目的 利用饲喂双链RNA（dsRNA）对家蝇拟除虫菊酯抗性相关基因CYP6D1的表达进行干扰，并对干扰效果进行评价。方法 化学合成家蝇CYP6D1基因，连接入载体质粒L4440，转化入大肠埃希菌DH5α扩增质粒。抽提质粒，将其转化到大肠埃希菌HT115（DE3），并在异丙基-D-硫代半乳糖苷（IPTG）的诱导下表达CYP6D1基因的dsRNA。将含有CYP6D1基因dsRNA的HT115（DE3）饲喂家蝇，72 h后通过荧光相对定量技术评价家蝇体内CYP6D1基因的表达变化。未进行质粒L4440转化的大肠埃希菌菌株HT115（DE3）饲喂家蝇作为对照。结果 采用2-△△Ct法对对照组和处理组的mRNA相对表达量进行定量。目的基因和内参基因标准曲线R2值均> 0.99，扩增效率M值均在0.90~1.10之间。与对照组相比，处理组CYP6D1基因mRNA相对表达量下降了40.1%，两者之间差异有统计学意义（t=-11.377，P=0.008）。结论 初步建立了饲喂dsRNA对家蝇拟除虫菊酯抗性基因CYP6D1表达进行干扰的方法，在mRNA水平上实现基因表达干扰效果。
Objective To interfere with the expression of pyrethroid resistance-related gene CYP6D1 in Musca domestica by feeding dsRNA, and to evaluate the interference effect. Methods The CYP6D1 gene of M. domestica was chemically synthesized, inserted into the vector plasmid L4440, and transformed into Escherichia coli DH5α to amplify the plasmid. The plasmids were then extracted and transformed into Escherichia coli HT115 (DE3) to express the dsRNA of CYP6D1 gene under the induction of isopropyl-D-thiogalactoside (IPTG). Escherichia coli HT115 (DE3) containing the dsRNA of CYP6D1 gene was fed to M. domestica, and relative fluorescence quantification was utilized to evaluate the expression changes of CYP6D1 gene in M. domestica 72 hours later. Escherichia coli strain HT115 (DE3) without plasmid L4440 transformation was fed to M. domestica, which was used as control group. Results The relative expression of mRNA in control group and treatment group was quantified by 2-△△Ct method. The R2 value of the standard curve of target gene and reference gene was greater than 0.99, and the amplification efficiency (M value) was between 0.90 and 1.10. The treatment group showed significantly lower (by 40.1%) relative expression of CYP6D1 mRNA than the control group (t=-11.377, P=0.008). Conclusion A method of interfering with the expression of pyrethroid resistance-related gene CYP6D1 in M. domestica was preliminarily established by feeding dsRNA, and the effect of interfering with gene expression was achieved at the mRNA level.
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