山葡萄VaCIPK18介导VaMYB4a磷酸化调控抗寒应答的分子机理

低温是严重影响北方寒地葡萄栽培生产的环境因子，开展葡萄低温应答机理研究有重要的理论意义和生产实践价值。类钙调素B亚基蛋白互作激酶(CIPK)在应对各种环境胁迫中起关键作用。然而，CIPK调控低温信号应答过程的研究甚少。我们前期鉴定了山葡萄CBL-CIPK网络的“信号枢纽”VaCIPK18正调控低温应答；发现与其互作、受冷诱导上调表达显著的转录抑制因子VaMYB4a。据此我们提出假设：低温促使VaCIPK18招募VaMYB4a为底物，诱导其磷酸化从CBFs启动子MYB位点解离，增强CBFs转录；磷酸化激活的VaMYB4a参与CBF-非依赖靶基因的抗寒应答调控。为证明这一假设，拟通过体外蛋白磷酸化、干扰/过表达（重要位点磷酸化）株系表型、ChIP-Seq等，解析VaCIPK18-VaMYB4a模块在葡萄抗寒应答中调控低温信号的作用模式，为激酶信号调控转录因子磷酸化参与植物低温应答提供理论依据。

Cold is an important environmental factor that severely affects grape cultivation and production in northern cold regions of China. It is of great theoretical significance and practical value to in-depth investigate the mechanism of low temperature response in grape plants. Calcineurin B-like protein-interacting protein kinases (CIPKs) play important roles in response to various environmental stresses. However, little is known about these regulatory process mediated by CIPKs in the low-temperature signal response. We have previously identified a Ser/Thr protein kinase VaCIPK18 as the “signal node” in the CBL-CIPK network of Vitis amurensis, which was involved in positive regulation of cold stress. Moreover, one MYB transcription repressor VaMYB4a, which was further confirmed as interactive component of VaCIPK18, was significantly induced in response to cold stress. Therefore, we hypothesized that low temperature activated and promoted VaCIPK18 to recruit VaMYB4a as substrate, induced VaMYB4a phosphorylation and dissociation from the MYB binding site of CBFs promoter, enhancing the transcriptional activity of CBFs gene. Concurrently, the phosphorylated VaMYB4a could regulate the CBF-independent downstream target genes to participate in cold-tolerant response. To test this hypothesis, in vitro protein phosphorylation, RNAi/overexpression (mutation in important phosphorylation site) phenotypes, ChIP-Seq and other studies will be carried out to further understand the role of VaCIPK18-VaMYB4a module in regulating cold signal in grape cold response, and provide theoretical basis for kinase signal regulation of transcription factor phosphorylation and participation in low temperature response of plants.