一份水稻抽穗延迟突变体的鉴定、基因克隆及功能研究

Rice is a model plant, also is one of the foremost crops. Stable and high yield always as a pivotal goal pursued in rice breeding. Heading date is the key to affect the rice planting area and seasonal adaptability, also is one of the main factor influencing the rice yield. At present, there are many reports about regulation of rice heading date in recently years, but the regulation network and molecular mechanism remains to be unclear. Based on previous work, we have identified a mutant with delayed heading date phenotype and then Hdl was cloned. Preliminary study found that heading date is delayed resulted from Hdl induced expression caused by promoter mutation. This project will uncover the function of Hdl by gene expression, subcellular localization and transgenic methods and so on. On the other hand, we will identify Hdl downstream genes according to the transcriptome sequencing and ChIP-seq technologies. Subsequently, through promoter activity analysis, key regulatory sequence identification, and then using yeast one-hybrid screening to find regulatory factors, and finally verify binding activity based on protein expression and EMSA. Additionally, though screening yeast library, identify Hdl interaction proteins, and verify its further function. Finally, by illustrating Hdl biology function and revealing the molecular mechanism of Hdl involved in rice heading date regulation, it also provided theoretical basis for new germplasm creation and rice molecular breeding.

水稻作为研究的模式植物，亦是重要的粮食作物，其稳产与高产一直是水稻育种追求的目标。抽穗期影响水稻的种植区域及季节适应性，同时也是影响水稻产量的主要因素之一。目前，尽管有关水稻抽穗期调控的研究陆续报道，但其调控网络及分子机制尚需进一步揭示。在前期工作基础上，我们获得并已鉴定了一份抽穗延迟突变体同时相关基因Hdl已被克隆, 研究发现该基因启动子突变造成基因诱导表达从而抑制水稻抽穗。本项目拟通过基因表达、亚细胞定位、转基因等分析其功能；利用转录组、ChIP-seq等技术挖掘Hdl的下游调控基因，通过启动子活性分析并鉴定其关键调控序列，采用酵母单杂等技术筛选上游调控因子，经蛋白质表达纯化及EMSA互作分析验证结合活性。此外，利用酵母文库淘洗HDL互作蛋白，并进一步验证其功能。通过阐明Hdl的生物学功能，初步揭示Hdl参与水稻抽穗期调控分子机制及通路，为水稻新材料创制与分子育种奠定基础。

水稻作为重要的粮食作物，其抽穗期的长短影响水稻的种植区域及季节适应性，同时也是影响水稻最终产量形成的主要因素之一。课题组在前期研究中筛选并获得了一份水稻抽穗延迟突变体，同时通过Tail-PCR方法结合测序分析我们成功鉴定出了候选基因Hdl，该基因编码一个含CCT结构域的蛋白质。进一步研究发现因该基因启动子上的插入突变造成了其表达量上调从而抑制水稻抽穗；另外，我们对Hdl启动子进行截短并融合荧光素酶和GUS报告基因进行了活性分析，发现启动子上的插入突变确实能够增强启动子的活性从而引起该基因的上调表达导致抽穗延迟。同时通过基因表达、亚细胞定位、转基因等方法对该基因的功能进行了解析；利用课题组前期建立的启动子免疫共沉淀（PIP）技术鉴定到了候选上游调控关键因子PIP10，并采用酵母单杂、EMSA等实验技术验证了其结合活性；同时我们还构建了PIP10的转基因的材料进行了功能解析与验证。此外，通过酵母文库筛选获得了HDL的互作蛋白，并通过酵母双杂、BiFC、Pull-down进一步验证其功能。利用转录组、荧光定量PCR等技术分析Hdl可能的下游调控基因，发现该基因通过调控并抑制Hd3a、Ehd1、RFT1的表达控制水稻的抽穗。综上所述，我们成功鉴定并阐明了Hdl参与水稻抽穗期调控分子机制及通路，为后续水稻新材料创制与分子育种奠定理论基础。