totally, 125 DnMYB genes were identified in the D. nobile genome, and they could be subdivided into 26 groups by further divided through phylogenetic analysis. Most genes with similar exon-intron structures and motif compositions in eaach subgroup exhibited similar functions. All of DnMYB genes were mapped on 19 chromosomes with the co-linearity relationship. Moreover, their expression patterns were analyzed in various tissues and abiotic stresses. The results showed that DnMYBs were significantly differential expressed in different tissues, following abiotic stresses and phytohormone treatments, indicating their possible roles in biological processes and some abiotic stress tolerance and adaptation. This work provides a comprehensive understanding of the R2R3-MYB family of D. nobile , and lays a foundation for future research on the potential function of DnMYB gene in the growth and development of D. nobile . Dendrobium nobile R2R3-MYB Genome-wide Gene family Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction Dendrobium nobile Lindl is a plant of the Dendrobium genus of Orchidaceae, which is a precious and high-grade Chinese medicinal material in China. Dendrobium has a medicinal history of more than 1500 years[ 1 ]. It is mainly distributed in the subtropical regions south of the Yangtze River such as Guizhou, Yunnan and Guangxi. D . nobile in Chinese Medicine was listed as the primary source of medicinal dendrobium in the Chinese Pharmacopoeia (2010). D . nobile contains several classes of interesting bioactive secondary metabolites including polysaccharides, alkaloids, flavonoids, coumarins, phenols, sesquiterpenes, etc . [ 2 ]. D . nobile comprises medicinal, nutritional, and ornamental functions, so it has great prospects for further developments. Transcription factors are located in the nucleus and can recognize and bind cis-acting elements in the promoter region of eukaryotic genes, thereby regulating the expression of downstream genes. Such as the SBP-box family[ 3 ], MYB[ 4 , 5 ], and BLH[ 6 , 7 ]. MYB TFs (transcription factors) are part of one of the largest families of TFs in eukaryotes, which contains a highly conserved DNA binding domain (R) in N-terminus[ 8 ]. According to the number and position of R sequences, the MYB transcription factor family can be divided into four categories, namely R1-MYB, R2R3-MYB, R1R2R3-MYB and 4R-MYB[ 9 , 10 ]. Among them, R2R3-MYB is the largest subfamily in the MYB family, which is widely involved in various physiological and biochemical processes, such as biotic and abiotic stress, metabolism, growth and development of plants[ 11 ]. To date, MYB TFs has been identified in many plants, including the orchid Dendrobium[ 8 ]. 101 DoMYB1 and 99 PaMYB R2R3-MYB genes were identified in the genomes of Dendrobium officinale and Phalaenopsis aphrodite , respectively[ 12 ]. A total of 99 R2R3-MYB proteins were identified in Dendrobium catenatum [ 13 ]. However, there is no report on the R2R3-MYB gene family in D . nobile . In this study, we conducted a genome-wide analysis of the R2R3-MYB gene family in D. nobile , a total of 125 R2R3-MYB proteins from D. nobile were identified and analyzed, including phylogenetic analysis, chromosome localization, gene duplication analysis, conserved domains and gene structures. We used real-time quantitative PCR (RT-qPCR) experiments to compare and analyze three stress treatments (cold, ABA and MeJA) with a control treatment. Our study will provide a foundation for future research into the molecular function of D. nobile R2R3-MYB genes. 2 Materials And Methods 2.1 Plant growth and treatment D. nobile were cultivated at the Anhui University of Chinese Medicine (Hefei, Anhui, China). D. nobile samples are stored in the Herbarium of Anhui University of Chinese Medicine (code: 20220601). It was identified as D. nobile by Professor Yang Qingshan of Anhui University of Traditional Chinese Medicine. In this study, the root, stem, leaf, flower of D. nobile were tested for tissue specificity. Each tissue was collected from three different plants, and the collected samples were immediately stored at − 80°C. For the stress treatment, the plants were treated with 100 µM abscisic acid (ABA), 100 µM methyl jasmonate (MeJA), and 4℃. And the leaves were then collected and frozen in liquid nitrogen at different time points (0, 1, 3, 6, 12, and 24 h) after treatment. 2.2 Identification and sequence analysis The genomes of D. nobile ( https://www.ncbi.nlm.nih.gov/genome/?term=dendrobium+nobile ) were downloaded to identify the candidate R2R3-MYB genes. We used the hidden Markov model (HMM) profile of the MYB domain (PF00249) from the Pfam database to search the protein sequences[ 14 , 15 ]. The putative DnMYB sequences were checked by the National Center for Biotechnology Information (NCBI). Redundant sequences were removed using CD-HIT online analysis (http : // www.bioinformiscs.org/CD-HIT/ ). The remaining sequences were submitted to HMMscan online analysis (https ༚// www.ebi.ac.uk/Tools/hmmer/search/hmmscan ) to determine the MYB domain and quantity, R2R3-MYB sequences were screened for structural features. A total of 126 R2R3-MYB transcription factor sequences of Arabidopsis were obtained from the TAIR (The Arabidopsis Information Resource) database ( https://www.arabidopsis.org/ ). The physicochemical properties of the protein sequences were analyzed with the ProtParam tool of ExPASy ( https://web.expasy.org/protparam/ ). TMHMM software was used to determine the number of transmembrane domains. The subcellular localization of MYB proteins was predicted using the PSORT tool. 2.3 Multiple Alignment and Phylogenetic Analysis The MEGA X software was used for sequence alignment, and the Neighbor-Joining method was used to construct the phylogenetic tree of D. nobile and Arabidopsis R2R3-MYB proteins. The 126 R2R3-MYB proteins of Arabidopsis was used as references, bootstrap analysis of 1000 repetitions were performed to compute the reliability of the phylogenetic tree. 2.4 Gene Structure and Conserved Motif Analysis For gene structure analysis, the corresponding genome sequences of DnMYB genes were obtained from the genome sequences of D. nobile which were downloaded from NCBI. The Gene Structure Display Server 2.04 (GSDS) ( http://gsds.gao-lab.org/ ) was employed to analyze the exons-introns structures of the obtained full sequences. For conserved motif analysis, The MEME suite (Multiple Expectation Maximization for Motif Elicitation, version 5.4.1) ( https://meme-suite.org/meme/tools/meme ) was used to analyze the conserved motif[ 16 , 17 ]. 2.5 Chromosomal Locations and Synteny analysis The physical position of the DnMYB genes on the chromosome was mapped by MG2G online software ( http://mg2c.iask.in/mg2c_v2.1/ )[ 18 ]. The synteny correlation analysis of DnMYB genes between the homologs in Dendrobium chrysotoxum and Arabidopsis thaliana was verified and visualized using TBtools software[ 19 ]. 2.6 Analysis of the cis-regulatory elements in the promoters of DnMYB genes The upstream 2 kb regulatory regions (from the translation start site) of DnMYB genes were obtained from the D. nobile genome database, and the analysis was then performed using PlantCARE software ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ). 2.7 Real-time Quantitative PCR (RT-qPCR) Analysis RT-qPCR analysis was performed using a SuperReal PreMix Plus SyBr Green PCR kit (Qiagen) on a Cobas z480 Real-Time PCR System as the method described by Wang et al. Candidate primers were designed using Primer 5.0 (Supplementary Table 1). Reactions contained 2.0 µL of diluted cDNA, 0.6 µL of each primer, 10 µL of 2 × SuperReal PreMix Plus, and 6.8 µL of RNase-free double-distilled water (ddH 2 O). All RT-qPCRs were performed as follows: denaturation at 95°C for 15 min, followed by 45 cycles of 95°C for 10 s, 58°C for 20 s, and 72°C for 30 s. Each sample contained three biological replicates, a housekeeping gene (18S) was used as a reference, and the relative expression level of each gene was calculated using the 2 −ΔΔCt approach[ 20 ]. 3 Results 3.1 Identification and Classification of R2R3-DnMYB Genes in D. nobile To identify MYB proteins in D. nobile , the genomic sequence was downloaded from NCBI. After searching through the Biolinux system using PF00249 as a seed sequence to perform HMMER alignment, the redundant sequences were removed by the CD-Hit online tool analysis, totally 276 candidate MYB genes were obtained. The number of MYB domains in the sequence of candidates were analyzed by HMMscan online analysis software, and 125 sequences were screened out which matched the characteristics of R2R3-MYB genes. The R2R3-MYB genes of D. nobile were terminated DnMYB1 to DnMYB125 (Supplementary Table 2). These DnMYBs were classified into four distinct groups: R1-MYB family (123 proteins, 44.57%), R2R3-MYB family (125 proteins, 45.29%), 3R-MYB (27 protein, 9.78%), and 4R-MYB families (1 protein, 0.36%), of which the R2R3-MYB family contains the most DnMYB genes, this result is consistent with previous reports, the R2R3-MYB subfamily is the most abundant transcription factor subfamily in plants [ 21 ]. The physicochemical properties of 125 R2R3-MYB genes, including gene locus, protein length, MW, pI, number of transmembrane regions, and subcellular localization of MYB proteins, are listed in Supplementary Table 2. The proteins encoded by these genes ranged from 88 (DnMYB50) to 2424 amino acids (DnMYB64), with an average number of 348 amino acids. The protein molecular weight (MW) and isoelectric point (pI) of predicted DnMYB proteins ranged from 10.32 to 74.13 kD and 4.88–9.01. All predicted DnMYB are not transmembrane proteins. Subcellular localization results show that all predicted DnMYB proteins are localized in the nucleus, which is consistent with the fact that transcription factors must act in the nucleus in order to regulate their expression [ 22 ]. 