OsGWT9调控水稻籽粒大小的分子机制研究

Rice (Oryza sativa L) is one of the most important crops in the world. Mining key genes of grain yield and understanding the function of these genes are being very important for marker assisted selection in rice breeding. In the early stage of this project, a mutant, gwt9, with high plant height, large grain shape and high 1000 grain weight, and the wild type were compared in the respects of morphological structure, contents of jasmonic acid and fatty acid. Though our previous efforts, the gene had finely mapped into a region of 60.5kb on Chr.9.Sequence analysis indicates that a single nucleotide mutation was found in the 2nd exon of Open Reading Frame 5, which caused a Pro amino acid changed to Ser amino acid in the protein. Sequence Blast analysis revealed that the candidate gene encoded an unknown protein. RNA-seq technology was used to analyze the changes of gene expression pattern of mutant and wild type. The results showed that the expression levels of genes involved in jasmonic acid synthesis and signal transduction were down regulated. In this proposed study, we will conduct functional complementary test and knock out the gene using CRISPR/cas to confirm the function of OsGWT9. The temporal and spatial expression patterns of OsGWT9 and protein subcellular localization will be analyzed. We will attempt to find out OsGWT9 interacting proteins by yeast two-hybrid system or/and OsGWT9 direct binding genes using ChIP-seq and GWCNA technology. Furthermore, the physicochemical properties of gwt9, such as JA, fatty acids and starch content will be compared with wild type. The expected goal of the project is to elucidate the physiological and molecular mechanisms of the OsGWT9 controlling rice grain size and involving in the jasmonic acid pathway.

水稻是世界主要的粮食作物之一，发掘控制产量的关键基因和研究水稻产量性状形成的分子机制，从而为水稻的高产分子设计育种提供技术支撑显得日益重要。本项目前期以一个表现为株高、大粒和千粒重显著增加的突变体gwt9为材料，利用图位克隆技术已经将突变基因定位在第9号染色体60.5kb的区域内，对定位区域内基因测序发现有一个基因出现单碱基的突变并引起编码氨基酸的改变，序列对比结果显示突变基因编码一个功能未知的蛋白，蛋白预测显示该蛋白具有核定位信号。转录组数据分析发现突变体差异表达基因中茉莉酸合成与信号转导相关的基因富集且表达水平下调。突变体茉莉酸含量下降但合成茉莉酸的前体物质积累。该项目拟对候选基因进行功能互补、时空表达分析、基因表达网络节点分析以及互作蛋白的筛选等实验，初步阐明突变基因调控水稻籽粒大小的生理和分子机制以及与茉莉酸途径的关系。

水稻( Oryza sativa L．) 是我国乃至世界主要的粮食作物之一， 但随着人口的增加和可耕种土地面积的逐渐减少，粮食安全成为一个突出问题，因此研究决定水稻产量相关基因的分子机制，对解决世界粮食安全问题具有重要意义。本项目所研究的突变体gwt9是一个粒宽、粒厚、粒重和株高都增加明显的突变体，基因定位表明突变基因为OsEMF2b。基因测序结果证实，突变体的转录本相比野生型第11个外显子缺失，导致蛋白缺失18个氨基酸，从而引起功能的改变。转录组和qRT-PCR数据分析表明，gwt9的茉莉酸合成与信号转导途径相关基因表达水平明显下调。生理实验表明,茉莉酸合成的前体油酸、亚油酸和亚麻酸含量增加。激素水平测定证实gwt9内源茉莉酸、茉莉酸-异亮氨酸、茉莉酸甲酯明显降低，但水杨酸含量增加。蛋白质组数据分析表明在gwt9突变体中有75个蛋白表达量上调，50个下调，GO和KEGG功能富集分析都表明，gwt9 的脂类、二萜类物质合成、苯丙烷类化合物和谷胱甘肽代谢途径较野生型发生了较大改变，相关蛋白明显富集。gwt9赖氨酸甲基化抗体检测结果发现，gwt9 突变体内组蛋白的赖氨酸三甲基化修饰没有明显变化，但赖氨酸二甲基化修饰状态明显比野生型低，这表明gwt9突变的分子机制与EMF2B功能缺失的分子机制不同。 .总之，本研究鉴定了OsEMF2b的一个新的突变形式，该突变通过改变组蛋白赖氨酸二甲基化修饰改变茉莉酸合成和信号转导相关基因的表达水平下调，进而影响水稻的脂质、萜类、苯丙烷类化合物和体内氧化还原状态的改变，导致水稻生物量增加。该研究阐明了JA信号转导途径调控水稻发育和产量的一种新的机制，同时为进一步研究禾谷类作物中同源基因并对其进行遗传工程操作提供理论依据。