Chuanshu Zhu

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 messageTwMDR1 transports sesquiterpene pyridine alkaloids, wilforine and wilforgine, into the hairy roots of T. wilfordii Hook.f. resulting in low secretion ratio of alkaloids.AbstractHairy 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

Abstract 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.

Cloning, Expression Analysis and Functional Characterization of Squalene Synthase (SQS) from Tripterygium wilfordii

Celastrol is an active triterpenoid compound derived from Tripterygium wilfordii which is well-known as a traditional Chinese medicinal plant. Squalene synthase has a vital role in condensing two molecules of farnesyl diphosphate to form squalene, a key precursor of triterpenoid biosynthesis. In the present study, T. wilfordii squalene synthase (TwSQS) was cloned followed by prokaryotic expression and functional verification. The open reading frame cDNA of TwSQS was 1242 bp encoding 413 amino acids. Bioinformatic and phylogenetic analysis showed that TwSQS had high homology with other plant SQSs. To obtain soluble protein, the truncated TwSQS without the last 28 amino acids of the carboxy terminus was inductively expressed in Escherichia coli Transetta (DE3). The purified protein was detected by SDS-PAGE and Western blot analysis. Squalene was detected in the product of in vitro reactions by gas chromatograph-mass spectrometry, which meant that TwSQS did have catalytic activity. Organ-specific and inducible expression levels of TwSQS were detected by quantitative real-time PCR. The results indicated that TwSQS was highly expressed in roots, followed by the stems and leaves, and was significantly up-regulated upon MeJA treatment. The identification of TwSQS is important for further studies of celastrol biosynthesis in T. wilfordii.