Cloning and overexpression of CYP6F1, a cytochrome P450 gene, from deltamethrin-resistant Culex pipiens pallens.
CYP6F1 (GenBank/EMBL accession No. AY662654), a novel gene with a complete encoding sequence in the cytochrome P450 family 6, was cloned and sequenced from deltamethrin-resistant 4th instar larvae of Culex pipiens pallens. The cDNA sequence of CYP6F1 has an open reading frame of 1527 bp, which encodes a putative protein of 508 amino acid residues. The deduced amino acid sequence of CYP6F1 indicated that the encoded P450 has conserved domains of a putative membrane-anchoring signal, putative reductase-binding sites, a typical heme-binding site, an ETLR motif and substrate recognition sites. Semi-quantitative RT-PCR analysis indicated that the CYP6F1 gene was expressed to a greater extent in the deltamethrin-resistant strain than in the susceptible strain of Cx. pipiens pallens. The expression levels of the CYP6F1 gene in the deltamethrin-resistant 1st, 2nd, 3rd, 4th instar larvae and adult female mosquitoes differed, with highest expression levels in the 4th instar larvae. In addition, the CYP6F1 gene was stably expressed in mosquito C6/36 cells, and the expected 61.2 kDa band was identified by Western blotting. The cells transfected with CYP6F1 had an increased resistance to deltamethrin as compared with control cells. These results indicate that CYP6F1 is expressed at higher levels in the deltamethrin-resistant strain, and may confer some insecticide resistance in Cx. pipiens pallens.
Gong
Mao-Qing
MQ
Department of Pathogenic Biology, Nanjing Medical University, Nanjing 210029, China.
Gu
Yan
Y
Hu
Xiao-Bang
XB
Sun
Yan
Y
Ma
Lei
L
Li
Xiu-Lan
XL
Sun
Li-Xin
LX
Sun
Jing
J
Qian
Jin
J
Zhu
Chang-Liang
CL
D000595:Amino Acid Sequence; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D003465:Culex / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000072472:Cytochrome P450 Family 6; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D007113:Immunity, Innate; D019476:Insect Proteins; D007306:Insecticides / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009570:Nitriles / Q000494:pharmacology*; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011722:Pyrethrins / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid
D019476:Insect Proteins; D007306:Insecticides; D009570:Nitriles; D011722:Pyrethrins; C017180:decamethrin; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; C406279:CYP6F1 protein, Culex pipiens pallens; D000072472:Cytochrome P450 Family 6