PCR-based gene targeting in Hanseniaspora uvarum.

IF 2.4 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Jennifer Badura, Niël van Wyk, Kerstin Zimmer, Isak S Pretorius, Christian von Wallbrunn, Jürgen Wendland
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引用次数: 1

Abstract

Lack of gene-function analyses tools limits studying the biology of Hanseniaspora uvarum, one of the most abundant yeasts on grapes and in must. We investigated a rapid PCR-based gene targeting approach for one-step gene replacement in this diploid yeast. To this end, we generated and validated two synthetic antibiotic resistance genes, pFA-hygXL and pFA-clnXL, providing resistance against hygromycin and nourseothricin, respectively, for use with H. uvarum. Addition of short flanking-homology regions of 56-80 bp to these selection markers via PCR was sufficient to promote gene targeting. We report here the deletion of the H. uvarum LEU2 and LYS2 genes with these marker genes via two rounds of consecutive transformations, each resulting in the generation of auxotrophic strains (leu2/leu2; lys2/lys2). The hereby constructed leucine auxotrophic leu2/leu2 strain was subsequently complemented in a targeted manner, thereby further validating this approach. PCR-based gene targeting in H. uvarum was less efficient than in Saccharomyces cerevisiae. However, this approach, combined with the availability of two marker genes, provides essential tools for directed gene manipulations in H. uvarum.

基于pcr的兔痘菌基因靶向研究。
缺乏基因功能分析工具限制了对葡萄和木薯上最丰富的酵母之一——葡萄芽孢酵母的生物学研究。我们研究了一种基于pcr的快速基因靶向方法,用于二倍体酵母的一步基因替换。为此,我们合成并验证了两个合成的抗生素耐药基因pFA-hygXL和pFA-clnXL,分别提供了对潮霉素和诺斯库霉素的耐药,用于uvarum。通过PCR在这些选择标记上添加56 ~ 80bp的短侧同源区域,足以促进基因靶向。我们在这里报道,通过两轮连续转化,uvarum的LEU2和LYS2基因与这些标记基因的缺失,每一轮都导致产生营养不良菌株(LEU2 / LEU2;lys2 / lys2)。由此构建的亮氨酸营养不良leu2/leu2菌株随后被有针对性地补充,从而进一步验证了该方法。在酿酒酵母中,基于pcr的基因靶向效率较低。然而,这种方法与两个标记基因的可用性相结合,为uvarum的定向基因操作提供了必要的工具。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
FEMS yeast research
FEMS yeast research 生物-生物工程与应用微生物
CiteScore
5.70
自引率
6.20%
发文量
54
审稿时长
1 months
期刊介绍: FEMS Yeast Research offers efficient publication of high-quality original Research Articles, Mini-reviews, Letters to the Editor, Perspectives and Commentaries that express current opinions. The journal will select for publication only those manuscripts deemed to be of major relevance to the field and generally will not consider articles that are largely descriptive without insights on underlying mechanism or biology. Submissions on any yeast species are welcome provided they report results within the scope outlined below and are of significance to the yeast field.
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