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

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。

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.