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Cytochrome P450 genes expressed in the deltamethrin-susceptible and -resistant strains of Culex pipiens pallens
Pesticide Biochemistry and Physiology 75 (2003) 19–26 www.elsevier.com/locate/ypest
Cytochrome P450 genes expressed in the deltamethrin-susceptible and -resistant strains of Culex pipiens pallens Bo Shen,a Hui-Qin Dong,b Hai-Sheng Tian,a Lei Ma,a Xiu-Lan Li,a Guan-Ling Wu,a and Chang-Liang Zhua,* a
Department of Parasitology, Nanjing Medical University, Nanjing 210029, People’s Republic of China b Ministry of Education, Key Laboratory for Biodiversity Science and Ecological Engineering, Institute of Biodiversity Science, Fudan University, Shanghai 200433, People’s Republic of China Received 12 August 2002; accepted 27 January 2003
Abstract cDNA sequences encoding cytochrome P450 were amplified, respectively, from deltamethrin-susceptible and -resistant strains of the mosquito, Culex pipiens pallens, with a pair of degenerate primers by RT-PCR and the Direct Cloning Method, and then identified by cDNA chip and Northern blot. Twenty-four positive clones obtained were shown to be new sequences. They had been placed in GenBank and were approved by the Cytochrome P450 Nomenclature Committee, classified to the subfamilies CYP4C, CYP4D, CYP4H, and CYP4J. The microarray signal values of five P450 sequences (CYP4H21, 4H22v1, 4J4v2, 4J6v1, and 4J6v2) were 3.1–7.7 times higher in resistant probe than in susceptible probe and the sequence 4H23v2 reacts only with resistant probe. The results of Northern blot of the above six P450 sequences were similar to that of microarray analyses. These results indicate that CYP4 may be related to deltamethrin resistance in Cx. pipiens pallens. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Culex pipiens pallens; Resistance to pesticide; Cytochrome P450; Microarray; Northern blot
1. Introduction The monovalent oxygenase system of cytochrome P450 is a primary detoxicating enzyme system to be involved in metabolisms of various insecticides and other exogenous and autogenous compounds in insects. The cytochrome P450 is the reductase that facilitates transfer of electrons from NADPH to the P450 oxidoreductase [10,20]. *
Corresponding author. Fax: +86-25-650-8960. E-mail address: [email protected] (C.-L. Zhu).
P450 in insects was discovered by Ray for the first time [14]. Following 20 years, because its content in insects was low and unstable, P450 was extremely difficult to purify and studies on it were limited on physico-chemical properties and enzymatic characteristics [19]. In recent years, the cytochrome P450s from over 20 kinds of insects were studied and more than 60 P450 genes have been cloned for Drosophila melanogaster alone [4,7,13,21]. Culex pipiens pallens is distributed widely and attain high populations in many regions of
0048-3575/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0048-3575(03)00014-2
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China. Since 1990s, the high populations of this insect have been controlled with deltamethrin, but the development of resistance to deltamethrin in mosquito has occurred much more quickly than resistance to organophosphates [11,16]. It is reported that P450 plays an important role in pyrethroid resistance [2,8]. Up till now, there has been no report on P450 gene in Cx. pipiens pallens. In order to investigate the relation between P450 gene and deltamethrin resistance in Cx. pipiens pallens, P450 specific fragments from both deltamethrin-susceptible and -resistant Cx. pipiens pallens were amplified by RT-PCR and were cloned by the direct cloing method. The expressions of positive clones were identified by microarray and Northern blot.
2. Materials and methods
2.3. RNA extraction and purification Eight hundred unfed female adult mosquitoes of the susceptible and resistant strains (about 1 g each) were collected within 3 days after its emergence. These mosquitoes were ground with liquid nitrogen before it was transferred to homogenizer. Isothicyanic acid guanidine one-step method was adopted to extract total RNA, and then 1% agarose-gel electrophoresis was performed. The total RNA purity was measured by spectrophotometry. With polyATtract mRNA Isolation System Kit, mRNA was purified from 0.5 mg total RNA according to the descriptions of the products. 2.4. cDNA synthesis With 2 lg mRNA obtained from the susceptible strain and resistant strain of Cx. Pipiens pallens, cDNA was synthesized using cDNA Synthesis System Kit according to the descriptions of the products.
2.1. Mosquitoes 2.5. Primer synthesis The susceptible strain of Cx. pipiens pallens (SS) was obtained from Shanghai Insect Institute of the Chinese Academy of Science in 1999 and was never exposed to any kind of pesticides. The strain was reared and bred at 26 1 °C, 80 5%RH) and 16L:8D photoregime and went down to further generation for more than 70 successive generations. The resistant strain of Cx. pipiens pallens (RR) was selected with deltamethrin generation by generation in our laboratory and the resistant ratio was 519 after 70 generations (Li et al., 2002).
2.2. Reagents Isothiocyanic acid guanidine was purchased from Shanghai Boau Biological Science and Technology Company. b-Sulfenyl alcohol was obtained from Fluka Company; SLS from Sigma Company; DEPC from Serva Company. PolyATtract Series 9600 RNA Isolation System Kit, cDNA Synthesis System Kit, reverse transcriptase (AMV), JM109 bacterial strain, Pinpoint Xa-1T plasmid carrier, and SmaI, Taq polymerase were the products of Promega Company.
According to Scott et al. [15], except for one inosine replaced by ACGT, a pair of P450 degenerate primers was synthesized by Dalian Bao Bio-Tech Company. The up-stream primer (P1) encoded a part of the hydrophobic helixes I of P450 protein and was considered to be a specific sequence in CYP4 family. The down-stream primer (P2) was located in the surrounding areas of cysterine residue combined with heme. The F–G– C–G structure found was the common characteristics of insect P450 superfamily
2.6. Amplification and purification of target fragments The synthesized primers were used to amplify P450 cDNA fragments by PCR from cDNA template. PCR system was primarily according to the reference documents [12]. Amplification was done under the following conditions: 94 °C predenaturation for 5 min, followed by 94 °C denaturation for 30 s, 55 °C renaturation for 30 s, and 72 °C extension for 1 min. After completion of 30 such cycles, the process was extended for 20 min at 72 °C.
