Cloning And Sequence Analysis Of Cytochrome Family 4 New Genes From The Deltamethrin-Resistant Mosquito, Culex Pipiens Pallens

The resistance to insecticide played a serious role in the control of pests.All the research gives more evidence that the resistance to insecticide in pest is determined by gene.To obtain the partial cDNA sequences encoding cytochrome P450 from the del tamethrin-res is tant mosquito, Cx. pipiens pallens. The conserved region in the alignment of insect P450 family 4 (CYP4) proteins served as a guide for the synthesis of degenerate oligonucleot ide primers to clone P450 cDNA. Primers were used in the reverse transcript ion-polymerase chain reaction(RT-PCR) of RNA from the 4th instar larvae of deltamethrin-resistant Cx. Pipiens pallens. With semi-quantitative PCR, we detected their differential expression in the resistant strain and the susceptible strain. The prokaryotic expression plasmid of CYP4E2r6 gene was constructed, and learned the prokaryotic expression of the gene.Part one: Cloning and sequence analysis of cytochrome family 4 new genes from the deltamethrin-resistant mosquito, culex pipiens pallensTo obtain the partial cDNA sequences encoding cytochrome P450 from the del tamethr in-res istant mosquito, Cx. pipiens pal lens. The conserved region in the alignment of insect P450 family 4 (CYP4) proteins served as a guide for the synthesis of degenerate oligonucleot ide primers to clone P450 cDNA. Primers were used in the reverse transcription-polymerase chain reaction (RT-PCR) of RNAfrom the 4th instar larvae of deltamethrin-resistant Cx. Plpiens paJlens. PCR products purified were cloned into pGEMR-T Easy Vector and sequenced.The results showed that nine of 36 positive clones obtained shown to be new sequences encoded cytochrome P450 and appraised belonging the subfamily CYP4H,CYP4J and CYP4E by the Nomenclature Committee of Cytochrome P450 (GeneBank CB074944-CB074951;CB270837, 2003). Their molecular weight were about 483-497bp.Part two: the identify of expression of CYP4 genes in the deltamethrin-resistant mosquito, Cx. piplens pallensIn order to definite high expression in the resistant strain, with the semi-quantitative PCR to reflect directly the quantity of the reverse transcription product,we can show the level of mRNA. The results showed that CYP4E2r6 gene was highly expressed in the resistant strain while the (3 -actin gene was expressed equivalencely in the resistant strain and the susceptible strain as house-keeping gene. We suspected that CYP4E2r6 gene was possible to relate the resistance of the Cx. pipiens pal lens.Part three: The prokaryotic expression and identification of CYP4E2r6 geneIn this paper CYP4E2r6 gene was amplified by PCR, and the prokaryotic expression plasmid pGex-6Pl/CYP4E2r6 is constructed. The polyclonal antibody of mouse anti-P450 of fly was prepared, and learned initially the prokaryotic expression of the gene.One: The construction of prokaryotic expression plasmid of CYP4E2r6 geneThe CYP4E2r6 gene was amplified by PCR, and the expression primerwas designed. The enzyme restriction sites were added considering the confluence express of the prokaryotic expression plasmid PGEX-6P1 when designing the primer. The PCR fragment was cloned into pGEM-T easy vector, and then identified by PCR, enzyme cutting and gene sequencing. Cut from the reconstruction plasmid pGEM-T/ CYP4E2r6 and from the super coil plasmid PGEX-6P1, the fragments CYP4E2r6 and PGEX-6P1 were obtained with the enzyme restriction site ends. The gene and plasmid fragments were recombined in the condition of T4 DNA ligase on the basis of some ratio. And then the reconstruction plasmid was identified using PCR, double enzyme cutting and gene sequencing.The results showed that the CYP4E2r6 gene sequence was the same as the reported, indicating that the express plasmid PGEX-6P1/ CYP4E2r6 was reconstructed successfully.Two: Prokaryyotic expression and identification of CYP4E2r6 gene The plasmids of PGEX-6Pl/CYP4E2r6 was transfected into the E. coil BL21 (DE3) by CaC12, and then induced by 1mM IPTG to obtain the protein in different time. Mixed with the 1 x Loading Buffer and degenerated in the condit...