油菜FOX-hunting突变体库构建及抗寒相关基因发掘和功能解析

Brassica rapa is one of the most important oil-producing crops in China and worldwide. Cold and freezing stress are the major environmental factors that limit the productivity of Brassica rapa. In Arabidopsis, although the ICE1-CBF-COR transcriptional cascade has been well studied. However, there still have many confuses. What are the receptor of cold signal? How the cold signal transduces from the plasma membrane to the nucleus to modulate the expression of CBF response to cold stress of plants? Why is there a need for multiple pathways leading from cold perception to response? Brassica rapa was planted normal in winter in many countries. In theory, Brassica rapa should have more stronger freezing resistance compare with Arabidopsis. However, Brassica rapa unlike Arabidopsis and Oryza sativa, due to lacked mutants resource, up to now, little is known about the molecular mechanism of cold response. .The generation of loss-of-function and gain-of-function mutant resources is one of the effective approaches for the identification of plant gene functions. Numerous loss-of-function resources have been produced by mutagenesis with chemicals, irradiation, or insertions of transposable elements or T-DNA. However, we may be unable to observe loss-of-function phenotypes for genes with functionally redundant homologs and for those essential for growth and development. To offset such disadvantages, gain-of-function transgenic resources have been exploited. Using the fl-cDNA clones, a novel gain-of-function strategy, FOX-hunting (Full-cDNA OvereXpressor gene) system, has been developed. A mutant phenotype in a FOX line can be directly attributed to the overexpressed fl-cDNA. Investigating a large population of FOX lines could reveal important genes conferring favorable phenotypes for crop breeding. .A long-term goal of Brassica rapa genetic improvement involves the identification of genes with broad-spectrum effects on various stress responses. Identification and elucidation of functions of plant genes is valuable for both basic and applied research. In the project, the modified FOX-hunting system was employed to investigate the cold response gene of Brassica rapa. We constructed a Brassica rapa cold stress cDNA gain-of-function mutants library using a cold stress cDNA population rather than a single cDNA, and the convenient Gateway technology was used to construct the cDNA expression library. In addition, the plant in vivo imaging system was used to large scale Brassica rapa cold stress response mutant. Then, the gene function of isolated from FOX-hunting mutant be characterized through genetics, molecular biology and biochemistry technology, and the molecular signal model of those gene in Brassica rapa be revealed. In the end, these valuable data could provide a theoretical basis for improving the quality and cold resistance of Brassica rapa through genetic improvement.

研究植物响应低温胁迫的分子机制，对改良农作物耐寒性状具有重要意义。油菜由于缺乏像拟南芥、水稻等模式植物有效的突变体库资源，虽然全基因组测序已经完成，但是我们对油菜参与抗寒的分子机制了解非常有限。对于世界范围内普遍冬种的油菜，尤其是强抗寒性的白菜型油菜，可能是一种研究作物抗寒分子机理理想的“模式”植物。以FOX-hunting技术为基础创建功能获得性突变体资源为大规模筛选油菜功能基因带来契机。本项目基于我们前期在棉花功能基因研究中的FOX-hunting基础，优化改进此系统，直接用冷冻胁迫下特异表达的cDNA群体，结合Gateway技术以及RNA-seq信息来创建一种经济、便捷、高通量的油菜冷冻胁迫功能获得性突变体库资源，利用高效的植物活体成像系统大规模筛选油菜响应冷冻胁迫功能基因。利用遗传、生理生化等多种手段揭示他们参与冷冻胁迫应答的分子机理，为油菜抗寒育种提供理论基础及种质资源。

油菜是重要的油料兼经济作物，低温冻害严重影响油菜的产量、品质和种植区域。因缺乏有效的突变体资源，目前对油菜参与低温应答的分子机制仍然知之甚少。项目基于FOX-hunting技术，构建了白菜型油菜FOX-Arabidopsis冷冻特异表达功能获得性突变体体库，已筛选到120多株有表型的突变体，并克隆到一些重要的调控靶基因。通过酵母双杂筛选到与油菜胞质受体激酶BrCRPK1互作蛋白280多个，其中包括一个亮氨酸受体激酶LRR-RLK，一些与BrCRPK1互作蛋白对低温胁迫积极响应。克隆白菜型油菜BrCRPK1/2研究发现，BrCRPK1/2是两个核定位的蛋白，冷胁迫不能改变其定位，说明BrCRPK1/2通过激酶活性参与低温应答。抗冻性分析表明BrCRPK1/2负调节植物的冷冻抗性，其冷冻抗性关联ROS积累并依赖下游的CBFs路径响应低温应答。利用CRISPR/Cas9技术获得了油菜BrCRPK1和phyC同源基因敲除突变体。对白菜型油菜重要调节子BrCRPK1/2, BrICE1/2, BrEBF1/2功能研究及转录组等分析发现，白菜型油菜可能通过细胞组分的变化来感知低温信号，进而激活MAPK激酶、相关激素及Ca2+信号等上游组分并级联放大低温信号，依赖类似BrCRPK1等受体激酶磷酸化传递信号到细胞核，并激活下游的转录因子，通过BrCRPK1/BrICE1/BrEBF1-ROS-CBFs的复杂路径，进而调节下游抗冻相关基因的表达来响应低温应答。本项目支持下，目前已发表基金标注见刊论文9篇，其中SCI论文3篇，CSCD收录北大核心期刊6篇。