COPI介导的核编码叶绿体蛋白胞质内运输途径分析

In plants, more than 90% of 3000 chloroplast proteins are encoded by nuclear genome, synthesized in the cytosol and subsequently imported into the chloroplast. The protein targeting into chloroplast is an essential cellular event for maintaining chloroplast function and plant life. But the question is, at present, few researches for how the proteins targeting to chloroplast's surface within the cytosol. To further explore the role of COPI-mediated the cytoplasmic transport of nuclear-encoded chloroplast protein, we utilized a strategy of RNA inference-mediated depletion of the β'-COP subunit of COP I. First, through the protein analysis of isobaric tags for relative and absolute quantitation (iTRAQ), differential expressed proteins of β'-cop-depleted plants were selected, and identified their biochemical pathways and signal transduction pathways by pathway analysis. Second, based on the proteomics result, we chose the proteins representing three general pathways of cytoplasmic transport for our study, such as Ribulose bisphosphate carboxylase/oxygenase small subunit (RbcS), α-amylase I and Ribulose bisphosphate carboxylase/oxygenase activase (RbcA). we showed that the COP I is participated in transport of nuclear-encoded chloroplast proteins directly according to the protoplast instantaneous expression, coimmunoprecipitation and laser confocal scanning microscope (LCSM) analysis. Together, our results demonstrated that COPI mediated the cytoplasmic transport of nuclear-encoded chloroplast protein, and maintaining chloroplast function.

植物的叶绿体含有约3000多种蛋白，其中90%以上的蛋白是由核基因组编码，这类蛋白在细胞质内合成后运输到叶绿体内部，对叶绿体的发育和功能起着重要作用。在这一运输过程中，核编码叶绿体蛋白如何在胞质内运输到叶绿体附近的研究较少，本项目采用RNAi方法获得水稻COPI亚基β'-COP表达下调的稳定突变体，研究COPI介导核编码叶绿体蛋白在胞质内运输的过程。首先通过蛋白iTRAQ分析，鉴定在Osβ'-cop RNAi突变体中差异表达的蛋白，利用pathway分析确定差异表达蛋白参与的最主要生化代谢途径和信号转导途径；其次在蛋白质组学分析结果的基础上，以代表3种胞内运输途径的蛋白（RuBisCO小亚基、α-淀粉酶和RuBisCO活化酶）为研究对象，采用原生质体瞬时表达、免疫共沉淀、激光共聚焦扫描分析等方法，研究COPI小泡参与核编码叶绿体蛋白运输的过程，揭示COPI在叶绿体发育与功能维持方面的作用。

本研究以水稻COP I亚基β’-COP表达下调的转基因植株（Osβ’-cop RNAi）为材料，研究COP I介导核编码叶绿体蛋白在胞质内运输的过程。首先，对Osβ’-cop RNAi植株的进行表型观察和超微结构分析。结果发现，COP I的减少使水稻叶绿体的结构发育异常，导致植株生长滞缓，叶片黄化，出现早衰现象，表明COP I在水稻的生长发育中起着重要作用。第二， Osβ’-cop RNAi植株与野生型植株相比，其净光合效率Pn，光饱和点和叶绿素荧光参数Fv/Fm、ФPS II、ETR和qP等显著下降。这表明COP I功能的缺失，使Osβ’-cop RNAi植株的光合效能下降，PS II受到光抑制，并影响其对光胁迫的调节能力，叶绿体的功能受损。第三，通过iTRAQ定量蛋白质组学分析发现，Osβ’-cop RNAi与ZH 11两者间表达呈显著性差异的蛋白共74个，主要参与光合作用，呼吸作用，新陈代谢等途径。其中，52.7%（39个）差异蛋白属于核编码的叶绿体蛋白，这表明COP I可能通过介导核编码叶绿体蛋白的运输，从而影响叶绿体的结构与功能。第四，为研究COP I是否直接参与核编码叶绿体蛋白的运输，选取2个差异表达显著的核编码叶绿体蛋白进行后续研究，分别为RuBisCO小亚基（Ribulose bisphosphate carboxylase/oxygenase small subunit，RbcS）和ATP合成酶γ亚基（ATP synthase gamma chain，AtpC）。通过PEG法瞬时转化原生质体、基因枪瞬时转化植株、Western blotting、免疫胶体金等方法研究差异蛋白在Osβ’-cop RNAi植株与野生型植株中的定位差异，结果显示，差异蛋白在Osβ’-cop RNAi植株不能正常运输到叶绿体中，表明由于 COP I功能的下降，影响了其胞质运输。第五，免疫共沉淀分析表明，COPI中β’-cop亚基和ε-COP亚基均能与细胞质中的核编码叶绿体蛋白的前体互作，证明其直接参与核编码叶绿体蛋白在胞质内的运输。