EMB17、EMB20和CRS1在玉米种子发育和内含子剪接过程中的功能和机理研究

Seed development is essential to seed vigor and yield. Hence, revealing the mechanism governing this process not only answers a fundamental question in plant science, but also provides new avenues for crop engineering. Recent studies indicated that PPR genes play critical roles in seed development, mostly by participating in RNA processing in mitochondria and chloroplasts. One major processing event is intron-splicing. Unfortunately thus far, genes required for intron-splicing are not fully identified, let alone the mechanism of intron-splicing. Here, we have cloned two embryo defective genes (Emb17 and Emb20) in maize by transposon tagging. Both genes are essential to maize seed development. Interestingly, both genes are required for the splicing of atpF intron in plastids. This function coincides with that of CRS1 which belongs to the CRM family of RNA-binding proteins, implying that all these three proteins are required for the splicing of atpF intron. Our preliminary results of yeast two hybrid assays suggest that EMB17, EMB20 and CRS1 may interact with each other, implying that a spliceosome-like complex may exist in chloroplasts. Building upon this foundation, here we propose a study to elucidate the function and mechanism of these three genes in intron splicing in plastids. This study is focused on the following objectives, 1) To confirm the physical relationship among these three proteins; 2) To determine the effects caused by the mutation of Emb17 and Emb20; 3) To identify additional components of the atpF intron spliceosome. While this study will reveal the molecular basis of three essential genes for seed development, it will provide a major advance in elucidating the intron-splicing mechanism in plastids.

种子发育不仅是植物学的基本问题，也是高产育种的关键。研究表明，PPR在植物胚胎发育过程中起重要作用，但由于其家族庞大、突变致死等原因，多数PPR基因的功能未知。目前，我们克隆了玉米种子发育的两个关键基因，Emb17和Emb20，二者均编码质体的P亚类PPR蛋白。EMB17和EMB20都参与atpF内含子的剪接，该功能又与CRM类RNA结合蛋白CRS1相同。酵母双杂结果表明，EMB17、EMB20和CRS1之间存在互作，暗示三个蛋白可能协同行使剪接功能，这为研究内含子的剪接机制提供了契机。因此，本课题将验证这三个蛋白之间的互作，解析atpF内含子剪接复合体的组分，以及Emb17、Emb20的突变造成的生理生化后果，目的是解析atpF内含子剪接机制并阐明其影响种子发育的分子基础。鉴于目前学术界对细胞器内含子剪接机制尚不清楚，本研究有可能率先阐明atpF内含子的剪接机制，具有重要理论意义。

种子发育直接影响粮食作物的产量。叶绿体作为植物的光合作用场所、线粒体作为氧化磷酸化的场所，其功能受损往往引起胚胎败育。我们利用转座子标签技术克隆了多个与玉米籽粒发育相关的基因，阐明了其调控玉米籽粒发育和细胞器基因表达调控的分子机理，具有重要的理论意义。.1)发现了调控叶绿体内含子剪接的调控因子EMB17和EMB20：EMB17、EMB20都定位于叶绿体, 其功能缺失导致叶绿体基因atpF的剪接缺失。EMB17、EMB20和CRS1蛋白互作参与atpF内含子的剪接。上述研究解释了内含子剪接影响叶绿体功能和种子发育的机制。.2)发现了在细胞色素c成熟和玉米种子发育过程中行使功能的RNA编辑因子PPR27：PPR27定位于线粒体，其功能缺失导致ccmFN-1357的编辑完全缺失，造成线粒体复合物III功能缺失。上述研究揭示了RNA编辑影响线粒体功能和玉米籽粒发育的机制。.3)发现了调控线粒体氧化磷酸化基因nad4内含子剪接的调控因子PPR18：PPR18参与线粒体复合物I亚基nad4的内含子剪接。其功能缺失导致了线粒体复合物I的组装和活性严重减少。上述研究揭示了内含子剪接影响线粒体功能和玉米籽粒发育的机制。.4)发现了调控线粒体氧化磷酸化基因nad2多内含子剪接的调控因子DEK47：DEK47编码线粒体定位的RCC1蛋白，其功能的丧失阻碍了玉米胚胎和胚乳的发育，导致线粒体复合物I基因nad2成熟转录本缺失，线粒体复合物I组装受阻。上述研究揭示了RCC1蛋白通过参与内含子剪接影响线粒体功能和玉米籽粒发育的机制。.5)发现了两个特殊的PPR蛋白SPR2与PPR-SMR1互作调控玉米线粒体内含子剪接的新机制：SPR2与PPR-SMR1互作形成剪接复合物，该复合物能够与不同类型剪接因子互作介导线粒体不同内含子的剪接，为阐明植物线粒体内含子剪接和玉米籽粒发育的机制提供了新的见解。