甘蓝杂种致死基因BoHL1和BoHL2的克隆与功能验证

Hybrid lethality is a kind of phenomenon which parents grow normally but their hybrid individuals die before sexual maturity. It plays a vital role during speciation as a mainly kind of reproductive isolation. The cloning and functional analysis of hybrid lethality genes are the basis for understanding the molecular mechanisms of hybrid lethality and will be helpful for adding some insight into the gene evolution and speciation. Based on previous the results of preliminary gene mapping, the cloning and functional analysis of BoHL1 and BoHL2 for hybrid lethality in cabbage will be carried out using the rare materials which the hybrid lethality between two cabbage inbreed lines was first reported in Brassica oleracea. Thus, the study includes the following aspects: 1. The lethality segregation populations with individuals over 10000 will be constructed by special twice-cross method which one inbreed line carried BoHL1 or BoHL2 crosses firstly with an introduced inbreed line without lethal gene and obtained F1 hybrid crosses secondly with the another inbreed line carried the complementary lethal gene. Used the data of parent genome re-sequencing and aimed for the genomic region of preliminary mapping, novel primers of InDel and SNP will be designed for fine mapping of BoHL1 and BoHL2. Finally, the complementary causal genes for hybrid lethality in cabbage will be mapped to around 50kb intervals, respectively. 2. Based on the mapping result, the candidate gene BoHL1 and BoHL2 for hybrid lethality in cabbage will be obtained and confirmed through several ways including annotation analysis of the candidate region of the cabbage, comparative genome analysis of Chinese cabbage and Arabidopsis thaliana, cDNA cloning of candidate genes and variation locus analysis using 20 different cabbage inbred lines. 3. Applied quantitative real-time PCR and a yeast two-hybrid system, the transcriptional analysis and the protein interaction of BoHL1 and BoHL2 will be carried out, respectively, and we'll verify the complementary function of BoHL1 and BoHL2 by transformation. This study aims to elucidate the molecular mechanism of hybrid lethality in Brassica oleracea and provides the guidance for cabbage breeding.

杂种致死是指双亲生长正常但杂交后代在性成熟前死亡的现象，是一种主要的生殖隔离类型，其对物种形成具有重要意义。杂种致死基因的克隆与功能分析是解析致死机理的分子基础，有助于理解其基因进化和在物种形成中的重要作用。本研究拟利用芸薹属作物中首次发现的稀有的甘蓝种内杂种致死材料，在初步定位基础上，进行甘蓝杂种致死基因BoHL1和BoHL2的克隆与功能验证。即通过二次杂交获得大样本分离群体（>10000株）和利用重测序信息针对亲本间的目标区段开发新的InDel引物，将致死基因精细定位于约50kb区段；利用甘蓝参考基因组的基因注释、与大白菜和拟南芥的比较基因组学分析、候选基因的扩增分析、20份自交系材料的变异位点分析等多种途径预测并确定目标基因，实现杂种致死基因的克隆；并分析致死基因的转录表达特征、蛋白水平的互补作用与转基因植株的基因互作。本研究的完成可阐明甘蓝杂种致死的分子机制，并为甘蓝育种提供指导。

杂种致死是指双亲生长正常但杂交后代在性成熟前死亡的现象，是一种主要的生殖隔离类型，其对物种形成具有重要意义。杂种致死基因的克隆与功能分析是解析致死机理的分子基础，有助于理解其基因进化和在物种形成中的重要作用。本研究拟利用芸薹属作物中首次发现的稀有的甘蓝种内杂种致死材料，在初步定位基础上，进行甘蓝杂种致死基因BoHL1和BoHL2的克隆与功能验证。首先通过扩大分离群体和加密分子标记，将杂种致死基因BoHL1定位在C1染色体末端70kb区段，将BoHL2定位在C4染色体88kb区段。根据TO1000基因组信息，BoHL1、BoHL2定位区段内分别包含17和11个基因。通过基因功能注释分析、重测序分析和转录组测序分析，并结合qRT-PCR以及基因扩增验证，将BoHL1定位区段内的Bo1g153280确定为BoHL1的候选基因:BoHL2定位区段内的Bo4g173720、Bo4g173780确定为BoHL2的候选基因。利用酵母双杂交系统分析BoHL1和BoHL2候选基因蛋白水平的互补作用，表明Bo1g153280和Bo4g173720之间存在互作；同时将2个基因构建至双分子荧光互补载体，进行进一步的互作验证。将Bo1g153280构建至CRISPR-Cas9载体进行遗传转化试验，初步获得Cas9检测阳性植株；将Bo1g153280、Bo4g173720、Bo4g173780构建至植物表达载体，转化至拟南芥，并对转基因植株进行互补杂交，验证二者的基因互作.