Expression Stability of Reference Genes in Larvae of Spodoptera frugipferda Under Azadirachtin Stress by Real-Time Quantitative PCR Analysis

Abstract

【Objective】Real-time quantitative PCR is one of the most conventional and efficient techniques for studying the mRNA expression level of target genes. The proper selection of reference genes is of great significance for the accuracy of the results. The study aimed to identify the most suitable reference genes by evaluating the expression stability of candidate reference genes in different samples of Spodoptera frugiperda larvae under azadirachtin stress. The results provided a basis for functional analysis of target genes and molecular toxicological mechanism analysis of azadirachtin.【Method】Eight candidate reference genes, including α-TUB, β-1-TUB, Actin, EF1α, EF2, GAPDH, RPL3 and RPL3, of S. frugiperda were selected. The expression stability of these genes in different samples of larvae, cuticle, midgut, fat body and malpighian tube under azadirachtin stress was evaluated by the ΔCt, GeNorm, NormFinder, BestKeeper and RefFinder software. And the optimal number of reference genes were determined by GeNorm software. Finally, the best reference gene combinations in different samples were comfirmed.【Result】When exposed to azadirachtin stress, in larvae samples, the expression stability of α-TUB and β-1-TUB was the highest by ΔCt and Normfinder analyses; the expression stability of RPL3 and RPL13 was the highest by GeNorm analysis; and the expression stability of α-TUB and RPL13 was the highest by BestKeeper analysis. In cuticle samples, β-1-TUB and α-TUB were rated as the most stable genes in ΔCt, GeNorm and Normfinder, while Actin and β-1-TUB were the most stable in BestKeeper. In the midgut tissue, ΔCt analysis found that EF2 and β-1-TUB expression was the most stable; GeNorm assessment showed that EF1α and RPL3 expression was the most stable; Normfinder considered that EF2 and β-1-TUB expression was the most stable, and BestKeeper analysis found that RPL13 and RPL3 expression was the most stable. ΔCt, GeNorm and Normfinder analyses showed that RPL3 and EF1α were the most stable in fat body tissue, while BestKeeper analysis showed that RPL13 and RPL3 were the most stable. In malpighian tube tissue, ΔCt analysis found that RPL3 and EF2 expression was the most stable, GeNorm assessment indicated that EF1α and EF2 expression was the most stable; Normfinder considered that β-1-TUB and EF2 expression was the most stable, and BestKeeper analysis found that RPL13 and β-1-TUB expression was the most stable. In addition, GeNorm software analysis found that the optimal number of reference genes in each sample was 2. Therefore, as assessed by RefFinder comprehensive analysis, α-TUB and β-1-TUB had the highest expression stability in larvae and cuticle tissue samples; EF2 and RPL3 had the highest expression stability in larval midgut and malpighian tube samples; and RPL3 and EF1α had the highest expression stability in fat body samples.【Conclusion】Under azadirachtin stress, the optimal reference genes for different samples of S. frugiperda larvae were shown as: α-TUB and β-1-TUB for larvae and cuticle samples; EF2 and RPL3 for midgut and malpighian tube samples; and RPL3 and EF1α for fat body samples.

Keywords

• real-time quantitative pcr
• spodoptera frugiperda
• azadirachtin
• reference gene
• expression stability