茶树CsbHLH024和CsbHLH133转录因子功能鉴定

doi:10.13305/j.cnki.jts.2022.03.001

摘要/Abstract

摘要： 茶树叶片毛状体含有多种次生代谢产物,在茶叶外观质量以及茶树响应生物和非生物胁迫方面起着重要作用。通过双荧光分子互补（BiFC）试验、GUS活性染色试验以及过表达试验对茶树叶片毛状体相关候选基因CsbHLH024和CsbHLH133的功能进行鉴定。结果表明,CsbHLH024/CsbHLH133和CsTTG1蛋白在植物中能够相互作用,并且它们的启动子能够在叶片组织中驱动下游基因的表达。进一步将它们分别过表达到野生型拟南芥Col和对应的拟南芥纯合突变体中,发现它们能够影响拟南芥叶片毛状体的形成,恢复突变体的表型,并引起毛状体相关基因表达水平的变化。本研究为进一步揭示茶树叶片毛状体形成的分子调控机制提供理论依据。

关键词: 茶树, 毛状体形成, CsbHLH024, CsbHLH133, 分子调控

Abstract: Tea plant leaf trichomes contain various secondary metabolites and play an important role in the tea appearance quality as well as the response of tea plants to biotic and abiotic stresses. In this study, the function of leaf trichome-related genes CsbHLH024 and CsbHLH133 were analyzed using Bimolecular Fluorescent Complimentary (BiFC), GUS staining and overexpression experiments. The results show that CsbHLH024/CsbHLH133 and CsTTG1 could interact in plants, and their promoters could drive downstream gene expression in leaf tissues. They were further transformed into wild Arabidopsis thaliana (Col) and corresponding homozygous mutants, respectively to get overexpression lines. Both genes could affect the leaf trichome formation in Arabidopsis thaliana, restore the phenotype of the mutants, and induce the expression levels of trichome-related genes. This study provided a theoretical basis for further research on the molecular regulation mechanism of trichome formation in tea leaves.

Key words: tea plant, trichome formation, CsbHLH024, CsbHLH133, molecular regulation

中图分类号:

S571.1

刘任坚, 王玉源, 刘少群, 舒灿伟, 孙彬妹, 郑鹏. 茶树CsbHLH024和CsbHLH133转录因子功能鉴定[J]. 茶叶科学, 2022, 42(3): 347-357. doi: 10.13305/j.cnki.jts.2022.03.001.

LIU Renjian, WANG Yuyuan, LIU Shaoqun, SHU Canwei, SUN Binmei, ZHENG Peng. Functional Identification of CsbHLH024 and CsbHLH133 Transcription Factors in Tea Plants[J]. Journal of Tea Science, 2022, 42(3): 347-357. doi: 10.13305/j.cnki.jts.2022.03.001.

