大白菜细胞核雄性不育突变基因msm1的克隆及功能验证

The hybrid seed production was performed using the male sterility line, which is an important way of heterosis utilization in Chinese cabbage. The applicant employed a Chinese cabbage inbred line ‘12A086’, which was also a backbone parent, as the plant material. A male sterility mutant was created using EMS mutagenesis method, its stamens were completely degenerated, and other characters were consistent with the wild-type ‘12A086’ except for the stamens, and thus the stamen fertility of excellent breeding material was transformed from fertility to sterility. Genetic analysis indicated that the mutant character was controlled by a single recessive nuclear gene msm1. The mutant gene msm1 has been located on the chromosome A09 using the BSR-Seq method. In this project, the study on fine mapping, clone and functional identification of msm1 will be performed. Fine mapping of msm1 will be conducted using the large-scale F2 segregation population; By virtue of the gene annotation information of Brassica Database, combined with the sequence alignment results, the candidate gene will be identified; The expression characteristics of the candidate gene will be explored by real-time quantitative PCR, promoter activity analysis and subcellular localization technology; The function of the candidate gene will be validated using RNAi technology. These research results contribute to reveal the role of the mutant gene msm1 in the stamen development of Chinese cabbage, and lay the foundation for developing and utilizing the precious genetic resources of male sterility.

利用雄性不育系配制杂交种，是大白菜杂种优势利用的重要途径。申请人以大白菜骨干亲本自交系‘12A086’为试材，采用EMS诱变方法，创制出1份雄性不育突变体，其雄蕊退化彻底，除雄蕊以外其他性状与野生型‘12A086’表现一致，实现了优异育种材料雄蕊育性由可育向不育的转变。遗传分析表明，其雄性不育性由单隐性核基因msm1控制。利用BSR-Seq方法已将msm1基因定位于A09染色体。本项目拟进一步开展msm1基因精细定位、克隆及功能验证研究：利用大规模F2分离群体精细定位msm1基因；参考大白菜基因组数据库的基因注释信息，结合序列比对结果，筛选确定候选基因；利用荧光定量PCR、启动子活性分析和亚细胞定位技术，探明候选基因的表达特性；利用基因沉默技术，验证候选基因的功能。研究结果有助于揭示msm1基因在大白菜雄蕊发育中的作用，为开发利用好这一珍贵的雄性不育遗传资源奠定基础。

大白菜具有明显的杂种优势，雄性不育是其杂种优势利用中最重要的制种途径之一。由于自然突变的雄性不育材料较为稀少，通过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。在本项研究中，以EMS诱变获得的雄性不育突变体msm1为试材，利用MutMap定位方法结合KASP基因分型技术，确定BraA09g012710.3C为雄性不育突变体msm1的候选基因，BraA09g012710.3C是拟南芥AT1G62940的同源基因，其编码酰基辅酶A合成酶（ACOS5），是花粉外壁孢粉素合成过程中起作用的关键酶。通过在野生型和雄性不育突变体msm1中分别克隆候选基因，序列分析表明与野生型相比，突变体msm1中BraA09g012710.3C基因在第3个外显子上发生了一个G-A的突变（A09: 7,453,637），导致编码的氨基酸由甘氨酸（GGA）变成了谷氨酸（GAA）。利用荧光定量PCR分析表明候选基因BraA09g012710.3C在花药中特异表达，与野生型相比，候选基因在突变体msm1中的表达量是显著降低的；亚细胞定位分析表明BraA09g012710.3C定位于内质网中。在前期利用EMS创制的大白菜突变体库中，共筛选出10份稳定遗传的雄性不育材料，在其两用系内采用不育株与可育株轮配杂交法，从中筛选到2份与雄性不育突变体msm1等位变异的突变体，利用等位变异互补验证候选基因BraA09g012710.3C的功能。通过在2份等位变异的雄性不育突变体中分别克隆候选基因，发现在2份等位突变体中BraA09g012710.3C基因的第4和第5个外显子上分别发生了G-A和C-G的突变，导致氨基酸的编码均发生了不同的变化，明确突变基因BraA09g012710.3C在大白菜雄蕊发育中的作用。研究结果为开发利用好这一珍贵的雄性不育遗传资源奠定基础。