红豆杉内生真菌产紫杉醇相关基因BAPT的鉴定及初步研究

从几种红豆杉中先后分离了30余种内生真菌,深入研究了三种能够产紫杉醇的内生真菌。形态学观察及18srDNA鉴定它们分属于Fusarium(属)和Pestalotiopsis(属),三个菌株均可以在离体培养的条件下产生紫杉醇,经两周培养产量可分别达到8.5,31.5,31.1μg/L。(其中Pestalotiopsis1分离于南方红豆杉,Fusarium1分离于东北红豆杉,Pestalotiopsis2分离于中国红豆杉)。对这些内生真菌产紫杉醇的初步机理作了研究。BAPT(C-13phenylpropanoidsidechain-CoAacyltransferase)是红豆杉中紫杉醇合成途径里支链合成的关键酶之一,我们根据其保守区序列设计了引物,首次在能产生紫杉醇的上述三种红豆杉内生真菌中克隆得到了BAPT基因片段,而分离的其它真菌并没有得到扩增。序列分析表明,来自内生真菌的BAPT基因片段序列与红豆杉BAPT基因片段序列具有非常高的相似性(98.9%)。推测红豆杉内生真菌之所以能够合成紫杉醇,相关基因可能直接源于其宿主植物,即其遗传学起源是基因转移而不是共进化。这同时也建立了一种快速经济的鉴定产紫杉醇真菌的辅助方法。内生真菌的遗传稳定性及改良在进一步研究中。

Identification and Pilot Study of Taxus Engophytic Fungi's Taxol-producing Correlation Gene BAPT

Three fungi that have the capability to produce taxol had been screened out from more than 30 endophytic fungi isolated from several taxus species. Via 18S rDNA sequence analysis and morphologic observation, the three fungi were primarily identified as species of Fusarium and Pestalotiopsis ( Pestalotiopsis 1 separated from Taxus Chinesis var mairei, Fusarium1 from Taxus cuspidate, and Pestalotiopsis2 from Taxus chinensis ). A pair of primers were designed to amplify for the first time from fungi a fragment of the gene coding BAPT (C-13phenylpropanoid side chain-CoA acyltransferase), one of the key enzymes in the taxol biosynthesis pathway. Sequence analysis showed high similarity(up to 98.9%) between fungal BAPT gene fragments and the counterparts from their yew hosts. The extraordinarily high sequence similarity indicated that the endophytic fungi might directly gain genes involved in taxol biosynthesis from their hosts and thus gain the ability to synthesize taxol, rather than a coevolution mechanism. Meanwhile, we had established an easy and economical method to screen taxol-producing endophytic fungus. An in-depth research of the mechanism and stability of such gene transfer from host to their endophytic fungus is under way.