大白菜持绿突变基因Brcl克隆及功能分析

持绿性是指植物衰老叶片长时间保持绿色而不黄化的特性。大白菜以叶片和叶球为产品器官，在生长发育后期和采后贮运保鲜过程中，外叶容易衰老变黄，造成产量损失和品质下降。申请者前期利用EMS诱变方法创制出一份大白菜持绿性突变体cl，其持绿性状由单隐性核基因控制。采用MutMap方法已将突变基因定位于A01染色体的2.8Mb区域，该区域内尚未见持绿性相关基因的报道。本项目拟进一步开展突变基因Brcl克隆及功能鉴定：通过突变体与野生型的叶绿素代谢、光合作用及衰老指标的对比分析，摸清该持绿性变异的生理特性；利用KASP技术筛选确定Brcl的候选基因；通过转基因功能互补及CRISPR/Cas9敲除实验，验证候选基因的功能；通过时空表达、亚细胞定位、转录组分析及转录调控和蛋白互作等实验，探索Brcl基因的作用机制。研究结果可望发掘出新的大白菜持绿基因，为创制大白菜持绿性品种提供新的基因资源。

Stay-green is a term refers to a trait whereby plants maintain their green leaves for a long time during leaf senescence. The leaf and the leafy head of Chinese cabbage were used as the product organs. At the end of growth period or in the process of storage and transportation after harvest, the outer leaves of Chinese cabbage are prone to senescence and yellowing, which results in yield loss and quality decline. A stay-green mutant cl of Chinese cabbage was created by EMS mutagenesis. Genetic analysis indicated that the stay-green trait of cl was controlled by a simple recessive nuclear gene. Using MutMap technology, Brcl was located within a distance of 2.8Mb, on the chromosome A01. There is no report of Chinese cabbage stay-green genes in this region. In this project, the gene cloning and functional identification of Brcl will be conducted. The chlorophyll metabolism, photosynthesis and senescence indexes of mutant and wild type were compared and analyzed to identified the physiological characteristics of the stay-green mutation. KASP genotyping will be performed to identify the mutation locus and predict the candidate gene. The function of Brcl will be validated by transgenic complementary experiment and CRISPR/Cas9 knockout experiment. The mechanism of Brcl gene was explored through spatiotemporal expression, subcellular localization, transcriptome analysis, transcription regulation and protein interaction experiments.The results are expected to identify a new stay-green gene in Chinese cabbage, and to provide a new gene resource for the breeding of new varieties of stay-green Chinese cabbage.