WRKY14与其互作蛋白WRKY55调控山葡萄（Vitis amurensis）耐旱性的机制解析

Drought is one of the main factors limiting the development of grapevine industry in north China. The early results the applicant acquired have shown that VaWRKY14 and VaWRKY55 are drought-response genes, and over-expression of the two genes improved the drought tolerance of Arabidopsis dramatically. And, the interaction of VaWRKY14 and VaWRKY55 was demonstrated. It has shown that the WRKY in the form of protein interaction would be more effectively to regulate the transcription of target genes. Therefore, we will get the plants over-expressing VaWRKY14-His and VaWRKY55-Flag simultaneously or respectively using the stable transformation of grapevine and determine whether or not the drought tolerance of the plant with two over-expressed genes will be better than the plant with one or no over-expressed gene. The plants over-expressing VaWRKY14-His and VaWRKY55-Flag simultaneously will be analyzed by RNA-seq and ChIP-seq using the antibody of Flag and His tag, and the candidate genes regulated by VaWRKY14 and VaWRKY55 will be screen out and demonstrated. Then，the interaction in vivo and in vitro between VaWRKY14 and VaWRKY55 will be confirmed again using Co-IP, BiFC and Pull-down. We expect that the mechanism of VaWRKY14 and VaWRKY55 regulating the drought tolerance of grapevine will be illustrated based on these studies above, and provide the necessary theoretical basis for breeding grapevine varieties with strong drought tolerance.

干旱是制约我国北方葡萄产业发展的主要因素之一。申请人前期结果表明：VaWRKY14和VaWRKY55同为葡萄干旱响应基因，过表达可有效提高拟南芥耐旱性，且两者之间存在蛋白互作。研究表明WRKY以蛋白互作形式能更有效地调控下游基因表达。因此，本研究拟通过稳定转化，获得VaWRKY14-His和VaWRKY55-Flag单过表达及双过表达葡萄植株，评价双过表达植株耐旱性是否显著高于单过表达植株及对照；对双过表达植株进行转录组分析以及利用His和Flag抗体对其进行ChIP-seq分析，进而筛选并验证VaWRKY14和VaWRKY55共同调控的下游靶基因；利用Co-IP、BiFC和Pull-down技术进一步在体内和体外验证两蛋白之间的互作。期望通过上述研究，阐明VaWRKY14和VaWRKY55以蛋白互作形式调控葡萄耐旱性的分子机理，为培育高耐旱葡萄品种提供必要的理论基础。