菠菜乙醇酸氧化酶编码区cDNA在酵母表达载体中的克隆

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，为酵母表达菠菜乙醇酸氧化酶提供了前题条件。

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

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.