苦荞麦WRKY15转录因子基因的分子克隆及其在叶斑病原与激素诱导下的表达分析  

Molecular cloning of WRKY15 transcription factor gene in Fagopyrum tataricum and analysis of its expression induced by leaf spot pathogens and hormones

作　　者：曾丽佩 彭喜旭[1,2,3] 邬清韬 田建红 唐新科 王海华[1,2,3] ZENG Lipei;PENG Xixu;WU Qingtao;TIAN Jianhong;TANG Xinke;WANG Haihua(School of Life and Health Sciences,Hunan University of Science and Technology,Xiangtan 411201,China;Key Laboratory of Genetic Improvement and Multiple Utilization of Economic Crops in Hunan Province,Xiangtan 411201,China;Key Laboratory of Ecological Remediation and Safe Utilization of Heavy Metal-polluted Soils,College of Hunan Province,Xiangtan 411201,China)

机构地区：[1]湖南科技大学生命科学与健康学院,湘潭411201 [2]经济作物遗传改良与综合利用湖南省重点实验室,湘潭411201 [3]重金属污染生态土壤修复与安全利用湖南省高校重点实验室,湘潭411201

出　　处：《植物病理学报》2023年第4期613-623,共11页Acta Phytopathologica Sinica

基　　金：科技部国家重点研发计划(2017YFE0117600);湖南省教育厅重点科研项目(19A176);国家大学生创新创业训练计划项目(202110534015)。

摘　　要：苦荞麦(Fagopyrum tataricum)叶斑病由链格孢(Alternaria alternata)和黑孢霉(Nigrospora osmanthi)引起。WRKY转录因子在植物免疫调节过程中发挥重要作用。研究苦荞麦FtWRKY15在叶斑病原与激素诱导下的表达,为深入解析FtWRKY15在植物抗病反应中的功能与机理奠定基础。采用逆转录PCR克隆获得了FtWRKY15的完整编码序列,长537 bp,编码178个氨基酸。FtWRKY15具有1个WRKY保守结构域,锌指类型为CCHH,属于WRKY IIc亚组。同源序列比对与系统进化分析表明,FtWRKY15与FtWRKY76的氨基酸序列同一性最高(64.0%),其次是藜麦(Chenopodium quinoa)WRKY50(52.3%)。原生质体瞬时表达分析表明FtWRKY15定位在细胞核中,与亚细胞定位预测结果一致。酵母单杂交实验表明FtWRKY15具有转录激活活性,其N端为酸性结构区(30~60位),富含丝氨酸、苏氨酸、酪氨酸磷酸化位点,可能负责转录激活。实时荧光定量PCR分析表明FtWRKY15在叶、花中的表达丰度最高,其次是茎、根,在果实中几乎无表达。病原N.osmanthi、A.alternata以及水杨酸(SA)、茉莉酸(JA)和乙烯(ET)激素显著诱导FtWRKY15的转录。上述结果提示,FtWRKY15具有转录因子的基本结构与生化特征,可能介导对苦荞麦叶斑病的防御反应。Tartary buckwheat(Fagopyrum tataricum)leaf spot is caused by Nigrospora osmanthi and Alternaria alternata.WRKY transcription factors play an important regulatory role in plant immunity.Expression analysis of F.tataricum WRKY15(FtWRKY15)induced by leaf spot fungi and hormones will provide a foundation for insights into functions and underlying mechanisms of FtWRKY15 in plant responses to pathogens.In this study,the entire coding sequence(CDS)of FtWRKY15 was cloned using reverse transcription PCR.The FtWRKY15 CDS is 537 bp in length and encodes a polypeptide of 178 amino acids.FtWRKY15 had a WRKY conserved domain,belonging toWRKY group IIc subgroup with zinc finger type CCHH.Homologous sequence alignment and phylogenetic analysis revealed that FtWRKY15 shared the highest identity at amino acid level with FtWRKY76(64.0%),followed by Chenopodium quinoa WRKY50(52.3%).Transient expression assay in protoplasts showed that FtWRKY15 was localized in nucleus,consistent with the prediction of subcellular localization.Yeast one-hybrid assay revealed that FtWRKY15 had transcription-activating activity.The acidic region(30th~60th amino acid residue)in FtWRKY15 N-terminus was rich in putative serine,threonine,and tyrosine phosphorylation sites,possibly responsible for the transcription-activating activity.Real-time fluorescence quantitative PCR analysis showed that the transcript abundance of FtWRKY15 was relatively higher in leaves and flowers,next was in stems and roots,whereas almost no expression in fruits.The expression of FtWRKY15 was significantly induced byleaf spot pathogens,N.osmanthi and A.alternata,and defense-related phytohormones,such as salicy-lic acid(SA),jasmonic acid(JA)and ethylene(ET).Taken together,FtWRKY15 possesses basic structural and biochemical characteristics as a putative transcription factor,andmay be involved in defense response to Tartary buckwheat leaf spot.

关 键 词：苦荞麦(Fagopyrum tataricum) 叶斑病原 WRKY转录因子 生物信息学分析 基因表达 亚细胞定位 转录激活活性 

分 类 号：S432.44[农业科学—植物病理学]