水稻ASP1基因参与DSB形成的功能研究

减数分裂过程中同源重组起始于DNA的双链断裂(Double-Strand Break，DSB)，并伴随DSB的修复而完成同源重组，但介导DSB形成和修复的关键基因在生物界并不保守。目前水稻中已经克隆和鉴定的DSB形成基因远少于酵母和哺乳动物。我们在前期工作中通过辐射诱变获得了一个水稻DSB形成缺陷突变体asp1。该植株表现为营养生长正常，但完全不育，并且减数分裂期染色体上检测不到γH2AX抗体的免疫荧光信号。已通过图位克隆方法获得了候选基因，它与已报道的水稻减数分裂基因均不等位。本项目拟在此基础上，进一步构建该基因与其它减数分裂关键基因的双突变体，表达目的蛋白的多克隆抗体，结合目的蛋白和减数分裂其它关键蛋白抗体的免疫荧光染色反应、荧光原位杂交、电泳迁移率实验、酵母双杂交、双分子荧光互补等手段，研究该基因在减数分裂过程中的生物学功能，为最终解析水稻减数分裂调控的分子机制提供新的证据。

Meiotic recombination plays an important role in maintenance of both genome stability and genetic variability for eukaryotes, which is initiated by DNA double-strand breaks (DSBs). Accompanying with repair of DSB, recombination is achieved. Spo11 is a homolog of a subunit of archaebacterial topoisomerase, catalyzing meiosis DSB together with its accessory proteins in budding yeast. But genes mediate DSB formation and repairing are not conserved across species. Only a few of these genes have been cloned and functionally identified in rice. In our previous studies, we have identified a rice DSB deficient mutant, asp1. It shows normal vegetative growth but abnormal homologous pairing, synapsis and γH2AX antibody immunofluorescence signals. Map-based clone technique was used to clone the target gene and we have got the sequence of the candidate gene. According to the sequences of the candidate gene and its prediction function, the genes affecting meiosis DSB formation reported in rice were excluded. In this proposal, we are going to over express and knock out the target gene to verify its function. A polyclonal antibody of the target gene would be prepared. Double mutants would be constructed between ASP1 and several other identified meiotic genes. We are also going to perform fluorescence in situ hybridization（FISH）, immunodetection of the ASP1 and other meiosis specific proteins, yeast two hybrid, bimolecular fluorescence complementation (BiFC) assay， electrophoretic mobility shift assay (EMSA) to study the biological function of the target gene in rice meiosis.