3.2 Phylogenetic analysis of the R2R3-MYB Proteins in D. nobile In order to explore the evolutionary relationship among these 125 R2R3-MYB proteins, a phylogenetic tree was constructed based on 125 and 126 MYB proteins from D. nobile and A. thalian , respectively (Fig. 1 ). The results showed most of the DnMYB in D. nobile were classified into different subclasses, which were supported by strong bootstrap. The phylogenetic tree of 125 DnMYB were divided into 26 subgroups with the number of members ranging from 1 to 26. The R2R3-MYB proteins has the most members in the S21 subfamily, including 14 DnMYBs. This result is consistent with the classification results of D. officinale and P. aphrodite . The S14 and S18 subfamily contained 7 DnMYBs respectively, followed by 5 DoMYBs were both divided into S4 and S17 subfamilies. The S13, S22, and S25 subfamilies contained 4 DnMYBs. The S1 and S11 subfamily contained 3 DnMYBs. Four subfamilies, including S2, S6, S10, S20, contained 2 DnMYBs. The remaining subfamilies, including S5, S9, S19, S23 and S24, contained only one DnMYBs. Interestingly, no R2R3-MYB proteins were classified into the S12 subfamilies. The Arabidopsis R2R3-MYB transcription factor contains 25 subclasses, of which the S1, S2, S20, and S22 subfamilies are involved in regulating plant responses to abiotic stresses[ 23 ]. The S4, S5 and S6 subfamilies of the Arabidopsis R2R3-MYB transcription factors are involved in plant anthocyanin synthesis[ 23 ]. Since some AtMYB proteins with similar functions are clustered in the same clade, this may indicate that DnMYB proteins in the same clade have similar functions[ 24 ]. However, the functions of these identified transcription factors need to be further studied. 3.3 Gene Structure Analysis of 125 DnMYB genes Exon/intron analysis showed that 125 DnMYB genes had introns in their coding regions. The 125 DnMYB genes have a high degree of diversity in the number and relative positions of introns and exons. As we know, genetic structural diversity may explain the evolution of polygenic families[ 24 ]. The gene structure map of these 125 DnMYB genes was constructed, and the results showed that the clustering pattern of 125 DnMYB genes was not clearly consistent with the exon/intron structure (Fig. 2 ) (Supplementary Table 3). Interestingly, most genes in the same subclass showed similar exon/intron structure. This phenomenon is similar to that mentioned in Liriodendron [ 24 ]. The number of exons ranged from 1 to 13, and most of these genes contained two exons (27 genes) and three exons (67 genes) based to the 125 DnMYB gene structure. 3.4 Motif composition of 125 DnMYB proteins There are many conserved sequences in MYB transcription factors and these conserved sequences may combine with some parts of DNA and play a role in expression regulation[ 25 ]. In order to deeply study the potentially conserved motifs in 125 DnMYB proteins of D. nobile , we systematically studied the evolutionary relationship of 125 DnMYB proteins, and analyzed the motif composition of 125 MYB-encoded proteins by MEME online analysis software. A total of 10 motifs were identified, of which motif 3 is the N-terminal domain of DnMYB proteins, which is present in almost all DnMYBs. Compared with the N-terminus, the C-terminus has a higher variability of motifs and different sequences, and these specific motifs indicate that different subgroups may have different functions[ 26 ] (Fig. 2 ). Diversity in the composition of DnMYB motifs explains the diversity of its functions. Moreover, most DnMYB had motifs 1, 2, 3, 4 and 6, and most members in the same subgroup contained more than one identical motif. Motifs 3, 6, and 2 constitute the complete R2 domain, of which the three tryptophan (W) residues are highly conserved. Motif 1 contains the complete R3 domain, the first tryptophan (W) residue is often replaced by phenylalanine (F), and the remaining two tryptophan (W) residues are highly conserved, and 19 amino acids are separated between the two adjacent tryptophan residues. These are consistent with the characteristics of MYB domain identified in soybean[ 26 ], Ginkgo biloba[ 27 ], Arabidopsis [ 28 ] and other species. Interestingly, the results also show that most members of the same subclass have similar motif composition. 3.5 Chromosomal location and gene duplication of R2R3-MYB gene family In order to understand the distribution of R2R3-DnMYB genes in the genome, MG2G online software was used to locate the 125 R2R3-MYB genes on the corresponding chromosomes. D. nobile has 19 chromosomes, shown ranging from Chr1 to Chr19 (Supplementary Table 4). DnMYBs were present on all chromosomes, while the arrangement and density of DnMYBs on every chromosome were uneven (Fig. 3 ). The maximal number of DnMYBs (12%) was located on chromosome 9, whereas the minimum number of DnMYBs (3.2%) was located on chromosome 4. There was a high density of DnMYBs located on chromosomes 6, 11, 12, 13, 17. Collinearity diagrams among DnMYBs were analyzed using gene duplication analysis. A total of 46 pairs of gene duplications were identified among DnMYBs (Fig. 4 ). The DnMYB genes were homologous to genes in other plants, and syntenic conservation was observed among D. chrysotoxum (174 orthologous gene pairs dispersed on all chromosomes). 28 collinear gene pairs between D. nobile and A. thaliana (Fig. 5 ). The number of orthologous events of D. nobile - D. chrysotoxum was far greater than that of D. nobile - Arabidopsis , and the closer evolutionary distance between D. nobile and D. chrysotoxum was confirmed. 3.6 Prediction of Cis-Acting Elements in the Promoters of 125 DnMYB Genes Promoter is a DNA sequence recognized and combined by RNA polymerase and starts the transcription of downstream genes. The cis-acting element in promoter can bind transcription factors and play an important role in the regulation of gene expression[ 29 ]. To analyze the cis-acting elements located in the 2000bp sequence upstream of the promoter region, we obtained 125 DnMYB promoter sequences and predicted them by PlantCARE online tool (Supplementary Table 5). In addition to the basic elements, these cis-acting elements could be divided into three groups, including hormone-responsive elements (ABA, salicylic acid, gibberellin, MeJA, auxin), abiotic stress elements (low temperature-responsive elements and drought-responsive elements), metabolism elements (regulation flavonoid biosynthetic) (Supplementary Fig. 1). MeJA-responsive element is the most abundant element, containing 350 instances. As we all know, Methyl jasmonic acid regulates physiological processes such as plant growth and development, especially the response of plants to biotic and abiotic stresses[ 30 ]. DnMYB23 contained 24 MeJA-responsive elements in its 2-k upstream regulatory region. In addition, hormone-related elements included 210 ABA-responsive elements, 87 gibberellin-responsive elements, 64 auxin-responsive elements, 58 salicylic acid-responsive elements. Abiotic stress elements contained 60 low temperature responsive elements, of which DnMYB 100 contained 3 low temperature responsive elements. Flowed by 80 drought responsive elements, DnMYB34 , DnMYB37 , DnMYB58 , DnMYB6 , DnMYB74 and DnMYB83 all harbored 3 drought responsive elements. Moreover, 14 regulation flavonoid biosynthetic-responsive elements in its 2-kb upstream regulatory region were identified. These results suggest that the expressions of DnMYB genes are controlled by complex regulatory networks. 3.7 Expression pattern of candidate DnMYB genes in different tissues MYB genes in different subgroups may perform different functions during the growth and development of D. nobile . To investigate the spatial expression levels of MYB genes in D. nobile , the expression levels of 8 genes from each type across four tissues, roots, stems, leaves, and flower, were analyzed by RT-qPCR. DnMYB8 , DnMYB14 , DnMYB26 , DnMYB27 , DnMYB81 , DnMYB90 , DnMYB108 , DnMYB116 belong to the S22, S2, S21, S1, S7, S4, S19, S20 subfamilies, respectively. As shown in Fig. 6 , 8 genes were expressed in all tissues. The results indicated that DnMYB8 , DnMYB14 , DnMYB26 and DnMYB81 shared similar expression patterns and were predominantly expressed in root. DnMYB27 had higher expression in the flower if compared with that in other tissues. Moreover, DnMYB108 showed tissue-specific expression patterns, with 30-fold higher expression levels in flowers than in other tissues. DnMYB90 was principally expressed in leaf and stem. DnMYB116 was classified in the S20 subfamilies with relatively high expression levels in leaf and/or stem. The results suggested that these genes had a possible role in the growth and development of D. nobile . 3.8 Expression pattern of candidate DnMYB genes in response to abiotic stress Based on the reported functions of R2R3-MYB in other plants, we screened out 8 DnMYBs belonging to eight subfamilies to study the role of R2R3-MYB in the regulation of secondary metabolism and the responses to abiotic stress for D. nobile . We performed the expression level of these genes by RT-qPCR after treatment with cold (4℃), ABA, and MeJA. Six DnMYB genes, DnMYB8 , DnMYB14 , DnMYB26 , DnMYB27 , DnMYB90 , and DnMYB116 showed increased expression levels at different times under the low temperature, ABA, and MeJA treatment (Fig. 7 ). While DnMYB81 was the most responsive to low temperature, and showed downregulated expression patterns under ABA and MeJA stress. DnMYB108 showed downregulated expression patterns under either cold or MeJA stress. The gene expression of DnMYB108 plummeted at the lowest value at 12 h and reached its peak at 24 h under ABA stress. 4 Discussion 4.1 Evolutionary Analysis of the DnMYB Gene Family R2R3-MYB transcription factors have been identified in many plants, including Arabidopsis [ 28 ], maize[ 31 ], soybean[ 26 ], etc. To date, there is no full analysis of the R2R3-DnMYB gene family and most functions remain unclear. In this study, 125 R2R3-MYB transcription factors were identified in the D. nobile genome and divided into 23 subfamilies. Gene structure, cis-acting element analysis, chromosomal distribution, phylogenetic analysis, gene duplication events, and expression level analysis were determined. This study offers new insights for future investigators to identify functional differences in the R2R3-DnMYB family genes. This study provides new insights for future researchers to determine the functional differences of R2R3-MYB family genes in D. nobile . 