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ISSN 1672-9145 Acta Biochim Biophys Sin 2005, 37(5): 317–326 CN 31-1940/Q

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Cloning and Overexpression of CYP6F1, a Cytochrome P450 Gene, from Deltamethrin-resistant Culex pipiens pallens

Mao-Qing GONG, Yan GU, Xiao-Bang HU, Yan SUN, Lei MA, Xiu-Lan LI, Li-Xin SUN, Jing SUN, Jin QIAN, and Chang-Liang ZHU*

Department of Pathogenic Biology, Nanjing Medical University; Key Laboratory of Modern Pathogenic Biology of Jiangsu Province, Nanjing 210029, China

Abstract 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.

Key words Culex pipiens pallens; insecticide resistance; cytochrome P450; RT-PCR

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Received: December 15, 2004 Accepted: March 9, 2005

This work was supported by the grants from the National Natural Science Foundation of China (No. 30170835, No. 30371755)

Abbreviations: RT-PCR, reverse transcription-polymerase chain reaction; RACE, rapid amplification of cDNA end; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

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