B. Shen et al. / Pesticide Biochemistry and Physiology 75 (2003) 19–26
With 1.5% low melting-point agarose-gel electrophoresis, target DNA fragments were recovered by purification attained by PCR amplification. 2.7. Cloning of target fragment Target DNA fragment was cloned with T/A direct cloning methods [17]. The purified target fragment was cloned with PinPoint Xa-1 T plasmid vector having T cohesive terminus and transferred to JM109. It remained and grew in AMPþ plate containing ampiciccin over night. Leukasmuses were selected and amplified to obtain positive clone with SP6/PinPoint primers by means of PCR. 2.8. Sequencing and analysis of target fragment Positive clones from the susceptible strain and resistant strain were randomly selected. With caustic cracking solving process, plasmid DNA was extracted. SP6/PinPoint primers were used to prepare duplex DNA target fragment They were purified with Quick PCR Purification Kit and sequenced with SP6 primer by Dalian Bao BioTech Company. The nucleotide sequences were compared to the protein sequence databases incorporated into GenBank using the BLASTX program on the NCBI BLAST Server [15]. Only those clones that had statistically significant similarity scores to numerous P450s and to no other genes were considered to be P450 genes. 2.9. Microarray analyses This was done in Shanghai Cell Biology Institute of The Chinese Academy of Sciences. About 810 subtractive cDNA sequences were cloned from deltamethrin-susceptible and -resistant strains of Cx. pipiens pallens by suppression subtractive hybridization (SSH) in our laboratory, a total of 24 P450 cDNA with the 810 clones was prepared for microarray analyses in this experiment. Twenty-four P450 cDNA samples were amplified by PCR, and after being tested by electrophoresis, the amplified productions were, respectively, dotted to 2 nylon membranes in array pattern with a high speed automaton made by TAS BioRobotics Company of UK. About 50 ng cDNA per dot (diameter 40 lm) was used and 2 dots per sample were made. The [a-33 P]dATP-
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cDNA probes were produced, respectively, with mRNAs from the susceptible and resistant strains (named as susceptible probe and resistant probe) and hybridized with the membranes [9,18]. Cx. pipiens pallens b-actin was used as control. Hybrid signals were analysed with FLA-300 Signal Tester made by Fuji Company, Japan. The discriminant standards were as follows: (A) The setting signal value was delineated in the two membranes. (B) The signal value difference of two dots per sample in a membrane was less than two times. (C) If the signal values of two dots per sample in a membrane were more than three times higher than those in the another one, significant difference between the two probes may be defined in the sample. (D) When the signal values of differentially expressed two lighter dots were more than 10, the difference was visible to the naked eye. 2.10. Northern blot analyses Gene expression of the divergence fragments screened with microarray were analysed by Northern blot [1,12]. Total RNA was extracted from the susceptible and resistant strains of Cx. mosquito. Five mg RNA from each strain was subjected to 1.5% agarose/formaldehyde gel electrophoresis and transferred to a nylon membrane. The PCR-amplified fragments of divergence P450 genes were labeled with [a-32 P]dCTP by the Random Prime DNA Kit (Boehringer–Mannheim). The probes were used to hybridize the target RNA on a membrane at 55 °C for approximately 20 h. The nylon membrane was firstly washed at room temperature for 15 min with wash solution (2 SSC and 0.1% SDS), then at 37 °C for 1 h with wash solution (0:1 SSC and 0.1% SDS), at last at 65 °C for 1 h with wash solution (0:1 SSC and 0.1% SDS). The membrane was exposed to X-ray film for 24 h.
3. Results 3.1. Target gene fragment amplification The first-chained cDNA was synthesized by reverse transcription of mRNA from both susceptible strain and resistant strain of Cx. pipiens
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pallens. A pair of degenerate primer transcript was used to amplify the template by RT-PCR. Two fragments of about 485 and 510 bp were obtained from both strains. After purification and PCR amplification for twice, stronger fragments were obtained. 3.2. Cloning of target gene fragment Using T4 DNA ligase, two target gene fragments of the susceptible strain and resistant srain were inserted into PinPoint Xa-1 T clone carrier, respectively. The up-stream and down-stream primers of muitlple clone locus of plasmid PinPoint Xa-1T (PinPoint sequencing primer: 50 -CG TGACGCGGTGCAGGGCG-30 ; SP6 sequencing primer: 50 -CATAGCGATTAGGTAGGTAACA CTATAG-30 ) were used as PCR primer. Leukasmuses were directly selected for PCR. The sum of the length of the two sequencing primers to the EcoRV clone locus is 144 bp. From the recombined clones, two segments of about 630 and 655 bp were obtained, which were identical in size to those before cloning.
3.3. Sequencing and analyses of target gene fragments Cloning was conducted twice and a total of 112 positive spots were obtained. Out of 37 clones sequenced, 31were shown to encode P450. Twenty-four of 31 sequences were not identified and the other seven ones were repeat copies, their molecular weight being 369–444 bp. Compared with P450 sequence databases incorporated into GenBank, all of the 24 sequence were new, of which 6 were obtained from the susceptible strain, and the other 18 were derived from the resistant strain. They had been lodged in Genbank. The 24 new genes (alleles) were approved by the Cytochrome P450 Nomenclature Committee, belonging to the subfamily CYP4C, CYP4D, CYP4H, and CYP4J in CYP4 family (Table 1). Para-position queuing graph of amino acid sequences was made, which were deduced from 24 P450 new genes (alleles) (Fig. 1). From the graph one dendrogram was obtained by unweighted pair-group method using arithmetic averages (UPGMA) (Fig. 2).
Table 1 Charactertics of 24 new cytochrome P450 genes (alleles) from Cx. pipiens pallens Genea (allele)
GenBank Accession No.
