【摘要】 利用分段RT-PCR法成功地从云南红豆杉叶片总RNA中分离出两条长约1.4kb和1.5kb的cDNA片段,克隆到pUCm-T载体中,序列拼接分析表明,该cDNA片段与WildungMR等发表的紫杉二烯合成酶基因编码区2586bp有41个核苷酸不同,同源性为98.42%,具有编码862个氨基酸蛋白质的能力.为了检测所克隆的基因编码产物是否具有活性,并进一步制备抗体以便于将来转基因植物的检测,构建了一个酵母表达载体GAPA-T14;为了转化植物构建了两个具有不同启动子的植物表达载体,pBI121-T14(含35S启动子)和pBIH-T14(在pBI121的基础上以胶乳特异启动子取代35S启动子).更多还原

【Abstract】 Twopairs of primers were designed a nd synthesized according to the analytical results of taxadiene synthase cDNA sequence by Wildung an d coworkers.1.4kb and 1.5kb cDNA fra gment were amplified from the total RNA of Taxus yunnanensis by reverse transcription-polymera se chain reaction(RT-PCR)and cloned into pUCm-T vector.After sequencin g,splicing of the two fragments,then alignment of the nucleotide se-quences of the cDNA coding fragment a nd taxadiene synthase gene revealed only 41mismatches in 2586coding regions homogeneous 98.42%.One yeast expression vector,GAPA-T14,was established in order to test whether the product has enzyme activ ity or not,two plant expression vectors,pBI121-T14(contains 35S pro-moter)and pBIH-T14(based on the pBI121,uses rubber special promoter instead of 35S promoter)containing different promoter were established in order to transfer to plant.更多还原