一个新的玉米隐性核不育基因克隆与功能分析

Male sterility is a common biological phenomenon in plants, which was widely used in hybrid seed production. However, for male sterility, the molecular mechanism was poorly understood and its application is very limited in maize. We isolated a novel recessive genic male sterile mutant ms (male sterility) from Mutator mutant library. Phenotypic comparison indicated that the development of anthers and pollen wall was abnormal. Through map-based cloning, we identified a MS gene which encodes a glucose-methanol-choline oxidoreductase. In this present project, we will apply transgenic technology to further verify the function of MS gene; the gene expression pattern will be analyzed by RT-PCR, qRT-PCR and mRNA in situ hybridization; the composition of fatty acids will be tested by gas chromatography; genetic networks regulated by MS gene will be preliminarily constructed with the analysis of double mutant and RNA-sequencing; the protein location will be determined using subcellular localization experiment, and the protein function will be confirmed through measuring enzymatic activity by spectrophotometer. The functional analysis of the MS gene will enrich our understanding of the molecular mechanism of male sterility in plants. This study will provide a new gene resource and theoretical basis for the future application of MS gene in hybrid maize seed production.

雄性不育是植物中普遍存在的生物学现象，广泛应用于杂交制种中。但目前对玉米雄性不育分子机制的研究较少，在生产上应用非常有限。申请者前期从Mutator突变体库筛选获得一份新的玉米隐性核不育突变体ms(male sterility)，表型分析表明突变体的花粉壁和花药发育异常而导致雄性不育；申请者利用图位克隆的策略分离了该目的基因，结果显示其编码葡萄糖-甲醇-胆碱氧化还原酶。本研究拟应用转基因等技术对MS基因作进一步功能验证；采用RT-PCR、qRT-PCR和组织原位杂交等方法分析基因表达模式；以气相色谱法测定脂肪酸组成；用双突变体和转录组测序分析初步构建目的基因的调控网络；通过亚细胞定位检测蛋白定位，分光光度计法测定酶活性，明确MS蛋白功能；阐明MS基因在玉米育性调控中的功能,丰富植物雄性不育分子机理。本研究将为进一步利用MS基因实现玉米雄性不育化制种提供基因资源和理论支撑。

雄性不育系在玉米杂交种子生产中具有重要应用价值，然而其分子遗传机制的研究却非常有限。前期，我们发现了一个玉米雄性不育突变体ms (ipe1)。本项目通过图位克隆、基因等位性测验、功能互补和CRISPR/Cas9的方法证明一个编码葡萄糖-甲醇-胆碱氧化还原酶的基因可以调控玉米雄性育性的形成。细胞学分析表明突变体花药角质层和花粉壁发育异常，进一步研究发现突变体花药中角质含量较野生型显著降低是突变表型产生的原因。原位杂交结果显示IPE1在单核前期花药的绒毡层中富集表达，IPE1蛋白定位于内质网。功能分析表明 IPE1可以调节脂肪酸合成和延长、蜡质合成和类黄酮代谢等基因的表达，与MS26和MS45协同调控玉米花药发育。研究结果揭示了一种新的植物花药发育的遗传调控路径，同时为玉米雄性不育化制种提供了重要的基因资源。