Tripterygium wilfordii subsp. suspension Hook.

4.7. Gene expression level network analysis for celastrol in T. wilfordii

The gene expression levels of all CYP450s and Tw OSCs in different T. wilfordii tissues were chosen for network regulation analysis associated with celastrol levels in different T. wilfordii tissues. Celastrol levels in different tissues were detected as previously described (Zhou et al., 2019). The celastrol level and gene expression data were normalized separately, and correlation network analysis was conducted with Cytoscape software (version 3.6.1). The Pearson correlation coefficient was calculated via the PCC method on the R platform between each set of variables (either metabolite or gene) across the profiles, and significant positive correlations with a p-value <0.05 were detected between the genes and celastrol.

4.8. RNAi knockdown and overexpression of TwCYP712K 1 in T. wilfordii suspension cells

For the RNAi knockdown of TwCYP712K1, the least homologous region of TwCYP712K1 was selected based on cDNA alignment, and specific primers were then designed according to the sequence of the region (Supplementary Table S4). A specific 300–500 bp fragment was amplified and inserted into the pK7GWIWG2D vector (Invitrogen) using the Gateway cloning system (Invitrogen). For the overexpression of TwCYP712K1, the full-length sequence of TwCYP712K1 was amplified and subcloned into the pH7WG2D vector (Invitrogen). The resulting vectors were separately transformed into T. wilfordii suspension cells based on particle bombardment-mediated transformation. The detailed processes of suspension cell preparation and transformation were the same as in a previous report (Zhao et al., 2017). The pK7GWIWG2D and pH7WG2D vectors were separately transformed into T. wilfordii suspension cells as controls, and at least three biological duplicates were included for all samples. The cell suspensions were cultured for another 7 days after transformation, and real-time PCR and UPLC analyses were conducted to measure gene expression and celastrol levels, respectively. The detailed process of UPLC analysis was the same as that described in our previous report (Zhou et al., 2019).