玉米CMS-C核恢复基因Rf4的克隆及育性恢复机理的解析

Cytoplasmic male sterility in plants is an important trait for its application to crop breeding and heterosis and for fundamental research of crosstalk between nucleus and mitochondrion. A candidate gene for restorer of fertility in CMS-C maize was conditioned into a 60k nt region in the short arm of chromosome 8 by map-based cloning combined with association analysis. Bioinformatic analysis showed that RF4 lacks signal peptides for mitochondrial localization and doesn't contain any PPR motif, which indicates that RF4 has a different mechanism of fertility restoration from those proteins with PPR motifs. Based on previous results, a new construct driven by the promoter of Rf4 gene itself and a RNAi construct will be developed and introduced into maize plant for further confirming the restoring ability of Rf4 gene. The expression pattern will be analyzed by northern blot, in situ hybridization; the cytological localizations by immunohistochemistry. Yeast two-hybrid will be applied to screen the potential protein(s) interacting with RF4. Combining the results from transcriptome analysis, the regulation network of Rf4 gene will be finally identified. The functional investigation of Rf4 gene will bring new insights into the biology of CMS and boost the application of maize CMS-C in its hybrid production.

细胞质雄性不育（CMS）是作物杂种优势利用和开展核-质互作分子生物学基础研究的重要性状。课题组前期利用图位克隆及关联分析策略，将玉米CMS-C育性恢复基因Rf4的候选基因Rf4_T03精细定位在Chr8短臂60Kb的区段内，并完成了其DNA及cDNA全长序列的克隆。生物信息学分析发现Rf4_T03编码的蛋白不含PPR模体，无靶向线粒体的信号肽，表明Rf4可能是一种新类型的CMS恢复基因。基于此，本研究拟在获得带有自身启动子的Rf4_T03正义链和35S驱动的反义链转基因植株的基础上，开展表型鉴定、Northern Blotting、原位杂交、免疫组化分析、花药转录组分析，Y2H筛选与Rf4_P03的互作蛋白等研究，以期阐明Rf4基因表达的调控网络及育性恢复的分子机制。研究结果不仅可为完善植物CMS育性机理提供理论依据，并将为玉米CMS-C不育化制种技术的应用提供理论支撑。

植物细胞质雄性不育（CMS）恢复基因的功能验证有助于探究育性恢复与核质互作分子机理。本课题利用RACE技术结合第三代测序技术发现候选恢复基因Rf4具有9条仅在转录起始位点不同的转录本；通过构建候选恢复基因Rf4的CRISPR/Cas9载体转化实验证明所选候选基因即为Rf4基因；western杂交显示恢复系中恢复基因的蛋白表达量比不育系中低，保持系中相应基因的表达量比不育系低；转录组数据分析显示氧化磷酸化途径中的多个基因的表达量在不育系中显著降低，在恢复系中表达量升高；花药线粒体iTRAQ蛋白分析表明除与能量代谢相关基因外，与蛋白翻译、异构成熟相关的蛋白在恢复系中表达升高；新构建的正向载体转化正在进行。综合上述结果，本研究初步验证了候选基因即玉米C型胞质不育的恢复基因，初步建立了玉米C型胞质恢复基因的恢复机制。