水稻雌雄不育基因XF2的克隆及其控制减数分裂的机制

Meiosis is the most important process of sexual reproduction. Cloning and functional analysis of several meiosis-related genes suggest that the plant meiosis is both conservative and special. We have previously discovered a rice sterility mutant, xf2. Reciprocal cross and cytological analysis showed that the male and female organs were both sterile (pollen grains were dead and embryo sacs were aborted without inner structure differentiation). Pollen development process investigation showed the pollen mother cell meiotic abnormalities. By employing a map-based cloning strategy, the XF2 gene was fine mapped to a 40-Kb fragment on rice chromosome. Candidate analysis showed that the mutant had a 2-bp-deletion on the 6th exon of a functional unknown gene, which causes a premature stop coden for the XF2 protein. Bioinformatics analysis found that the XF2 protein shared sequence homology to S, an arabidopsis protein involved in meiotic recombination; we thus speculate that XF2 is the rice ortholog of S. In this study, we aim of verifying the XF2 function by genetic complementary analysis and gene knock-out analysis. We also aim of confirming the specific function of the gene in rice meiosis process and revealing the similarities and differences between rice and arabidopsis thaliana. In addition, we aim to isolate and functional verify the partner of XF2 by employing an integrative method of protein-protein interaction (Yeast two-hybrid and Co-IP) and function analysis (knock-out analysis), in order to finally illustrate the exact role of XF2 and its partner in controlling of rice meiosis.

减数分裂是有性生殖最重要的过程。大量减数分裂功能基因克隆显示，植物的减数分裂既保守又特殊。我们前期发现了一个水稻不育突变体xf2，经正反杂交和细胞学分析显示其雌雄器官均不育（花粉不着色，胚囊无分化），花粉发育过程初步显示花粉母细胞减数分裂时期出现异常。利用图位克隆法，将XF2基因定位于水稻染色体约40 kb区域内。候选区间测序显示，突变体仅在一个未知功能基因的第6外显子上发生2 bp缺失，导致该蛋白大幅截短。比对分析发现，该蛋白与拟南芥减数分裂重组相关的S基因具有同源性，推测XF2为该基因在水稻中的同源基因。本研究拟利用功能互补、基因敲出等方法验证XF2的功能，并以细胞学等方法证实该基因在水稻减数分裂过程中的具体作用并揭示其与拟南芥的异同。同时，利用酵母双杂交等互作分析方法结合功能分析手段，鉴定与XF2发生直接作用的互作蛋白的功能，以期揭示XF2及其互作蛋白在控制水稻减数分裂过程中的机制。

减数分裂是真核生物有性繁殖所必需的过程，对植物育性及生产有重要影响。交叉(cross over, CO)不仅是减数分裂所必需的，也是配子中新的等位基因组合形成的基础。这项研究利用图位克隆法，成功分离了一个减数分裂关键基因OsSHOC1。在Osshoc1突变体中，雌、雄配子体发育均不正常，细胞观察发现该突变体在减数分裂过程中CO数量显著减少，导致未配对单价体大量增加。利用CRISPR/Cas9技术敲除该基因，敲除（KO）突变体能重演Osshoc1突变表型，说明OsSHOC1是水稻正常CO形成所必需的。进一步利用酵母双杂交（Y2H）、荧光双分子互补（BiFC）和免疫共沉淀（Co-IP）等实验证实，OsSHOC1能与OsPTD1发生直接相互作用，基因敲除OsPTD1后发现敲除植株与Osshoc1突变表型高度类似，也表现雌雄不育及CO数量减少。此外，该研究还利用q-PCR，GUS报告基因和原位杂交等手段研究了两个基因的表达模式，系统证实它们均主要在减数分裂期小花表达，与其功能基本一致。总之，该研究表明，OsSHOC1和OsPTD1是水稻雌雄育性和减数分裂CO形成的必要条件，并可能提示一种普遍保守的减数分裂机制。