一个特异介导水稻减数分裂DSB形成基因的克隆与功能分析

Meiosis is a critical process in eukaryotes that occupies a central role in the reproduction and life cycles of all sexually reproducing organisms. Meiosis differs from mitosis in that one round of DNA replication is followed by two sequential cell divisions, leading to the generation of four haploid cells from a single initial diploid cell. However, the mechanisms underlying meiosis remain largely obscure with respect to the series of events that occur in prophase I, including homologous chromosome recognition, pairing, synapsis and recombination. In the recent studies, we identified a rice mutant, which exhibits normal vegetative growth but complete male and female sterility. Cytological investigations revealed that homologous pairing and synapsis were severely disrupted in the mutant. No γH2AX signals in the mutant implied that double-strand break (DSB) formation was affected in the mutant meiocytes. In this proposal, we are going to identify the target gene by a map based cloning approach. We will also perform qRT-PCR and immunodetection to study its biological function in rice meiosis. According to the results of immunodetection, the biological functions of the target gene on homologous recombination would be investigated preliminarily. Together with the knowledge on the meiotic genes in rice isolated so far, those data will strength our understanding of the molecular mechanism on homologous recombination induced by DSB formation.

减数分裂DNA双链的主动断裂(Double-Strand Break，DSB)与程序化修复过程，是实现遗传重组的过程，目前对这一最基本遗传学过程的认识还非常初步。我们鉴定到了一不育突变体，其减数分裂期同源染色体不配对，特异指示DSB形成的γH2AX抗体在突变体花粉母细胞染色体上检测不到免疫荧光信号，表明该突变体DSB形成存在缺陷。对该基因的精细定位和功能预测表明它与已报道的水稻DSB形成基因均不等位。本项目拟克隆该目的基因，制备目的蛋白多克隆抗体、利用qRT-PCR和免疫荧光染色反应等手段，研究目的基因在水稻不同组织的表达模式、其他减数分裂相关蛋白在突变体染色体上定位情况，初步研究目的基因与其它减数分裂相关基因的作用关系，解析目的基因参与DSB形成和同源重组的分子机制。

减数分裂DNA双链的主动断裂(Double-Strand Break，DSB)与程序化修复过程，是实现遗传重组的过程，目前对这一最基本遗传学过程的认识还非常初步。且不同生物中介导DSB形成和修复的具体机制存在差异。我们通过本研究克隆了一个影响水稻减数分裂的关键基因LEPTOTENE1 (LEPTO1)。该基因编码蛋白为B型的响应调节蛋白，对减数分裂早期细线期染色体的形态建成致关重要，lepto1突变体中典型的细线期染色体和DSB不能形成，减数分裂特异蛋白OsAM1不能定位于染色体上，lepto1 Osam1 双突变体减数分裂期染色体表现和花药绒毡层不能正常降解的表型类似于lepto1，表明LEPTO1作用于OsAM1的上游；减数分裂特异的黏着蛋白OsREC8以及联会复合体的重要组分PAIR3和ZEP1均不能定位于染色体上，也进一步证明lepto1花粉母细胞减数分裂不能正常进行；LEPTO1编码626个氨基酸，它包括DDK结构域以及MYB型DNA结合结构域，酵母双杂交试验表明，LEPTO1通过DDK结构域与两种组氨酸磷酸转移蛋白OsAHP1和OsAHP2之间有相互作用；在lepto1突变体中，减数分裂的一些关键基因OsAM1，OsREC8，PAIR3, OsDMC1和 OsSPO11-1的表达量与未转化对照没有显著差异，但一个A型细胞周期相关基因CYCA1-1, 一个B型细胞周期相关基因CYCB1-3和OsSDS的表达量均显著降低，这表明LEPTO1对于细胞周期依赖的减数分裂过程是必需的，调节减数分裂细线期染色体的形态建成，影响DSB的形成。