关键词: 小菜蛾； 实时定量PCR, 内参基因； 抗药性； Bt毒素

Abstract: 【Aim】 To select the suitable reference genes in Plutella xylostella after exposure to Bt toxin by quantitative real-time PCR. 【Methods】 Eight candidate reference genes, including 18S ribosomal RNA (18S rRNA), beta actin gene (ACTB), elongation factor 1 gene (EF1), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), ribosomal protein L32 gene (RPL32), ribosomal protein S13 gene (RPS13), ribosomal protein S20 gene (RPS20) , and β-tubulin gene (TUB), were chosen. The stability of these candidate reference genes was investigated using three softwares (geNorm, NormFinder and BestKeeper). Then, the expression of aminopeptidase N2 gene (APN2) was analyzed by using different reference genes. 【Results】 Based on the results of geNorm analysis RPS13 and EF1 were the most suitable reference genes in P. xylostella after exposure to the Bt toxin Cry1Ac, while based on the results of the NormFinder and BestKeeper analysis RPS13 and RPL32 were the most suitable reference genes. Using new reference gene (EF1) or traditional reference gene (RPL32) for normalization, similar expression levels of APN2 were observed, whereas normalization with the unstable reference gene (18S rRNA) might lead to erroneous interpretations. 【Conclusion】 This work is contributable to the solid foundation for future gene expression studies in the diamondback moth, and may also serve as a resource to screen reference genes for expression studies in other insects.

Key words: Plutella xylostella, quantitative real-time PCR, reference gene, resistance, Bt toxin