Selection of suitable reference genes for quantitative RT-PCR normalization in the halophyte Halostachys caspica under salt and drought stress.

Xiaoya Yi,Suwei Zhang,Youling Zeng,Yufang Zhang

doi:10.1038/srep30363

Abstract

The plants are always subjected to various environmental stress, because of plant sessile growth. qRT-PCR is a sensitive and reliable technology, and the normalization of target gene expression with suitable reference genes is very important for obtaining accurate data. Halostachys caspica is an extremely salt-tolerant halophyte belonging to Chenopodiaceae and a good candidate to explore the stress-physiological and molecular mechanism. To get truly the expression profiles of coding genes and miRNAs in H. caspica in response to salt and drought stress using qRT-PCR, suitable reference genes need to be confirmed. In this study, 10 candidate genes including ACT, UBC10, UBC13, TUB2, TUB3, EF1α, 5S rRNA, tRNA, U6 and miR1436 from H. caspica are chosen, and among them, the former nine are commonly used as internal control genes, and miR1436 with high sequence copies is no significant difference expression in high salinity-treated and untreated small RNA libraries of this species. The three softwares are used to analyze expression stability. The results showed that EF1α and TUB3 were the most stable under salt and drought stress, respectively, and UBC10 was the most constant aross all the samples with the both stressed combination. This work will benefit deep studies on abiotic tolerance in H. caspica.

Highlights

Plants use multiple gene regulatory mechanisms in dealing with various environmental stress, and it improves stress tolerance by altering the expression levels of stress-responsive genes[1]
The target sequences of ACT, TUB2, TUB3, UBC10, UBC13 and EF1αin H. caspica were cloned with cDNA as template by specific primers, respectively
It is a good material to dig deeply importantly salt-resistant genes for function research, stress signal transduction and exploitation and utilization. qRT-PCR with suitable reference genes can truly detect the expressional profiles of candidate target genes from a suppression subtractive hybridization library (SSH) and small RNA libraries of this species H. caspica under high salt stress[25,34]

Summary

Introduction

Plants use multiple gene regulatory mechanisms in dealing with various environmental stress, and it improves stress tolerance by altering the expression levels of stress-responsive genes[1]. Arabidopsis thaliana NFYA5 is mainly regulated by miR169a and overexpression of NFYA5 confers enhanced drought tolerance in Arabidopsis by qRT-PCR and transgenic technologies[8] It is crucial for selecting the most appropriate reference gene to analysis the expression pattern of target genes in given speices under specific conditions. We constructed and evaluated the both small RNA libraries of the H. caspica roots under high salt stress (600 mM NaCl for 48 h)[25], and obtained the transcriptome data of this species To investigate exactly their expressions and correlations of miRNAs and coding genes under different abiotic stress, it is necessary to find a stable reference gene or a group for normalization the transcripts of the candidate targets in the H. caspica species. The data indicated that EF1αwas the most stable reference gene under salt stress in H. caspica and TUB3 was under drought stress, respectively; the expression level of UBC10 is the most constant aross all the H. caspica tested samples

Methods

Results

Conclusion