Identification of suitable reference genes for normalization of RT-qPCR data in eggplant fruit and shoot borer (Leucinodes orbonalis Guenée)

Abstract

The eggplant shoot and fruit borer, Leucinodes orbonalis Guenee (Lepidoptera: Crambidae) is a monophagous and destructive insect pest of eggplant. L. orbonalis developed multiple insecticide resistance that led to frequent field control failures in many countries. The possible reversal of resistance in L. orbonalis requires an understanding of the expression dynamics of individual genes conferring resistance to insecticides through gene expression analysis. The availability of reference genes (RG) to normalize transcript data of target genes, achieved by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), is mandatory. Hence, the present study investigated the stable expression pattern of nine putative reference genes viz., actin, 28S ribosomal protein S18c, mitochondrial isoform X2 (28SM), calnexin, β-tubulin, 28S ribosomal protein S3 mitochondrial (28SR3), TATA box binding protein (TATA), elongation factor-1α (EF-1α), glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), and ubiquitin 60S ribosomal protein (Ubiquitin) to select the most stable genes that could be used as reference genes for target transcript gene normalization. Different algorithms such as ΔCt, geNorm, NormFinder and BestKeeper were used to assess gene expression stability. By integrating these data into online RefFinder tool, 28SR3 and GAPDH, were chosen as highly suitable RGs based on their stable expression pattern.