药学学报, 2021, 56(4): 1170-1177
引用本文:
李元敏, 张娜, 李慧, 张明英, 彭亮, 颜永刚, 李依民, 张岗. 掌叶大黄转录因子RpMYB4基因克隆及分子表达特性[J]. 药学学报, 2021, 56(4): 1170-1177.
LI Yuan-min, ZHANG Na, LI Hui, ZHANG Ming-ying, PENG Liang, YAN Yong-gang, LI Yi-min, ZHANG Gang. Cloning and expression characteristics of a transcription factor gene RpMYB4 in Rheum palmatum L.[J]. Acta Pharmaceutica Sinica, 2021, 56(4): 1170-1177.

掌叶大黄转录因子RpMYB4基因克隆及分子表达特性
李元敏1, 张娜1,2, 李慧1, 张明英1, 彭亮1, 颜永刚1, 李依民1*, 张岗1*
1. 陕西中医药大学药学院/陕西省秦岭中草药应用开发工程技术研究中心, 陕西 西安 712046;
2. 陕西省天芪生物科技有限责任公司, 陕西 榆林 718400
摘要:
MYB转录因子在植物生长发育、次生代谢及逆境胁迫等过程中发挥重要的转录调控作用。本实验在掌叶大黄转录组数据库中筛选一个包含完整开放阅读框(ORF)的MYB家族成员序列,首次克隆获得RpMYB4基因ORF,编码一条245个氨基酸的多肽,分子质量为26.99 kDa,N端含有R2R3-MYB亚家族典型的2个DNA保守结合域(HTH-MYB),无信号肽或跨膜域,与其他植物物种MYB转录因子多序列比对均高达61%以上,系统进化分析显示与FtMYB8关系最近,共同聚在S4亚家族。亚细胞定位结果显示RpMYB4-GFP定位在烟草细胞核内。实时荧光定量分析表明RpMYB4的组织表达有差异,叶中表达量最高,依次为叶柄、根茎、根及种子;受200 μmol·L-1茉莉酸甲酯(MeJA)处理,RpMYB4表达在24 h内持续下调,200 μmol·L-1水杨酸(SA)处理在12和24 h显著上调,该基因对200 μmol·L-1脱落酸(ABA)处理未见明显变化;RpMYB4基因受干旱、高温及损伤胁迫诱导,分别在24、24、3 h处达峰值,低温胁迫抑制其表达,6 h呈谷值,其对盐胁迫响应不显著。首次获得掌叶大黄RpMYB4基因,在叶片和叶柄中表达量高,受激素SA与干旱、高温及损伤胁迫等诱导表达,为后续研究其在大黄次生代谢及逆境胁迫中的分子作用奠定基础。
关键词:    掌叶大黄      转录因子      MYB      基因表达      胁迫     
Cloning and expression characteristics of a transcription factor gene RpMYB4 in Rheum palmatum L.
LI Yuan-min1, ZHANG Na1,2, LI Hui1, ZHANG Ming-ying1, PENG Liang1, YAN Yong-gang1, LI Yi-min1*, ZHANG Gang1*
1. Shaanxi University of Chinese Medicine of Pharmacy/Shaanxi Qinling Chinese Herbal Medicine Application Development Engineering Technology Research Center, Xi'an 712046, China;
2. Shaanxi TianQi Biotechnology Limited Liability Company, Yulin 718400, China
Abstract:
MYB transcription factors play many important regulatory roles in plant growth and development, secondary metabolism, and stress adaptation processes. In this work, an MYB gene containing a complete open reading frame (ORF) was selected from the transcriptome database of R. palmatum L. RpMYB4 ORF and cloned, encoding a polypeptide of 245 amino acids with a molecular weight of 26.99 kDa. RpMYB4 lacks a signal peptide or transmembrane domain but contains two conserved DNA binding domains (HTH-MYB) of the R2R3-MYB subfamily at the N-terminus. Multiple-sequence alignment demonstrated that RpMYB4 shared as high as 61% identity with many MYB proteins from other species. Phylogenetic analysis showed that RpMYB4 had the closest relationship with FtMYB8 and was clustered in the S4 subfamily. Subcellular localization by confocal microscopy showed that an RpMYB4-GFP-fusion protein localized to the nucleus in tobacco. Real-time fluorescence quantitative PCR analyses revealed that RpMYB4 was differentially expressed in various tissues, with the highest expression in leaves, followed by petioles, rhizome, and roots, and with the lowest level in mature seeds. After treatment of R. palmatum L. seedlings with 200 μmol·L-1 MeJA, the expression of RpMYB4 in leaves was down-regulated within 24 h, and significantly up-regulated after 200 μmol·L-1 SA treatment at 12 h and 24 h. However, gene expression did not change with 200 μmol·L-1 ABA treatment. The transcripts of RpMYB4 under drought, high temperature, and mechanical injury stresses reached a peak at 24 h, 24 h, and at 3 h, respectively, while RpMYB4 expression was inhibited by low temperature stress, reaching its lowest value at 6 h. The gene showed no significant response to salt stress. Overall, RpMYB4 was cloned from R. palmatum L. for the first time, showed high expression in leaves, and was responsive to SA and various abiotic stress treatments including drought, high temperature, and mechanical injury. The results will be useful for further analysis of secondary metabolism and stress adaptations in R. palmatum L.
Key words:    Rheum palmatum L.    transcription factor    MYB    gene expression    stress   
收稿日期: 2020-10-26
DOI: 10.16438/j.0513-4870.2020-1654
基金项目: 国家自然科学基金资助项目(81973430);陕西中医药大学校级课题(2020PG29);陕西中医药大学思邈青年学者项目;陕西中医药大学新进博士科研启动经费(104080001);陕西中医药大学“秦药”品质评价与资源开发学科创新团队项目(2019-QN01).
