甘蓝型油菜萎蔫突变体Bnlew1基因克隆及分子机理研究

植物萎蔫通常是由于干旱或盐碱导致植物体内水分代谢失衡，叶片卷曲、上部下垂。我们利用EMS处理中双11号种子获得一个在正常生长条件下发生萎蔫的显性突变体植株(Bnlew1)。我们推测可能是由于突变体植株气孔密度增加，蒸腾作用增强导致植株过度失水而萎蔫，并响应干旱胁迫代谢途径。通过MutMap方法已将突变位点定位到10 个可能区域，总跨度为5.04 Mb。本项目拟：1)通过突变体植株混合池测序结果与608份甘蓝型油菜重测序结果比对寻找突变体混合池特有的SNP/InDel位点及对交换单株进行分析精细定位并克隆BnLEW1；2) GUS融合BnLEW1启动子及YFP融合BnLEW1的CDS，分析BnLEW1表达模式和亚细胞定位; 3)进行功能互补实验并利用CRISPR-Cas9方法进行功能缺失验证; 4)对转基因材料进行分子生理生化与耐旱性分析。项目研究可从不同的角度为油菜耐旱性育种提供理论基础。

Plants become wilted because of the water imbalance caused by drought or salinization, the plant leaves curved, and the upper stem bent in worse conditions. A dominant wilty mutant (Bnlew1) was found by EMS treated Zhongshuang11 seeds, which shows wilty phenotype under normal field condition. We proposed that the wilty phenotype was caused by the intense transpiration because of the increased stomatal density based on the water physiology, antanomy and transcriptome et al. analysis. The mutant locus was mapped to 10 possible regions, covered by 5.04Mb by Mutmap. Our research include: 1) Fine mapping and cloning BnLEW1 gene by the sequence alignment of mutant bulk genome sequence with the 608 lines resequence to identify the unique SNP/InDel and by recombinant plants identification; 2) Analyze BnLEW1 expression profiles and subcellular location by fusion BnLEW1 promotor and CDS to reporter gene GUS and YFP, respectively. 3) Gene function analysis will be conducted by CRISPR-Cas9 gene edit technology and gene complementation test. 4) Physiological analysis and drought tolerance test of the transgene lines. The research results will provide us the theoretical foundation in rapeseed breeding concern about the drought tolerance and high water use efficiency.