Molecular cloning, nucleotide sequence and gene expression of a cytochrome P450 (CYP6F1) from the pyrethroid-resistant mosquito, Culex quinquefasciatus Say
Molecular cloning, nucleotide sequence and gene expression of a cytochrome P450 (CYP6F1) from the pyrethroid-resistant mosquito, Culex quinquefasciatus Say
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DOI：
10.1016/s0965-1748(99)00114-9
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发表时间：
2000-02-01
影响因子：
3.8
通讯作者：
Yamakawa, M
中科院分区：
文献类型：
--
作者：
Kasai, S;Weerashinghe, IS;Yamakawa, M
为了分析致倦库蚊（Culex quinquefasciatus）的细胞色素P450 s，我们定量测定了致倦库蚊幼虫肠道和尸体微粒体中P450 s和B（5）的含量。结果表明，内脏中的含量是胴体的30倍。昆虫P450家族6（CYP 6）蛋白序列中的保守区域作为合成简并寡核苷酸引物以克隆P450 cDNA的指导。用引物对氯菊酯敏感和抗性的棉铃虫4龄幼虫肠道mRNA进行RT-PCR分析。致倦肌CA的PCR产物。克隆了250个碱基对（bp），并测定了来自敏感菌株的35个克隆和来自抗性菌株的28个克隆的核苷酸序列。从这些克隆推导的氨基酸序列的比对表明，它们可分为六个亚型。接下来，我们从肠道cDNA文库中筛选cDNA克隆（CYP 6 F1）并确定核苷酸序列。北方印迹分析表明，CYP 6 F1基因在氯菊酯耐药株中的表达比在敏感株中的表达更强。推导的氨基酸序列表明，CYP 6 F1在P450中具有膜锚定信号、还原酶结合位点、血红素结合位点、ETLR基序和底物识别位点等保守结构域。系统发育分析表明，CYP 6 F1与CYP 6D 1密切相关，参与拟除虫菊酯解毒。(C)2000爱思唯尔科技有限公司版权所有。
To analyze cytochrome P450s in the southern house mosquito, Culex quinquefasciatus, we quantified the content of P450s and b(5) in larval microsomes of guts and carcasses. Results indicated that content was 30 times higher in guts than in carcasses. A conserved region in the alignment of insect P450 family 6 (CYP6) proteins served as a guide for the synthesis of degenerate oligonucleotide primers to clone P450 cDNAs. Primers were used in the reverse transcription-polymerase chain reaction (RT-PCR) of gut mRNA from 4th-instar larvae of the permethrin-susceptible or resistant C. quinquefasciatus. PCR products of ca. 250 base pairs (bp) were cloned, and nucleotide sequences of 35 clones from susceptible and 28 from resistant strains determined. Alignment of the deduced amino acid sequences from these clones showed them to be classifiable into six isoforms. We next screened a cDNA clone (CYP6F1) from a gut cDNA library and determined the nucleotide sequence. Northern blot analysis showed that the CYP6F1 gene in the permethrin-resistant strain appeared to be expressed more strongly than in the susceptible strain. The deduced amino acid of CYP6F1 showed that it has conserved domains of a membrane-anchoring signal, reductase binding sites, a heme-binding site, ETLR motif and substrate recognition sites in P450s. Phylogenetic analysis showed that CYP6F1 is strongly related to CYP6D1 involved in pyrethroid detoxification. (C) 2000 Elsevier Science Ltd. All rights reserved.