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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.



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