二斑叶螨内参基因的筛选及解毒酶基因的表达水平

Selection of Reference Genes and Study of the Expression Levels of Detoxifying Enzymes of Tetranychus urticae(Acari:Tetranychidae)

【目的】筛选二斑叶螨(Tetranychus urticae)敏感(SS)和抗甲氰菊酯品系(Fe-R)中合适的内参基因,并通过基因相对表达定量PCR研究二斑叶螨抗性品系解毒酶基因表达水平的变化。【方法】采用实时荧光定量PCR(quantitative real time PCR,RT-qPCR)技术,分析二斑叶螨5.8S rRNA、α-tubulin、β-actin、ELF、GAPDH、RPL13a、SDHA和TBP共8个候选内参基因mRNA表达水平的稳定性,并分析二斑叶螨酯酶(Carboxyl/cholinesterases,CCEs)、谷胱甘肽转移酶(Glutathione s-transferase,GSTs)及细胞色素P450单加氧酶(Cytocheome P450 monooxygenases,P450s)等主要解毒酶基因相对表达量。用GeNorm和NormFinder软件进行内参基因稳定性评估,通过比较Ct值的方法进行基因相对表达量计算。【结果】8个内参基因中稳定性最高的为α-tubulin;以α-tubulin为内参基因,二斑叶螨Fe-R品系GSTs基因TuGSTo1、TuGSTd1及P450s基因CYP3D2相对表达量均显著高于SS品系,而CCEs基因TuCCE1的表达量有所降低。【结论】在二斑叶螨SS和Fe-R品系中α-tubulin为理想的内参基因,RT-qPCR研究表明TuGSTo1、TuGSTd1和CYP3D2相对表达量的升高可能是二斑叶螨对甲氰菊酯产生高抗性的分子机理。

Abstract： [Objective] The objective of this study is to select suitable reference genes for studying the expression levels of detoxifying enzymes on fenpropathrin susceptible （SS） and resistant strains （Fe-R） of Tetranychus urticae by using quantitative real-time PCR （RT-qPCR）. [Method] The mRNA expression stability of eight candidate reference genes 5.8S rRNA, a-tubulin, fl-actin, ELF, GAPDH, RPL13a, SDHA and TBP of T. urticae were detected by RT-qPCR, the relative expression of the main detoxifying enzymes CCEs, GSTs and P450s were analyzed. GeNorm and NormFinder softwares were used to analyze and evaluate the data for reference genes and comparison of Ct value method was used for the calculation of relative expression levels. [Result] a-tubulin was one of the most stable genes among the eight reference genes. When a-tubulin was used as a reference gene, the expression levels of TuGSTol, TuGSTdland CYP3D2 in Fe-R strain were significantly higher than that in SS strain while TuCCE1 was downregulated. [Conclusion] The gene a-tubulin can be used as an ideal reference in both SS and Fe-R strains of T. urticae. Futhermore, the elevated relative expression levels of TuGSTol, TuGSTdl and CYP3D2 may be the molecular mechanism of Z urticae that with high resistance to fenpropathrin.