玉米CMS-C育性恢复基因Rf5的功能验证及育性恢复机理解析

Hybrid seed production by male sterility is a key technique to crop heterosis utilization. In maize seed industry, C-type cytoplasmic male sterility (CMS-C) is utilized most popularly . The restoring of fertility in CMS-C maize was achieved independently by two duplicated genes, Rf4 and Rf5. In former work, our group had fine mapped the Rf5 gene to an 82Kb region in maize Chr.2. Two PPR-motif-containing genes were found in this region. The second PPR-motif-containing gene (named PPR2) was confirmed as the Rf5 candidate. The full length DNA and cDNA of the PPR2 gene were cloned. Protein sequence analysis showed that PPR2 have a signal peptide targeting to mitochondria. In this work, over-expression, RNAi and CRISP/Cas9 vectors will be constructed and transfer to maize materials to verify Rf5 gene function. In situ hybridization, immunohistochemical and transcriptome analysis will be processed to illuminate the Rf5 dependent gene regulation network that influencing the fertility of CMS-C maize. Combined with knowledge of the sterile gene atp6c, this work will clarify the mechanism of restoring of fertility. Our work will provide a solid molecular biological foundation to maize CMS-C male sterility hybrid seed production.

不育化制种是作物杂种优势利用的关键技术。玉米C型胞质雄性不育（CMS-C）是广泛应用的胞质不育类型。CMS-C育性恢复由两对具有重叠效应的基因Rf4和Rf5控制。课题组前期通过基因精细定位和候选基因分析，已确定一个含PPR结构域的基因（命名为PPR2）为Rf5的候选基因；完成了PPR2基因DNA及cDNA全长序列的克隆；该基因编码产物含有靶向线粒体的信号肽。本研究拟构建Rf5候选基因过表达载体、RNAi及CRISP/Cas9载体等，通过遗传转化结合表型鉴定验证Rf5候选基因的功能；开展原位杂交、免疫组化、花药样品转录组分析等研究，阐明Rf5候选基因影响CMS-C育性恢复的基因表达调控网络；结合课题组前期对CMS-C不育基因atp6c的研究成果，阐明重叠Rf5基因育性恢复的机制，进一步完善玉米CMS-C核质互作的育性恢复机理，为玉米不育化制种的应用提供理论依据。

细胞质雄性不育（CMS）恢复基因的功能验证有助于探究核质互作分子机理，促进不育技术在杂交制种中的利用。本课题通过构建候选恢复基因Rf5的过表达载体和CRISPR/Cas9敲除载体转化实验证明所选候选基因即为Rf5基因；qRT-PCR和半定量表达分析表明Rf5在多个组织中表达，在花粉母细胞中表达最高；亚细胞定位显示恢复基因在线粒体中发挥其作用；TUNNEL实验表明恢复基因抑制不育系过早的细胞程序性死亡；编辑检测显示恢复基因抑制了不育基因的编辑；凝胶电泳迁移实验发现恢复基因与不育基因不直接作用；转录组数据分析显示PPR基因与核酸代谢过程、大分子修饰、RNA 代谢过程、RNA 修饰生物学过程。综合上述结果，本研究验证了候选基因即玉米C型胞质不育的恢复基因Rf5，初步建立了玉米C型胞质恢复基因的恢复机制。