茶树R2R3-MYB转录因子CsTT2表达分析及功能初步鉴定

doi:10.13305/j.cnki.jts.2022.04.005

摘要/Abstract

摘要： 儿茶素是茶树中特色的次生代谢产物之一,是影响茶叶的品质与风味的主要组分,具有抗氧化、抗病毒、降脂减肥等药理功效。通过系统发育进化树分析、基因表达模式分析和分子生物学试验对茶树儿茶素生物合成相关调控因子CsTT2的功能进行初步鉴定。结果显示,CsTT2是R2R3-MYB转录因子,与拟南芥中调控次生代谢产物的MYB转录因子同在一个分支。在茶树品种顶芽组织中总儿茶素含量较高,CsTT2和儿茶素生物合成相关基因的表达水平也较高。亚细胞定位、酵母试验和双荧光素酶报告系统试验结果表明,CsTT2定位在细胞核中,其编码的蛋白是具有转录激活能力的调控因子,可以结合儿茶素生物合成关键基因ANR的启动子激活其表达。

关键词: 茶树, R2R3-MYB转录因子, CsTT2, 儿茶素生物合成

Abstract: Catechin is one of the most characteristic secondary metabolites in tea plants and the main component affecting tea quality and flavor. It possessesrich pharmacological effects, such as anti-oxidation, anti-virus, lipid lowering and weight loss, etc. In this study, the function of a catechin biosynthetic regulator CsTT2 was preliminarily identified using phylogenetic analysis, gene expression pattern analysis and molecular biology experiments. The results show that CsTT2 was a R2R3-MYB transcription factor that shares a branch withthe MYB transcription factor in Arabidopsis thaliana that regulates secondary metabolites. The expression levels of CsTT2 and catechin biosynthesis genes were relatively high in the apical bud tissue of tea plants with higher total catechin content. The results of subcellular localization, yeast assay and dual luciferase reporting system further reveal that CsTT2 was located into the nucleus and the protein it encodes possessed transcriptional activation capacity. CsTT2 could bind to the promoter of a key catechin biosynthetic gene ANR to activate its expression.

Key words: tea plant, R2R3-MYB transcription factor, CsTT2, catechin biosynthesis

中图分类号:

S571.1

王玉源, 刘任坚, 刘少群, 舒灿伟, 孙彬妹, 郑鹏. 茶树R2R3-MYB转录因子CsTT2表达分析及功能初步鉴定[J]. 茶叶科学, 2022, 42(4): 463-476. doi: 10.13305/j.cnki.jts.2022.04.005.

WANG Yuyuan, LIU Renjian, LIU Shaoqun, SHU Canwei, SUN Binmei, ZHENG Peng. Expression Analysis and Functional Identification of CsTT2 R2R3-MYB Transcription Factor in Tea Plants[J]. Journal of Tea Science, 2022, 42(4): 463-476. doi: 10.13305/j.cnki.jts.2022.04.005.