Strain
No. of nucleotide
No. of residues
No. of clone
CYP4C22v1 CYP4C22v2 CYP4C23 CYP4D18v1 CYP4D18v2 CYP4D19v1 CYP4D19v2 CYP4D19v3 CYP4H12v1 CYP4H12v2 CYP4H13 CYP4H21 CYP4H22v1 CYP4H22v2 CYP4H23v1 CYP4H23v2 CYP4H23v3 CYP4J4v1 CYP4J4v2 CYP4J6v1 CYP4J6v2 CYP4J7v1 CYP4J7v2 CYP4J8
AF157093 AF208659 AF157090 AF190775 AF191728 AF190786 AF190789 AF190777 AF157091 AF191731 AF160259 AF208660 AF190782 AF190784 AF190787 AF191729 AF190783 AF157092 AF190778 AF190776 AF190781 AF190780 AF190785 AF190788
R S S S R R R S R R R R R R R R R R S S R R R R
378 378 444 444 444 378 381 381 390 390 439 381 381 381 381 381 381 387 387 369 369 384 384 384
126 126 148 148 148 126 127 127 130 130 127 127 127 127 127 127 127 129 129 123 123 128 128 128
1 1 1 1 1 2 1 1 1 1 1 2 1 1 2 1 1 1 1 1 1 4 2 1
a
Named by the Cytochrome P450 Nomenclature Committee.
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Fig. 1. Alignment of deduced amino acid sequences for the 24 new cytochrome P450 genes (alleles) from Cx. pipiens pallens. + as termination codon (CYP4J4V1); ) as **KETLR*-P area and PERF area, respectively.
3.4. Microarray analyses After the 24 P450 cDNA samples reacted with susceptible probe and resistant probe separately, the microarray signal values of 22 samples were more than 10, while those of the other two samples were less than 10 in susceptible probe, but more than 10 in resistant probe, hybrid rate being 100%. The signal value ratios
between both chip membranes were less than three in 18 samples, the differences being not significant; the hybrid signal values of CYP4 H21, 4H22v1, 4J4v2, 4J6v1, and 4J6v2 were 3.1–7.7 times higher in resistant probe than in susceptible probe, and the hybrid signal value of 4H23v2 was three in susceptible probe, which corresponded to the setting signal value of the membrane, but 29.1 in resistant probe, the latter
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Fig. 2. UPGMA dendrogram of amino acid sequences deduced from 24 new cytochrome P450 genes (alleles) of Cx. pipiens pallens.
Table 2 Microarray signal value of 6 CYP4 cDNA from Cx. pipiens pallensa Gene
Site
4H21 4H22v1 4H23v2 4J4v2 4J6v1 4J6v2
E1 E7 G4 A3 A5 C3
a
F1 F6 G5 B2 B4 D2
S
R
R/S
124.8 33.2 3.0 8.2 96.3 15.4
383.3 109.6 29.1 63.3 530.4 53.4
3.1 3.3 9.7 7.7 5.5 3.5
S as susceptible probe; R as resistant probe; R/S as ratio between the both signal value.
being 9.7 times as more as the former (Table 2, Fig. 3). 3.5. Northern blot analyses The results of Northern blot of six P450 genes (CYP4H21, 4H22v1, 4H23v2, 4J4v2, 4J6v1, and 4J6v2) were consistent with those of the microarray analyses (Fig. 4), prompting that CYP4 may be related to deltamethrin resistance in Cx. pipiens pallens.
4. Discussion Though the sequence found in this study was only 1/3 length of P450, the hydrophobic helixes I, in which the up-stream primer 1 was located, had the common characteristics of CYP4, and heme combination, in which the down-stream
primer 2 was located, was also common in P450, which was regarded as the appraised characteristics of P450 family members. The structures of the 24 new sequences cloned in our laboratory contained two relatively conservative areas which CYP4 family had in common: **KETLR*-P area () indicate amino acid residue, * refers to hydrophobic residue) and PERF area [10,15,20]. These sequences belong to the subfamilies of CYP4C, CYP4D, CPY4H, and CYP4J of CYP4 family approved by the Cytochrome P450 Nomenclature Committee. It is commonly acknowledged that CYP6 family members have close relationship with the resistance to insecticide in the known superfamily of insect P450 [3,5]. In this investigation, two cDNA fragments were obtained from RNA of Cx. pipiens pallens with RT-PCR, the fragments in the susceptible strain being weaker than those in the resistant strain. After the electrophoretic
B. Shen et al. / Pesticide Biochemistry and Physiology 75 (2003) 19–26
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Fig. 3. Results of microarray analyses of 24 CYP4 from susceptible and resistant strains of Cx. pipiens pallens. S, susceptible strain; R, resistant strain.
Fig. 4. Northern blot of 6 CYP4 cDNA from deltamethrin-susceptible and -resistant Cx. pipiens pallens. (S) deltamethrin-susceptible strain, (R) deltamethrin-resistant strain, (1) CYP4H21, (2) CYP4H22v1, (3) CYP4H23v2, (4) CYP4J4v2, (5) CYP4J6v1, (6) CYP4J6v2, The bottom blot was obtained with Cx. pipiens pallens b-actin used as control.
