苦荞抗旱相关转录因子基因<em>FtWRKY10</em>的克隆及功能鉴定

doi:10.3969/j.issn.1674-7968.2020.04.006

摘要
图/表
参考文献
相关文章 (15)

全文: PDF (7477 KB) HTML (1 KB)
输出: BibTeX | EndNote (RIS)

摘要 WRKY转录因子是植物特有的一类锌指型蛋白。本研究基于苦荞(Fagopyrum tataricum)转录组数据,由苦荞中克隆1条WRKY转录因子基因FtWRKY10。序列分析表明,FtWRKY10定位于苦荞基因组第5号染色体,DNA全长4 303 bp,含6个外显子和5个内含子,ORF为1 647 bp可编码548个氨基酸残基的蛋白;推导的FtWRKY10蛋白为Ⅰ类WRKY转录因子,与拟南芥(Arabidopsis thaliana) AtWRKY20具有最近的系统发育关系,qRT-PCR显示其在苦荞中的表达受到干旱胁迫的诱导。分子鉴定结果表明,FtWRKY10在酵母(Saccharomyces cerevisiae)菌株AH109中显示出转录激活活性,在转基因拟南芥中定位于细胞核;该转录因子可分别在酵母和植物细胞中特异结合其靶DNA位点W-box并启动报告基因的表达。转基因拟南芥中,FtWRKY10的超表达可增强拟南芥萌发过程中对干旱和脱落酸(abscisic acid, ABA)处理的耐受;提高拟南芥植株在干旱胁迫下叶绿素(chlorophyll)和脯氨酸(proline, Pro)的含量,增加超氧化物歧化酶(superoxide dismutase, SOD)、过氧化物酶(peroxidase, POD)和过氧化氢酶(catalase, CAT)的活性,上调拟南芥抗逆相关基因脱水应答蛋白(responsive to dehydration protein, RD)RD29A、RD29B和RD22,ABA应答蛋白(responsive to ABA protein, RAB) RAB18及丝氨酸/苏氨酸蛋白激酶基因(serine/threonine protein kinase) KIN1的表达。本研究为苦荞优良生产性状的利用提供了了基础资料和思路,为进一步丰富WRKY转录因子家族的功能提供了基础素材。

	服务

	把本文推荐给朋友
	加入我的书架
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	王官凤
	吕兵兵
	王安虎
	赵海霞
	王晓丽
	吴琦
	陈惠
	李成磊

关键词 ：苦荞, WRKY转录因子, 分子克隆, 功能鉴定

Abstract：WRKY transcription factor is a zinc finger protein unique to plants. Based on the transcriptome data of tartary buckwheat, a WRKY transcription factor gene FtWRKY10 was cloned in this study. Sequence analysis results showed that FtWRKY10 was located on chromosome 5 of tartary buckwheat genome, its DNA length was 4 303 bp, which contained 6 exons and 5 introns, and its ORF was 1 647 bp. The deduced FtWRKY10 protein was a classic WRKY transcription factor of type I, and it had the closest phylogenetic relationship with AtWRKY20 of Arabidopsis thaliana. qRT-PCR showed that its expression was induced by drought stress in tartary buckwheat. Molecular identification results showed that FtWRKY10 protein showed transcriptional activation activity in yeast and located in the nucleus of transgenic Arabidopsis thaliana cells. The transcription factor could specifically bind with its target DNA motif of W-box in yeast and plant cells and initiate the expression of the reporter genes, respectively. In Arabidopsis, overexpression of FtWRKY10 could enhance the tolerance of Arabidopsis germination to drought and ABA treatments. Under drought stress, the overexpression of FtWRKY10 could increase the content of chlorophyll and proline, the activity of SOD (Superoxide dismutase), POD (Peroxidase) and CAT (Catalase), and also up regulate the expression of stress resistance related genes of responsive to dehydration protein genes (RD29A, RD29B, and RD22), responsive to ABA protein gene RAB18, and serine/threonine protein kinase gene KIN1. For conclusion, the FtWRKY10 encodes a type I WRKY transcription factor related to drought tolerance. This study could provide strategies for exploiting and utilizing the excellent stress resistance traits from tartary buckwheat, and supply basic materials for further enriching the functions of WRKY family.

