Cloning and disruption of the PpURA5 gene and construction of a set of integration vectors for the stable genetic modification of Pichia pastoris
Cloning and disruption of the PpURA5 gene and construction of a set of integration vectors for the stable genetic modification of Pichia pastoris
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DOI：
10.1002/yea.1049
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发表时间：
2003-11-01
期刊：
影响因子：
2.6
通讯作者：
Gemgross, TU
中科院分区：
文献类型：
--
作者：
Nett, JH;Gemgross, TU
利用一对简并引物扩增和克隆了含有巴氏酵母URA5和SEC65部分基因的DNA片段。利用巴斯德酵母部分基因组序列的额外信息，我们克隆并测序了一个1.9 kb的染色体片段，其中包含编码URA5基因的完整螺旋状磷酸核糖基转移酶。用卡那霉素抗性基因取代开放阅读框的一小部分，构建了一个破坏盒。利用裂解盒对野生型巴氏酵母NRRL Y-11430进行转化，鉴定出一株ura5营养不良菌株。为了产生可以在单个菌株的连续转化中重复使用的标记结构，我们构建了两个lacZ-PpURA3-lacZ和lacZ-PpURA5-lacZ磁带，并用它们来破坏PpOCH1。干扰物中的PpURA3和PpURA5基因通过筛选对5′-氟羟基乙酸的抗性成功回收。我们还组装了一组模块化质粒，可用于通过双交叉事件对巴氏酵母进行稳定的遗传修饰。这里展示的序列已提交给EMBL数据库，编号为Accession No.。AY303544。版权所有：John Wiley Sons， Ltd。
A pair of degenerate primers was used for amplification and cloning of a DNA fragment containing parts of the P. pastoris URA5 and SEC65 genes. Using additional information from a partial genomic sequence of P. pastoris, we cloned and sequenced a 1.9 kb chromosomal fragment containing the complete orotate-phosphoribosyltransferase-encoding URA5 gene. A disruption cassette was constructed by replacing a small part of the open reading frame with a kanamycin-resistance gene. The P. pastoris wild-type strain NRRL Y-11430 was transformed with the disruption cassette and an ura5 auxotrophic strain was identified. To generate marker constructs that can be reused in successive transformations of a single strain, we constructed two lacZ-PpURA3-lacZ and lacZ-PpURA5-lacZ cassettes and used them to disrupt PpOCH1. The PpURA3 and PpURA5 genes in the disruptants were then successfully recycled by selecting for resistance to 5'-fluoro-orotic acid. We also assembled a set of modular plasmids that can be used for the stable genetic modification of P. pastoris via a double cross-over event. The sequence presented here has been submitted to the EMBL data library under Accession No. AY303544. Copyright (C) 2003 John Wiley Sons, Ltd.