Cloning Of PR-XP1 Gene And Preliminary Research Of Its Insecticide-Resistance In C6/36 Drug-Resistant Lines

As the most widespread medical pest, mosquitoes could spread many deadly diseases, which threat human health seriously. Currently, chemical control is still the the primary way of mosquito control; and among them, Pyrethroid insecticides are used widely. However, mosquitos have formed more and more resistance to Pyrethroids for long-term and far-ranging using. Therefore, it is is very necessary to investigate the mechanism of resistance, expecially resistance related genes to pyrethroids. Because of the limited research strategy and technology, currently the permethrin-resistant genes and their functions of Culex pipiens pallens are still far to clarified. In order to investigate the new permethrin-resistant genes of Culex pipiens pallens and explore their Resistant function in cell, this study use resistant strain Culex pipiens pallens and mosquito C6/36 cell to carried out related work.Firstly, using five different method of guanidinium isothiocyanate, Trizol, advanced Trizol, Trizol plus CTAB, and CTAB to isloate the total RNA from Culex pipiens pallens, we have got a best strategy to isolate total RNA——our results suggested that the method of Trizol plus CTAB, invented by our lab——which provides a good technology for further researchin.Secondly, according to one Culex Pyrethroid-Resistance relevant EST(NCBI No. EC093826.1),we designed GSPs and used SMART-RACE technology to clone the gene 5'and 3'terminal sequence. And we tried to get the full length cDNA sequence by splicing, and after that we use NCBI ORF finder to position its open reading frame. The results showed that the full-length cDNA is 2004 bp, named the gene with PR-XP1, which encoding 249 amino acids. Additionally, bioinformatics software analysis, such as homologous comparison, phylogenetic tree construction, physical and chemical prop erties of protein-coding prediction, signal peptide sites prediction, phosphorylation site analysis and also on all suggested that gene PR-XP1 belongs to a more conservative"hypothetical protein"gene family,which only expressed in insects. The gene encodes a protein with a specific structure of U-hydrophobic transmembrane region, and has many phosphorylation sites; which revealed us to speculate that the function this gene is associated with insecticide resistance.And this was confirmed by the result of FQ-PCR monitoring, which showed that the expression level of PR-XP1 in resistant strains of mosquitoes was higher than 1.11 times in the sensitive strains.Thirdly, suitable cell model is necessary in studying the function of mosquito pesticide-resistance genes, in whether depending on the method of transgenic gene or virus induced gene silencing. Based on this, we used the MTT assay to detect the killing effect of permethrin on mosquito C6/36 cells(Lc50=517.64μg/ml), which could grow and passage stably in nutrient solution with the conentration of Pyrethroid is 300μg/ml, and further constructed resistant cell lines. Last, we used semi-quantitative RT-PCR to detect the expression level of resistant gene of PR-XP1, which increased more than 1.7 time in resistant cell lines to the control group .In summary, this study investigated the best strategy of total RNA extraction in Culex pipiens pallens; cloned permethrin-resistance related genes of PR-XP1 and analyzed by bioinformations, and then monitored the excessly expressional phenomenon. Based on these, this study further established mosquito C6/36 drug-resistant cell line to permethrin, and verified different expression level of resistance-related genes for the first time.And all of this provide an important clue for the studying and clarifying the mechanism of mosquito resistante gene.