Key words： Tartary buckwheat WRKY transcription factor Molecular cloning Functional identification

收稿日期: 2019-09-27

ZTFLH:

S336

基金资助:国家自然科学基金项目(No. 31871699)

通讯作者: ** lichenglei1998@163.com

作者简介: *同等贡献作者

引用本文:

王官凤, 吕兵兵, 王安虎, 赵海霞, 王晓丽, 吴琦, 陈惠, 李成磊. 苦荞抗旱相关转录因子基因FtWRKY10的克隆及功能鉴定[J]. 农业生物技术学报, 2020, 28(4): 629-644.
WANG Guan-Feng, LV Bing-Bing, WANG An-Hu, ZHAO Hai-Xia, WANG Xiao-Li, WU Qi, CHEN Hui, LI Cheng-Lei. Cloning and Functional Identification of Drought Resistance Related Transcription Factor Gene FtWRKY10 from Tartary Buckwheat(Fagopyrum tataricum). 农业生物技术学报, 2020, 28(4): 629-644.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2020.04.006 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2020/V28/I4/629

[1] 陈善福, 舒庆尧. 1999. 植物耐干旱胁迫的生物学机理及其基因工程研究进展[J]. 植物学报, 16(05): 555-560.
(Chen S F, Shu Q X Y.1999. Biological mechanism of and genetic engineering for drought stress tolerance in plants[J]. Chinese Bulletin of Botany, 16(05): 555-560.)
[2] 付乾堂, 余迪求. 2010. 拟南芥AtWRKY25, AtWRKY26和AtWRKY33在非生物胁迫条件下的表达分析[J]. 遗传, 32(8): 848-856.
(Fu Q T, Yu D Q.2010. Expression profiles of AtWRKY25, AtWRKY26 and AtWRKY33 under abiotic stresses[J]. Hereditas, 32(8): 848-856.)
[3] 侯伶俐, 杨雄榜, 董雪妮, 等. 2016. 逆境胁迫对苦荞花期总黄酮含量及关键酶基因表达的影响[J]. 核农学报, 30(01): 184-192.
(Hou L L, Yang X B, Dong X N, et al.2016. Effect of environmental stresses on the contents of total flavonoids and corresponding gene expression in Fagopyrum tataricum during florescence[J]. Journal of Nuclear Agricultural Sciences, 30(01): 184-192.)
[4] 黄云吉, 邓仁榆, 高飞, 等. 2015. 苦荞转录因子基因FtMYB21的克隆及其非生物胁迫下的表达分析[J]. 基因组学与应用生物学, 34(09): 1939-1945.
(Huang Y J, Deng R Y, Gao F, et al.2015. Cloning and expression analysis of transcription factor gene FtMYB21 from Tartary Buckwheat under abiotic stress[J]. Genomics and Applied Biology, 34(09): 1939-1945.)
[5] 刘为远, 刘桂男, 陈双, 等. 2018. 苦荞转录因子基因FtDREB1和FtDREB2的克隆及其对非生物胁迫的应答[J]. 基因组学与应用生物学, 37(05): 1985-1992.
(Liu W Y, Liu G N, Chen S, et al.2018. Cloning of FtDREB1 and FtDREB2 transcription factor genes from Fagopyrum tataricum and their responses to abiotic stress[J]. Genomics and Applied Biology, 37(05): 1985-1992.)
[6] 田云, 卢向阳, 彭丽莎, 等. 2006. 植物WRKY转录因子结构特点及其生物学功能[J]. 遗传, 28(12): 1607-1612.
(Tian Y, Lu X Y, Peng L S, et al.2006. Structural characteristics and biological functions of plant WRKY transcription factor[J]. Hereditas, 28(12): 1607-1612.)
[7] 王爱荣, 吴智芳, 张丽丽, 等. 2006. 影响拟南芥转化效率和激活标签丢失的因素分析[J]. 福建农林大学学报(自然科学版), 35(3): 298-302.
(Wang A R, Wu Z F, Zhang L L, et al.2006. Analysis of factors affecting transformation efficiency and loss of enhancer in generation of activation-tagged mutants of Arabidopsis thaliana[J]. Journal of Fujian Agriculture and Forestry University (Natural Science Edition), 35(3): 298-302.)
[8] 姚攀锋, 赵学荣, 李茂菲, 等. 2016. 苦荞转录因子基因FtbHLH3的克隆及其非生物胁迫下的表达分析[J]. 基因组学与应用生物学, 35(02): 429-435.
(Yao P F, Zhao X R, Li R F, et al.2016. Cloning and expression analysis of transcription factor gene FtbHLH3 from Fagopyrum tataricum under abiotic stress[J]. Genomics and Applied Biology, 35(02): 429-435.)
[9] Agarwal P K, Agarwal P, Reddy M, et al.2006. Role of DREB transcription factors in abiotic and biotic stress tolerance in plants[J]. Plant Cell Reports, 25(12) 1263-1274.
[10] Ashraf M, Foolad M.2007. Roles of glycine betaine and proline in improving plant abiotic stress resistance[J]. Environmental and Experimental Botany, 59(2): 206-216.
[11] Bai Y, Sunarti S, Kissoudis C, et al.2018. The role of tomato WRKY genes in plant responses to combined abiotic and biotic stresses[J]. Frontiers in Plant Science, 9: 801.
[12] Bai Y C, Li C L, Zhang J W, et al.2014. Characterization of two Tartary buckwheat R2R3‐MYB transcription factors and their regulation of proanthocyanidin biosynthesis[J]. Physiologia Plantarum, 152(3): 431-440.
