水稻雌性不育基因fsv1的克隆与功能分析

The fertility of female reproductive system is the basic of yields formation and heterosis utilizing, also an important research content of apomixes which is a main approach to fix heterosis. The varieties which are female sterility and male fertility as male parents are the best choice, mixed sowing with male sterile line for mechanization breeding. Now the female sterility varieties are few, and we know little about the molecular mechanism. A high frequency female-sterile mutant (fsv1) of rice was found primarily from a mutant library created by ear-stem-injecting genomic DNA of Panicum maximum into indica rice Gui99. The primary research showed that the female-sterility trait is stabilized and hereditable, the setting percentage under 15%; its pollens are fertility, and the female-sterility was controlled by a pair of recessive gene; embryo sac attributed to recession of functional megaspores; With molecular markers, this study fine-mapped fsv1 gene to a 21kb region on the long arm of chromosome 9. Sequencing analysis the candidate gene (FSV1) found that there is a single base transition (G to A) presenting in the extron13, and the mutation resulted in premature stop of protein translation. In this project, we will carry out complement experiment and functional analysis of FSV1 gene, and ascertain the function of FSV1 gene in female organ development. All these will provide theoretical basis for hybrid rice breeding and production of hybrid seeds.

雌性生殖系统的育性是产量形成、杂种优势利用的基础，也是固定杂种优势最可能途径无融合生殖的研究核心；而且雌性不育雄性可育恢复系可作为与母本混播混收机械化制种的极佳遗传工具。目前水稻雌性不育遗传材料不多、分子机理知之甚少。可见相关基因资源的发掘及其基础研究意义重大。本课题组利用穗茎注射法将大黍基因组DNA导入籼稻桂99获得已经遗传稳定的花粉可育但结实率低于15%的高度雌性不育系fsv1；发现fsv1雌性不育的原因是约85%胚珠中的功能大孢子衰退，雌不育性状受一对隐性基因FSV1控制；并将该基因定位在9号染色体的长臂端21kb区间，测序分析发现候选基因第13外显子中有一单碱基突变，翻译提前终止。本项目拟在此基础上开展突变体fsv1互补验证和FSV1基因功能分析等相关试验，探明FSV1基因在雌配子发育中的作用，为创新杂交水稻育种与制种途径提供理论依据。

雌性生殖系统是产量形成、杂种优势利用的基础，另外雌性不育雄性可育恢复系可用于混播混收机械化制种。目前水稻雌性不育遗传材料不多、分子机理知之甚少，因此相关基因资源的发掘及其基础研究具有重要意义。前期利用1份稳定的雌性不育突变体fsv1开展研究，发现fsv1 花粉可育但结实率保持15%左右，观察到突变体胚囊发育过程中约有85%的胚珠中的功能大孢子衰退；遗传分析发现雌不育性状受一对隐性基因FSV1控制；并将该基因精细定位在9 号染色体的长臂端20kb 的区间内，对区间内4个ORF测序，发现ORF1的第13 外显子中有一个C-A单碱基突变，造成翻译提前终止。在此基础上，开展了突变体fsv1互补验证和FSV1基因敲除实验，证实了ORF1（LOC_Os09g37930）控制雌性育性的基因，其编码DNA错配修复蛋白；启动子分析表明FSV1基因启动子为组成型表达，但在子房部位有强表达，同样FSV1基因的原位杂交试验也显示其在胚珠中表达；FSV1蛋白亚细胞定位结果表明其发挥作用的区域在细胞核中；Western-blot的结果说明突变体的FSV1蛋白因翻译提前终止而无法检测到；野生型和突变体转录组比较分析发现FSV1基因可能跟DNA错配修复途径中的关键基因OsRPA（Os01g0175300）存在互作；以上对FSV1基因的功能解析，表明其在胚珠中强表达，蛋白定位细胞核中，可能参与DNA错配修复，影响雌性育性，本研究将为创新杂交水稻育种与制种途径提供理论依据。