玉米淀粉分支酶sbe Ⅱ b基因启动子的克隆与特异表达

目的]构建玉米淀粉分支酶sbeⅡb基因种子特异表达启动子。方法]LA-PCR扩增了玉米淀粉分支酶sbeⅡb基因启动子序列,并克隆到pMD18-TVector上。再将启动子克隆到pBI121载体上,构建植物表达载体pBI121-sbeⅡb。把GUS基因连到pBI121-sbeⅡb上构建pSBE-GUS后利用农杆菌介导法转化烟草。分别进行PCR和Southern杂交检测,并对经基因枪轰击和农杆菌介导的烟草种子、根、茎及叶进行GUS组织化学分析和荧光检测。结果]测序结果与GenBank中发表的玉米sbeⅡb基因启动子同源性达98.52%。共培养和筛选培养后获得4株转pSBE-GUS植株,叶片仅有少量蓝斑出现,而根和茎部未见蓝斑,但烟草种子有大量蓝斑显出,初步推断sbeⅡb基因启动子能启动GUS在种子中特异表达。结论]成功构建了玉米淀粉分支酶sbeⅡb基因种子特异表达启动子。

Cloning and Expression of the Promoter of Maize Starch-Branching Enzyme sheⅡb Gene

Objective] The aim was to construct promoter of maize starch-branching enzyme sbeⅡb.Method] The promoter sequence of maize starch-branching enzyme sbeⅡb was amplified by using LA-PCR and cloned to pMD18-T Vector.Then the promoter was cloned into pBI121 vector to construct plant expression vector pBI121-sebⅡb.Subsequently,GUS gene was linked into pBI121-sebⅡb to construct recombinant plasmid pSBE-GUS,which was then transformed into tobacco mediated by Agrobacterium tumefaciens,and followed by PCR and Southern blot detection.The tissue chemical analysis and fluorescence detection of GUS were performed in different parts of the tobacco by Gene Gun Method and Agrobacterium tumefaciens transformation respectively.Result] The result indicated that the homology between promoter sequence cloned in this experiment and the corresponding sequence announced in the Genbank reached 98.52%.The four transformed tobaccos were detected by PCR and southern blot after co-culture and screening culture.A small number of blue spots appeared in leave,and the spots could barely been seen in the stems and roots,while a large number of blue spots were found in seeds.So,it could be indicated that the promoter of sbeⅡb gene was expressed specifically in seeds.Conclusion] Promoter of maize starch branching enzyme sbe Ⅱb expressed specifically in seed was constructed successfully.