[13] Bates L S, Waldren R P, Teare I.1973. Rapid determination of free proline for water-stress studies[J]. Plant and Soil, 39(1): 205-207.
[14] Birkenbihl R P, Kracher B, Ross A, et al.2018. Principles and characteristics of the Arabidopsis WRKY regulatory network during early MAMP‐triggered immunity[J]. The Plant Journal, 96(3): 487-502.
[15] Brighouse A, Dacks J B, Field M C.2010. Rab protein evolution and the history of the eukaryotic endomembrane system[J]. Cellular and Molecular Life Sciences, 67(20): 3449-3465.
[16] Cai M, Qiu D, Yuan T, et al.2008. Identification of novel pathogen‐responsive cis‐elements and their binding proteins in the promoter of OsWRKY13, a gene regulating rice disease resistance[J]. Plant, Cell & Environment, 31(1): 86-96.
[17] Chen L, Song Y, Li S, et al.2012. The role of WRKY transcription factors in plant abiotic stresses[J]. Biochim Biophys Acta, 1819(2): 120-128.
[18] Ciolkowski I, Wanke D, Birkenbihl R P, et al.2008. Studies on DNA-binding selectivity of WRKY transcription factors lend structural clues into WRKY-domain function[J]. Plant Molecular Biology, 68(1-2): 81-92.
[19] Comas L, Becker S, Cruz V M V, et al.2013. Root traits contributing to plant productivity under drought[J]. Frontiers in Plant Science, 4: 442.
[20] Dong J, Chen C, Chen Z.2003. Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response[J]. Plant Molecular Biology, 51(1): 21-37.
[21] Erpen L, Devi H S, Grosser J W, et al.2018. Potential use of the DREB/ERF, MYB, NAC and WRKY transcription factors to improve abiotic and biotic stress in transgenic plants[J]. Plant Cell, Tissue and Organ Culture (PCTOC), 132(1): 1-25.
[22] Eulgem T, Rushton P J, Robatzek S, et al.2000. The WRKY superfamily of plant transcription factors[J]. Trends in Plant Science, 5(5): 199-206.
[23] Eulgem T, Somssich I E.2007. Networks of WRKY transcription factors in defense signaling[J]. Current Opinion in Plant Biology, 10(4): 366-371.
[24] Fu Q, Yu D.2010. Expression profiles of AtWRKY25, AtWRKY26 and AtWRKY33 under abiotic stresses[J]. Hereditas, 32(8): 848-856.
[25] Gao F, Zhou J, Deng R Y, et al.2017. Overexpression of a Tartary buckwheat R2R3-MYB transcription factor gene, FtMYB9, enhances tolerance to drought and salt stresses in transgenic Arabidopsis[J]. Journal of Plant Physiology, 214: 81.
[26] Gill S S, Tuteja N.2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants[J]. Plant Physiology and Biochemistry, 48(12): 909-930.
[27] González R M, Iusem N D.2014. Twenty years of research on Asr (ABA-stress-ripening) genes and proteins[J]. Planta, 239(5): 941-949.
[28] Hammargren J, Rosenquist S, Jansson C, et al.2008. A novel connection between nucleotide and carbohydrate metabolism in mitochondria: Sugar regulation of the Arabidopsis nucleoside diphosphate kinase 3a gene[J]. Plant Cell Reports, 27(3): 529-534.
[29] Han Y, Zhang X, Wang Y, et al.2013. The suppression of WRKY44 by GIGANTEA-miR172 pathway is involved in drought response of Arabidopsis thaliana[J]. PLoS ONE, 8(11): e73541.
[30] He X, Li J, Chen Y, et al.2019. Genome-wide analysis of the WRKY gene family and its response to abiotic stress in buckwheat (Fagopyrum tataricum)[J]. Open Life Sciences, 14(1): 80-96.
[31] Hussain A, Noman A, Khan M I, et al.2019. Molecular regulation of pepper innate immunity and stress tolerance: An overview of WRKY TFs[J]. Microbial Pathogenesis,135(135): 103601.
[32] Ishiguro S, Nakamura K.1994. Characterization of a cDNA encoding a novel DNA-binding protein, SPF1, that recognizes SP8 sequences in the 5' upstream regions of genes coding for sporamin and β-amylase from sweet potato[J]. Molecular and General Genetics MGG, 244(6): 563-571.
