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本研究从赤霞珠(Vitis viniferaL.cv.Cabernet Sauvignon)葡萄果实中提取RNA,克隆得到葡萄蔗糖转运蛋白基因VvSUC27;从中蔬6号番茄子叶提取DNA,克隆得到番茄果实特异启动子基因E8。以实验室保存的pCAMBIA 1301为起始载体,分别构建了2个植物表达载体pCE8-SUC27vs和pCE8-SUC27。在这两个载体中,VvSUC27的表达均受番茄果实特异启动子E8的调控,pCE8-SUC27vs含有来自拟南芥葡萄糖转运蛋白基因AtVGT1的细胞液泡膜定位的信号肽编码序列和NOS终止子;pCE8-SUC27包含来自葡萄的蛋白质细胞质膜定位的信号肽编码序列和NOS终止子。在pCE8-SUC27vs质粒的基础上,构建了E8控制下的GUS植物表达载体pCE8-GUS。植物表达载体通过冻融法成功地转入到农杆菌EHA105菌株中。将包含不同质粒的根癌农杆菌注射到番茄果实中进行了启动子功能的瞬时表达验证,获得了阳性GUS染色结果。研究为下一步葡萄蔗糖转运蛋白基因VvSUC27在果实中的特异表达分析奠定了基础。
In this study, RNA was extracted from the grapevine of Cabernet Sauvignon (Vitis vinifera L.cv. Cabernet Sauvignon) to obtain the grapevine sucrose transporter gene VvSUC27. The tomato fruit-specific promoter gene E8 was cloned from Zhongcai No. 6 tomato cotyledon. Two plant expression vectors, pCE8-SUC27vs and pCE8-SUC27, were constructed using pCAMBIA1301 as a starting vector. In both vectors, VvSUC27 expression was regulated by the tomato fruit-specific promoter E8, pCE8-SUC27vs contained the signal peptide coding sequence and the NOS terminator from the vacuolar membrane of the Arabidopsis glucose transporter gene AtVGT1; pCE8 -SUC27 contains the signal peptide coding sequence for the plasma membrane protein localization from grapes and the NOS terminator. Based on the pCE8-SUC27vs plasmid, a GUS plant expression vector pCE8-GUS under the control of E8 was constructed. The plant expression vector was successfully transferred into the Agrobacterium EHA105 strain by freeze-thaw method. Agrobacterium tumefaciens containing different plasmids were inoculated into tomato fruits to verify the transient expression of promoter function, and positive GUS staining results were obtained. The research laid the foundation for the next specific expression analysis of VvSUC27, a glucose sucrose transporter gene in fruit.