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为了简化解脂耶氏酵母表达载体构建过程、消除抗生素污染,将mel基因(编码酪氨酸酶)作为新型报告基因用于构建新型酵母表达载体,利用组装PCR人工合成基因mel,并用重叠PCR将其与同源组成型强启动子pTEF、分泌性信号肽XPR2pre及强终止区LIP2t融合,构建新型胞外及胞内表达载体,并利用其在解脂耶氏酵母野生菌株中表达人源癌基因rho。成功获得mel全基因并将其与启动子、信号肽和终止区融合,得到融合片段TXML,用其替换原有表达载体的筛选标记基因ura3d4,构建得到新型胞外及胞内表达载体pINA1297-M和pINA1297-a-M,转化后的酵母阳性转化子性状明显,随后利用此新型表达系统获得可溶性异源蛋白Rho。首次实现了将mel作为一种便捷、价廉、无污染的新型筛选标记基因运用于非常规酵母表达系统中,更为mel在其它真核表达系统中的运用奠定了技术基础;获得的可溶性Rho蛋白可为研究其性质、结构、功能及与Rho癌基因家族其它成员的相互作用提供条件。
In order to simplify the construction of Y. lipolytica expression vector and eliminate the contamination of antibiotics, mel gene (coding tyrosinase) was used as a new reporter gene to construct a novel yeast expression vector. The assembled PCR was used to synthesize gene mel, It was fused with pTEF, XPR2pre, and LIP2t, and a novel extracellular and intracellular expression vector was constructed and expressed in human Yarrowia lipolytica rho. The mel gene was successfully obtained and fused with the promoter, signal peptide and terminator to obtain the fusion fragment TXML, which was used to replace the original expression vector ura3d4 to construct a novel extracellular and intracellular expression vector pINA1297-M And pINA1297-aM, the transformed yeast positive transformants showed obvious traits, and then the new expression system was used to obtain the soluble heterologous protein Rho. It is the first time that Mel has been applied as a convenient, cheap and non-polluting new selectable marker gene in unconventional yeast expression system, and mel has laid the technical foundation for the application of mel in other eukaryotic expression systems. The obtained soluble Rho Proteins may provide conditions for studying their properties, structure, function and interaction with other members of the Rho oncogene family.