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多站点 Gateway™ 载体组，用于对模式酿酒酵母中的基因进行功能分析。

A MultiSite Gateway™ vector set for the functional analysis of genes in the model Saccharomyces cerevisiae.

机构信息

Department of Plant Systems Biology, VIB, Technologiepark 927, B-9052, Gent, Belgium.

出版信息

BMC Mol Biol. 2012 Sep 20;13:30. doi: 10.1186/1471-2199-13-30.

DOI:10.1186/1471-2199-13-30
PMID:22994806
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3519679/
Abstract

BACKGROUND

Recombinatorial cloning using the Gateway™ technology has been the method of choice for high-throughput omics projects, resulting in the availability of entire ORFeomes in Gateway™ compatible vectors. The MultiSite Gateway™ system allows combining multiple genetic fragments such as promoter, ORF and epitope tag in one single reaction. To date, this technology has not been accessible in the yeast Saccharomyces cerevisiae, one of the most widely used experimental systems in molecular biology, due to the lack of appropriate destination vectors.

RESULTS

Here, we present a set of three-fragment MultiSite Gateway™ destination vectors that have been developed for gene expression in S. cerevisiae and that allow the assembly of any promoter, open reading frame, epitope tag arrangement in combination with any of four auxotrophic markers and three distinct replication mechanisms. As an example of its applicability, we used yeast three-hybrid to provide evidence for the assembly of a ternary complex of plant proteins involved in jasmonate signalling and consisting of the JAZ, NINJA and TOPLESS proteins.

CONCLUSION

Our vectors make MultiSite Gateway™ cloning accessible in S. cerevisiae and implement a fast and versatile cloning method for the high-throughput functional analysis of (heterologous) proteins in one of the most widely used model organisms for molecular biology research.

摘要

背景

利用 Gateway™ 技术进行重组克隆已成为高通量组学项目的首选方法，从而使 Gateway™ 兼容载体中可获得完整的 ORFeome。MultiSite Gateway™ 系统允许在一个单一反应中组合多个遗传片段，如启动子、ORF 和表位标签。迄今为止，由于缺乏合适的目的载体，这项技术在酵母酿酒酵母中不可用，而酿酒酵母是分子生物学中最广泛使用的实验系统之一。

结果

在这里，我们提出了一组用于酿酒酵母基因表达的三片段 MultiSite Gateway™ 目的载体，它们允许组合任何启动子、开放阅读框、表位标签排列，并与四个营养缺陷标记和三个不同的复制机制中的任何一个结合。作为其适用性的一个例子，我们使用酵母三杂交来提供证据，证明参与茉莉酸信号转导的植物蛋白的三元复合物的组装，该复合物由 JAZ、NINJA 和 TOPLESS 蛋白组成。

结论

我们的载体使酿酒酵母中的 MultiSite Gateway™ 克隆成为可能，并为在最广泛使用的分子生物学研究模式生物之一中进行（异源）蛋白质的高通量功能分析实施了一种快速而通用的克隆方法。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d774/3519679/61d84bf31b2f/1471-2199-13-30-3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d774/3519679/446ee71dd7af/1471-2199-13-30-1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d774/3519679/a7d474d5f1bc/1471-2199-13-30-2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d774/3519679/61d84bf31b2f/1471-2199-13-30-3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d774/3519679/446ee71dd7af/1471-2199-13-30-1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d774/3519679/a7d474d5f1bc/1471-2199-13-30-2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d774/3519679/61d84bf31b2f/1471-2199-13-30-3.jpg

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