Construction Of Expression Vector Driven By Tobacco Root-specific Promoter And Genetic Transformation Into Rhodiola Hairy Root

Plant cell-specific promoters can control exogenous genes to expression in particular tissue or organs, specific time, particular growing environment, and also can make sure the receptor plants grow regularly in the situation of the other part of receptor plants keep its physiological indexes intact, relying on the characteristics of cell-specific promoters,it is an effective way to use the cell-specific promoters correctly for plant gene engineering research.The root-specific promoter can drive exogenous genes to dysregulated in the root of the receptor plants, to reduce the waste which is caused by nonspecifical expression of the exopgenous genes in the receptor plants, and it can make exogenous genes expression in the root only in order to reach anticipation amount. Therefore, the root cell-specific promoters always get plant gene construction experts concentration, which achieve great progress.At present,make full use of the hairy root to product secondary metabolites is the most effective measure, because the short cycle of the hairy root,and have an genetic inherit stability, produce secondary metabolites matter which has high amount and is steady, which is_hormone autotrophic type included. Based above,we would take biotechnology measure to build up a high effective system of salidroside.The glycosylation reaction is catalyzed by glycosyltransferases and is the last step of biosynthesis from tyrosol to salidroside,and salidroside has been taken into effect in sports medicine,health medicine,Aerospace Medicine,army medicine. By now, how to increase the amount of salidroside from transgenetic hairy root become a hot topic. In the laboratory,the gene of glycosyltransferases was cloned from Rhodiola If we improve the glycosyltransferase gene expression, the salidroside in rhodiola hairy root would be also enhanced simultaneously. Therefore, in this study,we will use root-specific promoter of tobacco to drive the glycosyltransferase gene,and then excessive expression in rhodiola hairy root which was transformed.In this study,the 738bp fragment of root-specific promoter of tobaccoTobRB7 gene was cloned by PCR. The expression vector pCA-Tob7::UGTR,driven by tobacco root-specific promoter TobRB7, was constructed from pCAMBIA1301 by substituting the CaMV 35S promoter and GUS gene with TobRB7 and UGTR,a glycosyltransferase gene, respectively. The pCA-Tob7::UGTR vector was introduced into Agrobacterium tumefaciensstrain,and the explants of rhodiola hairy root were then transformed. PCR and PCR-Southern screening indicated that expression vector had integrated into rhodiola hairy root genome, the content of salidroside which is in transgenic hairy root is detected by HPLC.As a result, The specific expression vector and the rhodiola hairy root system was established,in the transgenic hairy root, the content of salidroside is 2.24 times larger than the wild type one's. The system is established successfully,it is not only settle theoretical foundation for researching plant cell-specific promoters,but also lead a new perspective in produce salidroside which is needed by humanity in large-scale by bioreactor.