4.2 DnMYB Genes Play Crucial Roles in D. nobile Growth and Development and Response to Phytohormone and Abiotic Stresses Low temperature, drought and abiotic stress seriously affected the yield of D. nobile . Secondary metabolites in plants, including flavonoids and anthocyanins, can help plants adapt to harsh environments. Meanwhile, flavonoid biosynthesis can be induced under a wide range of abiotic stresses. Therefore, the stress resistance of D. nobile can be improved by regulating the genes related to secondary metabolites, thereby increasing the yield. MYB gene can regulate the development of trichomes and root hairs, and plays a crucial role in regulating the synthesis of plant secondary metabolites such as flavonoids and anthocyanins and helping plants resist abiotic stress. In order to screen DnMYB genes that may be involved in regulating the growth, development and secondary metabolic synthesis of D. nobile , this study selected a total of 8 genes from different subgroups and measured their expression levels in different tissues and different stresses. Gene functions are closely related to tissue-specific expression. As the major pigments in plant, flavonoids contain various important bioactivities and have gained a lot of attention. It reported that flavonoids biosynthesis in plants could be induced under a wide range of abiotic stresses. The effects of these stresses on the contents of flavonoids have been investigated mainly in some crops and model plants. For example, in wheat, the key enzymes in the flavonoids’ biosynthesis pathway were induced by drought stress[ 32 ]. The S4 subfamily AtMYB4 , and AtMYB7 were related to the flavonol synthesis pathway[ 33 ]. The S7 subfamily AtMYB11 , AtMYB12 and AtMYB111 were involved in regulating flavonol biosynthesis in Arabidopsis [ 34 ]. Genes with high homology in the same branch of a phylogenetic tree generally have high sequence similarity and may also have similar functions. DnMYB90 also belonged to S4 subfamily, which is a homologous gene of AtMYB4 , AtMYB7 . RT-qPCR showed that DnMYB90 was expressed in all tissues of D. nobile , but the highest expression level was found in leaf. DnMYB90 was also expressed at very high levels under phytohormonal and abiotic stresses. DnMYB90 exhibited an elevated expression level under cold, ABA and MeJA stress. And it was the most responsive to low temperature. DnMYB81 belonging to the S7 subfamily had close homology with AtMYB11 , AtMYB12 , and AtMYB111 . DnMYB81 belonging to the S7 subfamily had close homology with AtMYB11 , AtMYB12 , and AtMYB111 . The RT-qPCR showed that DnMYB81 expressed in all tissues of D. nobile , but the highest expression level was found in the root. DnMYB81 was also responsive to phytohormonal and abiotic stresses. DnMYB81 was the most responsive to low temperature, and showed downregulated expression patterns under ABA and MeJA stress. In the future, we can verify whether DnMYB81 and DnMYB90 can regulate flavonoid synthesis through more in-depth experiments. It has been reported that several R2R3-MYB genes are involved in regulating responses to biotic and abiotic stresses. For example, overexpression of the S1 subfamily AtMYB94 , AtMYB96 gene enhances drought tolerance in plants[ 35 ]. The S2 subfamily AtMYB14 regulates cold tolerance in Arabidopsis [ 36 ]. Under phosphate starvation conditions, The S2 subfamily AtMYB20 , which plays a role in plant responses to salt and drought stress, directly regulates the expression of miR399f [ 37 ]. Overexpression of the S21 subfamily AtMYB52 confers ABA hypersensitivity and drought tolerance[ 38 ]. The S22 subfamily AtMYB44 was tested in response to salt, MeJA and drought[ 39 ]. In order to explore DnMYB genes involved in hormonal and abiotic stresses, we focused on the expression levels of DnMYB genes from S1, S2, S20, S21, and S22 under low temperature, ABA, and MeJA stress. Using RT-qPCR, we found that DnMYB27 of S1, DnMYB14 of S2, DnMYB116 of S20, DnMYB26 of S21, and DnMYB8 of S22, which were homologous to stress response genes, were expressed in all tissues. To further identify candidate genes involved in hormone signaling pathways or abiotic stress responses, we measured their expression levels under low temperature, ABA and MeJA stress. The results showed that the five genes showed increased expression levels at different times under different stress, which was consistent with the cis-acting element in its promoter (Fig. 2 , Fig. 7 ). Interestingly, the expression level of the S19 subfamily DnMYB108 gradually decreased after treatment with cold and MeJA stress. These results may suggest that members of the S19 subfamily play an important role in the response of D. nobile to abiotic stress. Some of the DnMYB genes screened in this study may be involved in abiotic stresses such as low temperature, ABA and MeJA stress, but the specific mechanism is still unclear and needs to be further verified in future experiments. Abbreviations TFs: transcription factors; RT-qPCR: real-time quantitative PCR; ABA: abscisic acid; MeJA: methyl jasmonate; HMM: hidden Markov model; NCBI: National Center for Biotechnology Information; MW: molecular weight; pI: isoelectric point. Declarations Acknowledgments We thank Dr. Shuai Liu from the University of Hawaii Cancer Center for helping to revise the manuscript. We also thank Dr. Xiaoxi Meng and Zhikai Liang from the Department of Horticultural Science at University of Minnesota for helpful comments on an earlier version of the manuscript. Statement Our experimental research on Dendrobium nobile was in accordance with the relevant national/institutional guidelines. Author contributions Conceived, designed, and implemented the study: LW, LZ, and SX; Statistics analysis: LW, JF, WJ, LZ, and XS; Reagents/materials/analysis tools: ZS, DP, and SX; Drafted the manuscript: LW, XS, WR, YD, YW, and SX; All authors participated in the editing of the manuscript and agreed to submit the final version. Funding This work was supported by was supported by National Natural Science Foundation of China (Grant No. U19A2009), Key Natural Science Research Projects in Anhui Universities (No. 2022AH050461, No. KJ2019A0453 and KJ2018A0275), Open Project of Provincial and Ministerial Scientific Research Platform, Fuyang Normal University (No. FSKFKT010D) and Anhui University Collaborative Innovation Project (Grant No. GXXT-2019-043, Grant No. GXXT-2019-049). Data availability statement All data generated or analyzed in this study are included in this article (Supplementary file). The genome sequences of D. nobile , D. chrysotoxum and Arabidopsis were downloaded from the https://www.ncbi.nlm.nih.gov/genome/?term=dendrobium+nobile, https://www.ncbi.nlm.nih.gov/genome/?term=Dendrobium+chrysotoxum, and https://www.arabidopsis.org/. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details 1 College of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China. 2 Institute of Traditional Chinese Medicine Resources Protection and Development, Anhui Academy of Chinese Medicine, Hefei 230012, China. 3 MOE-Anhui Joint Collaborative Innovation Center for Quality Improvement of Anhui Genuine Chinese Medicinal Materials, Hefei 230038, China. 4 Hunan Key Laboratory for Conservation and Utilization of Biological Resources in the Nanyue Mountainous Region, College of Life Sciences and Environment, Hengyang Normal University, Hengyang, 421008, China. 5 Anhui Province Key Laboratory of Environmental Hormone and Reproduction, Anhui Province Key Laboratory of Embryo Development and Reproductive Regulation, Fuyang Normal University, Fuyang, China. 6 Innovative Drug R&D Center, College of Life Sciences, Huaibei Normal University. 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R2R3-MYB transcription factor PpMYB17 positively regulates flavonoid biosynthesis in pear fruit. Planta. 2020;252(4):59. Du H, Yang SS, Liang Z, Feng BR, Liu L, Huang YB, et al. Genome-wide analysis of the MYB transcription factor superfamily in soybean. BMC Plant Biol. 2012;12:106. Liu X, Yu W, Zhang X, Wang G, Cao F, Cheng H. Identification and expression analysis under abiotic stress of the R2R3-MYB genes in Ginkgo biloba L. Physiol Mol Biol Plants. 2017;23(3):503–16. Stracke R, Werber M, Weisshaar B. The R2R3-MYB gene family in Arabidopsis thaliana . Curr Opin Plant Biol. 2001;4(5):447–56. Mikhaylichenko O, Bondarenko V, Harnett D, Schor IE, Males M, Viales RR, et al. The degree of enhancer or promoter activity is reflected by the levels and directionality of erna transcription. Genes Dev. 2018;32(1):42–57. Gomi K. Jasmonic acid pathway in plants 2.0. Int J Mol Sci. 2021;22(7):3506. Du H, Feng BR, Yang SS, Huang YB, Tang YX. The R2R3-MYB transcription factor gene family in maize. PLoS ONE. 2012;7(6):e37463. Colebrook EH, Thomas SG, Phillips AL, Hedden P. The role of gibberellin signalling in plant responses to abiotic stress. J Exp Biol. 2014;217(Pt 1):67–75. Fornalé S, Lopez E, Salazar-Henao JE, Fernández-Nohales P, Rigau J, Caparros-Ruiz D. AtMYB7, a new player in the regulation of UV-sunscreens in Arabidopsis thaliana . Plant Cell Physiol. 2014;55(3):507–16. Stracke R, Ishihara H, Huep G, Barsch A, Mehrtens F, Niehaus K, et al. Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. Plant J. 2007;50(4):660–77. Lee SB, Kim HU, Suh MC. MYB94 and MYB96 additively activate cuticular wax biosynthesis in Arabidopsis. Plant Cell Physiol. 2016;57(11):2300–11. Chen Y, Chen Z, Kang J, Kang D, Gu H, Qin G. AtMYB14 regulates cold tolerance in Arabidopsis. Plant Mol Biol Report. 2013;31(1):87–97. Baek D, Chun HJ, Kang S, Shin G, Park SJ, Hong H, et al. A role for Arabidopsis mir399f in salt, drought, and ABA signaling. Mol Cells. 2016;39(2):111–8. Park MY, Kang JY, Kim SY. Overexpression of AtMYB52 confers ABA hypersensitivity and drought tolerance. Mol Cells. 2011;31(5):447–54. Jung C, Seo JS, Han SW, Koo YJ, Kim CH, Song SI, et al. Overexpression of AtMYB44 enhances stomatal closure to confer abiotic stress tolerance in transgenic Arabidopsis. Plant Physiol. 2008;146(2):623–35. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigureS1ThecisactingelementsanalysisofDnMYBgenespromoters.jpg SupplementaryTableS1PrimersusedinrealtimequantitativePCRanalysis..xlsx SupplementaryTableS2PhysicochemicalpropertiesofDnMYBproteins..xlsx SupplementaryTableS3ExonsandintronsstructureofallDnMYBs..xlsx SupplementaryTableS4LengthandchromosomallocationoftheDnMYBs..xlsx SupplementaryTableS5Thecisactingelementsin2000bpsequenceupstreamofDnMYBspromoterregion..xlsx 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. We do this by developing innovative software and high quality services for the global research community. 