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为进一步研究紫杉二烯合成酶的作用机理和紫杉醇生物合成代谢调控机制,采用RT-PCR技术从东北红豆杉(Taxus cuspidata)树叶中获得了紫杉二烯合成酶基因片段,将该片段克隆在载体pGEMT-easy vector上,并转化到E.coliJM109中,经EcoRI酶切鉴定后,对阳性克隆进行序列测定,获得紫杉二烯合成酶基因片段长度为909 bp,编码303个氨基酸,与GenBank中收录的紫杉二烯合成酶的同源性达到98%;对获得的紫杉二烯合成酶基因和紫杉醇产生菌HQD33基因组DNA进行了Southern杂交。结果初步证实了紫杉二烯合成酶存在于通过内生真菌发酵生产紫杉醇的生物合成途径中。为利用分子生物学手段研究通过内生真菌发酵生产紫杉醇生物合成的代谢调控和构建高产紫杉醇基因工程菌株提供了强有力的理论指导。
In order to further study the mechanism of action of taxadiene synthase and the mechanism of taxol biosynthesis and metabolism, a taxadiene synthase gene fragment was obtained from the leaves of Taxus cuspidata by RT-PCR. The fragment was cloned The vector was cloned into pGEMT-easy vector and transformed into E. coli JM109. After digestion with EcoRI, the positive clones were sequenced and the length of the taxadiene synthase gene was 909 bp, encoding 303 amino acids. GenBank contains taxadiene synthase 98% identity; obtained taxane synthase gene and paclitaxel producing bacteria HQD33 genomic DNA Southern hybridization. Preliminary results confirm the presence of taxadiene synthase in the biosynthetic pathway for the production of paclitaxel by endophytic fungi. It provides a powerful theoretical guidance for the molecular biology to study the metabolic regulation of paclitaxel biosynthesis by endophytic fungi fermentation and the construction of high yield paclitaxel gene engineering strains.