参考文献

[1] Ming T, Bartholomew B.Theaceae[J]. Flora China, 2007, 12: 366-478.
[2] Zhao M, Zhang N, Gao T, et al.Sesquiterpene glucosylation mediated by glucosyltransferase UGT91Q2 is involved in the modulation of cold stress tolerance in tea plants[J]. New Phytol, 2020, 226(2): 362-372.
[3] 宛晓春. 茶叶生物化学[M]. 3版. 北京: 中国农业出版社, 2003.
Wan X C.Biochemistry of tea: the third edition [M]. 3rd ed. Beijing: China Agriculture press, 2003.
[4] Zhao M, Yu Y, Sun L, et al.GCG inhibits SARS-CoV-2 replication by disrupting the liquid phase condensation of its nucleocapsid protein[J]. Nature Communications, 2021, 12(1): 2114.doi: 10.1038/s41467-021-22297-8.
[5] Xiong L G, Chen Y J, Tong J W, et al.Epigallocatechin-3-gallate promotes healthy lifespan through mitohormesis during early-to-mid adulthood in Caenorhabditis elegans[J]. Redox Biology, 2018, 14: 305-315.
[6] Yuan H, Li Y, Ling F, et al.The phytochemical epigallocatechin gallate prolongs the lifespan by improving lipid metabolism, reducing inflammation and oxidative stress in high-fat diet-fed obese rats[J]. Aging cell, 2020, 19(9): e13199. doi: 10.1111/acel.13199.
[7] Lwxa B, Shang C, Tsza B, et al.Green tea derivative epigallocatechin-3-gallate (EGCG) confers protection against ionizing radiation-induced intestinal epithelial cell death both in vitro and in vivo[J]. Free Radical Biology and Medicine, 2020, 161: 175-186.
[8] Zhang Z, Zhang X, Bi K, et al.Potential protective mechanisms of green tea polyphenol EGCG against COVID-19[J]. Trends in Food Science & Technology, 2021, 114: 11-24.
[9] Zhao J, Blayney A, Liu X, et al.EGCG binds intrinsically disordered N-terminal domain of p53 and disrupts p53-MDM2 interaction[J]. Nature Communications, 2021, 12(1): 986. doi: 10.1038/s41467-021-21258-5.
[10] Bernier L P, York E M, Kamyabi A, et al.Microglial metabolic flexibility supports immune surveillance of the brain parenchyma[J]. Nat Commun, 2020, 11(1): 1559. doi: 10.1038/s41467-020-15267-z.
[11] Liu Z S, Cai H, Xue W, et al.G3BP1 promotes DNA binding and activation of cGAS[J]. Nat Immunol, 2019, 20(1): 18-28.
[12] Yang C S, Hong J.Prevention of chronic diseases by tea: possible mechanisms and human relevance[J]. Annual Review of Nutrition, 2013, 33: 161-181.
[13] 夏涛, 高丽萍. 类黄酮及茶儿茶素生物合成途径及其调控研究进展[J]. 中国农业科学, 2009, 42(8): 2899-2908.
Xia T, Gao L P.Research progress on biosynthesis pathway and regulation of flavonoids and catechins[J]. Scientia Agricultura Sinica, 2009, 42(8): 2899-2908.
[14] Weisshaar B, Jenkins G I.Phenylpropanoid biosynthesis and its regulation[J]. Current Opinion in Plant Biology, 1998, 1(3): 251-257.
[15] Furukawa T, Eshima A, Koiya M, et al.Coordinate expression of genes involved in catechin biosynthesis in Polygonum hydropiper cells[J]. Plant Cell Reports, 2002, 21(4): 385-389.
[16] 夏涛, 高丽萍, 刘亚军, 等. 茶树酯型儿茶素生物合成及水解途径研究进展[J]. 中国农业科学, 2013, 46(11): 2307-2320.
Xia T, Gao L P, Liu Y J, et al.Advances in biosynthesis and hydrolysis of catechins from tea tree[J]. Scientia Agricultura Sinica, 2013, 46(11): 2307-2320.
[17] 宛晓春. 茶树次生代谢[M]. 北京: 科学出版社, 2015.
Wan X C.Secondary metabolism of tea plant [M]. Beijing: Science Press, 2015.
[18] 陆建良, 林晨, 骆颖颖, 等. 茶树重要功能基因克隆研究进展[J]. 茶叶科学, 2007, 27(2): 95-103.
Lu J L, Lin C, Luo Y Y, et al.Advances in cloning of important functional genes from Camellia sinensis[J]. Journal of Tea Science, 2007, 27(2): 95-103.
[19] Stafford H A.Flavonoid metabolism pathway to proanthocyanindins (condensed tannins), flavan-3-ols, and unsubstituted flavans [M]. New York: CRC Press, 1990.
