多倍体水稻花粉育性稳定的细胞及分子机制研究

Polyploidy is a crucial pathway in rice breeding. However, the problem of low seed set rate due to disorganized meiosis has become a bottleneck for polyploidy rice breeding. The discovery and application of PMeS successfully overcame the low seed set rate of polyploidy rice. The PMeS line with the normal pollen fertility was selected because of its especially stable meiosis behavior and high seed set rate. The differential expression of anther proteins during meiosis of PMeS and no-background PMeS was explored by using a comparative proteomics approach, and PPFS of ABC family was screened in the PMeS(Polyploid-Pollen-Fertility-Stability) which was significantly high expressed confirmed by real time RT-PCR. PPFS pollen fertility declined distinctly and a lot of abnormal changes were discovered during meiosis and early pollen periods. The pollen fertility became normal after PPFS function compensated for ppfs. According to the former results, we will study the cytological events leading to the stable pollen fertility of polyploidy rice, the essential function of PPFS and the molecular mechanism of regulating PPFS expression. Meanwhile, more differential expression proteins would be identified, and their function in pollen fertility of polyploidy rice would be verified. All these will contribute to explain the cytological and molecular mechanism about the stable pollen fertility of polyploidy rice comprehensively and will supply the theoretical guidance for selection the key germ plasm of polyploidy rice breeding.

多倍化是水稻育种的重要途径，但因减数分裂紊乱导致的低结实率一直是制约多倍体水稻育种的瓶颈。减数分裂稳定品系PMeS的成功选育，突破了低结实的难关。前期研究证实PMeS花粉育性正常并高结实。采用比较蛋白质组学方法分析PMeS与非PMeS减数分裂时期花药蛋白质的差异表达，筛选出了在PMeS中显著高表达的ABC家族成员PPFS(Polyploid-Pollen-Fertility-Stability)，定量PCR证实其显著高表达。ppfs花粉育性显著下降，减数分裂和早期花粉出现异常，基因功能补偿后恢复正常。本项目拟在此基础上研究影响多倍体花粉育性稳定的细胞学事件；深入探讨PPFS在多倍体水稻花粉育性中的作用；解析调控PPFS表达的分子机制。同时进一步对其它差异蛋白进行鉴定，并验证其在花粉育性中的作用。以期较全面理解多倍体水稻育性稳定的细胞及分子机制，为多倍体水稻育种中关键种质的选择提供理论依据。

多倍化是水稻育种的重要途径，但因减数分裂紊乱导致的低结实率一直是制约多倍体水稻育种的瓶颈。多倍化影响花粉育性稳定的关键细胞学事件是减数分裂。PMeS（减数分裂稳定品系）具有类似二倍体水稻的稳定减数分裂行为，但没有PMeS背景的材料自身减数分裂紊乱，且杂交后代结实率低。减数分裂稳定品系PMeS的成功选育，突破了低结实的难关。前期研究证实PMeS花粉育性正常并高结实。采用比较蛋白质组学方法分析PMeS与非PMeS减数分裂时期花药蛋白质的差异表达，结果显示PMeS中上调表达的蛋白质152个，下调表达的蛋白质48个。已经筛选出了与花粉育性可能相关的基因多个。其中在PMeS中显著高表达的ABC家族成员PPFS(Polyploid-Pollen-Fertility-Stability)，定量PCR证实其在减数分裂时期显著高表达。GUS 染色显示该基因在花药和子房中优势表达，在营养体根、叶中未检测到表达。进化树分析表明，ABC1基因在21个物种中均有存在，同源性较高，说明该基因在进化上相对保守。ppfs花粉育性显著下降，减数分裂和早期花粉出现异常，基因功能补偿后恢复正常。PPFS在高低结实材料中显著差异表达的关键时期在减数分裂期。后续通过突变体、回补突变体，PPFS超量表达植株及amiRNA干扰植株结果分析表明，PPFS表达量高低与花粉发育之间存在明显的相关性。瞬时表达载体定位表明该基因主要在细胞膜上表达，与基因的定位预测基本一致。启动子比较分析表明PMeS和非PMeS材料在启动子区存在序列差异。该基因在PMeS和非PMeS、二倍体和四倍体材料中不存在甲基化修饰差异。RNA差别表达分析筛选出了多个在PMeS中显著差异表达的减数分裂相关基因，同时研究还获得了大量SNP、可变剪接预测以及新基因的发掘和注释信息。以期较全面理解多倍体水稻育性稳定的细胞及分子机制，为多倍体水稻育种中关键种质的选择提供理论依据。

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