comparison of increased template, the fragments in the susceptible strain were only 1/10 of those in the resistant strain. Our work observed more positive clones in resistant strain than in susceptible strain. There were repeat copies in five sequences of resistant strain but none in susceptible strain. The microarray signal values of CYP4H21, 4H22v1, 4J4v2, 4J6v1, and 4J6v2 were 3.1–7.7 times higher in resistant strain than in susceptible strain. The results of Northern blot of the six P450 genes were consistent with those of the microarray analyses. The results mentioned above prompted that CYP4 family may be related to deltamethrin resistance in Cx. pipiens pallens and further proved that over-expression of P450 mRNA is one of the molecular mechanisms of insect resistance to pyrethroid pesticide. It is interesting to observe that CYP4H23v2 could react with resistant probe, but not with susceptible probe. The two alleles of 4H23v2,
4H23v1 and 4H23v3, could react with susceptible probe and their hybrid signal values were 106.9 and 11.7, respectively. As compare to the nucleotide sequences of the two alleles, 4H23v2 had a T ! A difference, resulting in the replacement of Ser by Thr in position 75, and is worthwhile of further studies. It is wident from Fig. 1 that, the 24 new sequences commonly had 10 amino acid residues in the same positions, 5 of which were prolines (pro). In 1994, Scott et al. [15] reported 17 new sequences of CYP4. Among the 15 common amino acid residues, four were prolines. The above results suggested that proline was conservative compared with other amino acids and might play an important role in genetic stability of P450 genes. In this experiment, 2 or 3 alleles were cloned from nine genes, respectively, and replacement had taken place in 2–6 amino acid residues of them. Both CYP4D19 and CYP4H23 had three alleles. Residue replacement in the same positions was only found in one of the three and no different residue of each was found. CYP4D19v2 and 4D19v3 had 6 amino acid heteromorphoses, while in the same positions 4D19v1 had three same residues as 4D19v2 and three same residues as 4D19v3, suggesting 4D19v1 was the transition stage between 4D19v2 and 4D19v3. CYP4H23v1, 4H23v2, and 4H23v3 had 0, 1, and 2 residue replacement, respectively, indicating 4H23v1 was the oldest one among the three and 4H23v2 and 4H23v3 had been branched by 4H23v1 during evolution. Genes usually have only one terminator signal. In rare cases, two termination signals do exist, but the distance between the two terminator codons is
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usually shorter [6]. In this experiment, in the near 30 -terminus, CYP4J4v1 had a terminator codon T (U) AG, but followed by thorough reading. Compared with other full P450 sequence in GenBank, there is a length of about 70 amino acid residues after the position of T (U)AG in CYP4J4v1. UAG is a terminator codon that is less frequently used. Its exact function in peptide chain termination of CYP4J4v1 needs further studies.
Acknowledgments We are grateful to Prof. Zheng-Hua Tang and Mrs. Pei-Jun Zhuang working in Shanghai Insect Institute of the Chinese Academy of Science, for providing the susceptible strain of Cx. pipiens pallens. We thank Dr. David R. Nelson (University of Tennessee, USA) for his help in naming and sequence analysis of the P450 genes.This work was jointly supported by National Foundation of Natural Science of China (Nos. 39970666 and 30170835) and Natural Science Foundation of Jiangsu Province (BK02050).
References [1] F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Struhl, Current Protocols in Molecular Biology, Wiley, New York, 1994. [2] F.A. Carino, J.F. Koener, F.W. Plapp, R. Feyereisen, Constitutive overexpresion of the cytochrome P450 gene CYP6A1 in a house fly strain with metabolic resistance to insecticides, Insect Biochem. Mol. Biol. 24 (1994) 411–418. [3] M.B. Cohen, J.F. Koener, R. Feyereisen, Structure and chromosomal localization of CYP6A1, a cytochrome P450 encoding gene from the house fly, Gene 146 (1994) 267–272. [4] M.B. Cohen, R. Feyereisen, A cluster of cytochrome P450 genes of CYP6 family in the housefly, DNA Cell Biol. 14 (1995) 73–82. [5] P.B. Danielson, R.T. Macintyre, J.C. Fogleman, Molecular cloning of family of xenobiotic-inducible drosophlid cytochrome P450: evidence for involvement in host-plant allelochemical resistance, Proc. Natl. Acad. Sci. USA 94 (1997) 10797–10802. [6] N.G. Davis, P. Model, An artificial anchor domain: hydrophobicity suffices to stop transfer, Cell 41 (1985) 607–614.
[7] R. Feyereisen, Insect P450 enzymes, Annu. Rev. Entomol. 44 (1999) 507–533. [8] J.C. Fogleman, P.B. Danielson, R.J. Macintyre, The molecular basis of adaptation in Drosophila: the role of cytochrome P450, Evol. Biol. 30 (1998) 15–77. [9] T.R. Golub, D.K. Slonim, P. Tamayo, C. Huard, M. Gaasenbeek, J.P. Mesirov, et al., Molecular classification of cancer: class discovery and class predication by gene expression monitoring, Science 286 (1999) 531–537. [10] X.F. Leng, Z.H. Tang, Y.C. Wang, in: Molecular Toxicology of Pesticides and Insect Resistance to Pesticides, Chinese Agricultural Publishing House, Beijing, 1996, pp. 132–147. [11] X.L. Li, L. Ma, L.X. Sun, C.L. Zhu, Biotic characteristics in the deltamethrin-susceptible and resistant strains of Culex pipiens pallens (Diptera: Culicidae) in China, Appl. Entomol. Zool. 37 (2002) 305–308. [12] W.D. Liu, Mosquito resistance to pesticides in China, Chin. J. Vector. Biol. Cont. 1 (1990) 41– 43. [13] S.D. Lu, in: Current Protocols for Molecular Biology, Chinese Publishing House of Higher Education, Beijing, 1993, pp. 163–182. [14] D.R. Nelson, L. Koymans, T. Kamataki, J.J. Stegeman, R. Feyereisen, P450 superfamily: update on new sequences, gene mapping, accession numbers, and nomenclature, Pharmacogenetics 6 (1996) 1–42. [15] J.W. Ray, in: Pest Infestation Research, HMSO, London, 1965, pp. 69–74. [16] J.G. Scott, F.H. Collins, R. Feyereisen, Diversity of cytochrome P450 genes in the mosquito, Anopheles albimanus, Biochem. Biophys. Res. Commun. 205 (1994) 1452–1459. [17] Y.Q. Sun, J.G. Yuan, J. Li, The resistance mechanism of housefly against pyrethroids, Acta Entomol. Sinica. 33 (1990) 265–272. [18] Y. Wang, One new T/A cloning method, Prog. Biol. Tech. 16 (1996) 55–57. [19] G.P. Yang, D.T. Ross, W.W. Kuang, P.O. Brown, R.J. Weigel, Combining SSH and cDNA microarray for rapid identification of differentially expressed genes, Nucleic Acids Res. 27 (1999) 1517–1523. [20] B.H. Ye, in: Modern Medical Entomology, Chinese Publishing House of Higher Education, Beijing, 1996, pp. 179–182. [21] Q.H. Zhai, Some aspects of progress in insect biology: molecular mechanisms of insecticide resistance, Acta Entomol. Sinica 38 (1995) 493–501. [22] C.L. Zhu, G.L. Wu, Z.S. Zhang, Studies on insect cytochrome P450, Chin. J. Parasitol. Parasitic Dis. 17 (1999) 46–49.
