野生小米椒(Capsicum frutescens)CfWRKY转录因子遗传变异及干旱 调控机理研究

Wild pepper ‘Xiaomijiao’ (C.frutescens) is an endemic pepper species in China, which is highly tolerant to drought stress. However, little is known about the drought resistance mechanism in wild pepper ‘Xiaomijiao’. The proposer has been working on the functional identification and regulating mechanisms of WRKY transcription factors under biotic/abiotic stresses in plants. Based on the de novo sequencing data of wild pepper ‘Xiaomijiao’, we comparatively analyzed the WRKY families between wild pepper ‘Xiaomijiao’ and cultivated pepper (C.annuum), and identified 5 evolutionary independent CfWRKYs, which were potentially involved in the drought response regulation of wild pepper ‘Xiaomijiao’ plants. Thus, we proposed the hypothesis that CfWRKYs play critical roles in the transcriptional regulation of drought responding process in wild pepper ‘Xiaomijiao’. Based on the 5 identified evolutionary independent CfWRKYs, we intend to elucidate the interspecies variation of WRKYs between wild pepper ‘Xiaomijiao’ and cultivated pepper. Furthermore, functional analysis of CfWRKYs in drought resistance would be conducted using technical means including VIGS and transgene in wild pepper ‘Xiaomijiao’. Meanwhile, through transcriptome sequencing analysis and yeast hybrid system, the interactions between CfWRKY and other components in drought response pathway were inspected. In short, the implementation of this project would not only provide theoretical basis for the drought resisting regulating mechanism study of wild pepper ‘Xiaomijiao’, but also technical guidance for drought tolerance improvement of pepper germplasm resources.

野生小米椒(C.frutescens)是我国特有辣椒种质资源，耐旱性极强，然而关于野生小米椒耐旱机理的研究目前尚未开展。申请人一直从事辣椒WRKY转录因子抗逆功能鉴定和相关调控机理研究，在前期完成的野生小米椒denovo测序基础上，通过对野生小米椒CfWRKY和栽培椒(C.annuum) CaWRKY比较分析，发现5个独立进化，并潜在参与野生小米椒干旱应答调控的CfWRKY成员。基于此，提出CfWRKY参与野生小米椒抗旱转录调控的假设。项目拟在前期鉴定获得的特异CfWRKY基础上，阐明野生小米椒和栽培椒WRKY在种间遗传进化中的关系。同时，利用VIGS和转基因技术明确CfWRKY在野生小米椒抗旱应答中的功能，并结合转录组分析和酵母杂交等技术解析CfWRKY与干旱应答信号网络元件的相互作用及关键作用节点，以期为野生小米椒抗旱调控机制研究和辣椒种质资源抗旱性状改良提供理论依据和技术指导。

野生小米椒(C. frutescens)是我国特有的灌木辣椒种质资源，具备抗旱等多个优异性状，对辣椒的科学研究和遗传工程改良具有重要价值。辣椒属浅根性蔬菜作物，水分亏缺耐受性差，近年来，干旱胁迫严重影响辣椒栽培和生产，导致辣椒产量和品质显著降低。 . 本研究以野生小米椒（C. frutescens）为研究对象，在其全基因组de novo测序工作基础上，完成了基因组组装注释及比较基因组学分析工作，解析了小米椒野生种和栽培种辣椒基因组的遗传多样性，构建了野生小米椒全基因组序列图谱。在此基础上，研究以野生小米椒和栽培椒（C. annuum）为研究对象，通过生物信息学技术，对两者的WRKY转录因子家族进行了系统的挖掘、鉴定和比较分析，发现小米椒CfWRKY家族和栽培椒CaWRKY家族在成员数量、蛋白大小和亚族构成（集中在group IIb，group IIc，group IId和group III）等方面均存在一定变异。在前期鉴定获得的干旱应答小米椒CfWRKY和栽培椒CaWRKY转录因子中，通过病毒诱导基因沉默技术，分别挖掘得到正调控小米椒干旱抗性的CfWRKY27和负调控栽培椒干旱抗性的CaWRKY58。进一步对CfWRKY27干旱抗性调控机理的研究表明，CfWRKY27能够通过正向调控渗透调节物质脯氨酸合成关键基因CfP5CS1的表达，增强小米椒地上部分的脯氨酸积累，进而实现其对干旱的正向调控。CfP5CS1基因启动子上2个W-box序列的存在，暗示了CfWRKY27可能通过直接结合CfP5CS1基因启动子，激活该基因的表达。. 综上，本研究从基因组层面为研究我国特有辣椒种质资源（野生小米椒）的优异性状奠定了基础。对于挖掘辣椒抗旱调控功能基因，解析辣椒抗旱调控信号网络具有重要理论意义，也为辣椒种质资源抗旱性状改良提供了技术指导。