葡萄转录因子VlNAC60应答高温胁迫的功能及调控机制

Heat stress is one of the most important abiotic stresses, which affect grape production in the worldwide. Meanwhile, the thermostability of cultivated European grape species is weakly. Therefore, directional genetic improvement is necessary for European grape thermostability enhancement. Through assessing the thermostability of grape germplasm resources in south of China, we found V. labrusca × V. vinifera cv. Kyoho showed higher tolerance to heat stress in our previous research. According to our previous study, a up-regulated gene, VlNAC60, was selected through RNA-Seq in heat stress treated Kyoho. Deeply exploration for VlNAC60 biological function in grape and the regulation mechanisms are the further research. The biological function of VlNAC60 will be analyzed by gene over-expression and virus induced gene silencing (VIGS) in heat-intolerant ‘Red Globe’ (Vitis vinifera) and thermostable ‘Kyoho’ (V. labrusca ×V. vinifera) , respectively. The interaction proteins will be screened by yeast two-hybrid system, BiFC and GST pull-down will be used for proteins interaction verification. The downstream target gene will be selected by Chip-Seq, and EMSA will be used to verify the regulation ability, revealing the regulation mechanisms of VlNAC60 in response to heat stress. Our research will clearly elaborate the regulation mechanisms of VlNAC60 in response to heat stress, which will lay a solid theoretical foundation for grape abiotic stress resistance molucular breeding and germplasm improvement.

高温胁迫是世界范围内危害葡萄生产的重要逆境之一，现在广泛栽培的欧洲葡萄耐热性较差。因此，对欧洲葡萄进行定向改良以提高其耐热性成为葡萄抗逆育种的重要方向。项目组前期通过对我国南方地区葡萄种质资源的耐热性进行评价，发现欧美杂交后代巨峰较耐热。通过对巨峰葡萄在高温胁迫后不同时间点的转录组进行分析，筛选出一个上调表达的转录因子VlNAC60。本项目在此基础上，构建VlNAC60超表达载体和基因沉默载体，分别转化不耐热的欧洲葡萄‘红地球’和耐热的欧美杂种葡萄‘巨峰’，分析其应答高温胁迫的功能；通过酵母双杂交筛选其互作蛋白，通过BiFC、GST pull-down进行验证；通过ChIP-Seq筛选其下游靶基因，并利用EMSA进行验证，揭示其应答高温胁迫的调控机制，从而弄清VlNAC60基因在葡萄应答高温胁迫中的功能和调控机制，为葡萄抗逆分子育种及品种改良提供理论依据。

高温胁迫是世界范围内危害葡萄生产的主要逆境之一，但是目前对葡萄耐热机理的研究较少。项目前期筛选到一个在高温胁迫后上调表达的转录因子VlNAC60，在此基础上，本项目以耐热性较强的欧美杂交种“巨峰”葡萄为研究材料，挖掘VlNAC60的互作蛋白和下游靶基因，解析VlNAC60应答高温胁迫的功能及调控机制。主要研究结果如下：在“无核白”葡萄中过量表达VlNAC60会增加主根数量和根系重量，从而增加根系的生长量；高温条件下，过量表达VlNAC60会提高“无核白”组培苗叶片中的SOD、CAT和POD活性，降低MDA、H2O2和O2-含量，从而缓解高温胁迫造成的氧化损伤；利用酵母系统鉴定了VlNAC60的转录激活活性，发现其有自激活活性；通过酵母双杂交筛选到VlNAC60的互作蛋白GOLS1，发现高温条件下过量表达VlNAC60会提高“无核白”组培苗叶片中GOLS1基因的表达量，而在常温条件下过量表达VlNAC60对GOLS1的表达量没有影响；通过DAP-seq筛选到VlNAC60可能调控的下游靶基因CKX9，发现在常温和高温条件下，过量表达VlNAC60均会下调“无核白”组培苗叶片中CKX9基因的表达量，表明VlNAC60负调控CKX9基因的表达；正在继续利用转基因的过表达植株和RNAi植株对NAC60的下游基因进行挖掘和验证。项目执行期间共发表SCI论文1篇，中文核心期刊论文1篇。本研究初步鉴定了VlNAC60在葡萄应答高温胁迫中的功能及互作蛋白和下游靶基因，为葡萄耐热分子育种提供了理论依据。