重组马铃薯X病毒注射番茄果实高效表达胸腺素a1

High expression of thymosin alpha 1 by injecting recombinant PVX vector into the tomato fruit

摘要:

植物病毒载体表达外源蛋白表达水平高、表达速度快、宿主范围广。本研究用马铃薯X病毒(PVX)载体在番茄果实中表达胸腺素a1(Ta1), 是一种快速高效生产医用蛋白的新方法。将pGEM-T-Easy-Ta1质粒酶切, 获得大约100 bp大小的Ta1基因片段, 并将该片段与PVX载体(pGR-107)连接, 用Sal I和Cla I酶切鉴定, 筛选出阳性重组体。进一步将重组质粒转化根癌农杆菌GV3101, 用农杆菌注射法(Agroinjection)侵染不同生长阶段的番茄果实, 用ELISA检测外源蛋白Ta1的表达水平。结果表明已成功构建了pGR107-Ta1表达载体, 在农杆菌的菌液密度(OD600值)为1.0、番茄植株开花后2周半到3周时侵染番茄果实, Ta1的表达量最大。

Abstract:

For expression of foreign genes in plant, plant virus vector provides many advantages, such as high expression level, short expression period and wider plant hosts. In the present study, we report the expression of thymosin alpha 1 (Tα1) in tomato fruits by potato virus X (PVX) vector. Tα1 gene fragment from plasmid pGEM-T containing Tα1 gene was cloned into plant virus vector pGR107 and the resulting pGR107-Tα1 plasmid was confirmed by digestion with Sal I and Cla I. To express the Tα1 protein, Agrobacterium tumefaciens GV3101 transformed with pGR107-Tα1 was directly injected into tomato fruits through the fruit stylar apex at different developmental stages. The ELISA results showed that Tα1 protein was expressed successfully in fruits, and the highest expression level was obtained from 2.5–3 week-old tomato fruits inoculated by bacterium at 1.0 OD600 density.