MOLECULAR CLONING AND SEQUENCE ANALYSIS OF THREE NEW FULL‐LENGTH cDNAs OF CYTOCHROME P450 (CYP6N3v1‐v3)*FROM AEDES ALBOPICTUS**

Abstract The three new full‐length cDNA sequences including the complete 5′‐and 3′‐ untranslated regions (UTR) coding for cytochrome P450s from Aedes albopictus have been obtained. The P450 proteins deduced from the nucleotide sequences shared 58.6% ‐ 62.4% amino acid identity with CYP6N1 and CYP6N2 from Anopheles gambiae, and 99% with each other. The three new complete sequences have been submitted and named as CYP6N3v1, CYP6N3v2 and CYP6N3v3 by the P450 Nomenclature Committee. The original cDNAs were obtained by rapid amplification of cDNA ends (RACE) approach with several pairs of gene specific primers based on the cDNA fragment previously obtained from deltamethrin‐resistant strain of Ae. albopictus. Further analysis showed that the three new sequences are present in both resistant strain and susceptible strain and might be effectively translated. In addition, the 5′‐ and 3′‐UTRs were compared between the CYP6N3vl‐v3 and other known insect P450s. The multiplicity of trans‐lational control of insect P450 genes was discussed.     

Cloning and characterization of two cytochrome P450 CYP6AX1 and CYP6AY1 cDNAs from Nilaparvata lugens Stål (Homoptera: Delphacidae)

Two full‐length P450 cDNAs, CYP6AX1 and CYP6AY1, were cloned from the brown planthopper Nilaparvata lugens Stål (Homoptera: Delphacidae). Both CYP6AX1 and CYP6AY1 are typical microsomal P450s and their deduced amino acid sequences share common characteristics with other members of the insect P450 CYP6 family. CYP6AX1 and CYP6AY1 show the highest percent identity (36%) of amino acid to each other; both of them have 31–33% amino acid identity with CYP6B1 from Papilio polyxenes (Lepidoptera: Papilionidae), CYP6B4 from Papilio glaucus (Lepidoptera: Papilionidae), and CYP6B8 from Helicoverpa zea (Lepidoptera: Noctuidae). Phylogenetic analysis showed the clustering of CYP6AX1 and CYP6AY1 was in the clade including CYP6AE1 from Depressaria pastinacella (Lepidoptera: Oecophoridae) and the CYP6B family members from Helicoverpa and Papilio species. Northern blot analysis revealed that both of the P450s were induced by the resistant rice variety B5 (Oryza sativa L), and CYP6AY1 was expressed at a higher level than CYP6AX1. The results suggest that more than one P450s are likely involved in metabolism of rice allelochemicals and that they are possibly important components in adaptation of Nilaparvata lugens to host rice. Arch. Insect Biochem. Physiol. 64:88–99, 2007. © 2007 Wiley‐Liss, Inc.     

Metabolic imidacloprid resistance in the brown planthopper,Nilaparvata lugens,relies on multiple P450 enzymes

Target insensitivity contributing to imidacloprid resistance in Nilaparvata lugens has been reported to occur either through point mutations or quantitative change in nicotinic acetylcholine receptors (nAChRs). However, the metabolic resistance, especially the enhanced detoxification by P450 enzymes, is the major mechanism in fields. From one field-originated N. lugens population, an imidacloprid resistant strain G25 and a susceptible counterpart S25 were obtained to analyze putative roles of P450s in imidacloprid resistance. Compared to S25, over-expression of twelve P450 genes was observed in G25, with ratios above 5.0-fold for CYP6AY1, CYP6ER1, CYP6CS1, CYP6CW1, CYP4CE1 and CYP425B1. RNAi against these genes in vivo and recombinant tests on the corresponding proteins in vitro revealed that four P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, played important roles in imidacloprid resistance. The importance of the four P450s was not equal at different stages of resistance development based on their over-expression levels, among which CYP6ER1 was important at all stages, and that the others might only contribute at certain stages. The results indicated that, to completely reflect roles of P450s in insecticide resistances, their over-expression in resistant individuals, expression changes at the stages of resistance development, and catalytic activities against insecticides should be considered. In this study, multiple P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, have proven to be important in imidacloprid resistance.     

