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【目的】筛选出二斑叶螨Tetranychus urticae Koch抗甲氰菊酯、阿维菌素及螺螨酯混剂的实时定量PCR最优内参基因。【方法】选取5.8S rRNA,α-tubulin,TBP,β-actin,ELFn,RPL13a,GAPDH和SDHA 8个候选内参基因,以Ge Norm,Best Keeper和Normfinder 3个软件分析这8个基因在二斑叶螨多重抗性品系中的表达稳定性,并以筛选的内参基因分析二斑叶螨P450酶系CYP392A亚家族基因的表达水平。【结果】经Ge Norm,Best Keeper和Normfinder 3个软件综合评价确定ELFn基因为二斑叶螨敏感品系(susceptible strain,SS)和多重抗性品系(multipesticide resistant strain,Mp-R)各发育阶段的最优内参基因。以ELFn为内参基因对二斑叶螨CYP392A亚家族16个基因表达量进行分析,结果表明:经多重抗性选育40代后,Mp-R品系卵期CYP392A1表达量显著上调;CYP392A16基因在各发育阶段表达量极显著高于SS品系相应发育阶段;其他基因表达量在敏感品系和抗性品系之间差异不显著。【结论】筛选出了SS和Mp-R品系中各发育阶段最佳内参基因为EFLn;Mp-R品系CYP392A亚家族16个基因的表达量在幼螨和若螨阶段低于卵与成螨阶段,其中CYP392A16基因在二斑叶螨多重抗性的形成中起主要作用。该结果为二斑叶螨多重抗性研究奠定了一定基础。
【Objective】 The objective of this study was to screen out the best real-time PCR-based internal control gene for Tetranychus urticae Koch against fenpropathrin, avermectin and spirimate. 【Methods】 Eight candidate internal reference genes of 5.8S rRNA, α-tubulin, TBP, β-actin, ELFn, RPL13a, GAPDH and SDHA were selected and analyzed by Ge Norm, Best Keeper and Normfinder. Spider mite multi-resistant strains of the expression stability, and the internal control gene screening two-spotted spider mite P450 enzyme CYP392A subfamily gene expression levels. 【Result】 The results showed that the ELFn gene was identified as the susceptible strain (SS) and multipesticide resistant strain (Mp-R) at different developmental stages by the comprehensive evaluation of Ge Norm, Best Keeper and Normfinder. The best internal reference gene. The results showed that the expression level of CYP392A1 in Mp-R strain was significantly up-regulated after 40 passages of multi-resistance, while the expression level of CYP392A16 gene in each stage The expression level at the developmental stage was significantly higher than that at SS stage. The other gene expression was not significantly different between the susceptible and resistant lines. 【Conclusion】 The best internal reference gene of EFLn in each developmental stage of SS and Mp-R lines was screened out. The expression level of 16 genes of CYP392A subfamily of Mp-R strain was lower than that of egg and adult mite stages , In which the CYP392A16 gene plays a major role in the formation of multiple resistance to Tetranychus urticae. The results laid a solid foundation for the research on the multi-resistance of Tetranychus urticae.