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目的通过构建羟基桂皮酰辅酶A羟基桂皮酰转移酶(hydroxycinnamoyl CoA quinate hydrocycinnamoyl transferase,HQT)基因真核表达载体以及建立金银花的遗传转化系统,阐明金银花HQT基因在真核生物细胞中对绿原酸量的调控机制。方法应用通路克隆系统技术构建HQT基因真核表达载体,利用根癌农杆菌介导法,构建含金银花HQT基因的工程菌,对转染成功的外植体进行愈伤诱导,进而进行半定量RT-PCR检测转基因愈伤组织中HQT基因的相对表达量以及利用HPLC检测其绿原酸的量,并对二者相关性进行分析。结果金银花愈伤组织中绿原酸的量随HQT基因表达量的升高而升高。结论金银花HQT基因在真核生物细胞内对绿原酸的生物合成具备调控作用。
Objective To construct the eukaryotic expression vector of hydroxycinnamoyl CoA quinate hydrocinnamoyl transferase (HQT) gene and establish the genetic transformation system of honeysuckle, and to elucidate the effect of HQT gene in eukaryotic cells on the amount of chlorogenic acid The regulatory mechanism. Methods The eukaryotic expression vector of HQT gene was constructed by using the pathway cloning system technology. The Agrobacterium tumefaciens-mediated method was used to construct the engineering bacteria containing the HQT gene of honeysuckle, and the callus induction was performed on the transplanted explants. Semi-quantitative RT The relative expression level of HQT gene in transgenic callus was detected by PCR and the amount of chlorogenic acid was detected by HPLC. The correlation between them was analyzed. Results The amount of chlorogenic acid in callus increased with the increase of HQT gene expression. Conclusion The honeysuckle HQT gene has a regulatory effect on the biosynthesis of chlorogenic acid in eukaryotic cells.