通过细菌营养缺陷型互补和真菌营养缺陷型转化克隆黄孢原毛平革菌leu2基因
Cloning of Phanerochaete chrysosporium leu2 by complementation of bacterial auxotrophs and transformation of fungal auxotrophs.
作者信息
Zapanta L S, Hattori T, Rzetskaya M, Tien M
机构信息
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802, USA.
出版信息
Appl Environ Microbiol. 1998 Jul;64(7):2624-9. doi: 10.1128/AEM.64.7.2624-2629.1998.
A Phanerochaete chrysosporium cDNA library was constructed in an expression vector that allows expression in both Escherichia coli and Saccharomyces cerevisiae. This expression vector, lambda YES, contains the lacZ promoter for expression in E. coli and the GAL1 promoter for expression in yeast. A number of genes were cloned by complementation of bacterial amino acid auxotrophs. The cDNA encoding the beta-isopropylmalate dehydrogenase from P. chrysosporium was characterized further. The genomic clone (gleu2) was subsequently isolated and was used successfully as a selectable marker to transform P. chrysosporium auxotrophs for LEU2. Protoplasts for transformation were prepared with readily obtained conidiospores rather than with basidiospores, which were used in previous P. chrysosporium transformation procedures. The method described here allows other genes to be isolated from P. chrysosporium for use as selectable markers.
在一个允许在大肠杆菌和酿酒酵母中都能表达的表达载体中构建了黄孢原毛平革菌的cDNA文库。这个表达载体λYES，含有用于在大肠杆菌中表达的lacZ启动子和用于在酵母中表达的GAL1启动子。通过对细菌氨基酸营养缺陷型的互补作用克隆了许多基因。对编码黄孢原毛平革菌β-异丙基苹果酸脱氢酶的cDNA进行了进一步表征。随后分离出基因组克隆（gleu2），并成功地将其用作选择标记来转化黄孢原毛平革菌的LEU2营养缺陷型。用于转化的原生质体是用容易获得的分生孢子制备的，而不是用以前黄孢原毛平革菌转化程序中使用的担孢子。这里描述的方法可以从黄孢原毛平革菌中分离出其他基因用作选择标记。
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