紫杉醇合成途径中紫杉烯合成酶cDNA的克隆

cDNA Cloning of Taxadiene Synthase Functioning in Taxol Biosynthesis

【目的】研究紫杉烯合成酶超表达对紫杉醇代谢的影响,以提高红豆杉属植物细胞中紫杉醇的含量。【方法】从南方红豆杉(Taxus chinensis var.mairei)愈伤组织提取总RNA,根据已发表的中国红豆杉紫杉烯合成酶cDNA序列设计两对引物,通过RT-PCR技术扩增出该酶5′端长度为1 201 bp和3′端长度为1 388 bp的两个cDNA片段,将其克隆到pGEM-T Vector上并转化到E.coli DH5α中,5′端片段经XbaⅠ和BglⅡ双酶切鉴定,3′端片段经BglⅡ和SacⅠ双酶切鉴定后,对阳性克隆进行序列测定。两片段与双元载体pCAMBIA1300-35S::GUS以T4 DNA连接酶连接并转化到E.coli DH5α中,经XbaⅠ和SacⅠ双酶切鉴定。【结果】测序结果证实确为紫杉烯合成酶基因,酶切结果证实紫杉烯合成酶cDNA全长连接成功并插入到pCAMBIA130035S::GUS中。【结论】两cDNA片段拼接得到全长2 589 bp的紫杉烯合成酶基因,编码862个氨基酸,与中国红豆杉紫杉烯合成酶基因具有98%的同源性;成功构建了pCAMBIA130035S-TS载体,为下一步的转基因工作做好了准备。

[Objective] The objective was to study the effects of taxadiene synthase over expression in taxol metabolism, and to increase taxol production. [Method] Total RNA was extracted from Taxus chinensis var. mairei callus. Two primer pairs were designed according to the taxadiene synthase sequence of Taxus chinensis. 5＇- terminal 1 201 bp eDNA fragment and 3＇- terminal 1 388 bp eDNA fragment of taxadiene synthase were isolated by RT-PCR. The two eDNA fragments were cloned into pOEM-T vector and transformed into E. coli DH5α, After the 5＇- terminal fragment was confirmed by Xba Ⅰ and Bgl Ⅱ digestion and the 3＇- terminal fragment confirmed by BglⅡ and Sac Ⅰ digestion, they were sent to sequencing. After the two fragments and pCAMBIA1300-35S：：GUS vector were ligated and transformed into E. coli DH5α, they were digested with Xba Ⅰ and Sac Ⅰ . [Result] The sequences proved to be taxadiene synthase and the two fragments were successfully ligated and inserted into pCAMBIA1300-35S：：OUS. [Conclusion] The two fragments were ligated together as a 2 589 bp eDNA fragment, encoding 862 amino acid residues. The taxadiene synthase in T. chinensis var. mairei closely resembles the one in T. chinensis with 98% identity. pCAMBIA1300-35S-TS has been constructed and is ready to be transformed into cells.