大白菜核雄性不育基因ftms的克隆与鉴定

Male sterile mutants are the important genetic resources in heterosis utilization in Chinese cabbage, and the ideal materials for studying the male gametogenesis in plants. In previous research, an excellent Chinese cabbage DH line was employed as the plant material, and the isolated microspore culture and γ-ray mutagenesis were combined to create mutants. A male sterile mutant was obtained, which enjoyed thoroughly abortive stamens and no undesirable traits. Genetic analysis indicated that the male sterility was controlled by the recessive nuclear gene ftms. Using SLAF-seq technology and SSR marker, ftms was located within a genetic distance of 3.2cM on chromosome A05. In this project, cloning and functional identification of ftms will be performed. Fine mapping of ftms will be conducted using the large-scale F2 segregation population with the help of BAC library and the RNA-seq information. The biological function of encoding genes in the mapping region will be predicted and the sequence differences between the wild type and the mutant are analyzed to identify the candidate genes. The expression characteristics of the candidate gene will be analyzed by promoter activity analysis, subcellular localization of gene products and tissue in situ hybridization. The function of candidate genes will be validated using CRISPR-Cas9 technology, and its roles will be evaluated in the male gametogenesis of Chinese cabbage. These research results not only have important significance in the development and utilization of the new genetic male sterile resources in Chinese cabbage, but also can enrich the knowledge on gene regulation of the male gametogenesis in plants.

雄性不育突变体是大白菜杂种优势利用的重要遗传资源，也是研究植物雄配子发育的理想材料。申请者以大白菜优异DH系为试材，采用游离小孢子培养结合γ射线诱变处理，创制出1份雄蕊败育彻底、无不良性状伴生的雄性不育突变体，其不育性呈单基因隐性遗传，命名为ftms。利用SLAF-seq技术和SSR标记，将ftms基因定位于大白菜A05染色体的3.2cM区域内。本项研究拟进一步开展ftms基因克隆与功能鉴定研究。构建大规模F2精细定位群体，并借助大白菜基因组BAC文库，参考转录组测序信息，筛选确定ftms候选基因；通过启动子活性分析、基因产物亚细胞定位及组织原位杂交，分析候选基因的表达特性；利用CRISPR-Cas9技术，验证候选基因的功能。通过上述研究，探索ftms基因在大白菜雄配子发育中的作用。研究结果对于开发利用这一新的雄性不育遗传资源有重要意义，同时可以丰富植物雄配子发育基因调控方面的知识。

雄性不育广泛存在于高等植物中，对于具有显著杂种优势的十字花科蔬菜作物，雄性不育系的利用是配制杂交种的重要手段之一。因此，雄性不育性的研究一直受到育种专家的高度重视。由于自然突变的雄性不育材料较为稀少，利用人工诱变创制雄性不育材料，已经成为雄性不育研究的重要手段。本研究以优异大白菜DH系‘FT’试材，花药辐射诱变处理后利用小孢子培养，创制出1份遗传背景与原品系相同的核基因雄性不育突变体ftms，实现了优系雄蕊育性由可育向不育的快速转变。借助BSR-Seq技术和基因组重测序相结合的手段，成功筛选到雄性不育候选基因Bra010198，并为共分离试验所验证。候选基因Bra010198编码β-(1,3)-半乳糖基转移酶，其拟南芥同源基因AT1G33430参与花粉外壁形成。荧光定量分析发现，Bra010198在雄蕊中特异性表达。启动子活性分析表明，候选基因Bra010198的启动子只能启动该基因在拟南芥花药中特异表达。原位杂交试验证明，候选基因Bra010198主要在花药绒毡层和花粉粒上表达。亚细胞定位结果表明，Bra010198的翻译产物主要分布在细胞核上。利用RNA-Seq技术，对突变体ftms与野生型ʻFTʼ的花蕾进行比较转录组分析，在2,305个差异表达基因中，筛选出与绒粘层发育相关的AMS、与胼胝质合成相关的CALS5和CDKG1、与原外壁合成相关的RPG1、以及与花粉壁合成相关基因家族等，推测突变体ftms中候选基因Bra010198的单碱基突变，可能导致绒粘层、胼胝质以及花粉壁发育受阻而造成雄蕊败育。以上结果可以为探索候选基因Bra010198在大白菜雄配子发育中的作用奠定基础，有助于开发利用这一新的种质资源，丰富大白菜雄性不育分子调控机制。