| Mosquitoes are worldwide pests which spread various diseases and take a serious threat to people's lives and health. At present, pesticide is the primary means of prevention. In all types of pesticides, pyrethroid becomes the most commonly used one, because it is high efficiency and low toxicity synthetic. However, mosquitos have had a very strong resistance to pyrethroid. It makes prevention and control work for mosquitoes even more difficult. The rapid development of pesticide-resistance of insects has become a more and more serious public problem.[Objective] A pair of special primers were designed based on the different expression EST between Culex quinquefasciatus resistant and susceptible to permethrin, to verify whether were expressed in Culex Pipiens Pallens (Yang Ling, China) by the RT-PCR, and analyse homology of this 9 sequences. To amplify the full length cDNA from Culex Pipiens Pallens by RACE. To analyze the sequence by bioinformatics.To study the expression level of mRNA between Culex pipiens pallens resistant and susceptible to permethrin. Laid the foundation for clarifying pesticide-resistant mechanism causing by CYP450 gene encoding proteins.[Methods] According to one of 9 different expression EST between Culex quinquefasciatus resistant and susceptible to permethrin, a pair of primers were designed and the RT-PCR was performed to amplify this EST from Culex Pipiens Pallens.To analyze their homology after sequencing. A pair of special primers were designed based on one EST which GenBank accession number is EC093837. Rapid amplification of cDNA ends (RACE) was used for amplifying the 5'end and the 3'end sequences. Splice the sequencing results and get the full length cDNA. Find the ORF of the DNA sequence by ORF finder in NCBI. Predict some parameters of the encoding protein such as the number of amino acid residues, molecular composition, relative molecular mass, isoelectric point by ProtParam. Analyze hydrophobicity of encoded proteins by ProtScale. Predict the phosphorylation sites by NetPhos2.0 Server. Predict transmembranes of the encoding protein by SOSUI. Predict secondary structure of the encoding protein using a variety of methods providing by HNN. Use SignalP3.0 to predict the signal peptide. Use PSORTâ…¡to do the localization analysis. Analyze the protein domain by CDD (Conserved Domain Database). Do three-dimensional protein structure prediction by Swiss Model. Do homology comparison in nucleic acid databases (NR) and protein databases (Swissport) by BLAST. Do cluster analysis by ClustalW and DNAStar. Do a molecular evolutionary tree by MEGA. To study the expression level of mRNA between Culex pipiens pallens resistant and susceptible to permethrin by semi-quantity RT-PCR.[Results] Compared the nine cDNA sequences between two kinds of Culex mosquito. Homology analysis showed that identity of a pair of these sequences has more than 88%. It means that a pair of these sequences have the similar function, we can infer that these 9 gene fragment may be related to resistance of Culex Pipiens Pallens. One full length cDNA named XND-P450 has been cloned. The sequence has 1138bp including an ORF of 1005 bp and the deduced amino acid sequence has 305 amino acids. The physical and chemical properties and the structural characteristics have been predicted by sequence analysis. This gene was found in line with the general characteristics of the CYP9. This gene can be identified to belong to the CYP9 subfamily through evolution analysis. Semi-quantity RT-PCR showed that the expressed level in resistant Culex pipiens pallens was two times than susceptible Culex pipiens pallens. |
