High copy number integration into the ribosomal DNA of the yeast Phaffia rhodozyma
High copy number integration into the ribosomal DNA of the yeast Phaffia rhodozyma
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DOI：
10.1016/s0378-1119(96)00579-3
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发表时间：
1997-01-03
期刊：
影响因子：
3.5
通讯作者：
vanOoyen, AJJ
中科院分区：
文献类型：
--
作者：
Wery, J;Gutker, D;vanOoyen, AJJ
本文介绍了一种稳定的高拷贝数整合到虾青素生产酵母法夫酵母核糖体DNA(RDNA)中的转化系统。构建了转座子Tn5编码卡那霉素抗性基因(Km(R))与法夫酵母肌动蛋白基因5‘端融合的表达载体。该标记由法菲亚肌动蛋白启动子驱动，对G418(Geneticin)产生抗性。该质粒还含有一个包含18S rDNA的rDNA部分，并促进高拷贝整合，从而获得稳定的法夫酵母转化子，在15代非选择性生长后，该转化子保持了高拷贝数。Phaffia菌株CBS 6938含有分布在三条染色体上的rDNA单元，总拷贝数为61。法菲亚转化子被证明有超过50个拷贝的PGB-PH9串联整合在含有rDNA基因座的染色体上。如脉冲场电泳法所示，由于这些整合而发生的染色体移位强烈地表明法菲亚是单倍体。
This report describes a transformation system leading to stable high copy number integration into the ribosomal DNA (rDNA) of the astaxanthin-producing yeast Phaffia rhodozyma. A plasmid was constructed that contains the transposon Tn5 encoded kanamycin resistance gene (Km(R)) fused in frame to the 5'-terminal portion of the Phaffia actin gene. This marker, driven by the Phaffia actin promoter, confers resistance to G418 (Geneticin). The plasmid also contains a rDNA portion that comprises the 18S rDNA and promotes high copy integration leading to stable Phaffia transformants that maintained the plasmid at high copy number after 15 generations of non-selective growth. Phaffia, strain CBS 6938, was found to contain the rDNA units in clusters distributed over three chromosomes with a total copy number of 61. Phaffia transformants were shown to have over 50 copies of pGB-Ph9 integrated in tandem in chromosomes that contain rDNA loci. The chromosomal shifts that occur as a result of these integrations as shown by pulsed field electrophoresis strongly suggest that Phaffia is haploid.