水稻线粒体损伤型黄化突变体d82的基因克隆及功能研究

Leaves are the main organs for photosynthesis in plant. In the leaf cells, chloroplast, mitochondria and nuclear gene expression are controlled through synergistic signaling that regulates the growth and development of plants. Recently, more than 80 yellow-leaf genes have been identified in rice, but few of them are involved in mitochondria development. By chemical mutagenesis, we identified a yellow leaf mutant d82. Electron microscopy analysis showed that, besides chloroplasts, mitochondria was also serious impaired in d82. Genetic analysis showed that phenotype of d82 was controlled by a single recessive nuclear gene and the candidate gene was fine mapped to approximately 95 kb region on chromosome 6. A base mutation which led to a premature termination was found in the open reading frame of unknown function protein encoding gene. Protein localization signal prediction suggested that the protein was likely to locate in mitochondria. In this project, we intend to clone the target gene, complete complementary verification, observe leaf plastids microstructure, analyze expression pattern, detect intermediate products, as well as use protein expression and enzymology experiment, Western-blot experiment and protein subcellular localization, to better understand the function of the gene, explore the relationship between chloroplasts and mitochondria, and further provide theoretical and genetic support to cultivate improved varieties with high photosynthetic efficiency.

叶片是进行光合作用的主要器官，在叶肉细胞中，叶绿体、线粒体及细胞核基因协同表达，调控植物的生长发育，至今已经鉴定了80多个水稻叶色基因，但同时影响叶绿体和线粒体发育的还鲜有报道。我们通过化学诱变获得一份水稻黄化突变体d82，其突变性状受一对隐性核基因控制。经电镜观察，除叶绿体外，d82线粒体也损伤严重，嵴变少甚至消失。已将该基因精细定位于第6染色体约95 kb区域内，其中一个编码未知蛋白的候选基因读码框有1个碱基发生了突变，导致了编码蛋白的提前终止，蛋白定位信号预测表明该蛋白很可能定位在线粒体里。本项目拟克隆该基因，并通过转化互补验证、表达模式分析、相关中间产物检测、蛋白原核表达、酶学实验、Western blot检测、叶片质体显微结构观察及蛋白亚细胞定位等，深入了解该基因的功能，探讨水稻叶绿体与线粒体之间的互作关系，为培育高光合效率的水稻良种提供理论和基因资源支撑。

水稻作为世界上最重要的粮食作物之一，随着全球人口的增长，耕地面积的减少，培育高光合效率的高产水稻品种已经成为农业生产上的一项重要任务。植物进行光合作用的主要部位在叶片，近年来，叶色突变体越来越多地被应用于对植物功能基因组、光合作用的生理生化机制以及遗传育种应用等方面的研究。在叶肉细胞中，叶绿体、线粒体及细胞核基因协同表达，调控植物的生长发育，但同时影响叶绿体和线粒体发育的还鲜有报道。通过化学诱变，我们获得一份水稻黄化突变体d82，遗传分析表明其突变性状受一对隐性核基因控制。该突变体表型和农艺性状较野生型对照差异明显，除整个生长周期都表现出黄绿的表型外，株高、单株有效穗、穗长、剑叶长、结实率以及千粒重均显著降低。经电镜观察，除叶绿体外，d82线粒体也损伤严重，嵴变少甚至消失。叶绿素和光合速率测定表明，d82中叶绿素显著较少，并伴随着光合速率的降低。通过图位克隆的方法已将该基因精细定位于第6染色体约95 kb区域内，其中一个编码未知蛋白的候选基因读码框有1个碱基发生了突变，导致了编码蛋白的提前终止，蛋白定位信号预测表明该蛋白很可能定位在线粒体里。为了验证克隆基因的正确性，我们构建了D82基因两个不同靶位点的CRISPR-CAS9敲除载体，并通过农杆菌转化获得了敲除成功的转基因株系，通过自交获得了纯合敲除株系，验证了所克隆基因正确性。荧光定量RT-PCR表明，D82在水稻的整个生长周期中的叶片均有表达，在灌浆期表达量有一定程度的升高，此外，该基因在所检测的根、茎、叶、穗、胚乳中均有表达，在胚乳中的表达量较高。本研究深入研究了一个独特的叶色基因D82的生物学功能，为培育高光合效率的水稻良种提供理论和基因资源支撑。