[33] Jiang J, Ma S, Ye N, et al.2017. WRKY transcription factors in plant responses to stresses[J]. Journal of Integrative Plant Biology, 59(2): 86-101.
[34] Jiang Y, Zeng B, Zhao H, et al.2012. Genome‐wide transcription factor gene prediction and their expressional Tissue‐Specificities in Maize F[J]. Journal of Integrative Plant Biology, 54(9): 616-630.
[35] Kalde M, Barth M, Somssich I E, et al.2003. Members of the Arabidopsis WRKY group Ⅲ transcription factors are part of different plant defense signaling pathways[J]. Mol Plant Microbe Interact, 16(4): 295-305.
[36] Kasuga M, Miura S, Shinozaki K, et al.2004. A combination of the Arabidopsis DREB1A gene and stress-inducible rd29A promoter improved drought-and low-temperature stress tolerance in tobacco by gene transfer[J]. Plant and Cell Physiology, 45(3): 346-350.
[37] Kishor P K, Sangam S, Amrutha R, et al.2005. Regulation of proline biosynthesis, degradation, uptake and transport in higher plants: Its implications in plant growth and abiotic stress tolerance[J]. Curr Sci, 88(3): 424-438.
[38] Klein P, Dietz K J.2010 Identification of DNA-binding proteins and protein-protein interactions by yeast one-hybrid and yeast two-hybrid screen[J]. Methods in Molecular Biology (Clifton, N.J.), 639: 171-192.
[39] Kurkela S, Franck M.1990. Cloning and characterization of a cold-and ABA-inducible Arabidopsis gene[J]. Plant Molecular Biology, 15(1): 137-144.
[40] Lång V, Palva E T.1992. The expression of a rab-related gene, rab18, is induced by abscisic acid during the cold acclimation process of Arabidopsis thaliana (L.) Heynh[J]. Plant Molecular Biology, 20(5): 951-962.
[41] Li C, Bai Y, Li S, et al.2012a. Cloning, characterization, and activity analysis of a flavonol synthase gene FtFLS1 and its association with flavonoid content in Tartary buckwheat[J]. Journal of Agricultural and Food Chemistry, 60(20): 5161-5168.
[42] Li C, Zhao H, Li M, et al.2019a. Validation of reference genes for gene expression studies in Tartary buckwheat (Fagopyrum tataricum Gaertn.) using quantitative real-time PCR[J]. PeerJ, 7: e6522.
[43] Li C L, Bai Y C, Chen H, et al.2012b. Cloning, characterization and functional analysis of a phenylalanine ammonia-lyase gene (FtPAL) from Fagopyrum tataricum Gaertn[J]. Plant Molecular Biology Reporter, 30(5): 1172-1182.
[44] Li H, Lv Q, Ma C, et al.2019b. Metabolite profiling and transcriptome analyses provide insights into the flavonoid biosynthesis in the developing seed of Tartary buckwheat (Fagopyrum tataricum)[J]. Journal of Agricultural and Food Chemistry, 67(40): 11262-11276.
[45] Maleck K, Levine A, Eulgem T, et al.2000. The transcriptome of Arabidopsis thaliana during systemic acquired resistance[J]. Nature Genetics, 26(4): 403.
[46] Nagata T, Hara H, Saitou K, et al.2012. Activation of ADP-glucose pyrophosphorylase gene promoters by a WRKY transcription factor, AtWRKY20, in Arabidopsis thaliana L. and sweet potato (Ipomoea batatas Lam.)[J]. Plant Production Science, 15(1): 10-18.
[47] Nakashima K, Fujita Y, Katsura K, et al.2006. Transcriptional regulation of ABI3-and ABA-responsive genes including RD29B and RD29A in seeds, germinating embryos, and seedlings of Arabidopsis[J]. Plant Molecular Biology, 60(1): 51-68.
[48] Nakashima K, Shinwari Z K, Sakuma Y, et al.2000. Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration-and high-salinity-responsive gene expression[J]. Plant Molecular Biology, 42(4): 657-665.
[49] Park J, Cui Y, Kang B H.2015. AtPGL3 is an Arabidopsis BURP domain protein that is localized to the cell wall and promotes cell enlargement[J]. Frontiers in Plant Science, 6: 412.
[50] Phukan U J, Jeena G S, Shukla R K.2016. WRKY transcription factors: Molecular regulation and stress responses in plants[J]. Frontiers in Plant Science, 7: 760.
