二斑叶螨不同抗性品系最佳内参基因的筛选及CYP392E亚家族基因的表达分析

【目的】 筛选二斑叶螨Tetranychus urticae敏感品系（SS）、 抗阿维菌素品系(Av-R)和抗螺虫乙酯品系（Sp-R）中合适的内参基因， 研究二斑叶螨不同抗性品系CYP392E亚家族基因表达水平的变化。【方法】采用实时荧光定量PCR （quantitative real time PCR, qRT-PCR）技术， 对二斑叶螨α-tubulin， β-actin， ELFn， GAPDH， 5.8S rRNA基因和SDHA共6个看家基因的表达稳定性进行分析以期筛选出合适的内参基因， 并在此基础上进一步分析二斑叶螨不同品系P450酶系CYP392E亚家族基因的表达量差异。【结果】在二斑叶螨SS， Av-R和Sp-R品系中稳定性最高的内参基因为ELFn。以ELFn为内参基因， CYP392E7基因相对表达量在二斑叶螨Av-R品系中显著高于SS品系(P<0.05)， 为后者的2.18倍， 而在Sp-R品系和SS品系中差异不显著； 其余基因的相对表达量在2个抗性品系中均没有增加， Av-R品系的CYP392E4， CYP392E9和CYP392E10基因以及Sp-R品系的CYP392E1和CYP392E9基因相对表达量甚至显著下调， 分别为SS品系的48%， 74%， 65%， 63%和73%。【结论】在二斑叶螨SS， Av-R和Sp-R品系中ELFn为理想的内参基因， Av-R品系CYP392E4， CYP392E7， CYP392E9和CYP392E10基因相对表达量的显著变化可能与二斑叶螨对阿维菌素的抗性形成有关， Sp-R品系CYP392E1和CYP392E9基因也可能与二斑叶螨对螺虫乙酯的抗性形成有一定联系。

Screening of suitable reference genes and expression profiling of CYP392E subfamily genes in different resistant strains of Tetranychus urticae (Acari: Tetranychidae)

【Aim】 To screen suitable reference genes for studying the expression levels of CYP392E subfamily genes in susceptible (SS), avermectin-resistant (Av-R) and spirotetramat-resistant (Sp-R) strains of Tetranychus urticae. 【Methods】 The mRNA expression stability of six house keeping genes, i. e., α-tubulin, β-actin, ELFn, GAPDH, 5.8S rRNA gene and SDHA of T. urticae was detected by quantitative real-time PCR (qRT-PCR), and the relative expression level of CYP392E subfamily reference genes of cytochrome P450 monooxygenases (P450s) was analyzed. 【Results】 ELFn was the most stable reference gene in SS, Av-R and Sp-R strains of T. urticae. When ELFn was used as the reference gene, the expression level of CYP392E7 in Av-R strain (2.18-fold as high as that in SS strain) was significantly higher than that in SS strain, while there was no significant difference between Sp-R strain and SS strain. However, the relative expression levels of other genes did not increase, and even those of CYP392E4, CYP392E9 and CYP392E10 in Av-R strain and CYP392E1 and CYP392E9 in Sp-R strain decreased significantly (P<0.05) (48%, 74%, 65%, 63% and 73% of the expression level in SS strain, respectively). 【Conclusion】 ELFn could be used as an ideal reference in SS, Av-R and Sp-R strains of T. urticae. The significant change of the expression levels of CYP392E4, CYP392E7, CYP392E9 and CYP392E10 in Av-R strain may be related with the resistance of T. urticae to avermectin; CYP392E1 and CYP392E9 in Sp-R strain may also be related with the resistance of T. urticae to spirotetramat.