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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-2749425","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":189583572,"identity":"a679b916-d5b4-4bf1-8e32-90afe3ec0e4b","order_by":0,"name":"Liping Wu","email":"","orcid":"","institution":"Anhui University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Liping","middleName":"","lastName":"Wu","suffix":""},{"id":189583573,"identity":"e6a1a0cc-e320-4e1a-85c1-a6718aa93fc2","order_by":1,"name":"Jizhou Fan","email":"","orcid":"","institution":"Anhui University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jizhou","middleName":"","lastName":"Fan","suffix":""},{"id":189583574,"identity":"8923c5da-6343-4ca6-b990-b7c582cfcfa7","order_by":2,"name":"Xinglong Su","email":"","orcid":"","institution":"Anhui University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xinglong","middleName":"","lastName":"Su","suffix":""},{"id":189583575,"identity":"d80bc31b-379b-452a-8b28-6e0fcf75e562","order_by":3,"name":"Weiyi Rao","email":"","orcid":"","institution":"Anhui University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Weiyi","middleName":"","lastName":"Rao","suffix":""},{"id":189583576,"identity":"10dfb3cd-79e4-4a14-b690-8b770a86c203","order_by":4,"name":"Yingying Duan","email":"","orcid":"","institution":"Anhui University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yingying","middleName":"","lastName":"Duan","suffix":""},{"id":189583577,"identity":"3b72a50f-c761-4eee-a179-6f58ce564426","order_by":5,"name":"Yuqing Wang","email":"","orcid":"","institution":"MOE-Anhui Joint Collaborative Innovation Center for Quality Improvement of Anhui Genuine Chinese Medicinal Materials","correspondingAuthor":false,"prefix":"","firstName":"Yuqing","middleName":"","lastName":"Wang","suffix":""},{"id":189583578,"identity":"bfbcf4f1-55aa-44c2-b716-f8a8925046ac","order_by":6,"name":"Weimin Jiang","email":"","orcid":"","institution":"Hengyang Normal University","correspondingAuthor":false,"prefix":"","firstName":"Weimin","middleName":"","lastName":"Jiang","suffix":""},{"id":189583579,"identity":"0f05e9f1-74ee-4962-bb06-2ffe5651a324","order_by":7,"name":"Zongping Sun","email":"","orcid":"","institution":"Fuyang Normal University","correspondingAuthor":false,"prefix":"","firstName":"Zongping","middleName":"","lastName":"Sun","suffix":""},{"id":189583580,"identity":"3ed39ed5-522c-4f24-b5df-3c3d805291f6","order_by":8,"name":"Lei Zhang","email":"","orcid":"","institution":"Huaibei Normal University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Zhang","suffix":""},{"id":189583581,"identity":"dd0711ff-1704-4024-9eca-f328c876da6e","order_by":9,"name":"Daiyin Peng","email":"","orcid":"","institution":"Anhui Academy of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Daiyin","middleName":"","lastName":"Peng","suffix":""},{"id":189583582,"identity":"97ecd9c1-df09-4aba-8bf1-23189367f7cd","order_by":10,"name":"Shihai Xing","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYFACHgZmBoMDDGzsjY0PP5Cmhedws7EE8VoYDjAwSKS3CfAQo8Hg/NnDnwsK7sjzST5sY5BgsJPTbSCgRXJGXpr0DINnhm3SiW0PChiSjc0OENDCL8FjxsxjcJgRqKXdQILhQOI2QlrY+M8YfwZqsW+TPNgmwUOMFn6GHANpoJbENglGIrVIzsgxA2lJbuNJBAayARF+MTgPctifw7bz248/fPihwk6OoBZ0E0hTPgpGwSgYBaMABwAAWM8+AFl4EP4AAAAASUVORK5CYII=","orcid":"","institution":"Anhui Province Key Laboratory of Research \u0026 Development of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Shihai","middleName":"","lastName":"Xing","suffix":""}],"badges":[],"createdAt":"2023-03-29 02:29:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2749425/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2749425/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":35459402,"identity":"1049321f-5b68-4fd4-96df-d1ec95f9fba4","added_by":"auto","created_at":"2023-04-07 18:21:23","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1056366,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of R2R3-MYBs in \u003cem\u003eD. nobile\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e. The figure was generated by using MEGA-software coupled with a neighbor-joining method and a bootstrap of 1,000 replicate.\u003c/p\u003e","description":"","filename":"Figure1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2749425/v1/0cb6140f9a294c2607d3fd8a.jpeg"},{"id":35459407,"identity":"bd281a54-9a8b-4ef1-a8e7-f3d807d4ee45","added_by":"auto","created_at":"2023-04-07 18:21:23","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4946897,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic relationships, conserved motifs, and gene structures of R2R3-DnMYBs family member. (a) The phylogenetic tree was constructed based on the full-length sequences of 125 R2R3-DnMYB proteins using the neighbor-joining method in Mega X, with 1000 bootstrap replicates. (b) Conserved motifs of DnMYB proteins. Different motifs are represented by various colored boxes. (c) Exon/intron structures of \u003cem\u003eDnMYB\u003c/em\u003e genes. Exon(s), and intron(s) are represented by black boxes and black lines, respectively.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2749425/v1/bfcaa1a21d6763f97a3d930a.jpg"},{"id":35459409,"identity":"a1ea4541-9100-4e57-bd15-1939d7f331aa","added_by":"auto","created_at":"2023-04-07 18:21:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1296302,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of 125 R2R3-DnMYB genes on chromosomes of \u003cem\u003eD. nobile\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2749425/v1/bca783072129f071eacdda35.jpg"},{"id":35460401,"identity":"09af6079-dc90-4d41-9d8b-07c06b8ffd00","added_by":"auto","created_at":"2023-04-07 18:45:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2044461,"visible":true,"origin":"","legend":"\u003cp\u003eSynteny analysis of interchromosomal relationships of \u003cem\u003eDnMYB\u003c/em\u003e genes. Red lines indicate duplicated gene pair.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2749425/v1/b00a4ba0638e87a952883a07.jpg"},{"id":35459413,"identity":"ef97620e-eca1-41ed-a83a-8c6bc1dd2081","added_by":"auto","created_at":"2023-04-07 18:21:23","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2707917,"visible":true,"origin":"","legend":"\u003cp\u003eSynteny analysis of \u003cem\u003eDnMYBs\u003c/em\u003egenes in the genomes between \u003cem\u003eD. nobile\u003c/em\u003e and \u003cem\u003eD. chrysotoxum\u003c/em\u003eor \u003cem\u003eA. thaliana\u003c/em\u003e. (a) \u003cem\u003eD. nobile \u003c/em\u003eand \u003cem\u003eD. chrysotoxum\u003c/em\u003e; (b) \u003cem\u003eD. nobile \u003c/em\u003eand \u003cem\u003eA. thaliana\u003c/em\u003e. The gray lines show collinear blocks. The red and blue lines indicate the syntenic gene pairs between \u003cem\u003eD. nobile\u003c/em\u003e and \u003cem\u003eD. chrysotoxum\u003c/em\u003eor \u003cem\u003eA. thaliana\u003c/em\u003e, respectively.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2749425/v1/136079beb2c448d9c040c14f.jpg"},{"id":35459918,"identity":"6d922b20-9f86-44c5-8614-8c3c073a220f","added_by":"auto","created_at":"2023-04-07 18:37:23","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":666830,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression levels of 8 selected R2R3-DnMYB genes in different tissues. Error bars indicate the mean ± SE of three independent replicates. Different lowercase letters (a, b, and c) are significantly different (\u003cem\u003ep\u003c/em\u003e-value\u003cem\u003e \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2749425/v1/d8a0bb28f765cdbc18d954ce.jpg"},{"id":35459831,"identity":"7fdf1e5d-bf65-45fb-9804-2eb174da7f43","added_by":"auto","created_at":"2023-04-07 18:29:23","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1114732,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of 8 R2R3-DnMYB genes at 0, 2, 6, 12, and 24 h under various abiotic stresses. Error bars indicate the mean ± SE of three independent replicates. Different lowercase letters (a, b, and c) are significantly different (\u003cem\u003ep\u003c/em\u003e-value\u003cem\u003e \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2749425/v1/14a875cd027580759c632832.jpg"},{"id":38016339,"identity":"686b0d89-0b2b-46be-b44d-20fd6208596e","added_by":"auto","created_at":"2023-06-05 11:44:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1485901,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2749425/v1/651d02af-6970-49cb-a6d8-0456b5650734.pdf"},{"id":35459411,"identity":"d4fffaab-89fa-46d8-91fd-4829af7e3192","added_by":"auto","created_at":"2023-04-07 18:21:23","extension":"jpg","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4968491,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS1ThecisactingelementsanalysisofDnMYBgenespromoters.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2749425/v1/6a5d7c092486735c958d74d2.jpg"},{"id":35459827,"identity":"d2581ae4-a179-485b-a59a-1434cb932d9a","added_by":"auto","created_at":"2023-04-07 18:29:23","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10605,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1PrimersusedinrealtimequantitativePCRanalysis..xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2749425/v1/67e1ee543e4411fe23d8208b.xlsx"},{"id":35459826,"identity":"5cdb49f6-eb43-46f3-9ae2-1f28cf9ee581","added_by":"auto","created_at":"2023-04-07 18:29:23","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":42274,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS2PhysicochemicalpropertiesofDnMYBproteins..xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2749425/v1/847a3b046360a46d650bea40.xlsx"},{"id":35459829,"identity":"25497b69-96f8-4216-a0d3-505db8fcec3f","added_by":"auto","created_at":"2023-04-07 18:29:23","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":22530,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS3ExonsandintronsstructureofallDnMYBs..xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2749425/v1/534d118a67c900712efb5423.xlsx"},{"id":35459404,"identity":"52bd22ba-3945-4954-903d-c4d4686e2fc2","added_by":"auto","created_at":"2023-04-07 18:21:23","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":15660,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS4LengthandchromosomallocationoftheDnMYBs..xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2749425/v1/00c9ebf934238def74916c46.xlsx"},{"id":35459414,"identity":"3c4e4d1b-34d4-4b27-9e02-544e6a3ec9ad","added_by":"auto","created_at":"2023-04-07 18:21:23","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":33117,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS5Thecisactingelementsin2000bpsequenceupstreamofDnMYBspromoterregion..xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2749425/v1/d2ae1adabba4c78aa5d9aee2.