Overexpression and RNA interference of TwDXR regulate the accumulation of terpenoid active ingredients in Tripterygium wilfordii
To examine the putative regulatory role of TwDXR in terpenoid biosynthesis and terpenoid biosynthetic pathway-related gene expression, through overexpression and RNA interference with TwDXR.
We obtained 1410 and 454 bp TwDXR-specific fragments to construct overexpression and RNAi vectors. qRT-PCR was used to detect the expression of TwDXR and terpenoid biosynthesis pathway-related genes. The overexpression of TwDXR led to a 285% upregulation and the TwDXR RNAi led to a reduction to 26% of the control (empty vector-transformed cells) levels. However, pathway-related genes displayed different trends. When TwDXR was overexpressed, TwDXS expression decreased by 31% but increased to 198% when TwDXR expression was inhibited. The accumulation of terpenoids was also assayed. In the overexpression group, differences were not significant whereas the contents of triptolide and celastrol in the TwDXR RNAi samples were diminished by 27.3 and 24.0%, respectively.
The feedback regulation of gene transcription and the accumulation of terpenoids in terpenoid biosynthesis in Tripterygium wilfordii were verified by TwDXR overexpression and RNAi experiments.
KeywordsCelastrol Gene expression analysis Overexpression RNAi Terpenoid biosynthesis Triptolide TwDXR
This work was supported by the National Natural Science Foundation of China (81422053 and 81373906 to W.G., and 81325023 to L.H.); National High Technology Research and Development Program of China (863 Program: 2015AA0200908 to W.G.); the Support Project of High-Level Teachers in Beijing Municipal Universities in the Period of 13th Five-Year Plan (CIT&TCD20170324 to W.G.); and the Key Project at Central Government Level: The ability establishment of sustainable use for valuable Chinese medicine resources (2060302 to L.H.).
Supplementary Table 1—Primers used for RNAi expression vector construction.
Supplementary Table 2—Primers used for quantitative RT-PCR.
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