油菜是典型的越冬作物，抗寒性较强。为了充分利用长江流域大面积的冬闲田，湖南湖北等地近年来开始推广“稻-稻-油”三熟制栽培模式，种植早熟、特早熟甘蓝型油菜品种，但早熟品种往往抗寒性较弱，而创造和选育耐寒性强的早熟油菜品种可从根本上解决这一矛盾。本项目拟利用CRISPR/Cas9系统对抗寒性强的甘蓝型油菜中的9个BnFLC基因编码区及其启动子E-box（关键抗寒转录因子ICE1直接结合的顺式元件）进行定向编辑，以期筛选出早熟且抗寒性强的突变株系。通过比较分析各突变类型后代，明确9个BnFLC成员各自对甘蓝型油菜花期、抗寒性及其他农艺性状的影响；解析9个BnFLC成员在油菜花期调控中的相互效应，以及FLC介导的花期变化对植株抗寒性及其他农艺性状的影响；阐明E-box在BnFLCs响应低温以及春化应答反应中的功能。本项目的研究结果可为早熟、抗寒甘蓝型油菜分子设计育种提供理论依据和种质资源。

Brassica napus L. is a typical winter biennials plant, responses to vernalization and has strong cold-tolerance at vegetative stage. In recent years, as were Yangtze River area, more and more early-maturing rapeseed varieties start to be planted in the winter fallow field of Hunan and Hubei province, since the popularization of yearly ‘early rice-late rice-rapeseed’ or ‘rice-ratoon rice-rapeseed’ cropping system. Unfortunately, the weak cold-tolerance of early-maturing rapeseed varieties rises the risk of freeze injury in cold winter and spring, so it is vital to select early-maturing and cold/freeze resistant Brassica napus varieties. In this project, we will edit the ORF and cis-element E-box of 9 BnFLCs using the CRISPR/Cas9 system to generate early-maturing and cold/freeze resistant Brassica napus mutant lines. The functions of 9 BnFLCs in the rapeseed flowering time, cold-resistance and other agronomic traits will be characterized respectively, the similarities and differences of the 9 BnFLCs will be compared, and the coordination and interaction between the 9 BnFLCs will be evaluated. To further characterize the roles of cis-element E-box in the cold-response and vernalization of rapeseed, the expression patterns of 9 BnFLCs and cold-responsive genes in mutant lines and wild-type plants with or without cold treatments will be analyzed. Implementation of this project will be revealed the functions of BnFLCs in the rapeseed flowering time, cold-resistance and other agronomic traits, as well as to providing fundamental basis and genetic resource for cold-resistant, early-maturing Brassica napus vateries breeding.