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide identification of R2R3-MYB Family Genes and their Response to Stress in Dendrobium nobile","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003e \u003cem\u003eDendrobium nobile\u003c/em\u003e Lindl is a plant of the Dendrobium genus of Orchidaceae, which is a precious and high-grade Chinese medicinal material in China. Dendrobium has a medicinal history of more than 1500 years[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is mainly distributed in the subtropical regions south of the Yangtze River such as Guizhou, Yunnan and Guangxi. \u003cem\u003eD\u003c/em\u003e. \u003cem\u003enobile\u003c/em\u003e in Chinese Medicine was listed as the primary source of medicinal dendrobium in the Chinese Pharmacopoeia (2010). \u003cem\u003eD\u003c/em\u003e. \u003cem\u003enobile\u003c/em\u003e contains several classes of interesting bioactive secondary metabolites including polysaccharides, alkaloids, flavonoids, coumarins, phenols, sesquiterpenes, \u003cem\u003eetc\u003c/em\u003e. [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. \u003cem\u003eD\u003c/em\u003e. \u003cem\u003enobile\u003c/em\u003e comprises medicinal, nutritional, and ornamental functions, so it has great prospects for further developments.\u003c/p\u003e \u003cp\u003eTranscription factors are located in the nucleus and can recognize and bind cis-acting elements in the promoter region of eukaryotic genes, thereby regulating the expression of downstream genes. Such as the SBP-box family[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], MYB[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and BLH[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. MYB TFs (transcription factors) are part of one of the largest families of TFs in eukaryotes, which contains a highly conserved DNA binding domain (R) in N-terminus[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. According to the number and position of R sequences, the MYB transcription factor family can be divided into four categories, namely R1-MYB, R2R3-MYB, R1R2R3-MYB and 4R-MYB[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Among them, R2R3-MYB is the largest subfamily in the MYB family, which is widely involved in various physiological and biochemical processes, such as biotic and abiotic stress, metabolism, growth and development of plants[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. To date, MYB TFs has been identified in many plants, including the orchid Dendrobium[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. 101 \u003cem\u003eDoMYB1\u003c/em\u003eand 99 \u003cem\u003ePaMYB R2R3-MYB\u003c/em\u003e genes were identified in the genomes of \u003cem\u003eDendrobium officinale\u003c/em\u003e and \u003cem\u003ePhalaenopsis aphrodite\u003c/em\u003e, respectively[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. A total of 99 R2R3-MYB proteins were identified in \u003cem\u003eDendrobium catenatum\u003c/em\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, there is no report on the \u003cem\u003eR2R3-MYB\u003c/em\u003e gene family in \u003cem\u003eD\u003c/em\u003e. \u003cem\u003enobile\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we conducted a genome-wide analysis of the \u003cem\u003eR2R3-MYB\u003c/em\u003e gene family in \u003cem\u003eD. nobile\u003c/em\u003e, a total of 125 R2R3-MYB proteins from \u003cem\u003eD. nobile\u003c/em\u003e were identified and analyzed, including phylogenetic analysis, chromosome localization, gene duplication analysis, conserved domains and gene structures. We used real-time quantitative PCR (RT-qPCR) experiments to compare and analyze three stress treatments (cold, ABA and MeJA) with a control treatment. Our study will provide a foundation for future research into the molecular function of \u003cem\u003eD. nobile R2R3-MYB\u003c/em\u003e genes.\u003c/p\u003e"},{"header":"2 Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant growth and treatment\u003c/h2\u003e \u003cp\u003e \u003cem\u003eD. nobile\u003c/em\u003e were cultivated at the Anhui University of Chinese Medicine (Hefei, Anhui, China). \u003cem\u003eD. nobile\u003c/em\u003e samples are stored in the Herbarium of Anhui University of Chinese Medicine (code: 20220601). It was identified as \u003cem\u003eD. nobile\u003c/em\u003e by Professor Yang Qingshan of Anhui University of Traditional Chinese Medicine. In this study, the root, stem, leaf, flower of \u003cem\u003eD. nobile\u003c/em\u003e were tested for tissue specificity. Each tissue was collected from three different plants, and the collected samples were immediately stored at \u0026minus;\u0026thinsp;80\u0026deg;C. For the stress treatment, the plants were treated with 100 \u0026micro;M abscisic acid (ABA), 100 \u0026micro;M methyl jasmonate (MeJA), and 4℃. And the leaves were then collected and frozen in liquid nitrogen at different time points (0, 1, 3, 6, 12, and 24 h) after treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Identification and sequence analysis\u003c/h2\u003e \u003cp\u003eThe genomes of \u003cem\u003eD. nobile\u003c/em\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/genome/?term=dendrobium+nobile\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/genome/?term=dendrobium+nobile\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were downloaded to identify the candidate \u003cem\u003eR2R3-MYB\u003c/em\u003e genes. We used the hidden Markov model (HMM) profile of the MYB domain (PF00249) from the Pfam database to search the protein sequences[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The putative DnMYB sequences were checked by the National Center for Biotechnology Information (NCBI). Redundant sequences were removed using CD-HIT online analysis (http : //\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/genome/?term=dendrobium+nobile\" target=\"_blank\"\u003ewww.bioinformiscs.org/CD-HIT/\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.bioinformiscs.org/CD-HIT/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The remaining sequences were submitted to HMMscan online analysis (https ༚//\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/genome/?term=dendrobium+nobile\" target=\"_blank\"\u003ewww.ebi.ac.uk/Tools/hmmer/search/hmmscan\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.ebi.ac.uk/Tools/hmmer/search/hmmscan\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to determine the MYB domain and quantity, R2R3-MYB sequences were screened for structural features. A total of 126 \u003cem\u003eR2R3-MYB\u003c/em\u003e transcription factor sequences of \u003cem\u003eArabidopsis\u003c/em\u003e were obtained from the TAIR (The \u003cem\u003eArabidopsis\u003c/em\u003e Information Resource) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe physicochemical properties of the protein sequences were analyzed with the ProtParam tool of ExPASy (\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). TMHMM software was used to determine the number of transmembrane domains. The subcellular localization of MYB proteins was predicted using the PSORT tool.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Multiple Alignment and Phylogenetic Analysis\u003c/h2\u003e \u003cp\u003eThe MEGA X software was used for sequence alignment, and the Neighbor-Joining method was used to construct the phylogenetic tree of \u003cem\u003eD. nobile\u003c/em\u003e and \u003cem\u003eArabidopsis\u003c/em\u003e R2R3-MYB proteins. The 126 R2R3-MYB proteins of \u003cem\u003eArabidopsis\u003c/em\u003e was used as references, bootstrap analysis of 1000 repetitions were performed to compute the reliability of the phylogenetic tree.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Gene Structure and Conserved Motif Analysis\u003c/h2\u003e \u003cp\u003eFor gene structure analysis, the corresponding genome sequences of DnMYB genes were obtained from the genome sequences of \u003cem\u003eD. nobile\u003c/em\u003e which were downloaded from NCBI. The Gene Structure Display Server 2.04 (GSDS) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds.gao-lab.org/\u003c/span\u003e\u003cspan address=\"http://gsds.gao-lab.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was employed to analyze the exons-introns structures of the obtained full sequences. For conserved motif analysis, The MEME suite (Multiple Expectation Maximization for Motif Elicitation, version 5.4.1) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/tools/meme\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to analyze the conserved motif[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Chromosomal Locations and Synteny analysis\u003c/h2\u003e \u003cp\u003eThe physical position of the \u003cem\u003eDnMYB\u003c/em\u003e genes on the chromosome was mapped by MG2G online software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mg2c.iask.in/mg2c_v2.1/\u003c/span\u003e\u003cspan address=\"http://mg2c.iask.in/mg2c_v2.1/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The synteny correlation analysis of \u003cem\u003eDnMYB\u003c/em\u003e genes between the homologs in \u003cem\u003eDendrobium chrysotoxum\u003c/em\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e was verified and visualized using TBtools software[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Analysis of the cis-regulatory elements in the promoters of \u003cem\u003eDnMYB\u003c/em\u003e genes\u003c/h2\u003e \u003cp\u003eThe upstream 2 kb regulatory regions (from the translation start site) of \u003cem\u003eDnMYB\u003c/em\u003e genes were obtained from the \u003cem\u003eD. nobile\u003c/em\u003e genome database, and the analysis was then performed using PlantCARE software (\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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Real-time Quantitative PCR (RT-qPCR) Analysis\u003c/h2\u003e \u003cp\u003eRT-qPCR analysis was performed using a SuperReal PreMix Plus SyBr Green PCR kit (Qiagen) on a Cobas z480 Real-Time PCR System as the method described by Wang et al. Candidate primers were designed using Primer 5.0 (Supplementary Table\u0026nbsp;1). Reactions