大豆CMS育性恢复(Rf)基因的克隆与功能验证

It has been well documented that utilization of cytoplasmic-nuclear male sterility (CMS) in hybrid breeding is an effective way to increase soybean yield. However, the utilization of this system was greatly limited due to the unclear nature of soybean CMS. Cloning of the restorer-of-fertility (Rf) gene, which is a very important factor in plant CMS system, will greatly help us in understanding the cytoplasmic-nuclear interaction mechanism and in exploring the nature of plant CMS. Meanwhile, cloning and characterization of this Rf gene will greatly decrease the labor input in screening of the maintainer and restorer lines. Our early research shows that the ZD type CMS of soybean belongs to gametophytic CMS; the fertility restoration is controlled by one single dominant gene; and this gene is fine mapped in an about 228kb region on soybean chromosome 16 (linkage group J). Four mitochondria-targeted pentatricopeptide repeat (PPR) protein genes were found located consecutively in a 15kb region of this interested area. These PPR genes were considered to be the candidate soybean Rf genes due to their high similarity of protein sequences as well as their existing patterns when compared with already cloned Rf genes from other species. In this project, firstly, we are going to compare the gene expression features, CDS sequences and maybe the promoter sequences of the sterile, maintainer and restorer lines to further narrow down the number of candidate genes. After that, some gene specific markers will be developed and then be used for screening of CMS related germplasm, i.e. maintainer lines and restorer lines. Crosses between sterile lines and the screened maintainer or restorer lines will be made to verify the candidate genes. At the meantime, over-expression and RNAi of candidate genes will be conducted for the functional complement analysis and gene knock-out analysis, respectively. The objective of this project is to finally clone and characterize this Rf gene. The fulfillment of this project will greatly facilitate the discovery of mechanisms involved in soybean CMS, and will boost the complete using of this system in soybean hybrid breeding.

利用质核互作雄性不育（CMS）系统进行杂交育种是提高大豆单产水平行之有效的方法。对育性恢复Rf基因的克隆将有助于深入了解质核互作的过程，同时极大减少杂交育种过程中种质筛选的工作量。申请者前期研究发现大豆ZD型CMS属于配子体型雄性不育，育性恢复受显性单基因控制，并将Rf基因精细定位在大豆第16染色体上约228kb范围内。进一步生物信息学分析初步确定了该区域内几个线粒体靶向、成簇出现的PPR蛋白为候选Rf基因。本研究拟在此基础上，通过比较不育系、保持系和恢复系中候选基因的时空表达差异、编码区和启动子序列差异等进一步缩小候选基因范围；进而开发候选基因特异的分子标记用于CMS相关种质筛选；并通过田间杂交和转基因进行功能互补验证，以及进行RNAi基因敲除试验来验证候选基因的功能；最终达到鉴别、克隆该Rf基因的目的。以期为大豆CMS形成机理研究提供参考，并为大豆杂种优势育种的全面实现提供理论基础。