水稻渐绿型黄叶突变体grc的基因克隆及功能初步研究

Leaf color is an important trait directly related to photosynthesis and grain production. The rice green-revertible mutant generally shows non-green at the early stage and then gradually turns green . This kind of mutant plays an important role both in basic research of chloroplast differentiation and development and in the application research of variety purity in hybrid rice seed producion.From the previous studies we found that grc(green-revertible chlorine) mutant is independent controlled by a pair of recessive nuclear gene.Compared with other leaf mutants in the phenotype,each leaf of grc plant must undergo a process from yellow to green. So we presume that grc could be a new gene regulating the process of chloroplast differentiation and development at the early stage. And it was finally mapped between two SSR markers of 3.7cM.Based on the previous studies of the morphology,physiology,cellular,genetic,gene expression and mapping of grc,this project aims to map-based cloning of grc,clarify its mutation mechanism,analyze its structural features,spatial and temporal expression modules of and the subcellular localization of GRC protein ,and also to identify the possible sites regulated by grc in the chlorophyll synthesis pathway,Which will eventually help us to better understand the biological function of GRC gene and the molecular mechanism of GRC regulating the leaf color.

叶色是与光合作用、产量直接关联的重要性状。水稻转绿突变体一般表现为苗期失绿，而后逐渐复绿；这类突变体在叶绿体分化与发育等基础研究以及杂交稻制种中的除杂保纯等应用研究中起着重要的作用。通过前期研究，发现渐绿型黄叶突变体grc受一对隐性核基因独立控制，表型上区别于其他叶色突变体的最大特征是植株上生长的每一叶片都必须经历由黄转绿的过程，推断grc为一个新的调控叶绿体早期发育的关键基因，已将其定位在3.7cM内。在已明确grc的形态、生理、细胞、遗传特性及基因初定位区域等的基础上，本项目拟图位克隆水稻黄化转绿基因grc，研究该基因的突变机制、结构特点、时空表达特性、编码蛋白的亚细胞定位及其作用于叶绿素合成途径的可能位点，为深入认识GRC基因的生物学功能及其调控叶片转色的分子机理奠定基础。

叶色是与光合作用、产量直接关联的重要农艺性状。叶色突变体grc来自水稻保持系T98B的辐射诱变，其植株上的任一叶片在刚抽出时为黄色，在生长10d左右后变绿，具有单叶不依赖于植株特定发育阶段而独立转绿的特性；grc这类突变体在叶绿体分化与发育及叶色标记不育系育种中具有重要利用价值，但是grc的突变机制及其基础功能尚不清晰。. 为解决这一问题，本项目图位克隆了黄化转绿基因GRC，经遗传互补及测序分析明确了一个含保守肽uORF的转录产物新基因（LOC_Os06g02550）的表观变异导致了grc的突变表型；GRC含有14个外显子，cds全长1389bp，其蛋白产物由462个氨基酸残基构成，含有一个蛋白激酶功能域。功能分析发现，GRC具有泛表达特性，在叶片中表达量最高，其蛋白定位于叶绿体；在突变体中，黄化叶片的总叶绿素和叶绿素b 含量显著降低，叶绿体发育滞留在黄化质体阶段, 表明GRC 可能在叶片早期发育中起关键作用。转录组学及荧光定量PCR分析发现，GRC显著影响ribosome、aminoacyl-tRNA biosynthesis、porphyrin and chlorophyll metabolism等代谢通路，多个与质体发育、叶绿素合成的相关基因在突变体和野生型中发生了明显的表达差异。此外，还开展了将grc作为叶色标记应用于杂交稻育种的有益探索。. 已获得的grc突变机制信息有望为揭示表观遗传的具体作用方式提供新的线索，而对GRC基本功能的明确将为利用GRC或者其互作基因服务于高光效育种提供新的启示。共发表论文2篇，出版专著1部，申请发明专利2项，获得发明专利1项，培养博士1人。