E3泛素连接酶HOS1介导泛素化修饰调控葡萄抗寒应答的作用机制

Chilling injury at low temperature is an important adverse factor limiting the viticulture production. In-depth study on the mechanism of grape cold hardiness is the key to cultivate cold-resistant grape. The Arabidopsis RING-type E3 Ubiquitin Ligase HOS1 gene is an important negative regulator of low-temperature response and regulate cold-hardy metabolism via interacting with key factors of cold-temperature signal pathway. However, no reports have been reported on how this gene regulates cold tolerance in perennial Vitis plants at home and abroad. Our group found for the first time that the upstream promoter of VvHOS1 in Europen grapevine is 284-bp more than that of V. amurensis VaHOS1, and its nuclear localization is different from the previous reports. Based on these findings, we hypothesized that the activity HOS1 in grapes correlates with the promoter structure, and the interaction target protein enhances or attenuates ubiquitination modification through negative feedback regulation, thereby regulating the response to cold stress. To prove the hypothesis, this project is based on the overexpression and CRISPR knockout Chardonnay lines of VvHOS1, to extensively investigate molecular characterization, function and expression regulation mechanism of HOS1 in grape from 3 aspects of promoter, target protein interaction and ubiquitination modification. Collectively, the regulation mechanism involved in cold stress will be understood, thus putting forward new mechanism of cold-tolerant regulatory response in grape.

低温寒害是限制葡萄栽培生产的重要逆境因子，深入开展葡萄抗寒机制研究，是培育抗寒葡萄新品种的关键所在。拟南芥RING型E3泛素连接酶HOS1基因是低温应答的重要负调控因子，能与低温信号通路上关键因子互作调控抗寒代谢。但关于该基因在多年生葡萄属植物中是如何调控抗寒的机制迄今国内外尚无报道。本课题组首次发现欧洲葡萄霞多丽VvHOS1上游启动子比抗寒山葡萄VaHOS1多出284-bp；其核质定位特性不同于以往报道。据此我们提出假设：葡萄中HOS1活性与启动子结构相关，其互作靶蛋白通过负反馈调节加强或减弱泛素化修饰，调控低温胁迫应答。为证明这一假设，本项目拟在已构建的VvHOS1过表达、CRISPR 敲除霞多丽葡萄的基础上，从启动子、互作靶蛋白、泛素化修饰调控三个层面对HOS1基因的分子特征、功能和表达调控机制进行深入研究，明确HOS1在葡萄抗寒调控中的作用机理，提出葡萄抗寒调控应答的新机制。

本项目揭示了葡萄属植物关键低温调控因子VvHOS1参与低温胁迫应答调控的作用机制。经生物信息学及表达分析发现，VvHOS1定位于葡萄基因组第2条染色体，含有RING-finger（C3HC4型）和ELYS两个保守结构域，且受低温等多种非生物胁迫诱导表达。基于GUS定性、定量分析技术筛选VvHOS1基因启动子参与低温胁迫应答的核心区段，进一步采用DNA pull-down技术挖掘VvHOS1基因启动子上游调控因子，经验证发现VvHH1/2对VvHOS1基因启动子活性抑制作用最为显著，而VvPDS5B对VvHOS1基因启动子有显著的激活作用。HOS1作为E3泛素连接酶，其参与低温胁迫的生物学功能在其他植物中已得到充分验证，但其在葡萄中参与低温胁迫应答调控的生物学功能及作用机制尚不明确。因此，本项目制备了VvHOS1多克隆抗体；构建了VvHOS1过表达及基因编辑载体，并转化‘霞多丽’未成熟合子胚，获得‘霞多丽’过表达转化植株10株，基因编辑植株18株；为进一步解析VvHOS1参与低温胁迫应答调控的分子机制，采用酵母双杂交技术筛选获得VvHOS1下游互作组分，重点研究了其互作组分VvHIPP21参与低温应答的功能特点。经研究发现，VvHIPP21通过调控CBF及ABA信号通路关键基因表达负调控植物应答低温、干旱胁迫，正调控铜、镉胁迫。项目的开展为在基因、蛋白层面解析VvHOS1基因参与低温胁迫应答调控机制奠定了理论基础，为精准改良葡萄抗寒性状提供了新参考、新靶点。本项目开展期间共发表SCI论文4篇，中文核心期刊6篇，申请发明专利1项，共培养硕士研究生7名。