Cytochrome P450 genes expressed in the deltamethrin-susceptible and -resistant strains of Culex pipiens pallens Bo Shen,a Hui-Qin Dong,b Hai-Sheng Tian,a Lei Ma,a Xiu-Lan Li,a Guan-Ling Wu,a and Chang-Liang Zhua,* a
Department of Parasitology, Nanjing Medical University, Nanjing 210029, People’s Republic of China b Ministry of Education, Key Laboratory for Biodiversity Science and Ecological Engineering, Institute of Biodiversity Science, Fudan University, Shanghai 200433, People’s Republic of China Received 12 August 2002; accepted 27 January 2003
Abstract cDNA sequences encoding cytochrome P450 were amplified, respectively, from deltamethrin-susceptible and -resistant strains of the mosquito, Culex pipiens pallens, with a pair of degenerate primers by RT-PCR and the Direct Cloning Method, and then identified by cDNA chip and Northern blot. Twenty-four positive clones obtained were shown to be new sequences. They had been placed in GenBank and were approved by the Cytochrome P450 Nomenclature Committee, classified to the subfamilies CYP4C, CYP4D, CYP4H, and CYP4J. The microarray signal values of five P450 sequences (CYP4H21, 4H22v1, 4J4v2, 4J6v1, and 4J6v2) were 3.1–7.7 times higher in resistant probe than in susceptible probe and the sequence 4H23v2 reacts only with resistant probe. The results of Northern blot of the above six P450 sequences were similar to that of microarray analyses. These results indicate that CYP4 may be related to deltamethrin resistance in Cx. pipiens pallens. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Culex pipiens pallens; Resistance to pesticide; Cytochrome P450; Microarray; Northern blot
1. Introduction The monovalent oxygenase system of cytochrome P450 is a primary detoxicating enzyme system to be involved in metabolisms of various insecticides and other exogenous and autogenous compounds in insects. The cytochrome P450 is the reductase that facilitates transfer of electrons from NADPH to the P450 oxidoreductase [10,20]. *
Corresponding author. Fax: +86-25-650-8960. E-mail address: [email protected] (C.-L. Zhu).
P450 in insects was discovered by Ray for the first time [14]. Following 20 years, because its content in insects was low and unstable, P450 was extremely difficult to purify and studies on it were limited on physico-chemical properties and enzymatic characteristics [19]. In recent years, the cytochrome P450s from over 20 kinds of insects were studied and more than 60 P450 genes have been cloned for Drosophila melanogaster alone [4,7,13,21]. Culex pipiens pallens is distributed widely and attain high populations in many regions of
0048-3575/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0048-3575(03)00014-2
20
B. Shen et al. / Pesticide Biochemistry and Physiology 75 (2003) 19–26
China. Since 1990s, the high populations of this insect have been controlled with deltamethrin, but the development of resistance to deltamethrin in mosquito has occurred much more quickly than resistance to organophosphates [11,16]. It is reported that P450 plays an important role in pyrethroid resistance [2,8]. Up till now, there has been no report on P450 gene in Cx. pipiens pallens. In order to investigate the relation between P450 gene and deltamethrin resistance in Cx. pipiens pallens, P450 specific fragments from both deltamethrin-susceptible and -resistant Cx. pipiens pallens were amplified by RT-PCR and were cloned by the direct cloing method. The expressions of positive clones were identified by microarray and Northern blot.
2. Materials and methods
2.3. RNA extraction and purification Eight hundred unfed female adult mosquitoes of the susceptible and resistant strains (about 1 g each) were collected within 3 days after its emergence. These mosquitoes were ground with liquid nitrogen before it was transferred to homogenizer. Isothicyanic acid guanidine one-step method was adopted to extract total RNA, and then 1% agarose-gel electrophoresis was performed. The total RNA purity was measured by spectrophotometry. With polyATtract mRNA Isolation System Kit, mRNA was purified from 0.5 mg total RNA according to the descriptions of the products. 2.4. cDNA synthesis With 2 lg mRNA obtained from the susceptible strain and resistant strain of Cx. Pipiens pallens, cDNA was synthesized using cDNA Synthesis System Kit according to the descriptions of the products.
2.1. Mosquitoes 2.5. Primer synthesis The susceptible strain of Cx. pipiens pallens (SS) was obtained from Shanghai Insect Institute of the Chinese Academy of Science in 1999 and was never exposed to any kind of pesticides. The strain was reared and bred at 26 1 °C, 80 5%RH) and 16L:8D photoregime and went down to further generation for more than 70 successive generations. The resistant strain of Cx. pipiens pallens (RR) was selected with deltamethrin generation by generation in our laboratory and the resistant ratio was 519 after 70 generations (Li et al., 2002).
2.2. Reagents Isothiocyanic acid guanidine was purchased from Shanghai Boau Biological Science and Technology Company. b-Sulfenyl alcohol was obtained from Fluka Company; SLS from Sigma Company; DEPC from Serva Company. PolyATtract Series 9600 RNA Isolation System Kit, cDNA Synthesis System Kit, reverse transcriptase (AMV), JM109 bacterial strain, Pinpoint Xa-1T plasmid carrier, and SmaI, Taq polymerase were the products of Promega Company.
According to Scott et al. [15], except for one inosine replaced by ACGT, a pair of P450 degenerate primers was synthesized by Dalian Bao Bio-Tech Company. The up-stream primer (P1) encoded a part of the hydrophobic helixes I of P450 protein and was considered to be a specific sequence in CYP4 family. The down-stream primer (P2) was located in the surrounding areas of cysterine residue combined with heme. The F–G– C–G structure found was the common characteristics of insect P450 superfamily
2.6. Amplification and purification of target fragments The synthesized primers were used to amplify P450 cDNA fragments by PCR from cDNA template. PCR system was primarily according to the reference documents [12]. Amplification was done under the following conditions: 94 °C predenaturation for 5 min, followed by 94 °C denaturation for 30 s, 55 °C renaturation for 30 s, and 72 °C extension for 1 min. After completion of 30 such cycles, the process was extended for 20 min at 72 °C.
