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目的探讨家蝇溴氰菊酯抗性机制。方法使用高分辨率熔解曲线(highresolutionmelting,HRM)分析与半分析定量PCR相结合的方法,分析家蝇抗性基因cyp6d1突变和扩增情况。通过对扩增的PCR产物进行测序来验证HRM准确性。结果与敏感品系相比,抗性品系cyp6d1基因的mRNA表达量显著增加(P=0.0002),抗性品系cyp6d1基因的mRNA表达量是敏感品系的50.7倍;使用HRM对cyp6d1进行突变分析,将其分为3种基因型,分别为A型(Tm=85.5℃)、B型(Tm=87.3℃)和C型(Tm=88.5℃);经测序,该HRM分析结果分别与3类基因序列型相对应。A型基因型为纯合的1087G、1101T和1155A(对应GenBank序列号U22367.1);C型基因型为纯合的1087A、1101G和1155G;B型基因型为A型和C型的杂合型。生物信息学分析表明,该突变为无意义突变。结论杭州市家蝇抗性品系cyp6d1基因mRNA表达量显著增加,可能是家蝇抗性增加的原因之一。
Objective To investigate the resistance mechanism of housefly deltamethrin. Methods High-resolution melting curve (HRM) analysis and semi-quantitative PCR were used to analyze the mutation and amplification of cyp6d1. HRM accuracy was verified by sequencing the amplified PCR product. Results The mRNA expression of cyp6d1 in resistant strain was significantly increased (P = 0.0002) compared with that in susceptible strain. The mRNA expression of cyp6d1 in resistant strain was 50.7 times that of susceptible strain. The mutation of cyp6d1 was detected by HRM, There were three genotypes, which were type A (Tm = 85.5 ℃), type B (Tm = 87.3 ℃) and type C (Tm = 88.5 ℃) Corresponding. Type A genotypes were homozygous 1087G, 1101T and 1155A (corresponding to GenBank accession number U22367.1); genotype C was homozygous 1087A, 1101G and 1155G; genotype B was a type A and C heterozygosity type. Bioinformatics analysis showed that the mutation was a nonsense mutation. Conclusion The expression of cyp6d1 gene in housefly resistance strain in Hangzhou increased significantly, which may be one of the reasons for the increase of housefly resistance.