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[目的]构建1,4-丁二胺-氮-甲基转移酶(Putrescine N-methyltransferase,PMT)基因、托品酮还原酶-I(Tropinone reductase-I,TR-I)基因和莨菪碱6-β-羟化酶(Hyoscyamine 6β-Hydroxylase,H6H)基因的植物高效表达载体。[方法]采用RT-PCR方法从颠茄中克隆到PMT、TR-I和H6H基因编码区序列,并连接到植物表达载体pCAMBIA1304+(p1304+)中。[结果]成功构建了植物高效表达载体p1304+-PMT、p1304+-TR-I、p1304+-H6H、p1304+-PMT-H6H、p1304+-TR-I-PMT和p1304+-TR-I-H6H,并转化农杆菌,获得可直接用于遗传改良颠茄的工程菌。[结论]该研究为利用植物基因工程技术提高东莨菪碱的产量奠定了基础。
[Objective] The research aimed to construct the gene of Putrescine N-methyltransferase (PMT), Tropinone reductase-I (TR-I) and scopolamine 6 -β-hydroxylase (Hyoscyamine 6β-Hydroxylase, H6H) gene plant expression vector. [Method] The coding region sequences of PMT, TR-I and H6H genes were cloned from Belladonna davidiana by RT-PCR and ligated into plant expression vector pCAMBIA1304 + (p1304 +). [Result] The plant expression vectors p1304 + -PMT, p1304 + -TR-I, p1304 + -H6H, p1304 + -PMT-H6H, p1304 + -TR-I-PMT and p1304 + -TR-I-H6H were successfully constructed and transformed into Agrobacterium , Obtained engineering bacteria can be directly used for genetic improvement Belladonna. [Conclusion] This study laid the foundation for the improvement of scopolamine yield by plant genetic engineering.