联合调控对中国红豆杉细胞关键酶基因表达的影响  

Expression Profiling of Genes Involved in Taxuyunnanine C Biosynthesis in Cell Suspension Cultures of Taxus chinensis by Repeated Elicitation with a Newly Synthesized Jasmonate,in situ Absorption and Sucrose Feeding

作　　者：高明波[1,2] 张卫 李兴泰[2] 阮成江[1,2] 范圣第[1] 

机构地区：[1]生物化学工程国家民委一教育部重点实验室,大连116600 [2]大连民族学院生命科学学院,大连116600 [3]澳大利亚Flinders大学海洋分子生物过程及生物产品实验室,阿德莱德SA5042

出　　处：《中国生物工程杂志》2010年第8期31-36,共6页China Biotechnology

基　　金：国家自然科学基金(20676130)资助项目

摘　　要：红豆杉悬浮培养细胞可以持续提供抗癌药物紫杉醇及一些紫杉烷类。在中国红豆杉悬浮培养细胞中,云南紫杉烷C(Tc)是主要的紫杉烷。为了更理性地调控紫杉醇或有用紫杉烷的生产,有必要深入了解其生物合成过程。采用实时定量PCR(Real-time Quantitative PCR,即RQPCR)技术考察经调控后紫杉醇及紫杉烷代谢中关键酶基因—TASY,T5αH,TDAT,T10βH,TαH,T14βH表达水平的变化。在细胞培养的第7天和12天,分别以100μmol/L2,3-二羟丙基茉莉酸(DHPJA)诱导,同时在细胞培养第7天进行20g/L蔗糖饲喂、100g/LXAD-7HP的原位吸附。该联合调控处理使得细胞培养第30天时,Tc产量高达1517±37mg/L,是对照处理的11.1倍,是DHPJA重复诱导联合蔗糖饲喂处理的1.7倍。RQ-PCR结果显示:DHPJA的加入可使6个基因表达水平显著提高,但在12小时后快速下降,需补充DHPJA以再次提高基因表达水平。吸附剂同时引入会延缓基因表达水平的提高速度,但却能维持基因表达处于一个较高的水平,表现为在细胞培养中后期,基因表达水平将显著高于无吸附剂的调控体系。与13α-羟化相对应的TαH基因有所不同,吸附剂的存在更显著地抑制其表达,但仍有维持表达的功能。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. Quantitative real-time-PCR （RQ-PCR） to understand the dynamic profiling of key biosynthetic pathway genes of palcitaxel and taxoids during the culture process was employed. Six genes--TASY, TDAT, TSαH, TαH, TDAT and T10βH were quantified under a process condition of double elieitation by 100μmol/L 2,3-dihydroxylpropanyl jasmonate （DHPJA） on day 7 and day 12, 20g/L sucrose feeding and 100g/L XAD-7HP absorption on day 7. This process treatment led to a high accumulation of Tc at 1517 ± 37mg/L on day 30,11.1 folds of that of control and 1.7 folds of that of double elieitation with sucrose feeding. The RQ-PCR results showed that with DHPJA elicitation, the expression of all the six genes was greatly increased, but sharply declined after 12 hours. This rapid decline suggested that the second DHPJA elicitation was required to sustain gene expression. Simultaneous absorbents addition would delay the improvement of gene expression but maintain the gene expression at a higher level, resulting in higher gene expression over treatments without absorbents. TαH expression was somewhat different which was markedly inhibited by XAD-7HP absorption while absorbants still maintained its expression.

关 键 词：中国红豆杉 云南紫杉烷C 2 3-二羟丙基茉莉酸 XAD-7HP 实时定量PCR 

分 类 号：Q786[生物学—分子生物学]