B. Shen et al. / Pesticide Biochemistry and Physiology 75 (2003) 19–26
With 1.5% low melting-point agarose-gel electrophoresis, target DNA fragments were recovered by purification attained by PCR amplification. 2.7. Cloning of target fragment Target DNA fragment was cloned with T/A direct cloning methods [17]. The purified target fragment was cloned with PinPoint Xa-1 T plasmid vector having T cohesive terminus and transferred to JM109. It remained and grew in AMPþ plate containing ampiciccin over night. Leukasmuses were selected and amplified to obtain positive clone with SP6/PinPoint primers by means of PCR. 2.8. Sequencing and analysis of target fragment Positive clones from the susceptible strain and resistant strain were randomly selected. With caustic cracking solving process, plasmid DNA was extracted. SP6/PinPoint primers were used to prepare duplex DNA target fragment They were purified with Quick PCR Purification Kit and sequenced with SP6 primer by Dalian Bao BioTech Company. The nucleotide sequences were compared to the protein sequence databases incorporated into GenBank using the BLASTX program on the NCBI BLAST Server [15]. Only those clones that had statistically significant similarity scores to numerous P450s and to no other genes were considered to be P450 genes. 2.9. Microarray analyses This was done in Shanghai Cell Biology Institute of The Chinese Academy of Sciences. About 810 subtractive cDNA sequences were cloned from deltamethrin-susceptible and -resistant strains of Cx. pipiens pallens by suppression subtractive hybridization (SSH) in our laboratory, a total of 24 P450 cDNA with the 810 clones was prepared for microarray analyses in this experiment. Twenty-four P450 cDNA samples were amplified by PCR, and after being tested by electrophoresis, the amplified productions were, respectively, dotted to 2 nylon membranes in array pattern with a high speed automaton made by TAS BioRobotics Company of UK. About 50 ng cDNA per dot (diameter 40 lm) was used and 2 dots per sample were made. The [a-33 P]dATP-
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cDNA probes were produced, respectively, with mRNAs from the susceptible and resistant strains (named as susceptible probe and resistant probe) and hybridized with the membranes [9,18]. Cx. pipiens pallens b-actin was used as control. Hybrid signals were analysed with FLA-300 Signal Tester made by Fuji Company, Japan. The discriminant standards were as follows: (A) The setting signal value was delineated in the two membranes. (B) The signal value difference of two dots per sample in a membrane was less than two times. (C) If the signal values of two dots per sample in a membrane were more than three times higher than those in the another one, significant difference between the two probes may be defined in the sample. (D) When the signal values of differentially expressed two lighter dots were more than 10, the difference was visible to the naked eye. 2.10. Northern blot analyses Gene expression of the divergence fragments screened with microarray were analysed by Northern blot [1,12]. Total RNA was extracted from the susceptible and resistant strains of Cx. mosquito. Five mg RNA from each strain was subjected to 1.5% agarose/formaldehyde gel electrophoresis and transferred to a nylon membrane. The PCR-amplified fragments of divergence P450 genes were labeled with [a-32 P]dCTP by the Random Prime DNA Kit (Boehringer–Mannheim). The probes were used to hybridize the target RNA on a membrane at 55 °C for approximately 20 h. The nylon membrane was firstly washed at room temperature for 15 min with wash solution (2 SSC and 0.1% SDS), then at 37 °C for 1 h with wash solution (0:1 SSC and 0.1% SDS), at last at 65 °C for 1 h with wash solution (0:1 SSC and 0.1% SDS). The membrane was exposed to X-ray film for 24 h.
3. Results 3.1. Target gene fragment amplification The first-chained cDNA was synthesized by reverse transcription of mRNA from both susceptible strain and resistant strain of Cx. pipiens
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pallens. A pair of degenerate primer transcript was used to amplify the template by RT-PCR. Two fragments of about 485 and 510 bp were obtained from both strains. After purification and PCR amplification for twice, stronger fragments were obtained. 3.2. Cloning of target gene fragment Using T4 DNA ligase, two target gene fragments of the susceptible strain and resistant srain were inserted into PinPoint Xa-1 T clone carrier, respectively. The up-stream and down-stream primers of muitlple clone locus of plasmid PinPoint Xa-1T (PinPoint sequencing primer: 50 -CG TGACGCGGTGCAGGGCG-30 ; SP6 sequencing primer: 50 -CATAGCGATTAGGTAGGTAACA CTATAG-30 ) were used as PCR primer. Leukasmuses were directly selected for PCR. The sum of the length of the two sequencing primers to the EcoRV clone locus is 144 bp. From the recombined clones, two segments of about 630 and 655 bp were obtained, which were identical in size to those before cloning.
3.3. Sequencing and analyses of target gene fragments Cloning was conducted twice and a total of 112 positive spots were obtained. Out of 37 clones sequenced, 31were shown to encode P450. Twenty-four of 31 sequences were not identified and the other seven ones were repeat copies, their molecular weight being 369–444 bp. Compared with P450 sequence databases incorporated into GenBank, all of the 24 sequence were new, of which 6 were obtained from the susceptible strain, and the other 18 were derived from the resistant strain. They had been lodged in Genbank. The 24 new genes (alleles) were approved by the Cytochrome P450 Nomenclature Committee, belonging to the subfamily CYP4C, CYP4D, CYP4H, and CYP4J in CYP4 family (Table 1). Para-position queuing graph of amino acid sequences was made, which were deduced from 24 P450 new genes (alleles) (Fig. 1). From the graph one dendrogram was obtained by unweighted pair-group method using arithmetic averages (UPGMA) (Fig. 2).
Table 1 Charactertics of 24 new cytochrome P450 genes (alleles) from Cx. pipiens pallens Genea (allele)
GenBank Accession No.
