蔗糖转运蛋白基因VvSUC11和VvSUC12双价植物表达载体的构建及在甜菜中的遗传转化

Construction of a bivalent plant expression vector carrying VvSUC11 and VvSUC12 genes and its genetic transformation in sugar beet

摘要:

将葡萄Vitis vinifera L.的蔗糖转运蛋白基因VvSUC11和VvSUC12与甘薯Ipomoea batatas L. Lam.的甘薯贮藏蛋白 (Sporamin) 基因的根部特异性启动子命名为SP1和SP2重组。以pCAMBIA2301为起始载体，构建了pCAMBIA2301- SP1-VvSUC11-SP2-VvSUC12用农杆菌介导法转化了甜菜Beta vulgaris L.品种KWS-9103，发现预培养4 d，侵染时农杆菌的浓度OD600值为0.5，附加0.005％表面活性剂Silwet L-77，延迟筛选4 d，转化效率最高，可达42％。对在卡那霉素中分化并生根的甜菜植株进行PCR和RT-PCR检测，证明目的基因已整合到甜菜中并表达，为进一步研究该基因在甜菜Beta vulgaris中的功能奠定了基础。

Abstract:

We have recombined genes VvSUC11, VvSUC12 from Vitis vinifera L., and root-specific promoters of sweet potato storage protein gene from Ipomoea batatas L. Lam., named as SP1 and SP2. We have constructed a vector pCAMBIA2301-SP1-VvSUC11-SP2-VvSUC12 using pCAMBIA2301 as an original vector. VvSUC11 and VvSUC12 were under the control of root-specific promoters of sweet potato storage protein gene. We transformed the vector into KWS-9103 breeding line of Beta vulgaris L. with Agrobacterium-mediated transformation. We have established the optimal genetic transformation protocol of sugar beet as following: the explants pre-cultured for 4 days were immersed in Agrobacterium suspension of OD600=0.5, supplemented with 0.005% Silwet L-77, and followed by a 4-day culture on medium containing cefotaxime, then the buds were selected on medium containing kanamycin and cefotaxime. The percentage of kanamycin- resistant buds was as high as 42%. Results of PCR and RT-PCR proved that the target genes had integrated into sugar beet genome and expressed. It will lay a foundation for further studying their function in Beta vulgaris.