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拟南芥转录因子基因WRKY72的特性分析及其抗逆功能鉴定

Characterization and Stress-resistance Functional Identification of Transcription Factor Gene WRKY72 in Arabidopsis thaliana
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摘要 拟南芥(Arabidopsis thaliana)WRKY72转录因子属于Ⅱb亚组成员。为探明WRKY72基因的特性及其在非生物胁迫中的作用,本研究采用多种细胞分子生物学方法进行研究。亚细胞定位发现WRKY72转录因子定位在细胞核中。qRT-PCR检测WRKY72对多种逆境与激素处理的响应,结果表明在热害、盐害、脱落酸、低氮、葡萄糖等非生物胁迫处理3 h后,WRKY72的转录水平显著受到抑制。酵母体内转录活性分析以及双荧光素酶报告系统分析显示WRKY72是一个转录抑制子。酵母双杂交(yeast two-hybrid, Y2H)和双分子荧光互补(bimolecular fluorescence complementation, BiFC)发现,WRKY72不仅能与近源的WRKY9、WRKY36、WRKY47、WRKY61、WRKY72转录因子发生互作,还能与自身发生互作。利用该基因T-DNA插入突变体进行表型分析,结果显示,wrky72突变体在百草枯(methyl viologen,MV)和低氮(low nitrogen, LN)处理下主根延伸长度显著变短,说明WRKY72对MV和LN敏感,提示WRKY72参与调控活性氧耐受和氮营养吸收或利用过程。综上所述,WRKY72可能与自身及一些近源WRKY转录因子互作,以多聚体的形式发挥其转录抑制子的功能,并参与特定的非生物胁迫相关的信号转导过程。本研究为深入解析WRKY72转录因子的抗逆功能与分子调控机制提供了资料基础。 WRKY transcription factors constitute a large family in plants and play important roles in many different physiological processes. Many members of WRKY genes have been studied in various processes including abiotic and biotic stress tolerance in Arabidopsis thaliana. However, the functions and molecular mechanisms of many WRKY genes are still unknown. In this report, the WRKY72 gene in Arabidopsis, which is a member of group Ⅱb is studied. In order to explore the characteristics of WRKY72 gene and its role in abiotic stress tolerance, various cellular and molecular biology approaches were employed. Subcellular localization using green fluorescence protein reporter gene(GFP) in leaves of Nicotiana benthamiana showed that the WRKY72 transcription factor was localized in the nucleus of leaf epidermal cells. qRT-PCR was used toexamine its response to multiple stress and phytohormone treatments at 3 time-points. The results demonstrated that WRKY72 transcription was inhibited by cold, heat, salt, abscisic acid(ABA), methyl viologen(MV), low nitrogen(LN) and glucose(Glc) treatments, which showed that WRKY72 may play a role in these stress signaling pathways. The transcriptional activity of WRKY72 was assessed through in vivo yeast assay,revealing that WRKY72 was a transcriptional repressor. Through a dual luciferase reporter system, WRKY72 was identified to significantly repress the transcription of firefly luciferase gene under the control of tandem repeats of W-box cis-elements, compared to the control. Yeast two-hybrid(Y2H) and bimolecular fluorescence complementation(BiFC) assays were used to screen and confirm the interacting proteins of WRKY72 and the results showed that WRKY72 interacted with homologous WRKY9, WRKY36, WRKY47, WRKY61 and itself. These indicated that WRKY72 was able to form both heterodimers and homodimers in vivo. Finally, 2 TDNA insertion mutants of WRKY72 gene were identified through PCR, and semi qRT-PCR assay indicated lossof-function mutation. Phenotypic assay was done to compare the respo
作者 李琪 李烨 牛芳芳 郭小华 赵新杰 吴相民 杨博 江元清 LI Qi;LI Ye;NIU Fang-Fang;GUO Xiao-Hua;ZHAO Xin-Jie;WU Xiang-Min;YANG Bo;JIANG Yuan-Qing(College of Life Sciences,Northwest A&F University,Yangling 712100,China)
出处 《农业生物技术学报》 CAS CSCD 北大核心 2019年第2期191-203,共13页 Journal of Agricultural Biotechnology
基金 国家自然科学基金(No.31270293).
关键词 拟南芥 WRKY72 互作蛋白 抗逆 Arabidopsis thaliana WRKY72 Interacting proteins Stress resistance
作者简介 通讯作者:江元清,jiangyq@nwafu.edu.cn
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