盐胁迫下黄瓜TGase调节光化学效率的作用机制研究

Transglutaminases (TGase) are widely distributed enzyme in plants, play important roles in the regulation of plant growth and development, protein cross-linking, photosynthesis, and response to abiotic stresses. However, the roles of TGase in photochemical efficiency and their response mechanism to salt stress are not fully understood yet. In the present study, cucumber (Cucumis sativus L.) seedlings cultured in Hoagland nutrient solution were used as materials. The activity, protein expression, gene expression, and distribution of TGase were analyzed in salt-sensitive and salt-tolerant cucumber seedlings exposed to salt stress. Blue native polyacrylamide gel electrophoresis and yeast two-hybrid system were conducted to explore the photosynthetic membrane proteins interacted with TGase as well as their regulatory network. Furthermore, sense and anti-sense expressing vectors were constructed and transformed to obtain TGase over-expressing and suppressing transgenic cucumber plants. And then the structure and performance of photosynthetic apparatus, thylakoid membrane energy distribution, and changes of endogenous polyamines levels were examined to clarify the molecular mechanism of TGase in regulating the photochemical efficiency of cucumber under salt stress. These results would enrich plant photosynthetic physiology theory in adverse environmental conditions. Meanwhile, it would provide theoretical support and practical guidance for promoting growth of protected cultivation crops and breeding salt tolerant varieties.

转谷氨酰胺酶（简称TGase）广泛存在于植物体各个组织内，在调节植物生长发育、蛋白质交联、光合作用以及响应非生物胁迫过程中起重要作用。然而，TGase调节光化学效率的作用及其响应耐盐性机制仍不清楚。项目以设施主要栽培作物黄瓜为材料，采用营养液栽培，研究盐胁迫对耐盐和盐敏感黄瓜品种叶片TGase活性、分布、蛋白翻译和转录水平的影响；利用蓝绿温和胶电泳、质谱鉴定、酵母双杂等分子生物学技术，解析盐胁迫下与TGase互作的光合膜蛋白及其互作调控路径；通过构建黄瓜TGase基因正义和反义载体，转化黄瓜获得超表达和抑制表达的转TGase基因黄瓜株系，研究盐胁迫下转TGase基因黄瓜植株光合器官结构和性能、类囊膜光能分配以及内源多胺的变化，阐明TGase在调节盐胁迫下黄瓜光化学效率的分子作用机制。研究结果既能丰富植物逆境光合生理理论，也能对设施作物抗逆促长栽培和和耐性品种选育提供理论支撑。

转谷氨酰胺酶（TGase）广泛存在于植物体各个组织内，在调节植物生长发育、蛋白 质交联、光合作用以及响应非生物胁迫过程中起重要作用。然而，TGase调节光化学效率的作用及其响应耐盐性机制仍不清楚。本项目以黄瓜为试验材料，研究盐胁迫对耐盐和盐敏感黄瓜品种叶片TGase活性、分布、蛋白翻译和转录水平的影响；利用蓝绿温和胶电泳、质谱鉴定等分子生物学技术，解析盐胁迫下不同耐盐性品种中类囊体膜差异表达蛋白；利用酵母双杂交等技术筛选TGase互作蛋白并解析互作调控路径；构建烟草转黄瓜TGase过表达株系，研究盐胁迫下转TGase基因植株光合器官结构和性能、类囊膜光能分配及内源多胺（PAs）的变化，阐明TGase在调节盐胁迫下光化学效率的分子作用机制。.通过4年试验研究，顺利完成项目计划书的各项研究任务和目标，主要研究结论如下：（1）盐胁迫显著诱导黄瓜叶片TGase表达，提高其活性，TGase主要定位于类囊体膜上。（2）鉴定到不同耐盐性品种差异表达的类囊体膜蛋白，并利用酵母双杂交等技术证明黄瓜TGase与RAD23蛋白互作。（3）盐胁迫下，TGase过表达植株显著增加了PA含量，降低Na+含量和Na+/K+，提高质子泵ATP酶、液泡H+-ATP酶、液泡H+-焦磷酸酶和抗氧化酶活性，表明TGase通过调节盐胁迫下的PA代谢和Na+/K+平衡，提高植株耐盐性。（4）TGase过表达植株能有效地缓解盐胁迫诱导的光抑制、提高叶绿体PA水平、D1和D2蛋白含量；TGase还诱导光合作用相关基因的表达和类囊体的重组，表明TGase能够调节PA含量保护类囊体蛋白，提高植株耐盐性。研究结果既能丰富植物逆境光合生理理论，也能对设施作物抗逆促长栽培和和耐性品种选育提供理论支撑。.研究结果共发表标注项目资助的SCI论文18篇，培养研究生5名。