contained 2.0 \u0026micro;L of diluted cDNA, 0.6 \u0026micro;L of each primer, 10 \u0026micro;L of 2 \u0026times; SuperReal PreMix Plus, and 6.8 \u0026micro;L of RNase-free double-distilled water (ddH\u003csub\u003e2\u003c/sub\u003eO). All RT-qPCRs were performed as follows: denaturation at 95\u0026deg;C for 15 min, followed by 45 cycles of 95\u0026deg;C for 10 s, 58\u0026deg;C for 20 s, and 72\u0026deg;C for 30 s. Each sample contained three biological replicates, a housekeeping gene (18S) was used as a reference, and the relative expression level of each gene was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e approach[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Identification and Classification of \u003cem\u003eR2R3-DnMYB\u003c/em\u003e Genes in \u003cem\u003eD. nobile\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo identify MYB proteins in \u003cem\u003eD. nobile\u003c/em\u003e, the genomic sequence was downloaded from NCBI. After searching through the Biolinux system using PF00249 as a seed sequence to perform HMMER alignment, the redundant sequences were removed by the CD-Hit online tool analysis, totally 276 candidate \u003cem\u003eMYB\u003c/em\u003e genes were obtained. The number of MYB domains in the sequence of candidates were analyzed by HMMscan online analysis software, and 125 sequences were screened out which matched the characteristics of \u003cem\u003eR2R3-MYB\u003c/em\u003e genes. The \u003cem\u003eR2R3-MYB\u003c/em\u003e genes of \u003cem\u003eD. nobile\u003c/em\u003e were terminated \u003cem\u003eDnMYB1\u003c/em\u003e to \u003cem\u003eDnMYB125\u003c/em\u003e (Supplementary Table\u0026nbsp;2). These DnMYBs were classified into four distinct groups: R1-MYB family (123 proteins, 44.57%), R2R3-MYB family (125 proteins, 45.29%), 3R-MYB (27 protein, 9.78%), and 4R-MYB families (1 protein, 0.36%), of which the R2R3-MYB family contains the most \u003cem\u003eDnMYB\u003c/em\u003e genes, this result is consistent with previous reports, the R2R3-MYB subfamily is the most abundant transcription factor subfamily in plants [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The physicochemical properties of 125 \u003cem\u003eR2R3-MYB\u003c/em\u003e genes, including gene locus, protein length, MW, pI, number of transmembrane regions, and subcellular localization of MYB proteins, are listed in Supplementary Table\u0026nbsp;2. The proteins encoded by these genes ranged from 88 (DnMYB50) to 2424 amino acids (DnMYB64), with an average number of 348 amino acids. The protein molecular weight (MW) and isoelectric point (pI) of predicted DnMYB proteins ranged from 10.32 to 74.13 kD and 4.88\u0026ndash;9.01. All predicted DnMYB are not transmembrane proteins. Subcellular localization results show that all predicted DnMYB proteins are localized in the nucleus, which is consistent with the fact that transcription factors must act in the nucleus in order to regulate their expression [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Phylogenetic analysis of the R2R3-MYB Proteins in \u003cem\u003eD. nobile\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eIn order to explore the evolutionary relationship among these 125 R2R3-MYB proteins, a phylogenetic tree was constructed based on 125 and 126 MYB proteins from \u003cem\u003eD. nobile\u003c/em\u003e and \u003cem\u003eA. thalian\u003c/em\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The results showed most of the DnMYB in \u003cem\u003eD. nobile\u003c/em\u003e were classified into different subclasses, which were supported by strong bootstrap. The phylogenetic tree of 125 DnMYB were divided into 26 subgroups with the number of members ranging from 1 to 26. The R2R3-MYB proteins has the most members in the S21 subfamily, including 14 DnMYBs. This result is consistent with the classification results of \u003cem\u003eD. officinale and P. aphrodite\u003c/em\u003e. The S14 and S18 subfamily contained 7 DnMYBs respectively, followed by 5 DoMYBs were both divided into S4 and S17 subfamilies. The S13, S22, and S25 subfamilies contained 4 DnMYBs. The S1 and S11 subfamily contained 3 DnMYBs. Four subfamilies, including S2, S6, S10, S20, contained 2 DnMYBs. The remaining subfamilies, including S5, S9, S19, S23 and S24, contained only one DnMYBs. Interestingly, no R2R3-MYB proteins were classified into the S12 subfamilies. The \u003cem\u003eArabidopsis\u003c/em\u003e R2R3-MYB transcription factor contains 25 subclasses, of which the S1, S2, S20, and S22 subfamilies are involved in regulating plant responses to abiotic stresses[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The S4, S5 and S6 subfamilies of the \u003cem\u003eArabidopsis\u003c/em\u003e R2R3-MYB transcription factors are involved in plant anthocyanin synthesis[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Since some AtMYB proteins with similar functions are clustered in the same clade, this may indicate that DnMYB proteins in the same clade have similar functions[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, the functions of these identified transcription factors need to be further studied.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Gene Structure Analysis of 125 \u003cem\u003eDnMYB\u003c/em\u003e genes\u003c/h2\u003e \u003cp\u003eExon/intron analysis showed that 125 \u003cem\u003eDnMYB\u003c/em\u003e genes had introns in their coding regions. The 125 \u003cem\u003eDnMYB\u003c/em\u003e genes have a high degree of diversity in the number and relative positions of introns and exons. As we know, genetic structural diversity may explain the evolution of polygenic families[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The gene structure map of these 125 \u003cem\u003eDnMYB\u003c/em\u003e genes was constructed, and the results showed that the clustering pattern of 125 \u003cem\u003eDnMYB\u003c/em\u003e genes was not clearly consistent with the exon/intron structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (Supplementary Table\u0026nbsp;3). Interestingly, most genes in the same subclass showed similar exon/intron structure. This phenomenon is similar to that mentioned in \u003cem\u003eLiriodendron\u003c/em\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The number of exons ranged from 1 to 13, and most of these genes contained two exons (27 genes) and three exons (67 genes) based to the 125 \u003cem\u003eDnMYB\u003c/em\u003e gene structure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Motif composition of 125 DnMYB proteins\u003c/h2\u003e \u003cp\u003eThere are many conserved sequences in MYB transcription factors and these conserved sequences may combine with some parts of DNA and play a role in expression regulation[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In order to deeply study the potentially conserved motifs in 125 DnMYB proteins of \u003cem\u003eD. nobile\u003c/em\u003e, we systematically studied the evolutionary relationship of 125 DnMYB proteins, and analyzed the motif composition of 125 MYB-encoded proteins by MEME online analysis software. A total of 10 motifs were identified, of which motif 3 is the N-terminal domain of DnMYB proteins, which is present in almost all DnMYBs. Compared with the N-terminus, the C-terminus has a higher variability of motifs and different sequences, and these specific motifs indicate that different subgroups may have different functions[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Diversity in the composition of DnMYB motifs explains the diversity of its functions. Moreover, most DnMYB had motifs 1, 2, 3, 4 and 6, and most members in the same subgroup contained more than one identical motif. Motifs 3, 6, and 2 constitute the complete R2 domain, of which the three tryptophan (W) residues are highly conserved. Motif 1 contains the complete R3 domain, the first tryptophan (W) residue is often replaced by phenylalanine (F), and the remaining two tryptophan (W) residues are highly conserved, and 19 amino acids are separated between the two adjacent tryptophan residues. These are consistent with the characteristics of MYB domain identified in soybean[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], Ginkgo biloba[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], \u003cem\u003eArabidopsis\u003c/em\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and other species. Interestingly, the results also show that most members of the same subclass have similar motif composition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Chromosomal location and gene duplication of \u003cem\u003eR2R3-MYB\u003c/em\u003e gene family\u003c/h2\u003e \u003cp\u003eIn order to understand the distribution of R2R3-DnMYB genes in the genome, MG2G online software was used to locate the 125 \u003cem\u003eR2R3-MYB\u003c/em\u003e genes on the corresponding chromosomes. \u003cem\u003eD. nobile\u003c/em\u003e has 19 chromosomes, shown ranging from Chr1 to Chr19 (Supplementary Table\u0026nbsp;4). \u003cem\u003eDnMYBs\u003c/em\u003e were present on all chromosomes, while the arrangement and density of \u003cem\u003eDnMYBs\u003c/em\u003e on every chromosome were uneven (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The maximal number of \u003cem\u003eDnMYBs\u003c/em\u003e (12%) was located on chromosome 9, whereas the minimum number of \u003cem\u003eDnMYBs\u003c/em\u003e (3.2%) was located on chromosome 4. There was a high density of \u003cem\u003eDnMYBs\u003c/em\u003e located on chromosomes 6, 11, 12, 13, 17.\u003c/p\u003e \u003cp\u003eCollinearity diagrams among \u003cem\u003eDnMYBs\u003c/em\u003e were analyzed using gene duplication analysis.\u003c/p\u003e \u003cp\u003eA total of 46 pairs of gene duplications were identified among \u003cem\u003eDnMYBs\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The \u003cem\u003eDnMYB\u003c/em\u003e genes were homologous to genes in other plants, and syntenic conservation was observed among \u003cem\u003eD. chrysotoxum\u003c/em\u003e (174 orthologous gene pairs dispersed on all chromosomes). 