Selection and validation of reference genes for gene expression studies in Pseudomonas brassicacearum GS20 using real-time quantitative reverse transcription PCR

Bianxia Bai,Jiahong Ren,Fenling Bai,Lin Hao,Ulrich Melcher

doi:10.1371/journal.pone.0227927

Bianxia Bai, Jiahong Ren + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0227927

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Journal: PloS one	Publication Date: Jan 27, 2020
Citations: 20	License type: CC BY 4.0

Affiliation: Changzhi University, Shanxi Agricultural University

Abstract

Pseudomonas brassicacearum GS20 is an antagonistic strain of bacteria recently isolated from the rhizosphere of Codonopsis pilosula. No validated reference gene has been indentified from P. brassicacearum to use in the normalization of real-time quantitative reverse transcription-PCR data. Therefore, in this study, nine candidate reference genes (recA, gyrA, rpoD, proC, gmk, rho, 16S, ftsz, and secA) were assessed at different growth phases and under various growth conditions. The expression stability of these candidate genes was evaluated using BestKeeper, NormFinder and GeNorm. In general, the results showed rho, rpoD and gyrA were the most suitable reference genes for P. brassicacearum GS20. The relative expression of iron-regulated gene (fhu) was normalized to verify the reliability of the proposed reference genes under iron-replete and iron-limited conditions. The trend in relative expression was consistent with the change in siderophore production under different iron conditions. This study presents reliable reference genes for transcriptional studies in P. brassicacearum GS20 under the chosen experimental conditions.

Highlights

Real-time quantitative reverse transcription PCR is a technique that is widely used to monitor changes in gene expression [1]
C. pilosula can be affected by serious soil-borne diseases caused by phytopathogenic fungi; we found that P. brassicacearum GS20 can antagonize these phytopathogenic fungi
Based on previous reports on suitable internal control genes for Pseudomonas species, nine candidate reference genes were chosen for this study

Summary

Introduction

Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique that is widely used to monitor changes in gene expression [1]. The method is regarded to be sensitive, specific, reproducible and accurate [2], even though factors such as the quality of extracted RNA, primer selection and the efficiency of cDNA synthesis can affect the outcome [3]. These variables are minimized by relative normalization, whereby the expression of the target gene is normalized to the expression of an internal control gene [4]. Housekeeping genes are usually chosen as internal controls to normalize real-time qRT-PCR data; recent reports have claimed that the expression of housekeeping genes may vary according to the genes, cell types and experimental conditions [6].

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