The Impact Of The Overexpression Of Genes HMGR And DXR In Terpenes Biosynthesis In Tripterygium Wilfordii Hook. F.

Tripterygium is a perennial vine and Tripterygium in the Celastraceae family. In our country it has been used as traditional medicine for long. Modern scientific researches show that it can produce a variety of secondary metabolites with different biological activities, such as triptolide, triptolide alkaloids, which has the efficacy of anti-tumor. Meanwhile, in agriculture some of them can control a variety of pests with stomach poisoning, poor feeding, tag and so on. But these secondary metabolites contents in Tripterygium are very little,which is difficult to meet the medical and agricultural demands.The system of tripterygium hairy roots for the production of secondary metabolites is good means to increase the production. This study starts with the cloning of genes DXR and HMGR which are the key controlling synthases of terpene in tripterygium and destination plasmids are transferred into tripterygium hairy roots by overexpression technology and ends with detections of gene expression through RT-PCR and metabolites contents though HPLC, the main findings are founded as followings:1)With the premiers designed by Gateway the full-length of Tw DXR is 1709 bp, containing a 1410 bp open reading frame that encodes a peptide of 469 amino acids and the full-length of Tw HMGR is 1909 bp, containing a 1860 bp open reading frame that encodes a peptide of 579 amino acids;2) Through the Gateway technology,the entry vectors and the destination vectors of Tw DXR and Tw HMGR are built and transferred into agrobacterium which can induce hairy roots. After 3 times subculture, the genomic DNA is extracted and after verification it is got as 6 overexpression hairy root systems named as DXR-1, DXR-2,DXR-3,HMGR-1, HMGR-2 and HMGR-3;3)Detections of the expression of these two genes Tw DXR and Tw HMGR in tripterygium hairy root with specific primers are as below:Setting the ordinary tripterygium hairy roots as control, the real-time quantitative PCR results showed that these overexpression systems of Tw HMGR and Tw DXR has improved significantly: the relative expressions of DXR-1、DXR-2 and DXR-3 are3.66、4.81 and 5.81 times of control;the relative expressions of HMGR-1、 HMGR-2 and HMGR-3 is 4.73、3.48 and 2.68 times of control.4) Metabolites contents of the 6 systems though HPLC are as below:The content of triptolide、wilforgine and wilforine of DXR-1 is 100.36 μg/g, 604.53 μg/g and 99.86 μg/g, which is 1.49 times、1.38 times and 1.14 times of the control; The content of triptolide、wilforgine and wilforine of DXR-2 is 153.76 μg/g, 570.97μg/g and 128.30μg/g which is 2.28 times、1.30 times and 1.47 times of the control; The content of triptolide、wilforgine and wilforine of DXR-3 is 160.39 μg/g, 534.08 μg/g and 117.68 μg/g, which is 2.38 times and 1.35 times 1.22 times of the control. The content of triptolide、wilforgine and wilforine of HMGR-1 is 98.50 μg/g, 509.36μg/g and 210.58μg/g, which is 1.46 times、1.16 times and 2.41 times of the contro; The content of triptolide、wilforgine and wilforine of HMGR-2 is 71.29μg/g、390.86μg/g and 130.92μg/g, which is 1.06 times、0.89 times and 1.50 times of the control; The content of triptolide、wilforgine and wilforine of HMGR-3 is 84.97 μg/g,454.28 μg/g and 124.85 μg/g, which is 1.26 times、1.43 times 1.04 times of the control;The results that the relative expression and contents of 3 secondary metabolites both have improved showed that Tw DXR and Tw HMGR genes are the key genes to the metabolic pathways of terpenes in tripterygium,gained more to the study of the further research to improve the production of secondary metabolites.