28 collinear gene pairs between \u003cem\u003eD. nobile\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The number of orthologous events of \u003cem\u003eD. nobile\u003c/em\u003e-\u003cem\u003eD. chrysotoxum\u003c/em\u003e was far greater than that of \u003cem\u003eD. nobile\u003c/em\u003e-\u003cem\u003eArabidopsis\u003c/em\u003e, and the closer evolutionary distance between \u003cem\u003eD. nobile\u003c/em\u003e and \u003cem\u003eD. chrysotoxum\u003c/em\u003e was confirmed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Prediction of Cis-Acting Elements in the Promoters of 125 \u003cem\u003eDnMYB\u003c/em\u003e Genes\u003c/h2\u003e \u003cp\u003ePromoter is a DNA sequence recognized and combined by RNA polymerase and starts the transcription of downstream genes. The cis-acting element in promoter can bind transcription factors and play an important role in the regulation of gene expression[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. To analyze the cis-acting elements located in the 2000bp sequence upstream of the promoter region, we obtained \u003cem\u003e125 DnMYB\u003c/em\u003e promoter sequences and predicted them by PlantCARE online tool (Supplementary Table\u0026nbsp;5). In addition to the basic elements, these cis-acting elements could be divided into three groups, including hormone-responsive elements (ABA, salicylic acid, gibberellin, MeJA, auxin), abiotic stress elements (low temperature-responsive elements and drought-responsive elements), metabolism elements (regulation flavonoid biosynthetic) (Supplementary Fig.\u0026nbsp;1). MeJA-responsive element is the most abundant element, containing 350 instances. As we all know, Methyl jasmonic acid regulates physiological processes such as plant growth and development, especially the response of plants to biotic and abiotic stresses[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. \u003cem\u003eDnMYB23\u003c/em\u003e contained 24 MeJA-responsive elements in its 2-k upstream regulatory region. In addition, hormone-related elements included 210 ABA-responsive elements, 87 gibberellin-responsive elements, 64 auxin-responsive elements, 58 salicylic acid-responsive elements. Abiotic stress elements contained 60 low temperature responsive elements, of which \u003cem\u003eDnMYB\u003c/em\u003e100 contained 3 low temperature responsive elements. Flowed by 80 drought responsive elements, \u003cem\u003eDnMYB34\u003c/em\u003e, \u003cem\u003eDnMYB37\u003c/em\u003e, \u003cem\u003eDnMYB58\u003c/em\u003e, \u003cem\u003eDnMYB6\u003c/em\u003e, \u003cem\u003eDnMYB74\u003c/em\u003e and \u003cem\u003eDnMYB83\u003c/em\u003e all harbored 3 drought responsive elements. Moreover, 14 regulation flavonoid biosynthetic-responsive elements in its 2-kb upstream regulatory region were identified. These results suggest that the expressions of \u003cem\u003eDnMYB\u003c/em\u003e genes are controlled by complex regulatory networks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Expression pattern of candidate \u003cem\u003eDnMYB\u003c/em\u003e genes in different tissues\u003c/h2\u003e \u003cp\u003eMYB genes in different subgroups may perform different functions during the growth and development of \u003cem\u003eD. nobile\u003c/em\u003e. To investigate the spatial expression levels of MYB genes in \u003cem\u003eD. nobile\u003c/em\u003e, the expression levels of 8 genes from each type across four tissues, roots, stems, leaves, and flower, were analyzed by RT-qPCR. \u003cem\u003eDnMYB8\u003c/em\u003e, \u003cem\u003eDnMYB14\u003c/em\u003e, \u003cem\u003eDnMYB26\u003c/em\u003e, \u003cem\u003eDnMYB27\u003c/em\u003e, \u003cem\u003eDnMYB81\u003c/em\u003e, \u003cem\u003eDnMYB90\u003c/em\u003e, \u003cem\u003eDnMYB108\u003c/em\u003e, \u003cem\u003eDnMYB116\u003c/em\u003e belong to the S22, S2, S21, S1, S7, S4, S19, S20 subfamilies, respectively. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, 8 genes were expressed in all tissues. The results indicated that \u003cem\u003eDnMYB8\u003c/em\u003e, \u003cem\u003eDnMYB14\u003c/em\u003e, \u003cem\u003eDnMYB26\u003c/em\u003e and \u003cem\u003eDnMYB81\u003c/em\u003e shared similar expression patterns and were predominantly expressed in root. \u003cem\u003eDnMYB27\u003c/em\u003e had higher expression in the flower if compared with that in other tissues. Moreover, \u003cem\u003eDnMYB108\u003c/em\u003e showed tissue-specific expression patterns, with 30-fold higher expression levels in flowers than in other tissues. \u003cem\u003eDnMYB90\u003c/em\u003e was principally expressed in leaf and stem. \u003cem\u003eDnMYB116\u003c/em\u003e was classified in the S20 subfamilies with relatively high expression levels in leaf and/or stem. The results suggested that these genes had a possible role in the growth and development of \u003cem\u003eD. nobile\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Expression pattern of candidate \u003cem\u003eDnMYB\u003c/em\u003e genes in response to abiotic stress\u003c/h2\u003e \u003cp\u003eBased on the reported functions of R2R3-MYB in other plants, we screened out 8 \u003cem\u003eDnMYBs\u003c/em\u003e belonging to eight subfamilies to study the role of R2R3-MYB in the regulation of secondary metabolism and the responses to abiotic stress for \u003cem\u003eD. nobile\u003c/em\u003e. We performed the expression level of these genes by RT-qPCR after treatment with cold (4℃), ABA, and MeJA. Six \u003cem\u003eDnMYB\u003c/em\u003e genes, \u003cem\u003eDnMYB8\u003c/em\u003e, \u003cem\u003eDnMYB14\u003c/em\u003e, \u003cem\u003eDnMYB26\u003c/em\u003e, \u003cem\u003eDnMYB27\u003c/em\u003e, \u003cem\u003eDnMYB90\u003c/em\u003e, and \u003cem\u003eDnMYB116\u003c/em\u003e showed increased expression levels at different times under the low temperature, ABA, and MeJA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). While \u003cem\u003eDnMYB81\u003c/em\u003e was the most responsive to low temperature, and showed downregulated expression patterns under ABA and MeJA stress. \u003cem\u003eDnMYB108\u003c/em\u003e showed downregulated expression patterns under either cold or MeJA stress. The gene expression of \u003cem\u003eDnMYB108\u003c/em\u003e plummeted at the lowest value at 12 h and reached its peak at 24 h under ABA stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Evolutionary Analysis of the \u003cem\u003eDnMYB\u003c/em\u003e Gene Family\u003c/h2\u003e \u003cp\u003eR2R3-MYB transcription factors have been identified in many plants, including \u003cem\u003eArabidopsis\u003c/em\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], maize[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], soybean[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], etc. To date, there is no full analysis of the \u003cem\u003eR2R3-DnMYB\u003c/em\u003e gene family and most functions remain unclear. In this study, 125 R2R3-MYB transcription factors were identified in the \u003cem\u003eD. nobile\u003c/em\u003e genome and divided into 23 subfamilies. Gene structure, cis-acting element analysis, chromosomal distribution, phylogenetic analysis, gene duplication events, and expression level analysis were determined. This study offers new insights for future investigators to identify functional differences in the \u003cem\u003eR2R3-DnMYB\u003c/em\u003e family genes. This study provides new insights for future researchers to determine the functional differences of \u003cem\u003eR2R3-MYB\u003c/em\u003e family genes in \u003cem\u003eD. nobile\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e4.2\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eDnMYB\u003c/span\u003e \u003cb\u003eGenes Play Crucial Roles in\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eD. nobile\u003c/span\u003e \u003cb\u003eGrowth and Development and Response to Phytohormone and Abiotic Stresses\u003c/b\u003e \u003c/p\u003e \u003cp\u003eLow temperature, drought and abiotic stress seriously affected the yield of \u003cem\u003eD. nobile\u003c/em\u003e. Secondary metabolites in plants, including flavonoids and anthocyanins, can help plants adapt to harsh environments. Meanwhile, flavonoid biosynthesis can be induced under a wide range of abiotic stresses. Therefore, the stress resistance of \u003cem\u003eD. nobile\u003c/em\u003e can be improved by regulating the genes related to secondary metabolites, thereby increasing the yield. MYB gene can regulate the development of trichomes and root hairs, and plays a crucial role in regulating the synthesis of plant secondary metabolites such as flavonoids and anthocyanins and helping plants resist abiotic stress. In order to screen \u003cem\u003eDnMYB\u003c/em\u003e genes that may be involved in regulating the growth, development and secondary metabolic synthesis of \u003cem\u003eD. nobile\u003c/em\u003e, this study selected a total of 8 genes from different subgroups and measured their expression levels in different tissues and different stresses. Gene functions are closely related to tissue-specific expression. As the major pigments in plant, flavonoids contain various important bioactivities and have gained a lot of attention. It reported that flavonoids biosynthesis in plants could be induced under a wide range of abiotic stresses. The effects of these stresses on the contents of flavonoids have been investigated mainly in some crops and model plants. For example, in wheat, the key enzymes in the flavonoids\u0026rsquo; biosynthesis pathway were induced by drought stress[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The S4 subfamily \u003cem\u003eAtMYB4\u003c/em\u003e, and \u003cem\u003eAtMYB7\u003c/em\u003e were related to the flavonol synthesis pathway[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The S7 subfamily \u003cem\u003eAtMYB11\u003c/em\u003e, \u003cem\u003eAtMYB12\u003c/em\u003e and \u003cem\u003eAtMYB111\u003c/em\u003e were involved in regulating flavonol biosynthesis in \u003cem\u003eArabidopsis\u003c/em\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Genes with high homology in the same branch of a phylogenetic tree generally have high sequence similarity and may also have similar functions. \u003cem\u003eDnMYB90\u003c/em\u003e also belonged to S4 subfamily, which is a homologous gene of \u003cem\u003eAtMYB4\u003c/em\u003e, \u003cem\u003eAtMYB7\u003c/em\u003e. RT-qPCR showed that \u003cem\u003eDnMYB90\u003c/em\u003e was expressed in all tissues of \u003cem\u003eD. nobile\u003c/em\u003e, but the highest expression level was found in leaf. \u003cem\u003eDnMYB90\u003c/em\u003e was also expressed at very high levels under phytohormonal and abiotic stresses. \u003cem\u003eDnMYB90\u003c/em\u003e exhibited an elevated expression level under cold, ABA and MeJA stress. And it was the most responsive to low temperature. \u003cem\u003eDnMYB81\u003c/em\u003e belonging to the S7 subfamily had close homology with \u003cem\u003eAtMYB11\u003c/em\u003e, \u003cem\u003eAtMYB12\u003c/em\u003e, and \u003cem\u003eAtMYB111\u003c/em\u003e. \u003cem\u003eDnMYB81\u003c/em\u003e belonging to the S7 subfamily had close homology with \u003cem\u003eAtMYB11\u003c/em\u003e, \u003cem\u003eAtMYB12\u003c/em\u003e, and \u003cem\u003eAtMYB111\u003c/em\u003e. The RT-qPCR showed that \u003cem\u003eDnMYB81\u003c/em\u003e expressed in all tissues of \u003cem\u003eD. nobile\u003c/em\u003e, but the highest expression level was found in the root. \u003cem\u003eDnMYB81\u003c/em\u003e was also responsive to phytohormonal and abiotic stresses. \u003cem\u003eDnMYB81\u003c/em\u003e was the most responsive to low temperature, and showed downregulated expression patterns under ABA and MeJA stress. In the future, we can verify whether \u003cem\u003eDnMYB81\u003c/em\u003e and \u003cem\u003eDnMYB90\u003c/em\u003e can regulate flavonoid synthesis through more in-depth experiments.