[20] Stafford H A, Lester H H.The conversion of (L)-dihydromyricetin to its flavan-3, 4-diol (leucodelphinidin) and to (L)-gallocatechin by reductase extracted from tissue cultures of Ginkgo biloba and Pseudotsuga menziesii[J]. Plant Physiology, 1985, 78(4): 791-794.
[21] Punyasiri P, Abeysinghe I, Kumar V, et al.Flavonoid biosynthesis in the tea plant Camellia sinensis: properties of enzymes of the prominent epicatechin and catechin pathways[J]. Archives of Biochemistry & Biophysics, 2004, 431(1): 22-30.
[22] Niemetz R, Gross G G.Enzymology of gallotannin and ellagitannin biosynthesis[J]. Phytochemistry, 2005, 66(17): 2001-2011.
[23] Gross G G.From lignins to tannins: forty years of enzyme studies on the biosynthesis of phenolic compounds[J]. Phytochemistry, 2008, 69(18): 3018-3031.
[24] Liu Y, Gao L, Liu L, et al.Purification and characterization of a novel galloyltransferase involved in catechin galloylation in the tea plant (Camellia sinensis)[J]. Journal of Biological Chemistry, 2012, 287(53): 44406-44417.
[25] Zhong K, Zhao S Y, Jönsson L J, et al.Enzymatic conversion of epigallocatechin gallate to epigallocatechin with an inducible hydrolase from Aspergillus niger[J]. Biocatalysis, 2009, 26(4): 306-312.
[26] Wei C, Hua Y, Wang S, et al.Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality[J]. Proc Natl Acad Sci U S A, 2018, 115(18): E4151-E4158.
[27] Luo Y, Yu S, Li J, et al.Molecular characterization of WRKY transcription factors that act as negative regulators of O-Methylated catechin biosynthesis in tea plants (Camellia sinensis L.)[J]. J Agric Food Chem, 2018, 66(43): 11234-11243.
[28] Wang P, Zhang L, Jiang X, et al.Evolutionary and functional characterization of leucoanthocyanidin reductases from Camellia sinensis[J]. Planta, 2018, 247(1): 139-154.
[29] 牛义岭, 姜秀明. 植物转录因子MYB基因家族的研究进展[J]. 分子植物育种, 2016, 14(8): 2050-2059.
Niu Y L, Jiang X M.Research progress of plant transcription factor MYB gene family[J]. Molecular Plant Breeding, 2016, 14(8): 2050-2059.
[30] Martin C, Paz-Ares J.MYB transcription factors in plants[J]. Trends in Genetics, 1997, 13(2): 67-73.
[31] Verdonk J C, Haring M A, Tunen A J, et al.ODORANT1 regulates fragrance biosynthesis in Petunia Flowers[J]. Plant Cell, 2005, 17(5): 1612-1624.
[32] Bomal C, Bedon F, Caron S, et al.Involvement of Pinus taeda MYB1 and MYB8 in phenylpropanoid metabolism and secondary cell wall biogenesis: a comparative in planta analysis[J]. Journal of Experimental Botany, 2008, 59(14): 3925-3939.
[33] Schaart J G, Dubos C, Romero De La Fuente I, et al. Identification and characterization of MYB-bHLH-WD40 regulatory complexes controlling proanthocyanidin biosynthesis in strawberry[J]. The New Phytologist, 2013, 197(2): 454-467.
[34] An X H, Tian Y, Chen K Q, et al.MdMYB9 and MdMYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples[J]. Plant Cell Physiol, 2015, 56(4): 650-662.
[35] Tian J, Zhang J, Han Z Y, et al.McMYB12 transcription factors co-regulate proanthocyanidin and anthocyanin biosynthesis in Malus Crabapple[J]. Scientific Reports, 2017, 7(1): 43715. doi: 10.1038/srep43715.
[36] James A M, Ma D, Mellway R, et al.Poplar MYB115 and MYB134 transcription factors regulate proanthocyanidin synthesis and structure[J]. Plant Physiology, 2017, 174(1): 154-171.
[37] Wang N, Qu C, Jiang S, et al.The proanthocyanidin-specific transcription factor MdMYBPA1 initiates anthocyanin synthesis under low temperature conditions in red-fleshed apple[J]. The Plant J, 2018, 96(1): 39-55.
