| As a traditional pesticidal plant, Tripterygium wilfordii Hook.f. contains many secondary metaboliteshaving agricultural activities. However, the extremely low contents and high level of difficulty by chemicalsynthesis severely constrain its widely application. Optimization and scale up of stable and high-productionculture system and metabolic engeering of metabolic pathways provide an alternative strategy for thisproblem. In previous studies, in vitro culture systems of T. wilfordii Hook.f. had not been optimized andscaled up, on the other hand, the lackage of genes involved in active metabolites biosynthesis wasrestricting the application of metabolic engineering. This study systematically studied on these two subjectsand got the following results and conclusions:1. The suspension cell aggregate cultures were established to examine the relationship of cellaggregate size and type with the accumulation of active secondary metabolites. Results showed that thoughgrew fast, some kinds of aggregate cells contained little content of metabolites that far less than that in thenatural growing plants. Some green cell aggregates had higher production, however, grew too slow to beapplied for industrial production.2. Growth and production characterization of adventitious root of T. wilfordii Hook.f. wasconducted based on the construction of adventitious root culture system.The contents of triptolide,wilforgine and wilforing were higher than that in aggregate cells. MeJA could increase metaboliteproduction. The adsorption ratio of XAD-7for the three metabolites was high. Resin not only protectedalkaloids but also further increased the production maybe by decreasing the feedback inhibition. Theproductions of triptolide and wilforgine (652.14，1378.70μg/flask, respectively) were far more higher thannatural growing roots (132.77，454.00μg/g, respectively). The culture system described here was believedas the most successful one among others. Based on the flask experiment results, a modified bubble columnbioreactor was designed and applied. Similar results with the flask experiments were observed. However,adventitious root didn’t grow as well as in flasks, so, further experiments should be done for better rootgrowth and higher production.3. A suppression subtractive hybridization (SSH) cDNA library was successfully established withenriched genes related with MeJA elicitation and secondary metabolites biosynthesis and modulation.Because samples were treated by MeJA for as long as12days, many genes located on the downstream ofbiosynthesis pathway were enriched in this library, such as CYP450enzyme genes. In the library,11genesputatively involved in the biosynthesis of terpenoids and sesquiterpene pyridine alkaloids,9transcription factors putatively involved in regulation of biosynthesis and2putatively involved in secondary metabolitestransportion were identified. MeJA treatment changed the expressions of these genes significantlyindicating the relationship of these genes with MeJA elicitation. With this library, secondary metabolitebiosynthesis genes could be easily obtained by exclusion of paralogous genes.4. By prokaryotic expression and protein purification, fusion protein of TwHf714with His tags wasgot. For the lack of intermediates of triptolide biosynthesis, an innovative method of using crude extract asenzymatic substrates was applid. Experiment results showed that TwHf714could catalyze a chemical inmethylene dichloride and methanol (9:1, v/v) extract into a product with longer retention time. Thesubstrate and product did not emerge at the time of triptolide indicating TwHf714may not participate intriptolide biosynthesis as expected. To further identify the structures LC-MS analysis was conducted.However, though some character ion fragments of pentacyclic triterpene were observed, the specificmolecular ion peaks were not identified due to the complexity of the samples. The pentacyclic triterpenesalaspermic acid has the structure of five-membered oxoheterocyclic ring and may be formed by thecatalization of TwHf714. But further experiments should be done to prove this hypothesis.5. Using RNAi technology, the function of TwHfCYP-1, homological to TwHf362, wascharacterized. Experiment results showed that the level of TwHfCYP-1expression in interference linesdecreased similar extent with that of alkaloids contents, which indicated that TwHfCYP-1participated in acommon step. This study could not only helped understanding the biosynthesis mechanism of sesquiterpenepyridine alkaloids but also supplied a reliable method for studying gene functions for T. wilfordii Hook.f..6. Bioinformatical analysis showed that TwMDR1(TwHf70) located on plasma membrane of roottissue and mediated alkaloids influx. The release ratio of wilforine in adventitious root (AR) liquid cultureswas significantly higher than that of hairy root (HR). The transcript of TwMDR1in HR was3.56timeshigher than that in AR. However, the expression difference of TwMATE1, another transporter putativelyfunctioned as alkaloids efflux, was not significant indicating the lower release ratio of wilforine in HR maybe caused by higher expression of influx transporter, but not lower expression of efflux transporter.Additionally, MeJA could induce the expressions of both transporters. A TwMDR1/TwMATE1expressionratio change was basically coincident with that of wilforine influx transportation indicating a combinedeffect might exist.To sum up, this study established a complete set of solutions for industrial production of activemetabolites in T. wilfordii Hook.f., primarily illuminated the biosynthesis pathways of triptolide andsesquiterpene pyridine alkaloids. This study will guide industrial production and provide not onlyinformation but also protocols for further metabolic engineering. |
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