水稻种子特异表达基因OsEnS97的功能与作用机理研究

Rice is one of the most important foods for human. It is mainly from the endosperm of rice seeds. The development processes of endosperm were composed of different phases including mitosis, endoreduplication, accumulation of many nutrients, as well as the programmed cell death (PCD) of starch. Earlier studies have shown that auxin plays a key role in rice seed development and AUX/IAA protein involved in the signal transduction of auxin regulation, but the mechanism remained poorly understood. Our group identified a AUX/IAA protein OsEnS97, and the transgenic materials were obtained by RNAi, CRISPR and over-expression techniques. In T2 OsEnS97-RNAi lines, we found that the seed size and 1000-grain weight were significantly reduced compared with the WT, and a certain percentage of shrunken seeds were produced. Transcriptional profiling and in situ hybridization analysis confirmed that OsEnS97 was exclusively expressed in developing seed. Then study its biological function through bio-information analysis, RNAi, CRISPR and over-expression strategies. Further, the auxin treatment and flow cytometry were performed to analyze its function mechanism. Finally we propose to screen the interacting protein of OsEnS97 through the Yeast two-hybrid technology and Transcriptome Sequencing, and then proved by genetic experiments. Completion of this project will determine the molecular mechanism of the signal transduction of auxin in rice seed, and will provide resources for genetic improvement of yield and quality of rice.

稻米是人类最重要的粮食之一，它来自于水稻籽粒中的胚乳。水稻胚乳的发育主要包括细胞分裂与分化、储藏物质的合成与积累以及淀粉胚乳细胞的PCD等过程。生长素在种子发育过程中发挥着关键的作用，生长素受体基因（AUX/IAA）可能参与到生长素的信号转导调控。前期本课题组创建了一个种子特异表达AUX/IAA类基因OsEnS97的遗传材料，RNAi T2代家系的种子大小和千粒重都显著低于野生型，并产生一定比例的皱缩种子。本项目采用表达分析和原位杂交已证实OsEnS97为种子特异表达基因；将通过生物信息学分析、RNAi、CRISPR和超表达研究OsEnS97的生物学功能；从蛋白定位、IAA处理和流式细胞检测等分析其作用方式；最后对水稻种子酵母文库进行筛选，获得调控蛋白，并通过遗传学实验验证。研究OsEnS97在水稻种子中的功能，将有助于进一步解析生长素信号转导途径在胚乳发育中的作用和机制。

稻米是人类最重要的粮食之一，它源于水稻籽粒中的胚乳。水稻胚乳的发育主要包括细胞分裂与分化、储藏物质的合成与积累以及淀粉胚乳细胞的PCD等过程。生长素在种子发育过程也发挥着关键的作用，生长素受体基因（AUX/IAA）可能参与到生长素的信号转导调控，具体的分子机制有待进一步研究。本项目综合运用分子生物学、细胞学、生物化学和遗传学等方法研究了胚乳特异表达AUX/IAA类基因OsEns97的生物学功能与作用机理。首先，利用qRT-PCR、原位杂交和启动子连GUS报告基因实验，证实OsEnS97在授粉后7-10天的种子中特异表达；随后，我们创建了OsEnS97基因的突变体和过量表达材料并进行表型分析，发现osens97千粒重下降、异常籽粒比例增加，OsEnS97突变导致水稻胚和胚乳的发育异常；最后，激素处理实验显示在生长素处理后OsEnS97蛋白含量增加，OsEnS97可能为非经典AUX/IAAs；在授粉后7天的籽粒中，野生型胚乳细胞核可检测到3C、6C和12C三种倍性水平，而OsEnS97突变体材料的胚乳细胞只检测到少量的3C倍性，说明OsEnS97的突变导致水稻胚乳细胞的分裂和核内复制异常；酵母双杂交、GST-pull down和BiFC实验证实OsEnS97与OsARF17发生互作，并且影响到胚乳细胞周期和PCD等相关基因的表达。本研究有助于进一步解析生长素信号转导途径在胚乳发育中的作用和机制，为水稻产量和品质的遗传育种提供宝贵资源。