Study On Regulations Of HMGR And DXR Genes On Biosynthesis Of Terpenoids In Tripterygium Wilfordii Hook.f. Using RNAi

As a traditional pesticidal plant, Tripterygium wilfordii Hook.f. contains many secondary metabolites having agricultural activities. However, the extremely low contents and high level of difficulty by chemical synthesis severely constrain its widely application. Optimization and scale up of stable and high-production culture system and metabolic engeering of metabolic pathways provide an alternative strategy for this problem. In previous research,the hairy root culture has been established. A suppression subtractive hybridization(SSH) c DNA library was also successfully established with enriched genes related with Me JA elicitation and secondary metabolites biosynthesis and modulation,but the function of these genes remains to be validated.HMGR and DXR were considered as two key enzymes in the terpenoid biosynthesis pathway, so the genes expression of these two enzymes may directly affect the biosynthesis of terpenoids. RNA interference(RNAi) is exensively used for repressing of genes in functional genomies research. The principle of RNAi is that the homology-dependent mechanism is induced by ds RNA and results in the knockdown of gene function through the degradation of m RNA transcrips.Currently, RNAi becomes the reverse genetic technique for large-scale gene functional assays due to the specificity and efficiency.This reserch constucted the RNAi interfence expression vectors of Tw HMGR and Tw DXR by Gateway Cloning System and obtained the transgenic hairy roots of Tripterygium wilfordii Hook.f. through agrobaeterium-mediated transformation. The transgenic hairy roots phenotypes, the expressions of target genes and the contents of terpenoids were tested and analyzed to further validate the gene function and regulatory mechanism of Tw HMGR and Tw DXR. The mainresults are as follows:1,This experiment constructed the RNAi interfence expression vectors of Tw HMGR and Tw DXR by Gateway Cloning System and transferred them into Agrobacterium rhizogenes 15834.2,The hairy roots induced through agrobaeterium mediated method, and resistance selection,cultivation and PCR identification were used to screened the positive transgenic hairy root lines. The hairy roots appeared fast growth without adding any phytohormone into the medium and had a lot of branches and root hairs, both of which are classic characteristics of hairy root.3,Red fluorescence were detected in the transgenic hairy roots by fluorescent inverted microscope which therefore confirmed that the interference vectors were transferred into hairy roots and expressed.4,The results of Real-time PCR detecting showed that expression level of Tw HMGR in the three HMGR-RNAi transgenic lines were significantly reduced than contol.Same as the Tw DXR in the DXR-RNAi transgenic lines.The interference efficiency could reach 20%- 60%, which indicated that the RNAi effected the expression of target gene.5,The results of HPLC showed that the contents of triptolide, wilforgine, wilforine were decreased significantly in the transgenic hairy roots H-RNAi-1 and D-RNAi-3 compared with control.The contents of triptolide, wilforgine, wilforine in D-RNAi-3 were 13.24μg/g DW(34.61% of control), 243.83μg/g DW(51.30% of control), 81.36 μg/g DW(63.42% of control), respectively.The contents of triptolide, wilforgine, wilforine in H-RNAi-1 were17.81μg/g DW(46.55% of control),157.59μg/g DW(33.15% of control),40.84μg/g DW(31.83% of control). And in H-RNAi-1,the downtrend of the content of wilforgine and wilforine is higher than that of triptolide. Therefore it stated that the HMGR has a greater influence on the the synthesis of sesquiterpenes than diterpenes,and the DXR exhibited the opposite function.This research confirms the feasibility of large-scale ihp-RNA construction by Gateway Cloning System for gene silencing. This study revealed the regulations of HMGR and DXR genes on the bilsynthesis of terpenoids. This could not only supplied a reliable method for studying the functions of related genes in T. wilfordii Hook.f.,but also helped understanding the biosynthesis mechanism of sesquiterpene pyridine alkaloids.