The Full-length PR-XP2Gene’ Cloning From Culex Pipiens And The Analysis Of Its Expression Level In Permethrin-susceptible And Resistant C6/36Cells

The prevention and cure of mosquito is very difficult now.In recent years, the pesticidesof pyrethroid develops rapidly because of its high insecticidal efficacy. However, because ofits large and long-term using, many kinds of insects emerge to the resistance to pyrethroid.Therefore, it is particularly important to ascertain the mechanism of insect’ resistance toinsecticides drugs of pyrethroid. The study was done to obtain the full-length cDNA ofPR-XP2gene from Culex pipiens and to analysis the nr and protein sequences bybioinformatics, then detecte the expression level of PR-XP2mRNA in susceptible andresistant C6/36cells, respectively.Firstly, based on the EST（NCBI No. EC093833.1）got by our lab, the full-length genesequence was cloned by rapid amplification of cDNA ends method (RACE) from a resistantstrain of mosquito, then its characteristics were analysed by bioinformatics. The new gene,with1679bp, named PR-XP2. Bioinformatics analysis showed that the PR-XP2gene has ahigh homology which is up to75%with CYP450gene of aedes aegypti and Culex fatigans.And the protein of PR-XP2has the homology of40%with aedes and anopheles gambiaemosquitoes’ CYP450gene protein. In addition, we found that the protein of PR-XP2’50-342amino acid is completely consistent to and protein of the conservative field of cytochromeP450family. Thus we conjecture presumably that the PR-XP2gene belongs to the P450genes family.Secondly, we cultivate the C6/36cell line of culex intestinal epithelial, then screendrug-resistant cell line of permethrin. When we have got the permethrin-resistant cell line, thePR-XP2gene’ expression difference is detected from the resistant cell lines and sensitive celllines using the method of semi-quantitative RT-PCR to speculate the correlation between PR-XP2gene and the permethrin-resistance. At last, we got the permethrin-resistant cell linesuccessfully using the method of concentration ascending step by step, and C6/36cells wasscreened from the half lethal concentration (LC50) from200μg/L (PPM), to984μg/L byMTT assays. And the gene expression in the resistant cells is significantly higher than that ofsusceptible cells in RT-PCR, which showed that PR-XP2gene may be functional of resistanceto permethrin mechanism in C. pipiens. To sum up,(1) The research have cloned a full-length gene of C. pipiens named PR-XP2which belongs to the P450genes family by analysis of bioinformatics.(2) We got thepermethrin-resistant cell line successfully.(3) The expression amount of PR-XP2gene isdetected in susceptible and resistant C6/36cells by means of semi-quantitative RT-PCR. Andthe gene expression in the resistant cells is significantly higher than that of susceptible cells,which showed that PR-XP2gene may be functional of resistance to permethrin mechanism inC. pipiens. All what we do is important to the research of resistance mechanism of mosquito.