[38] Xu W, Dubos C, Lepiniec L.Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes[J]. Trends in Plant Science, 2015, 20(3): 176-185.
[39] Terrier N, Torregrosa L, Ageorges A, et al.Ectopic expression of VvMybPA2 promotes proanthocyanidin biosynthesis in grapevine and suggests additional targets in the pathway[J]. Plant Physiol, 2009, 149(2): 1028-1041.
[40] Gesell A, Yoshida K, Tran L T, et al.Characterization of an apple TT2-type R2R3 MYB transcription factor functionally similar to the poplar proanthocyanidin regulator PtMYB134[J]. Planta, 2014, 240(3): 497-511.
[41] Mellway R D, Tran L T, Prouse M B et al. The wound-, pathogen-, and ultraviolet B-responsive MYB134 gene encodes an R2R3 MYB transcription factor that regulates proanthocyanidin synthesis in poplar[J]. Plant Physiology, 2009, 150(2): 924-941.
[42] Stracke R, Werber M, Weisshaar B.The R2R3-MYB gene family in Arabidopsis thaliana[J]. Curr Opin Plant Biol, 2001, 4(5): 447-456.
[43] Liu R, Wang Y, Tang S, et al.Genome-wide identification of the tea plant bHLH transcription factor family and discovery of candidate regulators of trichome formation[J]. Sci Rep, 2021, 11(1): 10764. doi: 10.21203/rs.3.rs-148784/v1.
[44] Livak K J, Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta DeltaC(T)) method[J]. Methods, 2001, 25(4): 402-408.
[45] Chen C, Chen H, Zhang Y, et al.TBtools: an integrative toolkit developed for interactive analyses of big biological data[J]. Mol Plant, 2020, 13(8): 1194-1202.
[46] Dubos C, Stracke R, Grotewold E, et al.MYB transcription factors in Arabidopsis[J]. Trends Plant Sci, 2010, 15(10): 573-581.
[47] Stracke R, Ishihara H, Huep G, et al.Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling[J]. Plant J, 2007, 50(4): 660-677.
[48] Gonzalez A, Zhao M, Leavitt J M, et al.Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings[J]. Plant J, 2008, 53(5): 814-827.
[49] Lepiniec L, Debeaujon I, Routaboul J M, et al.Genetics and biochemistry of seed flavonoids[J]. Annu Rev Plant Biol, 2006, 57: 405-430.
[50] Zhong R, Lee C, Zhou J, et al.A battery of transcription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis[J]. Plant Cell, 2008, 20(10): 2763-2782.
[51] Zhou J, Lee C, Zhong R, et al.MYB58 and MYB63 are transcriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis[J]. Plant Cell, 2009, 21(1): 248-266.
[52] Sun B, Zhu Z, Cao P, et al.Purple foliage coloration in tea (Camellia sinensis L.) arises from activation of the R2R3-MYB transcription factor CsAN1[J]. Sci Rep, 2016, 6: 32534. doi: 10.1038/srep32534.
[53] Wang X C, Wu J, Guan M L, et al.Arabidopsis MYB4 plays dual roles in flavonoid biosynthesis[J]. Plant J, 2020, 101(3): 637-652.
[54] Ma D, Constabel C P.MYB repressors as regulators of phenylpropanoid metabolism in plants[J]. Trends Plant Sci, 2019, 24(3): 275-289.
[55] Agarwal M, Hao Y, Kapoor A, et al.A R2R3 type MYB transcription factor is involved in the cold regulation of CBF genes and in acquired freezing tolerance[J]. J Biol Chem, 2006, 281(49): 37636-37645.
[56] Kang Y H, Kirik V, Hulskamp M, et al.The MYB23 gene provides a positive feedback loop for cell fate specification in the Arabidopsis root epidermis[J]. Plant Cell, 2009, 21(4): 1080-1094.
[57] Jiang X, Huang K, Zheng G, et al.CsMYB5a and CsMYB5e from Camellia sinensis differentially regulate anthocyanin and proanthocyanidin biosynthesis[J]. Plant Sci, 2018, 270: 209-220.
[58] Wang P, Ma G, Zhang L, et al.A sucrose-induced MYB (SIMYB) transcription factor promoting proanthocyanidin accumulation in the tea plant (Camellia sinensis)[J]. J Agric Food Chem, 2019, 67(5): 1418-1428.