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IJSTR >> Volume 3- Issue 7, July 2014 Edition

International Journal of Scientific & Technology Research  
International Journal of Scientific & Technology Research

Website: http://www.ijstr.org

ISSN 2277-8616

Cloning Of Anopheles Gambiae CYP6M2 Gene Promoter And Construction Of Its Luciferase Reporter System

[Full Text]



Balarabe R. Mohammed, Craig S. Wilding, PhillipJ. Collier, Yusuf Y. Deeni



Index terms: Anophelesgambiae,CYP6M2, Drosophilamelanogaster, malaria, insecticide resistance, PCR, P450s



Abstract: CYP6M2 is a Cytochrome P450 gene involved in the detoxification of multiple classes of public health insecticidesin the malaria mosquito Anopheles gambiae. Some P450 genes are known to be up regulatedby the transcription factorsCnCC/ Keap 1 and (ss)/ (Tgo) in Drosophilamelanogaster. Whether this regulatory mechanism is involved inthe regulation of P450s in Anopheles gambiae is yet to be identified..In this study, we investigated cloning of 896 bp CYP6M2 promoter and construction of its luciferase reporter system in Anopheles gambiae. Bioinformatics tools were used to search for 5’ up stream region of CYP6M2 promoter sequence and specific primers designed. The promoter region of CYP6M2 (896 bp) was amplified using isolated the designed primers and isolated genomic DNA from insecticide resistant Tiassale and susceptible Kisumu strains of An. gambiae. The PCR products were cloned intopJET1.2/blunt vector. The resultant plasmid DNA was transformed into Escherichia coli XL1-Blue competent cells for propagation and purification, the plasmid constructs isolated and sequenced. Both cloned pJET1.2 and pGL3-Basic vectors were digested with BglII, purified and then ligated into the luciferase expression vector pGL3-Basic vector. The designed CYP6M2 promoter constructs were confirmed by sequencing. Results revealed that the retrieved CYP6M2 gene promoter sequence has 96% and 95% similarity for Kisumu and Tiassale cloned sequences respectively.Cloning of CYP6M2 gene promoter and construction of its dual luciferase reporter system were successfully established, this will be essential in further studies aimed at understanding the regulatory mechanisms involved in insecticide resistance, which is important in the control of malaria.



[1]. Estabrook, R.A. passion for P450s (remembrances of the early history of research on cytochrome P450). DrugMetabolism and Dispositon, 31(12): 1461-1473, 2003.

[2]. Guo, H, Zhenmin, B., Huixia, D., Lingling,Z.,Shi,W., Luyang, S., Xiaoyu, M., andXiaoli, H. Identification of Cytochrome P450 (CYP) Genes in Zhikong Scallop (Chlamysfarreri).J Ocean Univ .China (Oceanic and Coastal Sea Research) 12: 97-102, 2013.

[3]. Rongnoparut, P., Pethuan, S., Sarapusit, S. and PanidaLertkiatmongkol. Metabolism of Pyrethroids byMosquito Cytochrome P450 Enzymes: Impacton VectorControl,Insecticides– Pest Engineering, Dr. FarzanaPerveen(Ed.), ISBN: 978-953-307-895-3, 2012.

[4]. Zhu,F Timothy W Moural, Kapil Shah and Subba Reddy Palli. Integrated analysis of cytochrome P450gene superfamily in the red flour beetle, Triboliumcastaneum.BMC Genomics14:174, 2013.

[5]. Qin, Q., Li, Y., Zhong, D., Zhou1, N., Chang,X., Li, C., Cui,L., Yan, G. and Chen, X-G.Insecticide resistance of Anophelessinensisand An. Vagusin Hainan Island,a malaria-endemic area of China. Parasites &Vectors7:92, 2014.

[6]. Chauhan, R, R. Jones, P. Wilkinson, Y. Pauchet and R. H. ffrench-Constant. Cytochrome P450- encoding genes from the Heliconius genome as candidates for cyanogenesis. Insect Molecular Biology22(5):532–540, 2013. doi: 10.1111/imb.12042.

[7]. Hemingway, J., Field, L., Vontas, J. An Overview of Insecticide.Science : ( 298) 5591 pp. 96-97,2002, DOI: 10.1126/science.1078052

[8]. Wan, H, Liu, Y., Li,M., Zhu,S., Li,X.,Barry R Pittendrighd and Qiu, X. Nrf2/Maf-binding-site- containing functional Cyp6a2 allele is associated with DDT resistance in Drosophila melanogaster.Pest Manag Sci,2013

[9]. Munhenga, G and Lizette L. Koekemoer.Differen tial expression of cytochrome P450 genes in a laboratory selected Anopheles arabiensis. colony.African Journal of Biotechnology 10(59), pp. 12711-12716, 2011, doi: 10.5896/AJB11.363

[10]. Ranson, H.; Claudianos, C.; Ortelli, F.; Abgrall, C.; Hemingway, J.; Sharakhova, M.V.; Unger, M.F.; Collins, F.H. &Feyereisen, R. Evolution of super gene families associated with insecticide resistance. Science, 298, 179-181,ISS 0036 8075,2002.

[11]. Félix, R and Henrique Silveira (2012). The Role of Anophelesgambiae P450 Cytochrome in Insecticide Resistance and Infection, Insecticides - Pest Engineering, Dr. FarzanaPerveen (Ed.), ISBN: 978-953-307-895- 3, 2012.

[12]. Stevenson, B.J., Bibby, J., Pignatelli, P., Muangnoicharoen, S., O’ Neill, P.M., Lian, L.-Y., Müller, P., Nikou, D., Steven, A., Hemingway, J., Sutcliffe, M.J., Paine, M.J.I. Cytochrome P450 6M2 from the malaria vectorAnophelesgambiae metabolizes pyrethroids: sequential metabolism of deltamethrin revealed. Insect Biochem.Mol. Biol. 41, 492e 502, 2011.