\u003c/p\u003e \u003cp\u003eIt has been reported that several \u003cem\u003eR2R3-MYB\u003c/em\u003e genes are involved in regulating responses to biotic and abiotic stresses. For example, overexpression of the S1 subfamily \u003cem\u003eAtMYB94\u003c/em\u003e, \u003cem\u003eAtMYB96\u003c/em\u003e gene enhances drought tolerance in plants[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The S2 subfamily \u003cem\u003eAtMYB14\u003c/em\u003e regulates cold tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Under phosphate starvation conditions, The S2 subfamily \u003cem\u003eAtMYB20\u003c/em\u003e, which plays a role in plant responses to salt and drought stress, directly regulates the expression of miR399f [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Overexpression of the S21 subfamily \u003cem\u003eAtMYB52\u003c/em\u003e confers ABA hypersensitivity and drought tolerance[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The S22 subfamily \u003cem\u003eAtMYB44\u003c/em\u003e was tested in response to salt, MeJA and drought[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In order to explore \u003cem\u003eDnMYB\u003c/em\u003e genes involved in hormonal and abiotic stresses, we focused on the expression levels of \u003cem\u003eDnMYB\u003c/em\u003e genes from S1, S2, S20, S21, and S22 under low temperature, ABA, and MeJA stress. Using RT-qPCR, we found that \u003cem\u003eDnMYB27\u003c/em\u003e of S1, \u003cem\u003eDnMYB14\u003c/em\u003e of S2, \u003cem\u003eDnMYB116\u003c/em\u003e of S20, \u003cem\u003eDnMYB26\u003c/em\u003e of S21, and \u003cem\u003eDnMYB8\u003c/em\u003e of S22, which were homologous to stress response genes, were expressed in all tissues. To further identify candidate genes involved in hormone signaling pathways or abiotic stress responses, we measured their expression levels under low temperature, ABA and MeJA stress. The results showed that the five genes showed increased expression levels at different times under different stress, which was consistent with the cis-acting element in its promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Interestingly, the expression level of the S19 subfamily \u003cem\u003eDnMYB108\u003c/em\u003e gradually decreased after treatment with cold and MeJA stress. These results may suggest that members of the S19 subfamily play an important role in the response of \u003cem\u003eD. nobile\u003c/em\u003e to abiotic stress. Some of the \u003cem\u003eDnMYB\u003c/em\u003e genes screened in this study may be involved in abiotic stresses such as low temperature, ABA and MeJA stress, but the specific mechanism is still unclear and needs to be further verified in future experiments.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eTFs: transcription factors; RT-qPCR: real-time quantitative PCR; ABA: abscisic acid; MeJA: methyl jasmonate; HMM: hidden Markov model; NCBI: National Center for Biotechnology Information; MW: molecular weight; pI: isoelectric point.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Shuai Liu from the University of Hawaii Cancer Center for helping to revise the manuscript. We also thank Dr. Xiaoxi Meng and Zhikai Liang from the Department of Horticultural Science at University of Minnesota for helpful comments on an earlier version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur experimental research on \u003cem\u003eDendrobium nobile\u003c/em\u003e was in accordance with the relevant national/institutional guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceived, designed, and implemented the study: LW, LZ, and SX; Statistics analysis: LW, JF, WJ, LZ, and XS; Reagents/materials/analysis tools: ZS, DP, and SX; Drafted the manuscript: LW, XS, WR, YD, YW, and SX; All authors participated in the editing of the manuscript and agreed to submit the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by was supported by National Natural Science Foundation of China (Grant No. U19A2009), Key Natural Science Research Projects in Anhui Universities (No. 2022AH050461, No. KJ2019A0453 and KJ2018A0275), Open Project of Provincial and Ministerial Scientific Research Platform, Fuyang Normal University (No. FSKFKT010D) and Anhui University Collaborative Innovation Project (Grant No. GXXT-2019-043, Grant No. GXXT-2019-049).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed in this study are included in this article (Supplementary file). The genome sequences of \u003cem\u003eD. nobile\u003c/em\u003e, \u003cem\u003eD. chrysotoxum\u003c/em\u003e and \u003cem\u003eArabidopsis\u003c/em\u003e were downloaded from the https://www.ncbi.nlm.nih.gov/genome/?term=dendrobium+nobile, https://www.ncbi.nlm.nih.gov/genome/?term=Dendrobium+chrysotoxum, and https://www.arabidopsis.org/.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eCollege of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eInstitute of Traditional Chinese Medicine Resources Protection and Development, Anhui Academy of Chinese Medicine, Hefei 230012, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eMOE-Anhui Joint Collaborative Innovation Center for Quality Improvement of Anhui Genuine Chinese Medicinal Materials, Hefei 230038, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e4\u003c/sup\u003eHunan Key Laboratory for Conservation and Utilization of Biological Resources in the Nanyue Mountainous Region, College of Life Sciences and Environment, Hengyang Normal University, Hengyang, 421008, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e5\u003c/sup\u003e Anhui Province Key Laboratory of Environmental Hormone and Reproduction, Anhui Province Key Laboratory of Embryo Development and Reproductive Regulation, Fuyang Normal University, Fuyang, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e6\u003c/sup\u003eInnovative Drug R\u0026amp;D Center, College of Life Sciences, Huaibei Normal University. Huaibei 235000, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e7\u003c/sup\u003eAnhui Province Key Laboratory of Research \u0026amp; Development of Chinese Medicine, Hefei 230012, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhang X, Wang M, Zhang C, Liu Z, Zhou S. Clinical study of Dendrobium Nobile Lindl intervention on patients with metabolic syndrome. Med (Baltim). 2021;100(12):e24574.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrishnatreya DB, Baruah PM, Dowarah B, Bordoloi KS, Agarwal H. Agarwala N. Mining of mirnas from est data in \u003cem\u003eDendrobium nobile\u003c/em\u003e. Bioinformation. 2020;16(3):245\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhong H, Kong W, Gong Z, Fang X, Deng X, Liu C, et al. 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Plant Physiol. 2008;146(2):623\u0026ndash;35.\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":"Dendrobium nobile, R2R3-MYB, Genome-wide, Gene family","lastPublishedDoi":"10.21203/rs.3.rs-2749425/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2749425/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eR2R3-MYB is one of the largest and most important gene families, participating in the regulation of plant growth and development and response to abiotic stresses. However, the function of \u003cem\u003eR2R3-MYB\u003c/em\u003e genes in \u003cem\u003eDendrobium nobile\u003c/em\u003e remains largely unknown. Here, a comprehensive genome-wide analysis of \u003cem\u003eD. nobile\u003c/em\u003e R2R3-\u003cem\u003eDnMYB\u003c/em\u003es was performed, investigating phylogenic relationships, gene structures, motif composition, chromosomal locations, collinearity analysis, and cis-acting elements. Totally, 125 \u003cem\u003eDnMYB \u003c/em\u003egenes were identified in the \u003cem\u003eD. nobile \u003c/em\u003egenome, and they could be subdivided into 26 groups by further divided through phylogenetic analysis. Most genes with similar exon-intron structures and motif compositions in eaach subgroup exhibited similar functions. All of \u003cem\u003eDnMYB \u003c/em\u003egenes were mapped on 19 chromosomes with the co-linearity relationship. Moreover, their\u003cem\u003e \u003c/em\u003eexpression patterns were analyzed in various tissues and abiotic stresses. The results showed that \u003cem\u003eDnMYBs\u003c/em\u003e were significantly differential expressed in different tissues, following abiotic stresses and phytohormone treatments, indicating their possible roles in biological processes and some abiotic stress tolerance and adaptation. This work provides a comprehensive understanding of the R2R3-MYB family of \u003cem\u003eD. nobile\u003c/em\u003e, and lays a foundation for future research on the potential function of DnMYB gene in the growth and development of \u003cem\u003eD. nobile\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Genome-wide identification of R2R3-MYB Family Genes and their Response to Stress in Dendrobium nobile","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-07 18:21:18","doi":"10.21203/rs.3.rs-2749425/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":"fb378272-a8ce-4ef4-b209-e3c70b79fc16","owner":[],"postedDate":"April 7th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-06-05T11:44:37+00:00","versionOfRecord":[],"versionCreatedAt":"2023-04-07 18:21:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2749425","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2749425","identity":"rs-2749425","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}