菠菜乙醇酸氧化酶编码区cDNA在酵母表达载体中的克隆

Cloning of Spinach Glycolate Oxidase cDNA and Its Construction into Yeast Expression Vector

目的 :克隆菠菜乙醇酸氧化酶cDNA并构建其酵母表达载体。方法 :从新鲜菠菜叶子中提取总RNA ,用RT PCR法扩增菠菜乙醇酸氧化酶编码区cDNA并插入pMD T载体中进行序列测定 ;将此cDNA构建入P .Pastoris酵母表达体系中的pPIC3 5k上的SnaBI/NotI位点 ,进行PCR筛选和限制性酶切鉴定。结果 :测序表明获得的基因为乙醇酸氧化酶cDNA序列 ,与国外文献报道的序列相比有约 98%的同源性 ;重组质粒的SnaBI/NotI双酶切证明构建正确。结论 :菠菜乙醇酸氧化酶cDNA克隆及重组质粒pPIC3 5K GO的获得 。

METHOD:Using spinach to extract total RNA, the spinach GO cDNA was amplified by RT PCR. This cDNA was inserted into cloning vector pMD T and sequenced. The cDNA was then cloned into pPIC3 5K(P.Pastoris expression vector)by SnaBI/NotI double digestion. RESULT:Screening by PCR, we got the recombinant plasmid pPIC3 5K GO which was confirmed by SnaBI/NotI restriction enzyme analysis. Sequencing showed that this cDNA was spinach spinach glycolate oxidase cDNA. The SnaBI/NotI double digestion of pPIC3 5K GO ensured that the construction is correct.CONCLUSION:Obtained spinach glycolate oxidase cDNA and its recombinant plasmid pPIC3 5K GO which will be useful for further expression study.