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【目的】筛选出Bt毒素诱导后的小菜蛾Plutella xylostella(L.)的实时定量PCR最适内参基因。【方法】选取核糖体18S rRNA(18S rRNA)、肌动蛋白(ACTB)、延伸因子(EF1)、3-磷酸甘油醛脱氢酶(GAPDH)、核糖体蛋白L32(RPL32)、核糖体蛋白S13(RPS13)、核糖体蛋白S20(RPS20)和β-微管蛋白(TUB)基因作为候选内参基因,以geNorm、Normfinder和BestKeeper软件分析这8个基因在Bt毒素诱导后的小菜蛾不同品系中肠组织中的表达稳定性。并应用筛选出来的内参基因分析小菜蛾氨肽酶2(aminopeptidase N2,APN2)基因的表达水平。【结果】geNorm软件以RPS13和EF1为最稳定内参基因,NormFinder和BestKeeper软件均以RPS13和RPL32为最稳定基因。使用3种不同内参基因分析Bt毒素诱导后的小菜蛾Bt抗性和敏感品系中ANP2表达水平时,新的内参基因EF1和传统内参基因RPL32表现了良好的稳定性,二者作为标准化因子,ANP2表达量结果基本一致,而使用18S rRNA作为内参基因,却导致部分表达量分析结果有所误差。【结论】筛选出PRS13,RPL32和EF1可以作为小菜蛾某些试验条件下的内参基因,对小菜蛾基因表达研究奠定了一定基础,也对其他昆虫内参基因的筛选具有参考价值。
【Objective】 The objective of this study was to screen real-time quantitative PCR-optimized reference genes of Plutella xylostella (L.) induced by Bt toxin. 【Method】 The 18S rRNA, ACTB, EF1, GAPDH, ribosomal protein L32 (RPL32), ribosomal protein S13 RPS13, RPS20 and TUB genes were selected as candidate internal reference genes. The gene expression profiles of the eight genes were analyzed by geNorm, Normfinder and BestKeeper software in the midgut Stability of expression in tissues. The expression of aminopeptidase N2 (APN2) gene in diamondback moth Plutella xylostella was analyzed by using the selected reference genes. 【Results】 geNorm software was the most stable internal reference gene with RPS13 and EF1, and NormFinder and BestKeeper software with RPS13 and RPL32 as the most stable genes. Three different internal control genes were used to analyze the expression of ANP2 in Bt resistance and sensitive strains induced by Bt toxin. The new internal reference gene EF1 and the traditional internal reference gene RPL32 showed good stability. Both of them were used as normalizing factor, ANP2 The results of the expression were basically the same, but the use of 18S rRNA as an internal control gene, which led to some of the results of the amount of expression analysis of the error. 【Conclusion】 The results showed that PRS13, RPL32 and EF1 could be used as internal reference genes for some test conditions in Plutella xylostella, which laid a foundation for the gene expression study on Plutella xylostella and also had reference value for the screening of other insects’ reference genes.