玉米雄性不育基因SA1的克隆和功能研究

Male sterility of plant is a common genetic phenomenon in plants, which is widely used in hybrid seed production. However, the molecular mechanism of male sterility is poorly understood and its application is very limited in maize. We obtained a novel recessive genic male sterile mutant shrinking anther1 (sa1) from MuDR mutant library. Phenotypic comparisons suggested that the fomation of sa1 anthers was abnormal. Through map-based cloning, we isolated an SA1 gene encoding a putative glycerol-3-phosphate acyltransferase (GPAT) protein. This project will apply the transgenic technology to further verify the function of SA1 gene. The gene expression pattern will be analyzed by qRT-PCR and mRNA in situ hybridization. The protein location will be determined using subcellular localization experiment. The compositions of aliphatic polyester monomers in anthers and the substrate of the enzyme will be measured; yeast two-hybrid technique will be used for screening interaction proteins. Genetic networks regulated by SA1 gene will be preliminarily constructed with the RNA-seq data. The functional analysis of the SA1 gene will enrich the understanding of the molecular mechanism of male sterility in plants. This project will provide valuable gene resource and theoretical basis for the application of SA1 gene in hybrid maize seed production.

植物雄性不育是自然界中较为普遍的遗传现象，被广泛应用于作物杂交制种，但是目前对于玉米雄性不育分子机制的研究并不深入。shrinking anther1(sa1)突变体是申请者从MuDR突变体库中获得的一个新的玉米隐性核不育突变体，表现为不能形成成熟花药而导致雄性不育。申请者前期通过图位克隆的方法克隆了SA1基因，其编码一个甘油-3-磷酸酰基转移酶。本项目拟通过转基因等技术对SA1基因进行功能验证；使用qRT-PCR和mRNA原位杂交分析SA1基因的表达模式；采用洋葱表皮亚细胞定位确定SA1蛋白定位；通过花药中脂肪族聚酯单体组分测定和SA1蛋白底物测定初步分析SA1蛋白调控育性的分子机制；利用酵母双杂交技术筛选SA1的互作蛋白；通过转录组测序探索SA1基因的遗传调控网络。旨在阐明SA1基因在玉米育性调控中的功能，丰富植物雄性不育的分子机理，为玉米雄性不育化制种提供宝贵的基因资源和理论基础。

植物雄性不育是自然界中较为普遍的遗传现象，在玉米杂交制种中具有重要的应用价值，但是目前对于玉米雄性不育分子机制的研究并不深入。Shrinking anther1（sa1）突变体是申请者获得的一个新的玉米隐性核不育突变体，表现为花药角质层发育缺陷和小孢子提前降解。申请者前期通过图位克隆的方法克隆了SA1基因（后改称MS33基因），其编码一个甘油-3-磷酸酰基转移酶。本项目在前期研究的基础上，利用等位突变体材料对目的基因进行了功能验证。qRT-PCR的结果显示，MS33基因在四分体时期的花药中表达量最高。原位杂交实验表明，MS33基因在花药发育过程中主要在绒毡层细胞中表达。GC-MS分析显示，与野生型相比，突变体花药中的角质和蜡质含量显著降低，脂肪酸含量显著升高。转录组测序分析表明，MS33基因影响许多参与角质和蜡质生物合成的基因的表达。结合之前的细胞学研究基础，我们推测MS33通过影响脂肪族聚酯单体的生物合成调控花药角质层和小孢子的发育。本研究揭示了MS33基因在玉米育性调控中的功能，丰富了玉米雄性不育的分子机理，为玉米雄性不育化制种提供了基因资源。另外，我们还开展了玉米雄性不育基因Ms28的克隆和功能研究。在本项目支持下，发表SCI论文2篇。