参考文献

[1] Werker E.Trichome diversity and development[J]. Adv Bot Res, 2000, 31: 1-35.
[2] Serna L, Martin C.Trichomes: different regulatory networks lead to convergent structures[J]. Trends Plant Sci, 2006, 11(6): 274-280.
[3] Schilmiller A, Last R, Pichersky E.Harnessing plant trichome biochemistry for the production of useful compounds[J]. Plant J, 2008, 54(4): 702-711.
[4] Ishida T, Kurata T, Okada K, et al.A genetic regulatory network in the development of trichomes and root hairs[J]. Annu Rev Plant Biol, 2008, 59: 365-386.
[5] 曹敏, 张璐, 高新梅, 等. 植物表皮毛发育分子调控机制的研究进展[J]. 安徽农业科学, 2013, 41(10): 4231-4235.
Cao M, Zhang L, Gao X M, et al.Advances in molecular regulation of plant trichome development[J]. Journal of Anhui Agricultural Sciences, 2013, 41(10): 4231-4235.
[6] Wagner G, Wang E, Shepherd R.New approaches for studying and exploiting an old protuberance, the plant trichome[J]. Ann Bot, 2004, 93(1): 3-11.
[7] Fernandez V, Sancho-Knapik D, Guzman P, et al.Wettability, polarity, and water absorption of holm oak leaves: effect of leaf side and age[J]. Plant Physiol, 2014, 166(1): 168-180.
[8] Svetlikova P, Hajek T, Tesitel J.Hydathodetrichomes actively secreting water from leaves play a key role in the physiology and evolution of root-parasitic rhinanthoid Orobanchaceae[J]. Ann Bot, 2015, 116(1): 61-68.
[9] Dayan F, Duke S.Trichomes and root hairs: natural pesticide factories[J]. Pesticide Outlook, 2003, 14(4): 175. doi:10.1039/b308491b.
[10] Bleeker P, Mirabella R, Diergaarde P, et al.Improved herbivore resistance in cultivated tomato with the sesquiterpene biosynthetic pathway from a wild relative[J]. Proc Natl Acad Sci U S A, 2012, 109(49): 20124-20129.
[11] Luu V, Weinhold A, Ullah C, et al.O-acyl sugars protect a wild tobacco from both native fungal pathogens and a specialist herbivore[J]. Plant physiology, 2017, 174(1): 370-386.
[12] Yamasaki S, Murakami Y.Continuous UV-B irradiation induces endoreduplicationand trichome formation in cotyledons, and reduces epidermal cell division and expansion in the first leaves of pumpkin seedlings (Cucurbita maxima Duch.×C. moschata Duch.)[J]. Environmental Control in Biology, 2014, 52(4): 203-209.
[13] Weathers P, Arsenault P, Covello P, et al.Artemisinin production in Artemisia annua: studies in planta and results of a novel delivery method for treating malaria and other neglected diseases[J]. Phytochem Rev, 2011, 10(2): 173-183.
[14] Mellon J, Zelaya C, Dowd M, et al.Inhibitory effects of gossypol, gossypolone, and apogossypolone on a collection of economically important filamentous fungi[J]. J Agric Food Chem, 2012, 60(10): 2740-2745.
[15] Chalvin C, Drevensek S, Dron M, et al.Genetic control of glandular trichome development[J]. Trends Plant Sci, 2020, 25(5): 477-487.
[16] Schellmann S, Hulskamp M.Epidermal differentiation: trichomes in Arabidopsis as a model system[J]. Int J DevBiol, 2005, 49(5/6): 579-584.
[17] 杨君, 马峙英, 王省芬. 棉花纤维品质改良相关基因研究进展[J]. 中国农业科学, 2016, 49(22): 4310-4322.
Yang J, Ma S Y, Wang S F.Advances in studies on genes related to cotton fiber quality improvement[J]. Chinese Agricultural Sciences, 2016, 49(22): 4310-4322.
[18] 唐凯. 棉花磷脂酶D基因家族的分子特征及其GhPLDα1功能分析[D]. 北京: 清华大学, 2017.
Tang K.Molecular characterization of phospholipase D gene family and functional analysis of GhPLDα1 in cotton [D]. Beijing: Tsinghua University, 2017.
[19] Shi Y H, Zhu S W, Mao X Z, et al.Transcriptome profiling, molecular biological, and physiological studies reveal a major role for ethylene in cotton fiber cell elongation[J]. Plant Cell, 2006, 18(3): 651-664.
[20] Chen C, Liu M, Jiang L, et al.Transcriptome profiling reveals roles of meristem regulators and polarity genes during fruit trichome development in cucumber (Cucumissativus L.)[J]. J Exp Bot, 2014, 65(17): 4943-4958.
[21] Pan Y, Bo K, Cheng Z, et al.The loss-of-function GLABROUS 3 mutation in cucumber is due to LTR-retrotransposon insertion in a class IV HD-ZIP transcription factor gene CsGL3 that is epistatic over CsGL1[J]. BMC Plant Biology, 2015, 15: 302. doi: 10.1186/s12870-015-0693-0.
[22] Liu X, Bartholomew E, Cai Y, et al.Trichome-related mutants provide a new perspective on multicellular trichome initiation and development in cucumber (Cucumissativus L)[J]. Front Plant Sci, 2016, 7: 1187. doi: 10.3389/fpls.2016.01187.
[23] Zheng K, Tian H, Hu Q, et al.Ectopic expression of R3 MYB transcription factor gene OsTCL1 in Arabidopsis, but not rice, affects trichome and root hair formation[J]. Sci Rep, 2016, 6: 19254. doi: 10.1038/srep19254.
