芝麻隐性核不育保持系WB7-1D育性调控基因的发掘和分子机制研究

Sesame (Sesamum indicum L.) is an important oilseed crop with distinctive characteristics. The annual sesame consumption is beyond the domestic production capacity in China, and exploitation of heterosis is an effective way to significantly improve yield. Development of three-line system is one of the methods to solve the problem of high production cost in the present two-line system (dual purpose line, AB line). Our project team has bred a new sesame line WB7-1D, which could keep stable and thorough sterility of recessive genic male sterile line 0176A. The maintainer line WB7-1D is an excellent germplasm source for studying the molecular mechanism of fertility regulation in sesame，and makes it possible to develop three-line hybrid combination. Base on whole-genome resequencing, mapping of the loci that contribute to fertility traits of WB7-1D will be performed by constructing F2 segregating population and bulked segregant analysis (BSA). Meanwhile，the differentially expressed genes between fertile plants and sterile plants in the segregating population will be screened by transcriptome sequencing and comparative analysis. The candidate genes involved in fertility regulation will be obtained by the comprehensive analysis on the annotated genes in the mapped region and the transcriptome data. Moreover, the expression pattern of candidate genes will be assayed at different developmental stages of microspore by quantitative real-time PCR (qRT-PCR). Finally, through transgenic Arabidopsis lines and the yeast two-hybrid system, the functions of candidate genes will be identified and the pathways of fertility-related genes will be revealed. The results generated from this project will deep our knowledge of the molecular mechanism of fertility regulation in recessive genic male sterile maintainer line WB7-1D，and will provide a theoretical basis for utilization and improvement of new germplasms of hybrid sesame.

芝麻是重要的特色油料作物，目前中国芝麻生产面临着严重的供给不足，杂种优势的利用是提高产量的有效途径，而三系法制种是解决现在两用系法制种生产成本高的方法之一。项目组选育了一个新的品系WB7-1D，其对芝麻隐性核雄性不育株0176A具有完全保持特性，是研究芝麻育性调控分子机制和实现三系杂交组合的理想种质。本项目拟通过构建F2代分离群体，利用基因组重测序技术，使用BSA法定位保持系WB7-1D育性关联基因组区域，同时结合群体中可育株和不育株mRNA转录组比对信息获得差异表达基因，筛选育性调控相关的候选基因；利用qRT-PCR技术验证不同小孢子发育时期候选基因的表达模式；运用拟南芥转基因技术和酵母双杂交系统，研究候选基因的功能和其参与的信号通路。研究结果将加深对芝麻隐性核不育保持系WB7-1D育性调控分子机制的认知，并为芝麻杂交新种质的利用和改良提供理论依据。

芝麻是重要的特色油料作物，目前中国芝麻生产面临着严重的供给不足，杂种优势的利用是提高产量的有效途径，而三系法制种是解决现在两用系法制种生产成本高的方法之一。项目组选育了一个新的品系WB7-1D，其对芝麻隐性核雄性不育株0176A具有完全保持特性，是研究芝麻育性调控分子机制和实现三系杂交组合的理想种质。本项目通过构建F2代分离群体，利用基因组重测序技术开发SNP和InDel标记，使用BSA法将保持系WB7-1D育性调控基因定位到芝麻基因组1号连锁群360kb区域；利用转录组测序比对分析和实时荧光定量PCR技术，发现在定位区域内有4个基因（ZMwb58、ZMwb59、ZMwb66、ZMwb80）在保持系WB7-1D花蕾中显著上调表达，有2个基因（ZMwb49、ZMwb92）在保持系WB7-1D花蕾中显著下调表达，这6个显著差异表达基因应与育性调控密切相关；从保持系WB7-1D中克隆了一个新的育性调控基因ZMwb58，其编码蛋白含有一个保守的F-box结构域，通过构建过量表达载体和拟南芥遗传转化，获得了ZMwb58转基因拟南芥植株，ZMwb58转基因拟南芥植株性状上表现出较小的叶片和果荚，花粉活力降低，育性下降，种子数量减少。研究结果为进一步解析保持系WB7-1D的育性调控分子机制奠定了基础，并为芝麻杂交新种质的利用和改良提供理论依据。