[51] Rushton P J, Somssich I E.1998. Transcriptional control of plant genes responsive to pathogens[J]. Current Opinion in Plant Biology, 1(4): 311-315.
[52] Rushton P J, Somssich I E, Ringler P, et al.2010. WRKY transcription factors[J]. Trends in Plant Science, 15(5): 247-258.
[53] Rushton P J, Torres J T, Parniske M, et al.1996. Interaction of elicitor‐induced DNA‐binding proteins with elicitor response elements in the promoters of parsley PR1 genes[J]. The EMBO Journal, 15(20): 5690-5700.
[54] Sang-Won L, Sang-Wook H, Malinee S, et al.2009. Retraction. A type I-secreted, sulfated peptide triggers XA21-mediated innate immunity[J]. Science, 326(6155): 850-853.
[55] Seki M, Umezawa T, Urano K, et al.2007. Regulatory metabolic networks in drought stress responses[J]. Current Opinion in Plant Biology, 10(3): 296-302.
[56] Ueda M, Zhang Z, Laux T.2011. Transcriptional activation of Arabidopsis axis patterning genes WOX8/9 links zygote polarity to embryo development[J]. Developmental Cell, 20(2): 264-270.
[57] Ülker B, Somssich I E.2004. WRKY transcription factors: from DNA binding towards biological function[J]. Current Opinion in Plant Biology, 7(5): 491-498.
[58] Vierling E, Kimpel J A.1992. Plant responses to environmental stress[J]. Current Opinion in Biotechnology, 3(2): 164-170.
[59] Wang F, Jiang Y, Feng X C, et al.2007. Molecular cloning of a Glycyrrhiza uralensis F. aquaporin GuPIP 1 up-regulated in response to drought, salt and ABA stress[J]. Chemical Research in Chinese Universities, 23(1): 52-57.
[60] Wang H, Datla R, Georges F, et al.1995. Promoters from kin1 and cor6.6, two homologous Arabidopsis thaliana genes: Transcriptional regulation and gene expression induced by low temperature, ABA, osmoticum and dehydration[J]. Plant Molecular Biology, 28(4): 605-617.
[61] Wittenmayer L, Merbach W.2010. Plant responses to drought and phosphorus deficiency: Contribution of phytohormones in root-related processes[J]. Journal of Plant Nutrition & Soil Science, 168(4): 531-540.
[62] Wu K L, Guo Z J, Wang H H, et al.2005. The WRKY family of transcription factors in rice and Arabidopsis and their origins[J]. DNA research, 12(1): 9-26.
[63] Wu Q, Bai X, Zhao W, et al.2017. De novo assembly and analysis of Tartary buckwheat (Fagopyrum tataricum Garetn.) transcriptome discloses key regulators involved in salt-stress response[J]. Genes, 8(10): 255.
[64] Yamaguchi-Shinozaki K, Shinozaki K.1993. The plant hormone abscisic acid mediates the drought-induced expression but not the seed-specific expression of rd22, a gene responsive to dehydration stress in Arabidopsis thaliana[J]. Molecular and General Genetics MGG, 238(1-2): 17-25.
[65] Yang Y, Li R, Qi M.2000. In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves[J]. The Plant Journal, 22(6): 543-551.
[66] Yao H P, Li C L, Zhao H X, et al.2017. Deep sequencing of the transcriptome reveals distinct flavonoid metabolism features of black tartary buckwheat (Fagopyrum tataricum Garetn)[J]. Progress in Biophysics and Molecular Biology, 124: 49-60.
[67] Zhang J, Jia W, Yang J, et al.2006. Role of ABA in integrating plant responses to drought and salt stresses[J]. Field Crops Research, 97(1): 111-119.
[68] Zhao J, Zhang W, Zhao Y, et al.2007. SAD2, an importin β-like protein, is required for UV-B response in Arabidopsis by mediating MYB4 nuclear trafficking[J]. The Plant Cell, 19(11): 3805-3818.
[69] Zhu F.2016. Chemical composition and health effects of Tartary buckwheat[J]. Food Chemistry, 203: 231-245.
[70] Zou X, Shen Q J, Neuman D.2007. An ABA inducible WRKY gene integrates responses of creosote bush (Larrea tridentata) to elevated CO₂ and abiotic stresses[J]. Plant Science, 172(5): 997-1004.