葡萄蔗糖转运蛋白基因VvSUC27的克隆及其植物表达载体的构建  

Isolation of grape sucrose transporter VvSUC27 and construction of expression vectors

作　　者：冀敏[1] 曾磊[2] 赵凤霞[2] 陈尚武[2] 马会勤[1] 

机构地区：[1]中国农业大学农学与生物技术学院,北京100193 [2]中国农业大学食品科学与营养工程学院,北京100083

出　　处：《中国农业大学学报》2010年第3期15-21,共7页Journal of China Agricultural University

基　　金：国家自然科学基金资助项目(30471212;30500347)

摘　　要：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。Grape sucrose transporter VvSUC27 was cloned from Vitis vinifera L.cv.Cabernet Sauvignon berry RNA.Fruit-specific promoter E8 was cloned from genomic DNA of Lycopersicon esculentum Mill.cv.Zhong Shu No.6.Starting with pCAMBIA 1301,two plasmids pCE8-SUC27vs and pCE8-SUC27 were constructed.In both of the constructs,VvSUC27 was under the control of E8 promoter.In pCE8-SUC27vs,SUC27 was ligated with the vacuolar signal peptide coding sequence of Arabidopsis thaliana glucose transporter AtVGT1,while in pCE8-SUC27,SUC27 was recombined with the grape plasma membrane anchor signal peptide sequence,the expression of VvSUC27 was both controlled by NOS terminator.Furthermore,based on pCE8-SUC27vs,pCE8-GUS,where GUS expression was under the control of E8 promoter,was constructed.Each plant expressing plasmid was successfully transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method and the transient expression of GUS under E8 promoter was validated by tomato fruit agro-injection,and positive GUS stain was achieved.The results lay a sound base for the following study on the effect of specific expression of VvSUC27 in fruits.

关 键 词：VITIS VINIFERA L. VvSUC27 E8启动子 载体构建 GUS瞬时表达 

分 类 号：S663.1[农业科学—果树学]