[13]. Mitchell,S.N ., Stevenson, B.J.,Müllera,P.,Wilding, C.S.,Egyir-Yawsond,A.,Fielde,S.G., Hemingway,J., Mark J. I. P., Ranson, H. and Donnelly,J.Identifica- tion and validation of a gene causing cross-resista- nce between insecticide classes in An.gambiaefrom Ghana.PNAS | 109(16): 6147–6152, 2012.

[14]. Witzig, C., Wondji, C.S 1, Strode, C. 1, Djouka, R. and Ranson, H. Identifying permethrinresistance loci in malaria vectors by genetic mapping.Parasit. 140, 1468–1477, 2013.

[15]. Zimmera,C.T.,Martin, C.B.,Williamson,S.,Wölfel, K., Gutbrod,O., Kaussman, M., and Naue, R.Mole- cularand functional characterization of a CYP6QB- 23, cytochromeP450 conferring resistance to Pyrethroidsin European populations of pollen beetle,Meligethesaeneus. Insect Biochemistry and Molecular Biology.45 :18-29, 2014.

[16]. McMillan, B.J. and Bradfield, C.A. The Aryl Hydrocarbon Receptor sans Xenobiotics: Endogen- ousFunction in Genetic Model Systems. MolPharmacol,72(3):487–498, 2007.

[17]. Misra, J.R Geanette Lam, Carl S. Thummel. Constitutive activation of the Nrf2/Keap1 pathway in insecticide-resistant strains of Drosophila. Insect Biochemistry and Molecular Biology, 43,2013 1116e- 1124,

[18]. Wang-M; Jian-Ling Du, Zhi-Jun Duan, Shi-Bin Guo, Xiao-Yu Sun, Zhen Liu.Inhibiting heme oxygenase-1 attenuates rat liver fibrosis by removing iron

[19]. Phillipson, O.T. Management of the aging risk factor for Parkinson’s disease.Neurobiology of Aging .35 847e857, 2014.

[20]. Vorrink, S.U, Paul L. Severson, Mikhail V. Kulak, BernardW.Futscher, Frederick E. Domann.Hypoxia perturbs aryl hydrocarbon receptorsignaling and CYP1A1 expression induced by PCB 126 in human skin and liver- derived cell lines.Toxicology and Applied Pharmacology 274 408–416, 2014.

[21]. Maayah,Z.H • Mushtaq Ahmad Ansari • Moha med A. El Gendy • Mohammed N. Al Arifi Hesham M. Korashy. Development of cardiac hypertrophy by sunitinib in vivo and in vitro rat cardiomyocytes is influenced by the aryl hydrocarbon receptor signaling pathway.Arch Toxicol 88:725–738, 2013, doi: 10.1007/s00204-013- 1159-5.

[22]. Wan, H.,Liu,Y., Li,M., Zhu,S., Li,X. Pittendrighd,, B.R. and Qiua, X.Nrf2/Maf-binding-site-containing Functional Cyp6a2allele isassociatedwith DDT ResistanceinDrosophilamelanogaster. Pest ManagSci 70: 1048–1058,2014.

[23]. Wildinga,C.S., Smith,I., Lynd,A., Yawson,A.E., Weetman,D., Paine, M.J.I., Donnelly,M.J. A cis-regulatorysequencedrivingmetabolic insecticide resistance in mosquitoes: Functionalcharacterisation and signaturesof selection.InsectBiochemistryand MolecularBiology, 42 699-707, 2012.

[24]. Alout, H., Nicaise TuikueNdam,Marcel Maurice Sandeu, Innocent Dje´gbe, Fabrice Chandre1, RochKounbobr Dabire´ , Luc Salako Djogbe´nou, Vincent Corbel1, Anna Cohuet. Insecticide Resistance Alleles Affect Vector Competence of Anophelesgambiaes.s. for Plasmodiumfalciparum Field Isolates..PLoS ONE 8(5): e63849, 2013, doi:10.1371/journal.pone.0063849

[25]. Konan, K. G • A.B. Koné • Y.L. Konan • D. Fofana K.L. Konan • A. Diallo • J.C. Ziogba • M. Touré Jamet .Increase insusceptibility to insecticides with aging of wild Anopheles gambiae mosquitoesfrom Côte d’Ivoire.BMC Infectious Diseases, 12:214, 2012.

[26]. Kusumawati, AJoannes Sri Widada, Basuki Hidayat.Construction of an Expression Vector Based on pCDNA3.1(+) Expressing Jembrana Disease Virus ENV-TM Subunit Gene in Eukaryotic System.Jurnal Sain Veteiner. 22 (1):27-31, 2004.

[27]. You, F. M. Huo, N. Gu, Y. Q., et al. BatchPrimer3: A high throughput web application for PCR and sequencing primer design. BMC Bioinformatics.9(253), 2008, doi : 10.1186/1471-2105-9-253.

[28]. Chuang, L-Y,Yu-HueiCheng ,Cheng-Hong Yang. Specific primer design for the polymerase chain reaction. Biotechnol Lett 35:1541–1549, 2013.

[29]. Wilding, C.S D. Weetman, K. Steen and M.J. Donnelly. Accurate determination of DNA yields from individual mosquitoes for population genomic applications. Insect Science 16, 361–363, 2009, doi: 10.1111/j.1744-7917.2009.01260

[30]. Banerjee,A., Majumder, P., Sanyal, S, Singh, J, Das, K.J,C. Dasgupta, D. The DNA intercalatorsethidium bromide and propidium iodide also bind to core Histones.Elsevier.FEBS Open Bio 4: 251–259, 2014