title: Expression profiling of genes involved in Taxuyunnanine C biosynthesis in cell suspension cultures of Taxus chinensis by repeated elicitation with a newly synthesized jasmonate and sucrose feeding.
abstract: Taxus suspension cell culture has the potential to provide a sustainable source of anticancer drug paclitaxel (Taxol) and other taxoids. In the cell culture of Taxus chinensis, Taxuyunnanine C (Tc) is the primary taxoid. To design a rational strategy for redirecting the precursor fluxes from other taxoids into paclitaxel production, we employed Real-time Quantitative PCR (RQ-PCR) to understand the dynamic profiling of key biosynthetic pathway genes of palcitaxel and taxoids during the culture process. Six genes (TASY, TDAT, T5alphaH, TalphaH, T10betaH and T14betaH) were quantified under the process condition of double elicitation by 2,3-dihydroxylpropanyl jasmonate (DHPJA) (100 micromol/L on day 7 and day 12), and sucrose feeding (20 g/L) on day 7. This process treatment led to a high accumulation of Tc at (554.46 +/- 21.28) mg/L 8 days after the first elicitation. Then 9 days after the second elicitation, Tc production was as high as (997.72 +/- 1.51) mg/L. The early pathway genes TASY and TDAT were significantly up-regulated by 182-fold and 98-fold, respectively for the first DHPJA elicitation and by 208-fold and 131-fold, respectively for the second elicitation. The induction occurred after each elicitation lasted for about 24 h before their abundances decreased. Things are somewhat different in the case of the other four genes T5alphaH, TalphaH, T10betaH and T14betaH. For gene TalphaH, it was highly up-regulated by 3061-fold for the first DHPJA elicitation and by 1016-fold for the second elicitation. For the other three genes T5alphaH, T10betaH, T14betaH, they were up-regulated by 13-fold, 38-fold and 20-fold, respectively for the first DHPJA elicitation and by 7-fold, 16-fold and 6-fold, respectively for the second elicitation. The RQ-PCR results showed that there is tight correlation between gene expression and Tc accumulation. Gene expression was in accordance with Tc yield. Elicitation could improve expression of six genes. While along with culture course, high expression of the genes weakened. Elicitation for the second time would promote high expression of the genes again.
authors.affiliation: Key Laboratory of Biochemical Engineering (State Ethnic Affairs Commission-Ministry of Education), Dalian 116600, China. lily@dlnu.edu.cn
authors.forename: Mingbo
authors.initials: M
authors.lastname: Gao
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Xingtai
authors.initials: X
authors.lastname: Li
authors.forename: Chengjiang
authors.initials: C
authors.lastname: Ruan
authors.forename: Shengdi
authors.initials: S
authors.lastname: Fan
mesh_terms: D018929:Cell Culture Techniques; D003470:Culture Media; D003517:Cyclopentanes / Q000494:pharmacology*; D018506:Gene Expression Regulation, Plant; D054883:Oxylipins / Q000494:pharmacology*; D010937:Plant Growth Regulators / Q000494:pharmacology; D013395:Sucrose / Q000494:pharmacology*; D043823:Taxoids / Q000378:metabolism*; D020946:Taxus / Q000235:genetics* / Q000378:metabolism*; D059467:Transcriptome
chemical_list: D003470:Culture Media; D003517:Cyclopentanes; D054883:Oxylipins; D010937:Plant Growth Regulators; D043823:Taxoids; C494600:taxuyunnanine C; D013395:Sucrose; C011006:jasmonic acid