颠茄H6H基因的克隆及高效植物表达载体的构建

Cloning of H6H Gene from Atropa belladonna and Construction of the Efficient Plant Expression Vector

[目的]克隆颠茄(Atropa belladonna)H6H基因并构建高效植物表达载体。[方法]采用RT-PCR方法从颠茄中克隆莨菪碱-6β-羟化酶和1,4-丁二胺-氮-甲基转移酶基因编码区,插入经改造后获得双元三价植物高效表达载体p2301-gus,构建植物表达载体p2301-H6H,并将该表达载体导入根癌农杆菌LBA4404和发根农杆菌C58C1。[结果]获得了可直接用于遗传改良的颠茄工程菌p2301-H6H-LBA4404和p2301-H6H-C58C1。[结论]为利用植物基因工程技术提高颠茄中生物碱含量奠定了基础。

[ Objective ] The aim was to clone H6H gene from Atropa belladonna and construct an effective plant expression vector. [ Method J The coding sequence of H6H （Hyoscyamine 613-hydroxylase） was cloned from Atropa belladonna with RT-PCR. Then, the sequence was sub- cloned into the reconstructed plant binary expression vector p2301 to construct the recombinant vector p2301-H6H, which was then introduced into Agrobacterium tumefaciens strain LBA4404 and Agrobacterium rhizogenes strain C58C1, respectively. [ Result] The engineering bacteria p2301-//6H-LBA4404 and p2301-H6H-C58C1 which could be directly used in genetic improvement were obtained. [ Conclusion] The present research provided basis for the increasing of alkaloid content of Atropa belladonna by plant genetic engineering technology.