Strain
No. of nucleotide
No. of residues
No. of clone
CYP4C22v1 CYP4C22v2 CYP4C23 CYP4D18v1 CYP4D18v2 CYP4D19v1 CYP4D19v2 CYP4D19v3 CYP4H12v1 CYP4H12v2 CYP4H13 CYP4H21 CYP4H22v1 CYP4H22v2 CYP4H23v1 CYP4H23v2 CYP4H23v3 CYP4J4v1 CYP4J4v2 CYP4J6v1 CYP4J6v2 CYP4J7v1 CYP4J7v2 CYP4J8
AF157093 AF208659 AF157090 AF190775 AF191728 AF190786 AF190789 AF190777 AF157091 AF191731 AF160259 AF208660 AF190782 AF190784 AF190787 AF191729 AF190783 AF157092 AF190778 AF190776 AF190781 AF190780 AF190785 AF190788
R S S S R R R S R R R R R R R R R R S S R R R R
378 378 444 444 444 378 381 381 390 390 439 381 381 381 381 381 381 387 387 369 369 384 384 384
126 126 148 148 148 126 127 127 130 130 127 127 127 127 127 127 127 129 129 123 123 128 128 128
1 1 1 1 1 2 1 1 1 1 1 2 1 1 2 1 1 1 1 1 1 4 2 1
a
Named by the Cytochrome P450 Nomenclature Committee.
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Fig. 1. Alignment of deduced amino acid sequences for the 24 new cytochrome P450 genes (alleles) from Cx. pipiens pallens. + as termination codon (CYP4J4V1); ) as **KETLR*-P area and PERF area, respectively.
3.4. Microarray analyses After the 24 P450 cDNA samples reacted with susceptible probe and resistant probe separately, the microarray signal values of 22 samples were more than 10, while those of the other two samples were less than 10 in susceptible probe, but more than 10 in resistant probe, hybrid rate being 100%. The signal value ratios
between both chip membranes were less than three in 18 samples, the differences being not significant; the hybrid signal values of CYP4 H21, 4H22v1, 4J4v2, 4J6v1, and 4J6v2 were 3.1–7.7 times higher in resistant probe than in susceptible probe, and the hybrid signal value of 4H23v2 was three in susceptible probe, which corresponded to the setting signal value of the membrane, but 29.1 in resistant probe, the latter
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Fig. 2. UPGMA dendrogram of amino acid sequences deduced from 24 new cytochrome P450 genes (alleles) of Cx. pipiens pallens.
Table 2 Microarray signal value of 6 CYP4 cDNA from Cx. pipiens pallensa Gene
Site
4H21 4H22v1 4H23v2 4J4v2 4J6v1 4J6v2
E1 E7 G4 A3 A5 C3
a
F1 F6 G5 B2 B4 D2
S
R
R/S
124.8 33.2 3.0 8.2 96.3 15.4
383.3 109.6 29.1 63.3 530.4 53.4
3.1 3.3 9.7 7.7 5.5 3.5
S as susceptible probe; R as resistant probe; R/S as ratio between the both signal value.
being 9.7 times as more as the former (Table 2, Fig. 3). 3.5. Northern blot analyses The results of Northern blot of six P450 genes (CYP4H21, 4H22v1, 4H23v2, 4J4v2, 4J6v1, and 4J6v2) were consistent with those of the microarray analyses (Fig. 4), prompting that CYP4 may be related to deltamethrin resistance in Cx. pipiens pallens.
4. Discussion Though the sequence found in this study was only 1/3 length of P450, the hydrophobic helixes I, in which the up-stream primer 1 was located, had the common characteristics of CYP4, and heme combination, in which the down-stream
primer 2 was located, was also common in P450, which was regarded as the appraised characteristics of P450 family members. The structures of the 24 new sequences cloned in our laboratory contained two relatively conservative areas which CYP4 family had in common: **KETLR*-P area () indicate amino acid residue, * refers to hydrophobic residue) and PERF area [10,15,20]. These sequences belong to the subfamilies of CYP4C, CYP4D, CPY4H, and CYP4J of CYP4 family approved by the Cytochrome P450 Nomenclature Committee. It is commonly acknowledged that CYP6 family members have close relationship with the resistance to insecticide in the known superfamily of insect P450 [3,5]. In this investigation, two cDNA fragments were obtained from RNA of Cx. pipiens pallens with RT-PCR, the fragments in the susceptible strain being weaker than those in the resistant strain. After the electrophoretic
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Fig. 3. Results of microarray analyses of 24 CYP4 from susceptible and resistant strains of Cx. pipiens pallens. S, susceptible strain; R, resistant strain.
Fig. 4. Northern blot of 6 CYP4 cDNA from deltamethrin-susceptible and -resistant Cx. pipiens pallens. (S) deltamethrin-susceptible strain, (R) deltamethrin-resistant strain, (1) CYP4H21, (2) CYP4H22v1, (3) CYP4H23v2, (4) CYP4J4v2, (5) CYP4J6v1, (6) CYP4J6v2, The bottom blot was obtained with Cx. pipiens pallens b-actin used as control.
