水稻叶绿体发育和幼苗生长相关基因ASL4的克隆与功能分析

Chloroplasts are not only the exclusive place of photosynthesis in higher plants, but they are also responsible for the biosynthesis and storage of various metabolites. Abnormal chloroplast development would affect plant growth. Therefore, it is of great importance to study the mechanism of chloroplast function. The proposer previously isolated a chloroplast development deficient mutant named asl4, and identified the ASL4 gene which encodes the chloroplast ribosomal small subunit protein S1 as an essential regulator required for rice seedling growth. The chloroplast ribosome proteins play important roles in chloroplast ribosome biogenesis, plastid protein biosynthesis and chloroplast differentiation, however, the molecular functions and regulatory mechanisms of which are largely unknown. The project intends to clarify the function of ASL4 gene in expression and synthesis of plastid-encoded genes and proteins by using of genetics, genomics and molecular biology. This study will try to explore the relationship between ASL4 mutation and chloroplast differentiation and chloroplast ribosome biogenesis. The interacted protein of PRPS1 could be identified for exploration of the molecular biological pathways that PRPS1 may participate in. This project will try to elucidate the molecular mechanism of ASL4 that regulates chloroplast development and rice seedling growth.

叶绿体是光合作用以及多种代谢产物合成和积累的主要场所，叶绿体发育受阻将影响植物体的生长，因此，研究叶绿体发育机制具有重要的理论价值和实践意义。本研究利用叶绿体发育受阻突变体asl4，分离一个控制水稻幼苗生长发育基因ASL4，该基因编码一个叶绿体核糖体小亚基蛋白S1。叶绿体核糖体蛋白在叶绿体核糖体的形成、质体蛋白合成及叶绿体分化中行使重要的功能，然而，其分子机理和调控机制尚不清楚。本项目拟综合运用遗传学、基因组学、分子生物学等技术手段明确ASL4基因在质体编码基因表达和蛋白合成过程中行使的功能，探索该基因突变与叶绿体分化和叶绿体核糖体形成之间的关系，筛选PRPS1的互作蛋白，研究该蛋白可能参与的分子生物学途径，以期阐明ASL4基因调控叶绿体发育和植株幼苗生长的分子机理。

叶绿体核糖体负责质体编码蛋白的转录和翻译，对叶绿体发育和植物生长不可或缺。虽然质体中大多数核糖体蛋白已经被鉴定，但是其调控叶绿体发育的分子机制仍不够清楚。本研究我们鉴定一个白化致死突变体asl4，是由一个定位于叶绿体的30S核糖体蛋白S1突变获得，该突变体叶绿体发育和叶绿素合成受阻。通过定位克隆和基因组测序分析表明ASL4中存在一个2855bp的缺失，转基因互补验证其为目的基因。表达分析显示ASL4基因在叶片发育早期的第4叶部分和新展开的叶片中高度表达。黑暗处理后ASL4基因的表达水平随着光照时间的增加而提高。与野生型相比，一些涉及到叶绿体发育的基因在突变体中被上调和下调表达。同时质体编码的PEP光合基因和核编码的NEP持家基因在突变体中分别被下调和上调表达，暗示突变体中质体的转录受损。转录组和western杂交分析显示大部分质体编码的基因和蛋白在突变体中降低。降低的叶绿体核糖体RNA和核糖体蛋白表明asl4突变体中叶绿体核糖体收到损坏。这些研究结果表明ASL4基因对叶绿体核糖体的形成和早期叶绿体的发育必不可少，将有助于进一步阐明叶绿体发育的分子机制。