利用细胞表面gLUC报告基因的高通量酵母单杂交筛选。

Q1 Agricultural and Biological Sciences

Current protocols in plant biology Pub Date : 2019-03-01 Epub Date: 2019-02-11 DOI:10.1002/cppb.20086

Zheng Li, Katia Bonaldi, S Earl Kang, Jose L Pruneda-Paz

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引用次数: 5

摘要

以基因为中心的酵母单杂交(Y1H)筛选使用排列全基因组转录因子(TF)克隆集合提供了一个简单而有效的策略，以DNA片段为诱饵鉴定TF-启动子相互作用。为了改进检测方法，我们最近开发了一种使用细胞表面高斯荧光素酶报告基因(gLUC59)的Y1H系统。与其他可用的方法相比，这种基于荧光素酶的策略需要更短的处理时间，提高了吞吐量，并改善了以基因为中心的Y1H筛选的结果分析。在这里，我们描述了使用这种新策略进行高通量筛选的过程，其中包括两个单倍体酵母菌株的交配方案，分别携带一个排列的TF克隆集合和一个启动子::gLUC59报告子，以及一个分析二倍体细胞中gLUC59活性的方案。©2019 by John Wiley & Sons, Inc。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

High-Throughput Yeast One-Hybrid Screens Using a Cell Surface gLUC Reporter.

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High-Throughput Yeast One-Hybrid Screens Using a Cell Surface gLUC Reporter.

Gene-centered yeast one-hybrid (Y1H) screens using arrayed genome-wide transcription factor (TF) clone collections provide a simple and effective strategy to identify TF-promoter interactions using a DNA fragment as bait. In an effort to improve the assay we recently developed a Y1H system that uses a cell surface Gaussia luciferase reporter (gLUC59). Compared to other available methods, this luciferase-based strategy requires a shorter processing time, enhances the throughput and improves result analysis of gene-centered Y1H screens. Here, we described the procedure to perform high-throughput screens using this novel strategy, which involves a protocol for mating two haploid yeast strains carrying an arrayed TF clone collection and a promoter::gLUC59 reporter, respectively, and a protocol for analyzing gLUC59 activity in the resulting diploid cells. © 2019 by John Wiley & Sons, Inc.

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来源期刊

Current protocols in plant biology Agricultural and Biological Sciences-Plant Science

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期刊介绍： Sound and reproducible laboratory methods are the foundation of scientific discovery. Yet nuances that are critical for an experiment''s success are not captured in the primary literature but exist only as part of a lab''s oral tradition. Current Protocols in Plant Biology provides reproducible step-by-step instructions for protocols relevant to plant research. Furthermore, Current Protocols content is thoughtfully organized by topic for optimal usage and to maximize contextual knowledge. Quarterly issues allow Current Protocols in Plant Biology to constantly evolve to keep pace with the newest discoveries and developments. Current Protocols in Plant Biology is the comprehensive source for protocols in the multidisciplinary field of plant biology, providing an extensive range of protocols from basic to cutting edge. Coverage includes: Extraction and analysis of DNA, RNA, proteins Chromosome analysis Transcriptional analysis Protein expression Metabolites Plant enzymology Epigenetics Plant genetic transformation Mutagenesis Arabidopsis, Maize, Poplar, Rice, and Soybean, and more.