印度南瓜强雌基因sg1的克隆与功能分析

强雌性状是印度南瓜育种的重要目标性状，但其控制基因和基因功能尚不明确。本项目前期以强雌自交系2013-12和雌雄同株自交系9-6为亲本构建分离群体，遗传分析表明印度南瓜强雌性状为隐性单基因遗传，进而利用BSA-seq、KASP和CAPS技术将强雌控制基因sg1定位到2号染色体64kb区间内。本项目拟在前期研究基础上，扩大F2分离群体筛选重组单株，精细定位并克隆强雌基因sg1，通过组织解剖学观察雌花和雄花性别分化过程；采用原位杂交和亚细胞定位分析强雌基因sg1的表达模式；利用转基因技术确定该基因的生物学功能；明确强雌基因sg1与乙烯信号通路的相关性，同时借助酵母双杂交等技术鉴定sg1的互作蛋白，整合RNA-seq数据分析sg1基因在南瓜强雌性状形成过程的主要作用，并初步阐明其调控路径，为解析印度南瓜性别决定的分子机制奠定基础。

Subgynoecious is one of the important traits in Cucurbita maxima breeding, but the controlling genes and function is still not clear. In our previous research, segregation population derived from subgynoecious inbred strain 2013-12 and monoecious inbred strain 9-6 were constructed. Genetic analysis revealed that subgynoecious was controlled by a recessive single gene (sg1). We primary mapped and further more narrow down the sg1 gene into a 64 kb genomic region on chromosome 2 with the strategy of BSA-seq, Kompetitive Allele Specific PCR (KASP) method and cleaved amplified polymorphic sequence (CAPS) markers. Based on these results, a large F2 population will be constructed to select recombinants for sg1 fine mapping and gene cloning. The sex differentiation processes of Cucurbita maxima female and male flowers will be observed using histological anatomy. Expression patterns of sg1 gene will be analyzed using in situ hybridization and subcellular localization method. With transgene technology, biological function of sg1 gene will be determined. Through clearing the relevance between sg1 gene and ethylene signaling pathway, screening the interacting proteins of sg1 with Yeast two hybrid system and consolidating RNA-seq data, the main function of sg1 in Cucurbita maxima subgynoecious formation will be analyzed. The results of this project will be helpful to preliminary dissertate regulation pathway of sg1, and illuminate the molecular mechanism of sex determination in Cucurbita maxima.