comparison of increased template, the fragments in the susceptible strain were only 1/10 of those in the resistant strain. Our work observed more positive clones in resistant strain than in susceptible strain. There were repeat copies in five sequences of resistant strain but none in susceptible strain. The microarray signal values of CYP4H21, 4H22v1, 4J4v2, 4J6v1, and 4J6v2 were 3.1–7.7 times higher in resistant strain than in susceptible strain. The results of Northern blot of the six P450 genes were consistent with those of the microarray analyses. The results mentioned above prompted that CYP4 family may be related to deltamethrin resistance in Cx. pipiens pallens and further proved that over-expression of P450 mRNA is one of the molecular mechanisms of insect resistance to pyrethroid pesticide. It is interesting to observe that CYP4H23v2 could react with resistant probe, but not with susceptible probe. The two alleles of 4H23v2,
4H23v1 and 4H23v3, could react with susceptible probe and their hybrid signal values were 106.9 and 11.7, respectively. As compare to the nucleotide sequences of the two alleles, 4H23v2 had a T ! A difference, resulting in the replacement of Ser by Thr in position 75, and is worthwhile of further studies. It is wident from Fig. 1 that, the 24 new sequences commonly had 10 amino acid residues in the same positions, 5 of which were prolines (pro). In 1994, Scott et al. [15] reported 17 new sequences of CYP4. Among the 15 common amino acid residues, four were prolines. The above results suggested that proline was conservative compared with other amino acids and might play an important role in genetic stability of P450 genes. In this experiment, 2 or 3 alleles were cloned from nine genes, respectively, and replacement had taken place in 2–6 amino acid residues of them. Both CYP4D19 and CYP4H23 had three alleles. Residue replacement in the same positions was only found in one of the three and no different residue of each was found. CYP4D19v2 and 4D19v3 had 6 amino acid heteromorphoses, while in the same positions 4D19v1 had three same residues as 4D19v2 and three same residues as 4D19v3, suggesting 4D19v1 was the transition stage between 4D19v2 and 4D19v3. CYP4H23v1, 4H23v2, and 4H23v3 had 0, 1, and 2 residue replacement, respectively, indicating 4H23v1 was the oldest one among the three and 4H23v2 and 4H23v3 had been branched by 4H23v1 during evolution. Genes usually have only one terminator signal. In rare cases, two termination signals do exist, but the distance between the two terminator codons is
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usually shorter [6]. In this experiment, in the near 30 -terminus, CYP4J4v1 had a terminator codon T (U) AG, but followed by thorough reading. Compared with other full P450 sequence in GenBank, there is a length of about 70 amino acid residues after the position of T (U)AG in CYP4J4v1. UAG is a terminator codon that is less frequently used. Its exact function in peptide chain termination of CYP4J4v1 needs further studies.
Acknowledgments We are grateful to Prof. Zheng-Hua Tang and Mrs. Pei-Jun Zhuang working in Shanghai Insect Institute of the Chinese Academy of Science, for providing the susceptible strain of Cx. pipiens pallens. We thank Dr. David R. Nelson (University of Tennessee, USA) for his help in naming and sequence analysis of the P450 genes.This work was jointly supported by National Foundation of Natural Science of China (Nos. 39970666 and 30170835) and Natural Science Foundation of Jiangsu Province (BK02050).
References [1] F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Struhl, Current Protocols in Molecular Biology, Wiley, New York, 1994. [2] F.A. Carino, J.F. Koener, F.W. Plapp, R. Feyereisen, Constitutive overexpresion of the cytochrome P450 gene CYP6A1 in a house fly strain with metabolic resistance to insecticides, Insect Biochem. Mol. Biol. 24 (1994) 411–418. [3] M.B. Cohen, J.F. Koener, R. Feyereisen, Structure and chromosomal localization of CYP6A1, a cytochrome P450 encoding gene from the house fly, Gene 146 (1994) 267–272. [4] M.B. Cohen, R. Feyereisen, A cluster of cytochrome P450 genes of CYP6 family in the housefly, DNA Cell Biol. 14 (1995) 73–82. [5] P.B. Danielson, R.T. Macintyre, J.C. Fogleman, Molecular cloning of family of xenobiotic-inducible drosophlid cytochrome P450: evidence for involvement in host-plant allelochemical resistance, Proc. Natl. Acad. Sci. USA 94 (1997) 10797–10802. [6] N.G. Davis, P. Model, An artificial anchor domain: hydrophobicity suffices to stop transfer, Cell 41 (1985) 607–614.
[7] R. Feyereisen, Insect P450 enzymes, Annu. Rev. Entomol. 44 (1999) 507–533. [8] J.C. Fogleman, P.B. Danielson, R.J. Macintyre, The molecular basis of adaptation in Drosophila: the role of cytochrome P450, Evol. Biol. 30 (1998) 15–77. [9] T.R. Golub, D.K. Slonim, P. Tamayo, C. Huard, M. Gaasenbeek, J.P. Mesirov, et al., Molecular classification of cancer: class discovery and class predication by gene expression monitoring, Science 286 (1999) 531–537. [10] X.F. Leng, Z.H. Tang, Y.C. Wang, in: Molecular Toxicology of Pesticides and Insect Resistance to Pesticides, Chinese Agricultural Publishing House, Beijing, 1996, pp. 132–147. [11] X.L. Li, L. Ma, L.X. Sun, C.L. Zhu, Biotic characteristics in the deltamethrin-susceptible and resistant strains of Culex pipiens pallens (Diptera: Culicidae) in China, Appl. Entomol. Zool. 37 (2002) 305–308. [12] W.D. Liu, Mosquito resistance to pesticides in China, Chin. J. Vector. Biol. Cont. 1 (1990) 41– 43. [13] S.D. Lu, in: Current Protocols for Molecular Biology, Chinese Publishing House of Higher Education, Beijing, 1993, pp. 163–182. [14] D.R. Nelson, L. Koymans, T. Kamataki, J.J. Stegeman, R. Feyereisen, P450 superfamily: update on new sequences, gene mapping, accession numbers, and nomenclature, Pharmacogenetics 6 (1996) 1–42. [15] J.W. Ray, in: Pest Infestation Research, HMSO, London, 1965, pp. 69–74. [16] J.G. Scott, F.H. Collins, R. Feyereisen, Diversity of cytochrome P450 genes in the mosquito, Anopheles albimanus, Biochem. Biophys. Res. Commun. 205 (1994) 1452–1459. [17] Y.Q. Sun, J.G. Yuan, J. Li, The resistance mechanism of housefly against pyrethroids, Acta Entomol. Sinica. 33 (1990) 265–272. [18] Y. Wang, One new T/A cloning method, Prog. Biol. Tech. 16 (1996) 55–57. [19] G.P. Yang, D.T. Ross, W.W. Kuang, P.O. Brown, R.J. Weigel, Combining SSH and cDNA microarray for rapid identification of differentially expressed genes, Nucleic Acids Res. 27 (1999) 1517–1523. [20] B.H. Ye, in: Modern Medical Entomology, Chinese Publishing House of Higher Education, Beijing, 1996, pp. 179–182. [21] Q.H. Zhai, Some aspects of progress in insect biology: molecular mechanisms of insecticide resistance, Acta Entomol. Sinica 38 (1995) 493–501. [22] C.L. Zhu, G.L. Wu, Z.S. Zhang, Studies on insect cytochrome P450, Chin. J. Parasitol. Parasitic Dis. 17 (1999) 46–49.