紫杉醇及其前体在中国红豆杉植株中合成和积累部位探讨

Sites of Taxol and Its Precursor Biosynthesis and Accumulation in Taxus chinensis

研究紫杉醇及其重要前体物质巴卡亭Ⅲ在中国红豆杉(Taxus chinenesis)中合成和积累部位,通过HPLC分析代谢产物在中国红豆杉植物中的空间分布,定量PCR技术研究代表紫杉醇合成途径中不同步骤的6种关键酶基因在植株中的表达特征。结果表明,巴卡亭Ⅲ主要分布在针叶中,而紫杉醇主要分布在树皮和树根皮中;针叶中,紫杉醇代谢途径上游步骤的关键酶基因表达水平较高,树皮中催化紫杉醇侧链连接的酶转录表达水平高,而树皮中能检测到较高含量的6种关键酶基因的表达。表明紫杉醇的骨架结构主要在针叶中合成,而树皮中转化这些前体物质生成紫杉醇的能力较强,树根中含有完整的紫杉醇代谢酶系,暗示根组织培养是生产紫杉醇很有前景的方法。进一步建立了组织扦插培养红豆杉离体组织体外生产紫杉醇的系统。在茉莉酸甲酯的诱导下,红豆杉针叶在体外能够生产紫杉醇;树皮中由于缺乏足够的前体物质,离体培养中紫杉醇含量不断下降;枝叶培养中,产生的紫杉醇主要集中在树皮中。上述研究结果暗示,紫杉醇及其前体物质在中国红豆杉体内存在转移现象。

Location of taxol and its important precursor baccatin biosynthesis and accumulation in Taxus chinensis intact plants was studied. The transcription expression characteristics of genes encoding six enzymes in taxol biosynthesis were investigated by real time PCR, using 18S rDNA as internal standard. In six enzymes,3-hydroxy-3-methylglutaryl-Coenzyme A reductase （HMGR） and 1-deoxy-D-xylulose-5-phosphate reducto-isomerase （DXR） were the dedicated enzyme in mevalonate and no-mevalonate pathway,and play important role in the biosynthesis of the terpenoid precursor isopentenyl diphosphate （IPP）; geranylgeranyl diphosphate synthase （GGPPS） and taxadiene synthase （TS） were responsible for the carbon skeleton formation of taxol; cytochrome P450 taxane-10β-hydroxylase （C10 β-hydroxy- lase） and 10-deacetyl baccatin - 10-acetyltransferase （DBAT） catalyze the decoration of taxol core structure and functional group connection, respectively. Data showed that baccatin mainly located in fresh needle, and taxol mainly accumulated in bark and root; a highly transcript of genes encoding the enzymes in responsible to the upstream pathway of taxol biosynthesis were found in fresh needle; dabt which en- coding the downstream enzyme was mainly detected in bark transcripts of genes encoding all six enzymes were abundant in root. Results indicated that the precursors of taxol was mainly formed in needles; the remaining downstream reaction, was mainly run in bark; and root contained whole enzymes for taxol biosynthesis, which suggested that root culture for taxol production is a promising strategy. And then,cuttage tissue culture for in vitro taxol production was designed. Data indicated that needle could produce taxol induced by methyl jasmonate; the taxol content in bark （in vitro） decreased due to no enough pre cursors. And the taxol produced in twig cuttage tissue culture system was accumulated mainly in bark. Resuhs indicated that taxol and its precursors translocated in intact yew plant.