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.

A MDR transporter contributes to the different extracellular production of sesquiterpene pyridine alkaloids between adventitious root and hairy root liquid cultures of Tripterygium wilfordii Hook.f.

KEY MESSAGE: TwMDR1 transports sesquiterpene pyridine alkaloids, wilforine and wilforgine, into the hairy roots of T. wilfordii Hook.f. resulting in low secretion ratio of alkaloids. Hairy roots (HRs) exhibit high growth rate and biochemical and genetic stability. However, varying secondary metabolites in HR liquid cultures mainly remain in root tissues, and this condition may affect cell growth and cause inconvenience in downstream extraction. Studies pay less attention to adventitious root (AR) liquid cultures though release ratio of some metabolites in AR liquid cultures is significantly higher than that of HR. In Tripterygium wilfordii Hook.f., release ratio of wilforine in AR liquid cultures reached 92.75 and 13.32% in HR on day 15 of culture. To explore potential roles of transporters in this phenomenon, we cloned and functionally identified a multidrug resistance (MDR) transporter, TwMDR1, which shows high expression levels in HRs and is correlated to transmembrane transportation of alkaloids. Nicotiana tabacum cells with overexpressed TwMDR1 efficiently transported wilforine and wilforgine in an inward direction. To further prove the feasibility of genetically engineered TwMDR1 and improve alkaloid production, we performed a transient RNAi experiment on TwMDR1 in T. wilfordii Hook.f. suspension cells. Results indicated that release ratios of wilforine and wilforgine increased by 1.94- and 1.64-folds compared with that of the control group, respectively. This study provides bases for future studies that aim at increasing secretion ratios of alkaloids in root liquid cultures in vitro.

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.

Effects of plant stress signal molecules on the production of wilforgine in an endophytic actinomycete isolated from Tripterygium wilfordii Hook.f.

The endophytic actinomycete F4-20 was isolated from Tripterygium wilfordii Hook.f. and was confirmed to produce wilforgine, a secondary metabolite discovered in its host. F4-20 showed a close phylogenetic relationship to Streptomyces species. To seek elicitors that may enhance the production of wilforgine in F4-20, four plant stress molecules were applied to the in vitro liquid cultures. Results showed that methyl jasmonate (MeJA), salicylic acid (SA), and hydrogen peroxide (H2O2) inhibited bacterial growth, whereas glutathione (GSH) treatment significantly increased bacterial growth. The wilforgine contents in the mycelia of F4-20 were reduced by MeJA and GSH but were induced by SA and H2O2. When added in the end of the culture period (7 day), 1 mM SA and 5 mM H2O2 resulted in 69.35 ± 1.71 and 71.80 ± 3.35 µg/g DW of wilforgine production, 1.55 and 1.60 fold to that of control (44.83 ± 1.35 µg/g DW), respectively. Though this improved production was about 6.5 times lower than that of the natural root (454.00 µg/g dry root bark), it provided an alternative method for the production of valuable plant secondary metabolites.