【摘要】 【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。更多还原

【Abstract】 【Aim】This study aims to select suitable reference genes in the multi-pesticide( abamectin,fenpropathrin and spirodiclofen) resistant strain( Mp-R) of T. urticae by quantitative real-time PCR( RT-q PCR). 【Methods】Eight candidate reference genes including 5. 8S rRNA,α-tubulin,TBP,β-actin,ELFn,RPL13 a,GAPDH and SDHA were chosen. The stability of these candidate reference genes was investigated by using three softwares( Ge Norm,Norm Finder and Best Keeper). Then the expression of CYP392 A subfamily gene in P450 enzyme system of T. urticae was analyzed by using different reference genes. 【Results】EFLn was confirmed the most suitable reference gene in the sensitive strain( SS) and the multi-pesticide resistant strain( Mp-R) of T. urticae by ge Norm,Norm Finder and Best Keeper softwares. The expression level of CYP392A1 was up-regulated significantly in the eggs of the Mp-R strain,and the expression levels of CYP392A16 gene in various developmental stages of the Mp-R strain was significantly higher than the same stage of the sensitive strain of T. urticae after 40-generation selection with the multi-pesticides. The expression levels of CYP392A16 gene in various developmental stages of the Mp-R strain was significantly higher than those in the same stage of the sensitive strain of T.urticae. The expression levels of other genes were not significantly different between the Mp-R strain and the sensitive strain. 【Conclusion】EFLn gene is confirmed the most suitable reference gene in the multipesticide resistant strain and the susceptible strain of T. urticae. This work is contributable to the foundation for future gene expression studies in the resistant strain of T. urticae.更多还原