关键词: 南方红豆杉, 紫杉醇, 紫杉烯合成酶, cDNA克隆

Abstract: Total RNA was extracted from Taxus chinensis var. mairei callus. According to taxadiene synthase sequence of Taxus chinensis, two primer pairs were designed. The 5’- terminal 1201 bp cDNA fragment and the 3’- terminal 1388 bp cDNA fragment of taxadiene synthase were isolated by RT-PCR. The two cDNA fragments were cloned into pGEM-T vector and transformed to E. coli DH5α. After the 5’- terminal fragment was confirmed by digesting with XbaⅠand BglⅡand the 3’- terminal fragment with BglⅡand SacⅠ, they were sequenced and proved to be taxadiene synthase. The two fragments were ligated together and gave a 2589 bp cDNA fragment, encoding 862 amino acid residues. The taxadiene synthase of T. chinensis var. mairei most closely resembles the one of T. chinensis (98% identity). The two fragments and pCAMBIA1300 vector were ligated and transformed into E. coli DH5α. After digested with XbaⅠand SacⅠ, the two fragments were proved to be successfully ligated and inserted into pCAMBIA1300, which was prepared for being transformed into the cells.

Key words: Taxus chinensis var. mairei, taxol, taxadiene synthase, cDNA cloning