Ricaurte ML, Govind NS
Department of Marine Sciences, University of Puerto Rico, P.O. Box 9013, Mayaguez 00681, Puerto Rico
Mar Biotechnol (NY). 1999 Jan;1(1):15-19. doi: 10.1007/pl00011745.
: We have constructed two plasmid vectors (pMR95 and pMR96) with selectable markers for the marine yeast Debaryomyces hansenii. Plasmid pMR95 contains an autonomously replicating sequence previously isolated from Debaryomyces and a hygromycin B resistance gene from the plasmid pLG90 under the control of the isocytochrome C1 promoter and terminator sequences, while pMR96 has, in addition, the Saccharomyces URA3 gene. Transformation in Debaryomyces was accomplished by electroporation. Plasmid pMR95 was capable of transforming both Saccharomyces cerevisiae and D. hansenii to hygromycin resistance at low frequencies; pMR96 transformed both yeasts at low frequencies when selected for hygromycin B resistance and at very high efficiencies when selected for uracil prototrophy. The presence of the plasmids in the transformed yeast was confirmed by polymerase chain reaction. The plasmids could be recovered back in Escherichia coli when transformed with total DNA from the yeast transformants, indicating at least a partial autonomous existence of the plasmids in the marine yeast. To our knowledge this is the first successful attempt to transform D. hansenii.
我们构建了两种带有海洋酵母汉逊德巴利酵母选择标记的质粒载体（pMR95和pMR96）。质粒pMR95含有先前从德巴利酵母中分离出的自主复制序列以及来自质粒pLG90的潮霉素B抗性基因，该抗性基因受异细胞色素C1启动子和终止子序列的控制，而pMR96还含有酿酒酵母URA3基因。通过电穿孔法实现了在德巴利酵母中的转化。质粒pMR95能够以低频率将酿酒酵母和汉逊德巴利酵母转化为潮霉素抗性；当选择潮霉素B抗性时，pMR96以低频率转化两种酵母，而当选择尿嘧啶原养型时，转化效率非常高。通过聚合酶链反应证实了转化酵母中质粒的存在。当用来自酵母转化子的总DNA进行转化时，质粒可以在大肠杆菌中回收，这表明质粒在海洋酵母中至少部分自主存在。据我们所知，这是首次成功转化汉逊德巴利酵母的尝试。
