中国红豆杉细胞经重复诱导和蔗糖饲喂后云南紫杉烷C生产的相应基因表达变化

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

摘要:

红豆杉悬浮培养细胞具有可持续生产抗癌药物紫杉醇及其他紫杉烷的潜力。在中国红豆杉悬浮培养细胞中，云南紫杉烷 C(Tc) 是主要的次生代谢产物。为促使代谢前体由生成其他紫杉烷的代谢支路转到生产紫杉醇，实验采用实时定量PCR 技术 (RQ-PCR) 揭示细胞培养过程中紫杉醇及紫杉烷合成关键基因的动态变化。在细胞培养的第7 天和第12 天，以100 μmol/L 2,3-二羟丙基茉莉酸 (DHPJA) 进行诱导，同时在第7 天饲喂20 g/L 的蔗糖，在此过程中考察6 个关键基因 (TASY，TDAT，T5αH，TαH，T10βH 和T14βH) 的表达变化。上述联合调控手段使得Tc 产量在第1 次诱导8 d 后达 (554.46±21.28) mg/L，第2 次诱导9 d 后高达 (997.72±1.51) mg/L。代谢早期基因TASY 和TDAT 在第1 次诱导后表达量分别提高了182 和98 倍，在第2 次诱导后表达量分别提高了208 和131 倍。在每次诱导后基因表达量提高约持续24 h，之后下降。其他4 个基因 (T5αH、TαH、T10βH 和T14βH) 的情况有所不同。基因TαH 在2 次诱导后表达量分别提高了3 061 和1 016 倍。其他3 个基因T5αH、T10βH、T14βH 在第1 次诱导后表达量分别提高13、38、20 倍，在第2 次诱导后分别提高7、16、6 倍。RQ-PCR 结果表明基因表达和Tc 积累之间存在紧密相关性：基因表达的变化与Tc 产量的变化相一致，诱导可提高6 个基因的表达量。基因的高表达随着培养过程逐渐衰减，再次诱导可再次促使基因的高表达。

关键词:中国红豆杉; 云南紫杉烷C; 2; 3-二羟丙基茉莉酸; 实时定量PCR

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, T5αH, TαH, T10βH and T14βH) were quantified under the process condition of double elicitation by 2,3-dihydroxylpropanyl jasmonate (DHPJA) (100 μmol/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 T5αH, TαH, T10βH and T14βH. For gene TαH, 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 T5αH, T10βH, T14βH, 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.

Key words:Taxus chinensis; Taxuyunnanine C; 2;3-dihydroxylpropanyl jasmonate; Real-time Quantitative PCR