As an important part of mature floral organs, ovary of soybean has an important impact on number of sub⁃room cell division in early stage of development. It may directly affect seed develop⁃ment and quality formation. So researches on specific expression gene in ovary will provide basis for molecular basis of formation of soybean pods with multiple seeds. In this study, significantly over⁃ex⁃pressing HSP17�4Ⅲ gene was selected as target gene, and soybean pods with multiple seeds as ex⁃perimental materials. Throughout RT-PCR, HSP17�4Ⅲ gene full length CDS sequence was ampli⁃fied and similarity of its coded sequence with HSP17�4Ⅲgene sequence in the NCBI was 99%. Ex⁃pression vector pCAMBIA3300 was connected with the method of double enzyme digestion and over⁃expression vector was constructed;Core region of HSP17�4Ⅲ gene sequence with 246 bp by PCR amplification was inserted to transition carrier, and intron-SSR was inserted between anti⁃sense gene and sense gene. Interference elements were inserted to expression vector pCPB by double enzyme di⁃ gestion with BamHⅠand SacⅠto construct RNA interference expression vector and then appraised by PCR and enzyme digestion. Sequence analysis showed that over⁃expression vector pCPB-QC3 and RNA interference expression vector pCPB-ZSF-RNAi were constructed, providing a basis for the study of HSP17�4Ⅲ protein function in soybean ovary.%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pCAMBIA3300，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pBluescript SK，再利用BamHⅠ和SacⅠ双酶切将其整个干扰元件连入表达载体pCPB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pCPB－QC3和RNA干扰植物表达载体pCPB－ZSF－RNAi，为研究HSP17�4Ⅲ蛋白在大豆子房中的功能奠定基础。
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