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大豆子房作为成熟花器官的重要组成部分,发育初期对子房内细胞分裂数有着重要的影响,可能直接影响子粒发育以及品质形成,因此对子房特异表达基因的研究将可能为多粒荚形成的分子基础提供依据。该研究以大豆多粒荚突变体为试验材料,选取上调表达差异显著的HSP17.4Ⅲ基因为目标基因,通过RT-PCR扩增HSP17.4Ⅲ基因全长CDS序列,编码序列与NCBI中HSP17.4Ⅲ基因序列相似性为99%。双酶切连入表达载体p CAMBIA3300,构建超表达载体;亚克隆法扩增该基因246 bp大小的核心保守序列,依次将其正义片段、功能性间隔序列Intron-SSR和反义片段双酶切连接到中间载体p Bluescript SK,再利用Bam HⅠ和SacⅠ双酶切将其整个干扰元件连入表达载体p CPB上,构建RNA干扰植物表达载体。质粒PCR、酶切鉴定以及测序结果表明,成功构建了超表达载体p CPB-QC3和RNA干扰植物表达载体p CPB-ZSF-RNAi,为研究HSP17.4Ⅲ蛋白在大豆子房中的功能奠定基础。
Soybean ovary, as an important part of mature flower organ, has an important influence on the number of cell division in the early stage of development and may directly affect the development and quality of the seed. Therefore, the study on the gene specific expression in the ovary will probably be a multi- The basis for the formation of the molecular basis. In this study, soybean pod pod mutant was used as the experimental material, the HSP17.4Ⅲ gene with significant up-regulated expression was selected as the target gene, and the full-length CDS sequence of HSP17.4Ⅲ gene was amplified by RT-PCR. The coding sequence was identical with HSP17.4Ⅲ The gene sequence similarity is 99%. Double digestion into the expression vector p CAMBIA3300 to construct an over-expression vector; subcloning to amplify the 246 bp core conserved sequence of the gene, followed by its positive fragment, the functional spacer sequence Intron-SSR and antisense fragments double digestion Was ligated into the intermediate vector p Bluescript SK, and then the entire interfering element was ligated into the expression vector p CPB by restriction enzymes Bam HI and Sac I to construct RNA interference plant expression vectors. Plasmid PCR, restriction enzyme digestion and sequencing results showed that overexpression vector p CPB-QC3 and RNA interference plant expression vector p CPB-ZSF-RNAi were successfully constructed, which laid the foundation for studying the function of HSP17.4Ⅲ in soybean ovary.