利用家蚕杆状病毒表达系统表达葡萄白藜芦醇芪合酶基因

Using Baculovirus Expression System of Silkworm to Express Vitis Vinifera's Gene of Resveratrol Synthase

通过提取葡萄幼叶总RNA,用RT-PCR的方法获得白藜芦醇芪合酶基因,并将其成功克隆到pFastBac HTA中,获得重组pFastBac HTA-STS载体,转化DH10Bac大肠杆菌感受态细胞,筛选获得重组Bacmid DNA后转染家蚕BmN细胞,进一步筛选获得重组杆状病毒,重组病毒接种家蚕蛹,工程蚕蛹匀浆后经初步纯化收获粗酶液,利用粗酶液对葡萄提取液进行转化,体外生物合成白藜芦醇,提取液中的白藜芦醇含量显示出明显的上升。该研究为体外生物合成白藜芦醇提供了新途径。

In this study, total RNA was extracted from the young leaves of Vitis vinifera, and the gene of resveratrol synthase was obtained according to the method of RT-PCR, then the target was successfully connected to the vector pFastBac HTA and the recombinant vector pFastBac HTA-STS was attained. Then the right recombinant vector pFastBac HTA-STS was transformed into DH10Bac competent cells, and the positive recombinant Bacmid DNA was screened out to be transfected into BraN cells of silkworm （Bombyx mori） ,so the recombinant silkworm baculovirus could be further screened out and be vaccinated into the pupae of silkworm. The engineering pupae were evenly grinded and the prime enzyme solution of resveratrol synthase was acquired through a preliminary purification. Then the extrxcting solution of Vitis vinifera could be transformed throght the function of the prime enzyme solution, so the resveratrol could be biologically synthesised in vitro. The result revealed that the content of resveratrol in the extrxcting solution was markedly elevated. Thus ,the study can provide a new method to synthesize biologically synthesis resveratrol in vitro.