岷江百合WRKY转录因子基因的克隆与功能分析  

Cloning and Functional Analysis of WRKY Transcription Factor Gene from Lilium regale Wilson

作　　者：王自娥 邓婕 梁婷婷 苏琳琳 葛锋[1] 刘迪秋[1] WANG Zi'e;DENG Jie;LIANG Tingting;SU Linlin;GE Feng;LIU Diqiu(Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China)

机构地区：[1]昆明理工大学生命科学与技术学院,昆明650500

出　　处：《西北植物学报》2021年第12期1983-1993,共11页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(31760586);云南省“万人计划”青年拔尖人才项目。

摘　　要：WRKY转录因子家族在植物应对非生物和生物胁迫的防卫反应中起重要作用。岷江百合(Lilium regale Wilson)是高抗枯萎病的野生百合,该研究基于前期转录组测序分析,采用RT-PCR方法从岷江百合中克隆得到WRKY转录因子基因LrWRKY4并分析其功能,以探讨岷江百合对枯萎病菌尖孢镰刀菌(Fusarium oxysporum)侵染的转录调控机制,为进一步研究岷江百合WRKY基因家族的功能奠定基础。结果显示:(1)LrWRKY4开放阅读框为993 bp,编码330个氨基酸,LrWRKY4含有一个高度保守的‘WRKYGQK’七肽序列和一个C2H2锌指基序,属于Ⅱc类WRKY转录因子。(2)成功构建GFP-LrWRKY4融合载体并通过根癌农杆菌介导转化洋葱表皮细胞,激光共聚焦显微观察发现,GFP-LrWRKY4融合蛋白表达的绿色荧光特异性地分布在洋葱表皮细胞的细胞核中。(3)成功构建了过表达载体pCAMBIA2300s-LrWRKY4并转化烟草,得到11个T2代转基因烟草株系,且转LrWRKY4基因烟草与野生型烟草(WT)在表型上无明显差异;尖孢镰刀菌接种根部和叶片的实验结果显示,转LrWRKY4基因烟草对尖孢镰刀菌的抗性较WT明显增强;qRT-PCR分析显示,岷江百合LrWRKY4基因在11个转基因烟草株系中均有表达,且转基因烟草中JA/SA信号途径相关基因的表达上调,并诱导了部分病程蛋白相关基因以及抗氧化相关基因的表达上调。(4)岷江百合鳞片浸染LrWRKY4 RNAi载体后的腐烂程度和病变面积均远大于RNAi空载转化的鳞片;瞬时表达LrWRKY4 RNAi载体的岷江百合鳞片中LrWRKY4基因的表达水平较对照下降了约45.7%,接种尖孢镰刀菌72 h后表达水平下降了93.8%;瞬时表达LrWRKY4 RNAi后,一些JA/SA信号途径相关基因的表达水平明显下降。研究表明,岷江百合LrWRKY4编码一个定位于植物细胞核的Ⅱc类WRKY转录因子;LrWRKY4基因能够在转基因烟草中稳定表达,且过表达LrWRKY4基因提高了烟草对尖孢镰刀菌的抗性;但瞬时表达LrWRKYWRKY transcription factors(TFs)have been proven to play vital roles in responses to biotic and abiotic stresses.The Lilium regale Wilson is a wild lily with a high level of resistance to fusarium wilt.A WRKY TF gene LrWRKY4 was cloned from L.regale Wilson by RT-PCR based on previous transcriptome sequencing data in this study,and its function was analyzed to explore the transcriptional regulatory mechanism of L.regale during response to fusarium wilt pathogen Fusarium oxysporum infection.This study lays the foundation for subsequent further studying the function of the WRKY gene family in L.regale.The results showed that:(1)the open reading frame of LrWRKY4 was 993 bp,encoding 330 amino acids.LrWRKY4 contained a highly conserved‘WRKYGQK’heptapeptide and a C2H2 zinc finger motif,belonging to the groupⅡc WRKY.(2)The GFP-LrWRKY4 fusion vector was successfully constructed and transformed into onion by Agrobacterium tumefaciens,and the laser confocal microscopy observed that the green fluorescence expressed by the GFP-LrWRKY4 fusion protein was specifically distributed in the nucleus of onion epidermal cells.(3)The overexpression vector pCAMBIA2300s-LrWRKY4 was constructed and transformed into tobacco,and 11 lines of T2 LrWRKY4 transgenic tobacco seedlings were obtained.There was no significant phenotype difference between transgenic tobacco and wild-type(WT)tobacco;the root and leaf inoculation assays showed that LrWRKY4 transgenic tobacco was more resistant to F.oxysporum than the WT;qRT-PCR analysis showed that the LrWRKY4 gene of L.regale was expressed in 11 transgenic tobacco lines,and the expression of JA/SA signaling pathway-related genes were up-regulated in LrWRKY4 transgenic tobacco compared with WT as well as some pathogenesis-protein related genes and antioxidant-related genes.(4)The decay and lesion area of L.regale scales infected with LrWRKY4 RNAi vector were much larger than those of RNAi empty vector infected scales;the expression level of LrWRKY4 in L.regale scales transiently expressing LrWRKY

关 键 词：岷江百合 尖孢镰刀菌 枯萎病 WRKY转录因子 

分 类 号：Q785[生物学—分子生物学] Q786