高温胁迫下拟南芥miR400及其靶基因PPRP的功能及作用机理研究

In the past decade,high temperature became a crucial factor which seriously limits the yield of crops. Recently, numerous studies have demonstrated that microRNA is one of the most important regulators in controlling gene expression. It has been also reported that PPRPs play important roles in plant growth, development and stress responses. However, to our knowledge,there is no report about miRNAs and PPRPs in response to high temperature stress in plants to date. Our previous study indicated that the expression of miR400 was repressed at high temperature in Arabidopsis seedlings. The transgenic lines overexpressing of miR400 showed a sensitive phynotype，whereas miR400 T-DNA insertion mutant showed a resistant phynotype when treated with high temperature. About 25 PPRP genes were predicted to be the targets of miR400 in Arabidopsis. Therefore,in this project, we will detect the regulation of miR400 expression in Arabidopsis,and then identify its target PPRPs under heat stress. By using the transgenic Arabidopsis overexpressing miR400 and target PPRP genes, we will determine their biological function in response to heat stress. Furthermore, the interaction proteins and target RNAs with the selected PPRPs will be identified by coimmunoprecipitation and CHIP sequencing technologies. All together,we will reveal the biological function and melacular mechanism of miR400 and its target PPRP genes in response to heat stress in Arabidopsis. These results obtained from this investigation will likely provide a novel pathway for plant response to the heat stress and will be very helpful for better understanding the regulatory mechanism in higher plants.

高温已成为影响作物产量形成的重要环境因素。microRNA是近年来备受关注的基因表达调控因子，PPRP在植物生长发育和胁迫响应过程中具有非常重要的生物学功能，但植物应答高温胁迫的miRNA和PPRP及其作用机制尚未见报道。我们的研究表明拟南芥miR400的表达受高温抑制，miR400超表达对高温敏感而突变体对高温有明显抗性，初步分析发现miR400的靶基因可能是25个功能未知的PPRPs。因此本项目通过深入研究miR400的表达调控和生物学功能、鉴定miR400的靶PPRPs、分析miR400和靶PPRP转基因株系的高温响应，结合Affymetrix芯片分析、免疫共沉淀和CHIP测序等技术鉴定PPRP的互作蛋白及靶RNAs，明确miR400和靶PPRP参与的植物高温响应信号途径，阐明高温下拟南芥miR400及其靶PPRP的功能及其分子调控机制。为作物抗高温遗传改良提供新思路。

MicroRNA是近年来备受关注的基因表达调控因子，其作用涉及动植物的生长发育及外界逆境响应等多种生物学过程，具有重要的生物学功能。但人们对于miRNA自身功能调控的机制却知之甚少。近期研究首次发现高温胁迫诱导的可变剪切可以作为一种新的调节机制调控植物中miRNA的加工，可变剪切发生导致miRNA400所在内含子3′末端出现100bp碱基丢失，直接影响miR400成熟体加工，初步分析发现可变剪切信号和剪切调控因子可能在加工过程中起重要作用。到目前为止，可变剪切调控miRNA的机制却未见报道。研究表明拟南芥miR400的表达受高温抑制，miR400超表达对高温敏感而突变体对高温有明显抗性，初步分析发现miR400的靶基因是PPR蛋白。因此本项目通过深入研究miR400的表达调控和生物学功能、鉴定miR400的靶PPRPs、分析miR400和靶PPRP转基因株系的高温响应，结合Affymetrix芯片分析、免疫共沉淀和CHIP测序等技术鉴定PPRP的互作蛋白及靶RNAs，明确miR400和靶PPRP参与的植物高温响应信号途径，阐明高温下拟南芥miR400及其靶PPRP的功能及其分子调控机制。为深入理解miRNA的作用机理提供全新的理论依据和应用工具。