VvUFD1基因在葡萄盐胁迫反应中的功能研究

Salinity is an environmental factor that greatly affects agriculture sustainable development. The research and utilization of salt tolerance related gene in plant is important to improve crop yield and quality. A salt induced UFD1-like gene was identified through cDNA-AFLP and RACE-PCR from a high salt tolerant grape rootstock 'Kangzhen No.5' in our previous study. UFD1 is best characterized as ubiquitin-recognition protein facilitates clearance of misfolded proteins through the endoplasmic reticulum associated degradation (ERAD) pathway and related to different stress responses in yeast and mammals. Grape and Arabidopsis will be used in this study. To test whether ERAD is regulated by the grape salt stress response, we explore in detail the function of putative ERAD component VvUFD1 using a combination of molecular and cellular biological approaches. To test whether VvUFD1 is involved in salt stress response by the ERAD pathway, genetic transformations were performed using the T-DNA insertion mutant of Arabidopsis UFD1 homolog, rootstock varieties of grape with different salt tolerances. In a word, this study could not only clarify the function of VvUFD1 in salt stress response, but also define the relationship between ERAD and the salt stress response, which will be useful for grape salt tolerance improvement by genetic engineering.

盐害是影响农业可持续发展的重要因素，植物耐盐相关基因的研究和利用对于稳定作物产量、提高作物品质具有重要意义。课题组前期通过cDNA-AFLP和RACE-PCR，从我国自主选育的极耐盐葡萄砧木"抗砧5号"中克隆了盐胁迫响应的泛素融合降解蛋白基因VvUFD1，编码内质网相关蛋白降解(ERAD)途径必需的泛素识别元件，在酵母和哺乳动物中参与调控抗胁迫过程。本项目以葡萄和拟南芥为材料，利用分子生物学和细胞生物学技术，从研究VvUFD1基因编码蛋白在ERAD途径中的作用着手，探索VvUFD1基因参与的ERAD途径与盐胁迫反应的关系；利用拟南芥T-DNA插入突变体和不同耐盐力的葡萄砧木，通过遗传转化技术，研究VvUFD1基因对植株耐盐性和ERAD途径的影响。项目完成后不仅能明确VvUFD1基因在葡萄盐胁迫反应中的功能，还可揭示盐胁迫与ERAD途径间的调控关系，为利用基因工程方法改良葡萄砧木耐盐性提供理论依据。

对于发生错误折叠和组装的蛋白，真核生物中存在精密的内质网相关蛋白降解系统（Endoplasmic reticulum associated degradation, ERAD）来降解这些蛋白，以确保其免受非正常蛋白的损害，维持机体生理平衡和内环境稳定。本研究结果表明ERAD途径在葡萄盐胁迫反应中发挥调节作用。盐胁迫诱导下，错误折叠蛋白迅速积累，通过ERAD途径泛素化降解，并诱发内质网应激反应。ERAD元件VvUFD1作为泛素识别因子介导泛素化的靶蛋白至26S蛋白酶体降解。其N端含有泛素结合位点，C端分别含有Npl4蛋白和p97蛋白结合位点。VvUFD1基因能够通过ERAD途径调节细胞中错误折叠蛋白降解水平，提高转基因拟南芥植株的耐盐性。优化了葡萄砧木遗传转化体系，通过农杆菌介导法获得了潜在的转基因葡萄砧木再生植株。