MOLECULAR CHARACTERIZATIONS OF TWO CYTOCHROME P450 GENES ENCODING CYP6A41 AND CYP6EK1 FROM THE ORIENTAL FRUIT FLY,Bactrocera dorsalis (DIPTERA: TEPHRITIDAE)

Two P450 genes encoding CYP6A41 and CYP6EK1 were cloned from the oriental fruit fly using polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) techniques. CYP6A41 and CYP6EK1 contained open reading frames of 1,530 and 1,524 nucleotides that encode 510 and 508 amino acid residues, respectively. The putative proteins shared 44% identity with each other. Phylogenetic analysis showed that CYP6A41 and CYP6EK1 were most closely related to Ceratitis capitata CYP6A10 and CYP6A subfamily. Expression patterns of the two genes in different geographical populations (Yunnan, Hainan, Dongguang, and Guangzhou), developmental stages (eggs, larvae, pupae, and adults), and tissues (midguts, fat bodies, and Malpighian tubules) were analyzed by real‐time quantitative PCR (RT‐qPCR) methods. The results showed that the expression levels of CYP6EK1 were significantly different among the four populations, but were not different for CYP6A41. Both the expressions of CYP6A41 and CYP6EK1 were development specific and had significantly higher levels in the larval stage. The expression of CYP6A41 did not vary among the midgut, fat body, or Malpighian tubules; however, CYP6EK1 expression was higher in the Malpighian tubules. The results suggest that CYP6A41 and CYP6EK1 might be involved in detoxification of xenobiotic compounds that were harmful to larval flies or development. Moreover, high expression of CYP6EK1 in the Malpighian tubules also implied participation in detoxification.     

STUDY ON THE DIVERSITY OF CYTOCHROME P450 GENES FROM AEDES ALBOPICTUS*

Abstract Several pairs of specific primers according to the obtained cDNA sequence fragment from deltamethrin‐resistant Aedes albopktus were designed to amplify new CYP6 genes from total RNA of Aedes albopictus by rapid amplification of cDNA ends (RACE) technique. The products of RACE were cloned and selected for sequencing. The deduced amino acid sequences were subjected to homologous analysis. The results indicated that the identities of clone GZS331 sequence from 5′‐RACE products and clone GZG033 sequence from 3′‐RACE products to CYP6N3vl ‐ v3 are 83.9% ‐ 84.3% and 98.2% ‐ 99.1% respectively; while the identities of the others from 3′‐RACE products to CYP6N3v1 ‐ v3 are 84.3% ‐ 85.6%. All of these obtained cDNA sequences have a higher homology to CYP3A1 in mouse and CYP9A1 in moth. The dendrogram constructed by PC/GENE software showed similar results to homologous analysis. These obtained sequences were submitted and named by the P450 Nomenclature Committee. The diversity of cytochrome P450 genes in Culicidae species was discussed.     

Characterization of multiple <Emphasis Type="Italic">CYP9A</Emphasis> genes in the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

Based on the advances in the silkworm genome project, a new genome-wide analysis of cytochrome P450 genes was performed, focusing mainly on gene duplication. All four CYP9A subfamily members from the silkworm, Bombyx mori, were cloned by RT-PCR and designated CYP9A19–CYP9A22 by the P450 Nomenclature Committee. They each contain an open reading frame of 1,593 bp in length and encode a putative polypeptide of 531 amino acids. Both nucleic acid and amino acid sequences share very high identities with one another. The typical motifs of insect cytochrome P450, including the heme-binding region, helix-C, helix-I, helix-K, and PERF, show high sequence conservation among the multiple proteins. Alignment with their cDNA sequences revealed that these paralogues share identical gene structures, each comprising ten exons and nine introns of variable sizes. The locations of their introns (all nine introns follow the GT–AG rule) are absolutely conserved. CYP9A19, CYP9A20, and CYP9A21 form a tandem cluster on chromosome 17, whereas CYP9A22 is separated from the cluster by four tandem alcohol-dehydrogenase-like genes. Their phylogenetic relationships and structural comparisons indicated that these paralogues arose as the results of gene duplication events. RT-PCR detected their mRNAs in different “first line of defense” tissues, as well as in several other organs, suggesting diverse functions. Tissue-selective expression also indicates their functional divergence. The identified CYP9A genes have not yet been found outside the Lepidoptera, and are probably unique to the Lepidoptera. They show high sequence and structural similarities to each other, indicating that the Lepidoptera-specific P450s may be of functional importance. This analysis constitutes the first report of the clustering, spatial organization, and functional divergence of P450 in the silkworm.     

