大白菜叶球叠抱与舒心性状基因定位及关键基因功能解析

The head-forming leaf embrace pattern at top region of Chinese cabbage is important trait to leafy head shape. One DH plant was obtained by microspore culture, and rooted plantlet was transferred to a greenhouse after seven months tissue culture. The traits of self-crossed progenies were consistant except head-forming leaf embrace pattern at top region. There are overlapping and curling outwards two types of head-forming leaf pattern at top region. Overlapping and curling outwards plants from self-crossed progenies of one microspore regeneration plant are used as materials, Candidate region is identified by whole-genome resequencing combined BSA. key genes is identified by gene annotation, gene expression and pathway enrichment. The gene expression model and regulation network of aim gene are analysed by qRT-PCR. Subcellular localization is studied by expressing GFP fusion technology. Gene function is verified by overexpression and CRISPR knockout, gene biochemical characteristics is analysed by prokaryotic expression and eukaryotic expression. The effection of auxin etc. supplying on gene modulation of transgenic plants. The molecular mechanism on the formation of overlapping and curling outwards of head-forming leaf will be comprehensively analysed, which has important scientific value to realize molecular design breeding of head-forming pattern of Chinese cabbage, as well as promote an understanding of the genetic mechanism underlying leafy head formation in Chinese cabbage.

叶球抱合方式是决定大白菜商品器官形状的主要性状。项目组对二倍体大白菜小孢子培养获得的1个DH植株，经组培继代9代，历时7个月，定植于田间，其自交后代在叶球抱合方式上发生了叠抱和舒心两类分离，而其它性状一致。为能够开展大白菜叠抱与舒心的分子机制研究提供了机遇。本研究以上述叠抱和舒心的两类植株为材料，利用基于全基因组重测序的BSA分析，确定大白菜叶球叠抱和舒心形成相关位点，通过基因功能注释、基因表达及差异基因富集通路分析等，定位控制大白菜叠抱与舒心抱合方式的关键基因；分析目的基因表达模式及其调控网络；通过基因过表达、CRISPR敲除等方法，验证基因的生物学功能和生化特性；分析转基因植株外施生长素等植物激素对该基因的调控作用。多水平全方位解析大白菜叶球叠抱、舒心形成分子机制，为阐明大白菜叶球形成与发育奠定基础，对实现大白菜叶球抱合方式这一重要性状的分子设计育种具有重要科学价值。

叶球抱合方式是决定大白菜商品器官形状的主要性状。研究团队在大白菜和甘蓝杂交种的基础上，通过回交、自交和游离小孢子培养获得了添加甘蓝片段的易位系B3-29，该易位系自交后代在叶球抱合方式上发生了叠抱和舒心两类分离，其它性状一致。本研究以上述叠抱和舒心的两类植株为材料，利用基于全基因组重测序的BSA-Seq和转录组分析，确定大白菜叶球叠抱和舒心形成关键基因BraHt1；BSA-Seq和转录组分析均表明激素信号转导通路是影响该性状的关键通路，外施生长素时大白菜叶片卷曲发生变化；分析了叶球抱合方式相关基因的不同时期、不同部位叶片和同一叶片不同部位的基因表达情况；克隆了BraHt1基因及上游序列，该序列与大白菜、甘蓝参考基因组和亲本序列都有较大差异，表明该序列是非同源染色体间重组形成的，启动子活性分析表明该序列具备启动子活性，启动子活性受温度调控，26℃时的启动子活性高于13℃；亚细胞定位分析显示BraHt1蛋白行使功能的位置在细胞核；构建了BraHt1基因RNA干涉表达等载体, BraHt1基因干涉表达的转基因植株较对照叶片明显向外翻卷，表明BraHt1基因与叶片卷曲相关，影响大白菜叶球顶端的抱合形态。该研究成果对大白菜叠抱、舒心叶球抱合方式这一重要性状的分子设计育种具有重要的科学价值，同时为阐明大白菜叶球形成与发育奠定基础。