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Selection and evaluation of reference genes for qRT-PCR analysis of different developmental stages in Chilo suppressalis

https://doi.org/10.1016/j.aspen.2021.08.008Get rights and content

Highlights

  • The stabilities of reference genes in different developmental stages and sexes of C. suppressalis were systematically studied.

  • H3 was the most stable reference gene for normalizing expression in different developmental stages.

  • It was not required to select and validate reference genes in different sexes of C. suppressalis.

Abstract

Quantitative real-time RT-PCR is considered to valuable method for the analysis of gene expression. But for the data accuracy of gene expression, the normalization of data using reference genes is essential. In this study, we focus on the analysis of the relative expression of nine commonly used candidate reference genes in the different developmental stages and female and male pupae/adults of Chilo suppressalis, and expression stabilities were analyzed by the ΔCt method, geNorm, NormFinder, and RefFinder. The results showed that H3 was the most stable reference genes in the different developmental stages while EF1 was the optimal genes in pupae/adult of different sexes of C. suppressalis. To validate, the expression profile of the gene encoding heat shock protein 60 (Hsp60) was investigated. The results showed that the transcription of target gene Hsp60 varied depending on the reference gene selected for normalization in different developmental stages, but the trend of expression of Hsp60 between the most stable reference gene combination and the least stable reference gene in pupae/adults of different sexes was the same. Therefore, it is imperative to select the reference genes to estimate target gene expression accurately in the specific experiment. This study provides scientific basis on functional analyses of the important genes contributed to the development and sex differentiation of C. suppressalis.

Introduction

The striped stem borer, Chilo suppressalis, an important rice pest widely distributed in Asia, southern Europe and North Africa, has caused the huge damage to rice and water oats (Khan et al., 1991, Gao et al., 2019, Lu et al., 2014, Song et al., 2020). In recent years, the population of C. suppressalis has gradually increased in some provinces of China, for example, Jiangsu, Jiangsu, Jianxi, Hunan, Hubei provinces (Lu et al., 2012, Lu et al., 2014, Song et al., 2020). This insect pest includes four developmental stages (egg mass, larvae, pupae and adults), and different developmental stages of C. suppressalis have evolved different adaption to the environment that confronted with. For example, overwintering larvae could suffer at −21 °C, and adults didn’t all die under certain high temperature conditions until after exposure to 46 °C (Lu et al., 2012, Lu et al., 2014). Therefore, it is necessary to study the molecular mechanisms of the resistance of C. suppressalis in the different developmental stages, which are inseparable from the research of the expression of related genes. And accurate analysis of the expression of related genes is essential. At the same time, the genome and multiple transcription of C. suppressalis were available (http://www.insect-genome.com/data/detail.php?id=7) (Yin et al., 2014, Xia et al., 2015, Ma et al., 2020) and these data will provide basic information to promote these related genes studies.

Quantitative real-time PCR (qRT-PCR) has become the main technique of choice to measure and validate gene expression (Bustin et al., 2010, Huggett et al., 2005). It has the advantages of high sensitivity, strong specificity, less time and ease of operation (Andersen et al., 2004, Bustin et al., 2009). However, inconsistencies in the quality and integrity of total RNA, enzymatic efficiencies as well as PCR efficiency have contributed to irregularities in qPCR data accuracy (Bustin et al., 2005, Strube et al., 2008). To ensure the data accuracy of gene expression by qRT-PCR, it is essential to normalize data using internal reference genes. Generally, reference genes were assumed that their expression remained constitutive and were unaffected under different condition during the experiments (Vandesompele et al., 2002a, Vandesompele et al., 2002b, Li et al., 2013, Zhu et al., 2014).

However, previous studies have shown that these reference genes were not always stably expression all developmental and experimental conditions (Ruan and Lai, 2007). Therefore, it is important to systematically validate the suitability of reference genes to improve the reliability of qRT-PCR data across various samples. Recently, the selection of suitable reference genes for qRT-PCR has been carried out in some studies of insect development. For example, EF1a was one of the most stable reference genes for developmental stages both in Dichelops melacanthus and Harmonia axyridis (Yang et al., 2018, Pinheiro et al., 2020). And the research showed that EF-1 and rpl32 were the most stable reference genes in different developmental stages in Frankliniella occidentalis (Zheng et al., 2014). However, S27Ae and Reep5 were selected to be the most stable reference gene under developmental stages of Aquatica leii (Ren et al., 2019); RPL13A and GAPDH were suitable for developmental stages (Hu et al., 2018); β-Tubulin was found to be most stable reference genes followed by rpl32 and α-Tubulin under developmental stages of Phenacoccus solenopsis (Arya et al., 2017); for different developmental stages of Lysiphlebia japonica, the comprehensive ranking was DIMT, 18SrRNA, RPL13, TBP, TUB, EF1A, AK, RPII3, RPL27, PPI, ACTB, and RPS18 (Gao et al., 2017). And for different sexes, RPL13A were suitable for sexes of Helopeltis theivora (Hu et al., 2018); FAU and RPS23 were the most stable for sex of Dichelops melacanthus (Pinheiro et al., 2020); in Aedes albopictus, for embryo-derived samples up to 24 h post-oviposition, PGK1 and ILK was the most stable, and RPL32 and RPS17 for late embryos, all four larval instars, and pupae (Dzaki and Azzam, 2018). Above list of studies indicated because different kinds of insect subject to environmental changes throughout their development, they exhibited different kinds of stable reference genes under different developmental stages and different sexes. Therefore, it is crucial to validate the stability of reference genes with respect to the development of specific kind of insect before using them to normalize the qRT-PCR.

In this study, we evaluated nine candidate reference genes for expression stability in different developmental stages and sexes (pupae and adults) of C. suppressalis. And the expression patterns of the heat shock protein 60 gene (Hsp60) using different reference genes was also evaluated. Our results will be conducive to future study the function of development related genes of this important pest and explore related method to control them.

Section snippets

Insects

C. suppressalis were reared by artificial diet in the chamber with 28 ± 1 °C, 16:8 (light/dark) photoperiod, and 70 ± 5 % relative humidity. And the population of C. suppressalis reared successively to the more than third generation was used in the experiment.

Sample collection

All samples were randomly collected in the experiment. Developmental stages included the egg, the first, second, third, fourth, fifth instar larvae, pupae (male and female), and one-day adults (male and female). Each developmental stage

Total RNA quality and PCR amplification efficiencies specificity

The expression of ten genes (nine potential reference genes and Hsp160; Table 1) was investigated in different developmental stages of C. suppressalis by qRT-PCR. The integrity, concentration and purity of total RNA isolated from different samples was evaluated by spectrophotometric analysis. The A260/280 ratios ranged from 1.80 to 2.30, indicating a high level of purity for all RNA samples. The ten primer pairs resulted in amplicons of the sample in this study that exhibited a single,

Discussion

Even though qRT-PCR is a sensitive, accurate and reproducible technique for transcription level and gene expression (Andersen et al., 2004, Nolan et al., 2006, Gachon et al., 2004, Wong and Medrano, 2005), it is necessary to screen stable reference genes to offset variation in experimental data and ensure the reliability and accuracy of the results due to the great differences in the quality of RNA and the efficiency of reverse transcription (Vandesompele et al., 2002a, Vandesompele et al.,

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This research was funded by the Natural Science Foundation of Jiangsu Province (BK20191440), the National Natural Science Foundation of China (31401733, 31371937).

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