Agrobacterium rhizogenes-mediated RNAi of Tripterygium wilfordii and application for functional study of terpenoid biosynthesis pathway genes

Abstract

Tripterygium wilfordii is an important medical plants which is rich in several valuable bioactive terpenoids, such as triptolide and wilforine. Developing a fast and efficient transformation method is imperative for exploring these metabolites biosynthesis pathway by using RNA interference (RNAi) technology. In the present study, an efficient transformation system using Agrobacterium rhizogenes was established by optimizing four parameters including vacuum infection time, pre-culture duration, appropriate concentration of Agrobacterium, and co-cultivation duration. The transformation efficiency could reach to 62.22% by using leaf sections pre-cultured 5 days as explants, vacuum infection with Agrobacterium at optical density (OD600) of 0.6 for 3 min and co-cultivation for 3 d. The T. wilfordii 3-hydroxy-3-methylglutaryl- coenzyme A reductase (TwHMGR) RNAi hairy root was induced to validate the applicability of this system. The identification of transgenic hairy root was monitored by fluorescence microscopy and verified by PCR analysis. Real-time quantitative PCR analysis showed that the expression of TwHMGR was significantly decreased in transgenic hairy roots compared with control lines. The detection of metabolites showed that the silence of TwHMGR could highly decrease the content of triptolide and wilforine. These results indicated that this A. rhizogenes-mediated RNAi system is an effective protocol for characterizing the function of genes involved in the terpenoid biosynthetic pathway in Tripterygium wilfordii.