曼地亚红豆杉植株中GGPP合成酶的克隆与分析

Cloning and Analysis of a cDNA Fragment Encoding Geranylgeranyl Diphosphate Synthase in Taxus media

从 5年生曼地亚红豆杉 (Taxusmedia)的当年生新鲜枝叶中提取分离出mRNA ,然后根据已知植物的牛儿基牛儿基焦磷酸合成酶基因 (GGPPS基因 )DNA序列保守区设计特异简并引物。RT PCR获得了一条大小约 60 0bp的扩增谱带 ,回收该特异谱带并进行TA克隆 ,蓝白斑筛选 ,得到若干阳性克隆。经过质粒大小比较和PCR验证后 ,进行序列测定和同源性比较。发现该序列属于GGPP合成酶的片断 ,与Taxuscanadensis (AAD 1 60 1 8 1 )和Abiesgrandis (AAL1 761 4 2 )的GGPP合成酶相应区段的氨基酸序列一致性为 98%和 86%。蛋白质序列分析发现该序列含有一个特征的异戊二烯合成酶保守的结构域。进化树分析表明 ,曼地亚红豆杉GGPPS在进化上位于植物类 ,靠近古细菌类。曼地亚红豆杉GGPPS基因的克隆为研究红豆杉生产紫杉醇的分子机理和转基因植株的构建奠定了良好的基础。

Total RNA was extracted from fresh leave and twigs of 5-year old Taxus media and then,mRNA was isolated by polyATtract mRNA isolation syatem Ⅲ (Promega).cDNA encoding GGPPS was cloned using reverse transcription-PCR strategy on the design of degenerated oligonuclotides.A bright band of about 600 bp was acquired.After PCR product recovery,TA cloning,white/blue screening,recombinant plasmid electrophoresis analysis and PCR testification,one clone was selected for sequencing.Searching in the GenBank database using BLAST revealed that the deduced amino acid sequence of this clone shared 98% and 86% identity with Taxus canadensis (AAD 16018.1) and Abies grandis (AAL 17614.2).and a characterized domain area of isoprenyl synthase family was found in this deduced amino acid sequence.Deduced amino acid sequences analysis by blastP,PROSITE,ClustalX(1.81) and Phylio(3.6 alpha) showed GGPPS cDNA was cloned from gymnosperm Taxus media.;