水稻减数分裂同源重组起始因子MRIF1的功能研究

Homologous recombination is a key event of meiosis. It helps connect homologous chromosomes to promote their accurate segregation and shuffles alleles between homologous chromosomes to increase genetic variability. Meiotic recombination is initiated by the generation of DNA double-strand breaks (DSBs), catalyzed by topoisomerase VI. In this study, we screened the interaction protein MRIF1 of OsMTOPVIB by yeast two-hybrid assays. The mrif1 mutant was obtained by CRISPR-Cas9 technique. Its vegetative growth was normal, but it showed complete sterility after flowering. Homologous pairing was aborted, which was consistent with the mutants that are in defective in DSB formation. Moreover, there is no signal of γH2AX (DSB indicator protein) in the mutant, which indicates that MRIF1 may be involved in the initiation of homologous recombination. The cytological analysis of the double mutant of mrif1 with other meiotic mutants was carried out. The localization pattern of MRIF1 protein on meiotic chromosome was analyzed. The localization of other meiotic proteins were detected in mrif1 mutant. The biochemical activity of OsMTOPVIB-MRIF1 was detected by means of gel electrophoresis migration test and topoisomerase activity assay, to clarify the specific functions of MRIF1 in homologous recombination. Thereafter, other MRIF1 interacting proteins were screened by yeast two-hybrid assays and protein immunoprecipitation, which provided important theoretical support for elucidating the molecular regulatory network of rice homologous recombination, and laid a theoretical foundation for the application of homologous recombinant genes in rice breeding.

同源染色体重组是减数分裂的关键事件，其确保染色体的准确分离并且促进遗传多样性发生。本研究中，我们借助酵母双杂交实验，筛得同源重组起始关键因子OsMTOPVIB的互作蛋白MRIF1。通过CRISPR-Cas9技术，获得mrif1突变体，突变体完全不育，同源染色体不配对，且花粉母细胞中无DSB指示蛋白 γH2AX 的信号，说明MRIF1可能参与DSB形成。通过对MRIF1与其它减数分裂基因配制的双突变体进行表型分析，观察MRIF1蛋白在染色体上的定位模式，检测mrif1中其它减数分裂蛋白的定位情况，借助凝胶电泳迁移实验和拓扑异构酶活性检测技术检测OsMTOPVIB-MRIF1复合体的生物化学活性，来阐明MRIF1在减数分裂重组过程中的具体功能。此外，借助酵母双杂交和蛋白质免疫沉淀等手段筛选MRIF1的其它互作蛋白，最终为阐明水稻同源重组起始的分子调控网络提供依据，进而为同源重组基因在水稻育种中的应用奠定理论基础。

同源染色体重组是减数分裂的关键事件，同源重组起始于DSB的产生。减数分裂DSB形成过程中一个关键酶是Spo11。Spo11是II型拓扑异构酶VI家族的成员，是古细菌TopVIA的同源蛋白。古细菌中DSB是由II型拓扑异构酶TopVIA和TopVIB所形成的异源四聚体介导完成，拟南芥和小鼠中报道了TopVIB的同源蛋白MTOPVIB和TOPOVIBL通过和SPO11互作参与DSB形成。我们证明了水稻OsMTOPVIB是TopVIB的同源蛋白，其参与DSB形成。鉴于TopVIA和TopVIB在DSB形成中的重要地位。借助酵母双杂交实验，筛得OsMTOPVIB的互作蛋白MRIF1 (Meiotic Recombination Initiation Factor 1)。对候选基因MRIF1进行功能验证；通过对突变体和双突变体的细胞学表型分析，以及免疫荧光实验明确目标基因MRIF1参与DSB产生；对MRIF1的细胞学定位及互作蛋白进行定位探究，解析水稻减数分裂同源重组起始机理和可能的分子调控网络。研究结果显示mrif1突变体完全不育，同源染色体不配对，且花粉母细胞中无DSB指示蛋白γH2AX 的信号，并且MRIF1的突变可以抑制重组缺陷突变体Osrad51c的染色体修复缺陷问题，此外，MRIF1和OsMTOPVIB共定位于染色体线圈上，说明二者在DSB形成中起到重要作用。. 本研究为阐明水稻同源重组起始的分子调控网络提供依据，为解析同源重组过程，调控重组率提供必要的理论支撑。进而为同源重组基因在水稻育种中的应用奠定理论基础。