水稻高温胁迫诱导OsNCED1基因的功能分析与调控

In recent years, the impact on rice production caused by high temperature in southern rice regin has become the bottleneck of limiting rice safety production in Yangtze River Basin in China. Mining and utilizing heat resistant gene has important theoretical and practical significance for the genetic improvement of heat resistance in rice. Abscisic acid (ABA) is one of the important plant hormones to regulate plant growth and development and stress resistance response,9-cis-epoxycarotenoid dioxygenase（NCED） is a key enzyme in controlling the synthesis and metabolism of ABA.Preliminary study on anther proteomics of rice under high temperature stress found OsNCED1 of anther induced by high temperature significantly up-related,But for function of OsNCED1 in response to heat resistance in rice has been remained unclear.The expression characteristics of OsNCED1 in space-time in rice is analyzed by real-time fluorescence quantitative PCR and Northern hybridization to understand the model of OsNCED1 in response to high temperature stress, and to clone target gene.Using CRISPR/Cas9 system site directed mutagenesis rice gene OsNCED1,and combined with overexpression technology to analyze biological function for target gene in response to high temperature stress, the regulation of target gene on rice heat resistance is analyzed from protein location, transcriptional level, physiological and biochemical and interaction protein,the aim of the research is to illuminate biological function and regulation mechanism of OsNCED1,The results will provide a basis for revealing the mechanism of genetic and regulation of heat tolerance in rice.

近年来发生在我国南方稻区的高温危害已成为限制我国长江流域水稻安全生产的瓶颈，挖掘和利用水稻耐高温基因对水稻耐热性遗传改良具有重要的理论和实践意义。脱落酸（ABA）是植物生长发育、抗逆反应调控的重要植物激素之一，9-顺式-环氧类胡萝卜素双加氧酶（NCED）是控制ABA合成代谢的关键酶。项目前期研究高温胁迫下水稻花药蛋白质组学，发现水稻花药中OsNCED1受高温诱导显著上调，但目前对于OsNCED1如何参与水稻高温响应的功能尚不清楚。本项目拟通过分析OsNCED1在水稻中的时空表达特性，明确OsNCED1对高温胁迫的响应模式，并克隆目标基因，利用CRISPR/Cas9系统定点突变OsNCED1，结合超表达技术分析目标基因的功能，并从蛋白定位、转录水平、生理生化及互作蛋白方面分析目标基因对水稻耐热性调控，明确目标基因的生物学功能及调控机制，为揭示水稻耐热性遗传调控机理提供研究基础。

近年来发生在我国南方稻区的高温危害已成为限制我国长江流域水稻安全生产的瓶颈，挖掘和利用水稻耐高温基因对水稻耐热性遗传改良具有重要的理论和实践意义。本项目对高温胁迫诱导基因OsNCED1 进行功能分析及调控机制研究，取得的重要结果如下：（1）OsNCED1基因长度为2160bp，没有内含子，只由一个外显子组成；OsNCED1蛋白由638个氨基酸组成，没有信号肽和跨膜结构，具有NCED家族典型的RPE65保守结构域，进化树分析班该基因与二穗短柄草亲缘关系较近。（2）OsNCED1在高温下被诱导表达，在叶片和叶鞘中表达量较高；亚细胞定位分析OsNCED1蛋白定位在细胞质细胞核。（3）OsNCED1过表达增加了ABA含量，减少了膜损伤，提高了抗氧化酶活性、花粉活力与结实率，基因敲除株系则相反，表明OsNCED1正向调控水稻抽穗开花期耐热性。（4）OsNCED1过表达增加了高温下幼苗ABA含量，减少了膜损伤和ROS水平，提高了抗氧化酶活性与幼苗存活率，基因敲除株系则相反，表明OsNCED1正向调控水稻苗期耐热性。高温胁迫下OsNCED1正向激活了防御、抗氧化、热和ABA应答相关基因的表达。（5）对野生型和突变体nced1的幼穗进行转录组分析发现，252与nced1在高温处理第1天、第3天和第5天分别共有差异基因187个和173个，252和nced1共有差异基因有39个。GO分析发现高温下252的转运活性高，nced1的转运调节活性高，说明高温下252可能会影响离子转运和物质运输来调节高温，而nced1可能会影响植物的转运调节过程。（6）用酵母双杂交系统筛选水稻转录因子文库，筛选出一个转录因子OsWRKY51，利用酵母双杂交试验与体外pull-down实验验证了OsNCED1与OsWRKY51互作。研究结果不仅为揭示水稻耐热性遗传调控机理提供研究基础，而且为培育耐热水稻品种提供新的基因。