中华按蚊CYP6Y亚家族基因的鉴定和生物信息学分析

【目的】鉴定中华按蚊Anopheles sinensis CYP6Y亚家族基因,分析它们的结构和特征,推测其可能的功能。【方法】以冈比亚按蚊An. gambiae CYP6Y1作为询问序列,通过双向Blast方法检索中华按蚊转录组中CYP6Y亚家族基因,并通过生物信息学方法分析基因结构、特征及可能的功能。【结果】从中华按蚊转录组测序数据中鉴定出2条CYP6Y亚家族基因,分别命名为AsCYP6Y1（GenBank登录号：KF709397）和AsCYP6Y2（GenBank登录号：KF709398）。序列分析显示,AsCYP6Y1和AsCYP6Y2全长分别为1 713 bp和1 815 bp,分别编码502和526个氨基酸。基因结构分析显示,该亚家族基因仅含有1个相位1型内含子并与其他P450基因形成保守的共线性分布。蛋白结构分析显示,这2个基因编码的蛋白含P450特有的5个特征序列和6个底物结合位点,且均不存在信号肽,其亚细胞定位为细胞质。3D结构分析显示,AsCYP6Y1有18条α螺旋和13股反向平行的β折叠,AsCYP6Y2有19条α螺旋和11股反向平行的β折叠。通过同样的方法,在达林按蚊An. darlingi中也鉴定出2个CYP6Y亚家族基因。系统进化分析显示,AsCYP6Y1和AsCYP6Y2分别与其他3种按蚊的CYP6Y1和CYP6Y2聚成一支,Bootstrap值均大于90%。替换率分析显示,中华按蚊AsCYP6Y1和AsCYP6Y2与其他3种按蚊同源基因的Ka/Ks均小于1。相对进化速率分析显示,中华按蚊CYP6Y和CYP6M亚家族的相对进化速率均显著快于CYP6P亚家族,而CYP6Y和CYP6M亚家族之间没有显著差异。【结论】在中华按蚊和达林按蚊中存在2个CYP6Y亚家族基因,之前在冈比亚按蚊和不吉按蚊An. funestus中也发现2个CYP6Y亚家族基因,表明CYP6Y亚家族基因可能在按蚊属广泛存在,且可能为按蚊属昆虫所特有。     

CYP9E2, CYP4C21 and related pseudogenes from German cockroaches, Blattella germanica: implications for molecular evolution, expression studies and nomenclature of P450s

The cDNAs of two novel P450s (CYP9E2 and CYP4C21) were isolated from German cockroaches, Blattella germanica. Both CYP9E2 and CYP4C21 are typical microsomal P450s and their deduced amino acid sequences share a number of common characteristics with other members of the P450 superfamily. Northern blot analyses using a CYP9E2 or CYP4C21 probe showed that 'CYP9E2' and 'CYP4C21' were expressed at all life stages. Two pseudogenes related to CYP9E2 and three pseudogenes related to CYP4C21 were also isolated. These represent the first P450 pseudogenes from an insect other than Drosophila melanogaster. The relative number of P450 pseudogenes in B. germanica is apparently higher than in D. melanogaster. The implications of these results for the molecular evolution, expression studies and nomenclature of P450s are discussed.     

An overexpressed cytochrome P450 CYP439A1v3 confers deltamethrin resistance in Laodelphax striatellus Fallén (Hemiptera: Delphacidae)

Deltamethrin resistance in Laodelphax striatellus had been associated with its oxidative detoxification by overexpression of four cytochrome P450 monooxygenases like CYP353D1v2, CYP6FU1, CYP6AY3v2, and CYP439A1v3. The first three P450s have been validated for insecticide‐metabolizing capability and only CYP6FU1 was found to degrade deltamethrin. In this study, an investigation was conducted to confirm the capability of CYP439A1v3 to degrade deltamethrin. The CYP439A1v3 was first expressed in Sf9 cell line and its recombinant enzyme was tested for metabolic activity against different insecticides using substrate depletion assay combined with metabolite identification. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and carbon monoxide (CO)‐difference spectra analysis showed that the intact cytochrome P450 protein was successfully expressed. Tests with probe substrates proved its enzyme activity, as p‐nitroanisole, ethoxycoumarin, and ethoxyresorufin were preferentially metabolized (specific activity 7.767 ± 1.22, 1.325 ± 0.37, and 0.355 ± 0.37 nmol/min per mg of protein, respectively) while only luciferin‐HEGE was not. In vitro incubation of the recombinant CYP439A1v3 protein with deltamethrin revealed hydroxylation by producing hydroxydeltamethrin. On the contrary, no metabolite/metabolism was seen with nonpyrethroid insecticide, including imidacloprid, buprofezin, chlorpyrifos, and fipronil. To the best of our knowledge, this is the first study to link a CYP450 from family 439 to confer pyrethroid resistance to L. striatellus. This finding should help in the design of appropriate insecticide resistance management for control of this strain of L. striatellus.