通讯作者: 李依民,E-mail:jay_gumling2003@aliyun.com;张岗,E-mail:2051058@sntcm.edu.cn
Email: jay_gumling2003@aliyun.com;2051058@sntcm.edu.cn
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参考文献:
[1] Hai D, Li Z, Liu L, et al. Biochemical and molecular characterization of plant MYB transcription factor family[J]. Biochem (Mosc), 2009, 74:1-11.
[2] Rosinski JA, Atchley WR. Molecular evolution of the MYB family of transcription factors:evidence for polyphyletic origin[J]. J Mol Evol, 1998, 46:74-83.
[3] Katiyar A, Smita S, Lenka S, et al. Genome-wide classification and expression analysis of MYB transcription factor families in rice and Arabidopsis[J]. BMC Genomics, 2012, 13:544.
[4] Dubos C, Stracke R, Grotewold E, et al. MYB transcription factors in Arabidopsis[J]. Trends Plant Sci, 2010, 15:573-581.
[5] Li CL, Lu SF. Genome-wide characterization and comparative analysis of R2R3-MYB transcription factors shows the complexity of MYB-associated regulatory networks in Salvia miltiorrhiza[J]. BMC Genomics, 2014, 15:1-12.
[6] Li SY, Li TT, Li L, et al. Genome-wide identification and bioinformatics analysis of Dendrobium officinale R2R3-MYB transcription factor[J]. Mol Plant Breeding (分子植物育种). https://kns.cnki.net/kcms/detail/46.1068.S.20200923.0903.002.html.
[7] Preston J, Wheeler J, Heazlewood J, et al. AtMYB32 is required for normal pollen development in Arabidopsis thaliana[J]. Plant J, 2004, 40:979-995.
[8] Li Y, Chen X, Wang J, et al. Two responses to MeJA induction of R2R3-MYB transcription factors regulate flavonoid accumulation in Glycyrrhiza uralensis Fisch[J]. PLoS One, 2020, 15:e0236565.
[9] Afrin S, Zhu J, Cao H, et al. Molecular cloning and expression profile of an abiotic stress and hormone responsive MYB transcription factor gene from Panax ginseng[J]. Acta Biochim Biophys Sin, 2015, 47:267-77.
[10] Chinese Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China (中华人民共和国药典)[S]. Part Ⅰ. 2020 ed. Beijing:China Medical Science Press, 2020:24.
[11] Yang Y, Shui PX, Chen Y, et al. Efficacy of rhubarb in clinical application and its pharmacological effects[J]. Genom Appl Biol, 2017, 36:1226-1231.
[12] Hei XB, Li YY, Li H, et al. Investigation on seeds characteristics and establishment of aseptic cultural system for Rheum palmatum[J]. Chin Tradit Herb Drugs (中草药), 2019, 50:4430-4437.
[13] Sun ML, Wang YS, Yang DQ, et al. Reference genes for real-time fluorescence quantitative PCR in Camellia sinensis[J]. Chin Bull Bot (植物学报), 2010, 45:579-587.
[14] Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR[J]. Nucleic Acids Res, 2001, 29:e45.
[15] Xie DJ, Ye YJ, Yang DM, et al. Identification and analysis of the R2R3-MYB transcription factor based on a transcriptome database in Morinda officinalis[J]. Acta Pharm Sin (药学学报), 2020, 55:160-167.
[16] Zhou M, Zhang K, Sun Z, et al. LNK1 and LNK2 corepressors interact with the MYB3 transcription factor in phenylpropanoid biosynthesis[J]. Plant Physiol, 2017, 174:1348-1358.
[17] Jin H, Cominelli E, Bailey P, et al. Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis[J]. EMBO J, 2000, 19:6150-6161.
[18] Huang Y, Wu Q, Wang S, et al. FtMYB8 from Tartary buckwheat inhibits both anthocyanin/proanthocyanidin accumulation and marginal trichome initiation[J]. BMC Plant Biol, 2019, 19:263.
[19] Liu X, Yu W, Zhang X, et al. Identification and expression analysis under abiotic stress of the R2R3-MYB genes in Ginkgo biloba L.[J]. Physiol Mol Biol Plant, 2017, 23:503-516.
[20] Chen J, Tang XH, Ren CX, et al. Cloning and expression analysis of MYB transcription factor genes in safflower[J]. Acta Pharm Sin (药学学报), 2018, 53:144-149.
[21] Zhao H, Baloch SK, Kong LR, et al. Molecular cloning, characterization, and expression analysis of LeMYB1 from Lithospermum erythrorhizon[J]. Biol Plant, 2014, 58:436-444.
[22] Lu B, Zhang WZ, Li CY, et al. The MYB transcription factor OsMYB84confers salt stress response trough the ABA pathway[J]. J Fudan Univ (Nat Sci), (复旦大学学报自然科学版), 2015, 54:591-600.
[23] Qiu WY, Wang SY, Li XF, et al. Functions of plant MYB transcription factors in response to abiotic stress and plant hormones[J]. Acta Agric Zhejiang (浙江农业学报), 2020, 32:1317-1328.
[24] Chen BJ, Wang Y, Hu YL, et al. Cloning and characterization of a drought-inducible MYB gene from Boea crassifolia[J]. Plant Sci, 2005, 168:493-500.
[25] Bohlmann J, Crock J, Jetter R, et al. Terpenoid-based defenses in conifers:cDNA cloning, characterization, and functional expression of wound-inducible(E)-α-bisabolene synthase from grand fir (Abies grandis)[J]. Proc Natl Acad Sci U S A, 1998, 95:6756-6761.
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