红豆杉香叶基香叶基焦磷酸合成酶基因克隆分析

CLONING AND CHARACTERIZATION OF THE GERANYLGERANYL DIPHOSPHATE SYNTHASE GENE IN TAXUS WALLICIANA

20碳单位的香叶基香叶基焦磷酸是紫杉醇生物合成的直接前体,它由香叶基香叶基焦磷酸合成酶催化生成。采用RT-PCR技术从高原植物西藏红豆杉中克隆了编码香叶基香叶基焦磷酸合成酶基因,命名为TwGGPPS(Gen- Bank登录号:DQ 364604)。该基因编码区长为1 182 bp,编码393个氨基酸的多肽;生物信息学分析表明TwGGPPS定位于质体,在其氨基端具有长为18个氨基酸的质体转运肽;TwGGPPS属于异戊烯基转运酶家族,具有该酶家族特有的2个天冬氨酸富集基序;分子系统进化分析表明植物的GGPPS分为被子植物和裸子植物2种类型,TwGGPPS属于裸子植物类型的GGPPS;对TwGGPPS的蛋白质结构模拟分析结果表明,TwGGPPS由大量的α-螺旋通过随机卷曲连接而成,这些α-螺旋环绕形成一个袋状空腔,其空腔口部为底物结合与碳链延伸的活性部位。对TwGGPPS基因组结构的分析表明该基因没有内含子。该基因的克隆和分析为实现紫杉醇的基因工程提供了一个重要的基因和作用靶点。

RT-PCR was performed to clone the functional gene encoding GGPPS （geranylgeranyl diphosohate synthase） from Tibet specific yew （ Taxus wallichiana）, which is designated as TwGGPPS （ GenBank Accession number： DQ 364604）. TwGGPPS is 1183 bp in length encoding a 393-amino acid pelypeptide. There exists an 18-amino acid plastidial transit peptide in the N-terminus and is followed by the catalytic region. In the catalytic region of TwGGPPS, two Asp-rich motifs axe found that are considered as the active sites for mediate binding and C-chain extension. Phylogenetic analysis demonstrated that the plant GGPPSs had two groups： one is angiosperm GGPPS and the other is gymnosperm GGPPS and that TwGGPPS belongs to the group of the latter. When the homology-based modeling was applied to construct the 3-D structure of TwGGPPS, it was found that TwGGPPS＂ is composed of numerous α-helixes connected with random coils. The helixes are circled to form a cave in which the Asp-rich motifs axe localized. The genomic DNA sequence of TwGGPPS was isolated and proved to be the same as that from cDNA, thus suggesting that TwGGPPS is an intron-free gene.