葡萄VvRMA1H1泛素化修饰VvBAP1参与低温应答的研究

Grape is sensitive to cold stress, Thus it attracted lots attention on the mechanism of grape cold resistance. Our previous studies identified a target protein VvRMA1H1（RING Membrane-Anchor E3 Ubiquitin Ligase Homolog）, using cold resistance VvBAP1 as bait protein by screening the yeast two hybrid library. VvRMA1H1 gene was clonedand the gene expression was induced by cold stress. This study will verify the protein interaction between VvBAP1 and VvRMA1H1 using BiFC, Pull-down and CoIP techniques. The VvRMA1H1 expression mode and subcellular localization will be studied further. We will obtain the VvRMA1H1-overexpression and VvRMA1H1-knockdown plants by transgene and Crispr/Cas9 technique. We will identify VvRMA1H1 biological function in cold stress resistance by detecting the physiological indicators in these transgenic plants under cold stress. Moreover, we will identify E3 ubiquitin ligase activity of VvRMA1H1 and explore whether VvRMA1H1 participate in cold stress response by VvBAP1 ubiquitination. This project will lay the foundation for characterization and enhancement of cold stress tolerance in grapes.

葡萄属于低温敏感的植物，因此探明其抗冷机制一直备受关注。我们前期利用抗冷相关蛋白VvBAP1筛选酵母双杂交文库，得到一个RING型E3泛素连接酶VvRMA1H1。克隆得到VvRMA1H1基因，并发现低温能够诱导VvRMA1H1基因表达量升高。本项目拟利用BiFC、Pull-down和CoIP技术验证VvBAP1和VvRMA1H1存在相互作用；进一步研究VvRMA1H1的表达特性、亚细胞定位。以VvRMA1H1基因过表达和缺失植株为材料，检测低温后抗冷相关生理指标，明确其响应低温应答和抗性中的功能；鉴定VvRMA1H1的E3泛素连接酶活性并探明其是否通过泛素化VvBAP1参与葡萄响应低温的过程，从而揭示其作用机制。本项目为深入研究葡萄抗冷机制，进一步利用基因工程提高葡萄的抗冷性提供理论基础。

葡萄属于低温敏感的植物，因此探明其抗冷机制一直备受关注。我们前期利用抗冷相关蛋白VvBAP1筛选酵母双杂交文库，得到VvRMA1H1，推测为RING型E3泛素连接酶。本项目在此基础上，克隆得到VvRMA1H1基因，发现低温以及相关信号分子和激素能够诱导VvRMA1H1基因表达量升高。亚细胞定位结果显示VvRMA1H1定位于细胞质和内质网中。利用原核表达系统纯化得到VvRMA1H1-MBP蛋白，经过体外泛素化实验分析发现其具有E3泛素连接酶活性。进一步以VvRMA1H1基因过表达拟南芥和葡萄愈伤组织为材料，低温处理后，转基因拟南芥和葡萄抵御低温胁迫能力均明显增强，且H2O2 、O2.-含量和MDA含量均低于野生型，SOD、POD和CAT三种抗氧化酶活性以及低温相关marker基因表达量均高于野生型，因此明确VvRMA1H1参与葡萄响应低温应答过程。同时，利用酵母双杂交和BiFC技术证明VvBAP1和VvRMA1H1存在相互作用，进而影响葡萄相关生理指标来抵御低温胁迫，揭示了其低温应答的机制。此外，VvRMA1H1为诱饵蛋白，在酵母双杂交文库中筛选得到了81种互作蛋白，为研究其低温作用机制奠定了基础。以本项目为深入研究葡萄抗冷机制，进一步利用基因工程提高葡萄的抗冷性提供理论基础。