Identification and functional characterization of a PDR transporter in Tripterygium wilfordii Hook.f. that mediates the efflux of triptolide.

KEY MESSAGE: TwPDR1, a PDR transporter from Tripterygium wilfordii Hook.f., was proved to efflux triptolide and its stability could be enhanced by A1033T mutation. Triptolide, an abietane-type diterpene in Tripterygium wilfordii Hook.f., possesses many pharmacological activities. However, triptolide is in short supply and very expensive because it is present at low amounts in natural plants and lack alternative production methods. Transporter engineering, which increases the extracellular secretion of secondary metabolites in in vitro culture systems, is an effective strategy in metabolic engineering but is rarely reported. In this study, TwPDR1, a pleiotropic drug resistance-type ATP binding cassette transporter, was identified as the best efflux pump candidate for diterpenoids through bioinformatics analysis. TwPDR1 was located in the plasma membrane, highly expressed in adventitious roots, and induced by methyl jasmonate. The triptolide efflux function of TwPDR1 was confirmed by transient expression in tobacco BY-2 cells and by downregulation via RNA interference in the native host. However, the overexpression of TwPDR1 had a limited effect on the secretion of triptolide. As shown by previous studies, a single amino acid mutation might increase the abundance of TwPDR1 by increasing protein stability. We identified the A1033 residue in TwPDR1 by sequence alignment and confirmed that A1033T mutation could increase the expression of TwPDR1 and result in the higher release ratio of triptolide (78.8%) of the mutants than that of control (60.1%). The identification and functional characterization of TwPDR1 will not only provide candidate gene material for the metabolic engineering of triptolide but also guide other transporter engineering researches in the future.

Validation of suitable reference genes for quantitative gene expression analysis in Tripterygium wilfordii.

Validation of suitable reference genes is critical in quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Suitable and reliable reference genes for the normalization of gene expression data are characterized by high gene expression stability across tissues and different experimental conditions. This study evaluated the gene expression stability of ten reference genes commonly used in Arabidopsis thaliana for their suitability in qRT-PCR analysis in Tripterygium wilfordii Hook.f. The orthologous sequences of these ten candidate genes were identified from T. wilfordii transcriptomic data (Project No. SRX472292). Five algorithms including GeNorm, NormFinder, BestKeeper, ΔCt, and RefFinder were used to assess the gene expression stability of these putative reference genes in different plant tissues and different stress conditions. The results identified ACTINT7 and TBP as the most suitable reference genes across all samples. The gene expressions of TwHMGR (3-hydroxy-3-methylglutaryl coenzyme A reductase, KU246037.1) and of TwDXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase, KJ174341.1) were investigated to validate the suitability of the reference genes. The validation analysis confirmed the suitability of ACTINT7 and TBP as the best reference genes for elucidating secondary metabolite biosynthesis pathway in T. wilfordii. In summary, this study identified the most suitable and reliable reference genes for future qRT-PCR- based studies in T. wilfordii.

Differential expressed analysis of Tripterygium wilfordii unigenes involved in terpenoid backbone biosynthesis.

Tripterygium wilfordii Hook. f. is the traditional medicinal plants in China. Triptolide, wilforgine, and wilforine are the bioactive compounds in T. wilfordii. In this study, the contents of three metabolites and transcription levels of 21 genes involved in three metabolites biosynthesis in T. wilfordii were examined using high-performance liquid chromatography and reverse transcription PCR after application of methyl jasmonate (MeJA) on hairy roots in time course experiment (3-24 h). The results indicated that application of MeJA inhibited triptolide accumulation and promoted wilforgine and wilforine metabolites biosynthesis. In hairy roots, wilforgine content reached 693.36 µg/g at 6 h after adding MeJA, which was 2.23-fold higher than control. The accumulation of triptolide and wilforine in hairy roots increased the maximum at 9 h, which was 1.3- and 1.6-folds more than the control. Most of the triptolide secretes into the medium, but wilforgine and wilforine cannot secrete into the medium. The expression levels of unigenes which involved terpenoid backbone biosynthesis exist the correlation with marker metabolites (triptolide, wilforgine and wilforine) after induction by MeJA, and can be then used to infer flux bottlenecks in T. wilfordii secondary metabolites accumulation. These results showed that these genes may have potential applications in the metabolic engineering of T. wilfordii metabolites production.