[24] Qin B, Tang D, Huang J, et al.Rice OsGL1-1 is involved in leaf cuticular wax and cuticle membrane[J]. Mol Plant, 2011, 4(6): 985-995.
[25] Ming T, Bartholomew B.Theaceae[J]. Flora China, 2007, 12: 366-478.
[26] 陈亮, 虞富莲, 童启庆. 关于茶组植物分类与演化的讨论[J]. 茶叶科学, 2000, 20(2): 89-94.
Chen L, Yu F L, Tong Q Q.Discussion on the classification and evolution of tea plants[J]. Journal of Tea Science, 2000, 20(2): 89-94.
[27] Li P, Xu Y, Zhang Y, et al.Metabolite profiling and transcriptome analysis revealed the chemical contributions of tea trichomes to tea flavors and tea plant defenses[J]. J Agric Food Chem, 2020, 68(41): 11389-11401.
[28] Zhu M, Li N, Zhao M, et al.Metabolomic profiling delineate taste qualities of tea leaf pubescence[J]. Food Res Int, 2017, 94: 36-44.
[29] Wei C, Yang H, 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]. PNAS, 2018, 115(18): E4151-E4158.
[30] Gilbert N.The science of tea's mood-altering magic[J]. Nature, 2019, 566(7742): S8-S9.
[31] Cao H, Li J, Ye Y, et al.Integrative transcriptomic and metabolic analyses provide insights into the role of trichomes in tea plant (Camellia sinensis)[J]. Biomolecules, 2020, 10(2): 311. doi: 10.3390/biom10020311.
[32] Barman T, Baruah U, Saikia J.Seasonal changes in metabolic activities of drought tolerant and susceptible clones of tea (Camellia sinensis L.)[J]. Journal of Plantation Crops, 2008, 36(3): 259-264.
[33] Konrad W, Burkhardt J, Ebner M, et al.Leaf pubescence as a possibility to increase water use efficiency by promoting condensation[J]. Ecohydrology, 2015, 8(3): 480-492.
[34] Das S, Zaman A, Borchetia S, et al.Genetic relationship in tea germplasms with drought contrasting traits[J]. Plant Breeding and Biotechnology, 2016, 4(4): 484-494.
[35] 杨丽丽, 郑高云, 梁丽云, 等. 茶树抗病虫机制的研究进展[J]. 福建茶叶, 2008(2): 8-11.
Yang L L, Zheng G Y, Liang L Y, et al.Research progress on resistance mechanism to disease and insect in tea plant[J]. Tea in Fujian, 2008(2): 8-11.
[36] Dutta M.Morphological resistance of certain tea clones to red spider mite (Oligonychus coffeae) in tea[J]. Journal of Entomology and Zoology Studies, 2015, 4(3): 454-457.
[37] Bandyopadhyay T, Gohain B, Bharalee R, et al.Molecular landscape of helopeltis theivora induced transcriptome and defense gene expression in tea[J]. Plant molecular biology reporter, 2015, 33(4): 1042-1057.
[38] Yue C, Cao H, Chen D, et al.Comparative transcriptome study of hairy and hairless tea plant (Camellia sinensis) shoots[J]. J Plant Physiol, 2018, 229: 41-52.
[39] Sun B, Zhu Z, Liu R, et al.TRANSPARENT TESTA GLABRA1 (TTG1) regulates leaf trichome density in tea Camellia sinensis[J]. Nordic Journal of Botany, 2020, 38(1): 1-10.
[40] 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: 10.1038/s41598-021-90205-7.
[41] Livak K, Schmittgen T.Analysis of relative gene expression data using real-time quantitative PCR and the <inline-graphic xlink:href="1000-369X-42-3-347/img_1.wmf"/> method[J]. Methods, 2001, 25(4): 402-408.
[42] Rerie W, Feldmann K, Marks M.The GLABRA2 gene encodes a homeo domain protein required for normal trichome development in Arabidopsis[J]. Genes Dev, 1994, 8(12): 1388-1399.
[43] Luo D, Oppenheimer D.Genetic control of trichome branch number in Arabidopsis: the roles of the FURCA loci[J]. Development, 1999, 126(24): 5547-5557.
[44] Payne C, Zhang F, Lloyd A.GL3 encodes a bHLH protein that regulates trichome development in Arabidopsis through interaction with GL1 and TTG1[J]. Genetics, 2000, 156(3): 1349-1362.
[45] Zhang F, Gonzalez A, Zhao M, et al.A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis[J]. Development, 2003, 130(20): 4859-4869.
[46] Zhao M, Morohashi K, Hatlestad G, et al.The TTG1-bHLH-MYB complex controls trichome cell fate and patterning through direct targeting of regulatory loci[J]. Development, 2008, 135(11): 1991-1999.
[47] Mondal T, Bhattacharya A, Laxmikumaran M, et al.Recent advances of tea (Camellia sinensis) biotechnology[J]. Plant Cell, Tissue and Organ Culture, 2004, 76(3): 195-254.
[48] Szymanski D, Jilk R, Pollock S, et al.Control of GL2 expression in Arabidopsis leaves and trichomes[J]. Development, 1998, 125(7): 1161-1171.
[49] Boeglin M, Fuglsang A, Luu D, et al.Reduced expression of AtNUP62 nucleoporin gene affects auxin response in Arabidopsis[J]. BMC Plant Biol. 2016, 16: 2. doi: 10.1186/s12870-015-0695-y.
[50] 张晨光. 苹果核孔蛋白MdNup54/62调控开花和高温胁迫响应功能研究[D]. 杨凌: 西北农林科技大学, 2021.
Zhang C G.Study on the function of apple nuclear pore protein MDNUP54/62 in regulation of flowering and heat stress response [D]. Yangling: Northwest A&F University, 2021.