Evaluation of Reference Genes for Real-Time Quantitative PCR Analysis in Larvae of Spodoptera litura Exposed to Azadirachtin Stress Conditions

Abstract

Azadirachtin is an efficient and broad-spectrum botanical insecticide against more than 150 kinds of agricultural pests with the effects of mortality, antifeedant and growth regulation. Real-time quantitative polymerase chain reaction (RT-qPCR) could be one of the powerful tools to analyze the gene expression level and investigate the mechanism of azadirachtin at transcriptional level, however, the ideal reference genes are needed to normalize the expression profiling of target genes. In this present study, the fragments of eight candidate reference genes were cloned and identified from the pest Spodoptera litura. In addition, the expression stability of these genes in different samples from larvae of control and azadirachtin treatments were evaluated by the computational methods of NormFinder, BestKeeper, Delta CT, geNorm, and RefFinder. According to our results, two of the reference genes should be the optimal number for RT-qPCR analysis. Furthermore, the best reference genes for different samples were showed as followed: EF-1α and EF2 for cuticle, β-Tubulin and RPL7A for fat body, EF2 and Actin for midgut, EF2 and RPL13A for larva and RPL13A and RPL7A for all the samples. Our results established a reliable normalization for RT-qPCR experiments in S. litura and ensure the data more accurate for the mechanism analysis of azadirachtin.

Keywords: RT-qPCR; Spodoptera litura; azadirachtin; reference gene; stability.

Figure 1

The expression profiles of the eight candidate reference genes. (A) Agarose gel of eight candidate reference genes PCR products amplified with the RT-qPCR primers. Lanes: M, Marker 2000, lane 1-8, Actin, EF-1α, EF2, GAPDH, RPL7A, RPL13A, α-Tubulin and β-Tubulin. (B–F) The expression profiles of the eight candidate reference genes in cuticle, fat body, midgut, larva, and all samples. (Actin), actin; (EF-1α), elongation factor 1alpha; (EF2), elongation factor 2; (GAPDH), glyceraldehyde 3-phosphate dehydrogenase; (RPL7A), ribosomal protein L7A; (RPL13A), ribosomal protein L13A; (α-Tubulin), alpha-tubulin; (β-Tubulin), beta-tubulin.

Figure 2

The calculation of eight candidate reference genes expression stability by NormFinder. Abbreviations were showed as in Figure 1. Panels (A–E) were represented as the stability validation results of reference genes expression in cuticle, fat body, midgut, larva, and all samples.

Figure 3

Expression stability of eight candidate reference genes evaluated by Delta CT method. Abbreviations were showed as in Figure 1. Panels (A–E) were represented as the stability validation results of reference genes expression in cuticle, fat body, midgut, larva, and all samples.

Figure 4

The stability of reference genes expression and the optimal number for normalization analyzed by geNorm software. (A,C,E,G) The stability validation of reference genes expression in cuticle, fat body, midgut, and larva. (B,D,F,H) pairwise variation values in cuticle, fat body, midgut, and larva. Abbreviations were showed as in Figure 1.

Figure 5

Stability values of the candidate reference genes in different experiment sets generated by RefFinder algorithm. Abbreviations were showed as in Figure 1. Panels (A–E) were represented as the stability validation results of reference genes expression in cuticle, fat body, midgut, larva, and all samples.