sHSP25响应温度胁迫及调控乌菜叶绿体膜蛋白表达的作用机理

During creation process of breeding genotypes, we found a special genotype WS-1, which could resist both high and low temperature. The chloroplast membrane system was relatively intact under high and low temperature stress. It has been proved that sHSPs play a vital role in response to several stresses by regulating membrane system stability. In our study, further analysis showed that the transcription level of BccrsHSP25 was significantly up-regulated under high and low temperature stress; the location of sHSP25 protein was predicted in chloroplast, which significantly affected the PSII photochemical activity. However, the regulation mechanism of sHSP25 on chloroplast membrane proteins is still unclear. In the present study, the chloroplast was treated with sHSP25 antibody in WS-1. Differently expressed proteins of chloroplast-complex-proteins were identified to explore the protein types and functions induced by high and low temperature. Immunoprecipitation and yeast two-hybrid were used to analyze the key proteins interacting with CP-sHSP25. Westernblot and qRT-PCR were utilized to detect the proteins. The purpose of this study is to reveal the difference of regulation mechanism in WS-1 response to high and low temperature, and elucidate the effect of Cp-sHSP25 on chloroplast proteins, providing the theoretical foundation for fully excavating WS-1 germplasm characteristics.

课题组前期研究发现乌菜特异材料WS-1基因型既耐高温，也耐低温，在高低温胁迫下，WS-1叶绿体膜系统较为完整。已有研究证实sHSPs通过稳定生物膜结构调控植物响应多种逆境，是植物细胞耐逆性提高的重要原因之一。转录水平分析表明，高低温胁迫下乌菜BccrsHSP25的表达显著上调，预测该蛋白定位于叶绿体，且显著影响PSII光化学活性，但该蛋白如何调控乌菜叶绿体膜蛋白表达以响应高低温胁迫的机理尚不明确。本项目利用sHSP25抗体处理WS-1叶绿体，采用双向电泳技术分离膜蛋白，研究不同处理诱导差异表达蛋白的种类和功能；采用免疫共沉淀和酵母双杂交技术解析高低温胁迫下与叶绿体sHSP25（Cp-sHSP25）互作的关键蛋白；采用蛋白免疫印迹和qRT-PCR技术，进一步验证Cp-sHSP25作用的蛋白种类，揭示WS-1响应高低温胁迫的调控途径，为充分挖掘WS-1种质特性奠定理论基础。

乌菜是江淮流域秋冬季特有的叶菜，然而乌菜响应温度胁迫的过程和机制尚不明确。本项目首先研究了乌菜热激蛋白HSP90和HSP20家族基因在温度胁迫下的响应机理及相关功能。鉴定了14个HSP90和48个HSP20基因家族成员，并分析了其理化性质、基因结构、染色体分布、进化关系、启动子元件以及在不同模式下的表达模式。荧光定量分析表明这些基因在温度胁迫下存在显著差异，表明这些基因广泛参与对温度胁迫的响应。同时，采用串联质谱标签（TMT）与液相色谱-质谱联用（LC-MS / MS）的蛋白质组学方法，鉴定出在高低温胁迫处理后的差异表达蛋白（DEPs）1022种，这些差异蛋白参与氧化还原稳态，光合作用，碳水化合物代谢，热激蛋白和信号转导等途径。而且，温度胁迫后乌菜中与热激蛋白、氧化还原稳态、碳水化合物代谢及光合作用相关的基因表达水平是上升的，这与相关蛋白质的变化一致。此外，与常温对照相比，我们发现了高温下有10702个差异基因，低温胁迫下有7267个差异基因发生变化。大部分基因参与到光合及光合-天线蛋白路径。高温明显抑制了光合途径，BrLHC超家族成员和Psa家族等基因的表达，而低温下这些基因上调表达。而且，高温导致大部分HSFA及HSFB家族成员上调，其中HSFA7A表达量上调最多，我们推测其可能参与sHSP功能蛋白的表达。我们将围绕此结果展开进一步的讨论。