Study On The Relationship Between Iron Metabolism Related Protein Gene And Drug Resistance Mechanism Of Culex Pipiens Pallens

Objective The large number and continuous use of insecticides have led to the emergence and development of insecticide resistance.It is imperative to study the mechanism of mosquito resistance and appropriate management.With the continuous development of modern biotechnology,more and more drug-resistant genes have been discovered for different species.Culex pipiens pallens is the carrier of lymphatic filariasis and encephalitis,and its drug resistance has become a major problem in the prevention and control of vector diseases.Culex pipiens pallens is mainly distributed in the northern regions of China.The experimental study on the mechanism of the resistance of C.pipiens pallens to cypermethrin and the iron metabolism-related protein genes(ferritin and iron-regulating protein)has been conducted experimentally,which is an in-depth study of mosquitoes.The mechanism of drug resistance has important theoretical significance.Methods Primers were designed to amplify the full length of the target gene,purification and sequencing,and sequence analysis.The expression level of ferritin gene and iron regulatory protein gene was detected by fluorescence quantitative PCR.Using the t-test in the hypothesis test,the expression levels of ferritin and ferroregulatory protein genes in two differently treated samples of Culex pipiens pallens were statistically analyzed.The gene fragments of different developmental stages of Culex pipiens pallens were amplified,and the changes of gene expression of ferritin and iron regulatory proteins were observed.CCK-8 was used to detect the viability of C6/36 cells.Vector recombinant plasmids were constructed and transfected into C6/36 cells.The viability of transfected recombinant plasmids and empty plasmids was measured.The viability of C6/36 cells loaded with the target gene was observed.Result Through the above experimental design,we successfully amplified the fragments of the ferritin and iron regulatory protein genes of Culex pipiens pallens.Quantitative fluorescent PCR assay revealed that the Cx.pipiens pallens sensitive strains were ferritin quantified(Fn-sensitive)and resistant population ferritin fluorochromes.Quantitative(Fn anti-)results showed that ferritin gene expression in the resistant strain Culex pipiens pallens was about 10 times that of the sensitive strain.The results of fluorescence quantification of iron regulatory protein genes showed that the expression level of the iron regulatory protein gene of Culex pipiens pallens decreased in the resistant strain Culex pipiens pallens.The t test showed that there was a statistically significant difference in the expression of ferritin and iron regulatory protein genes between the two samples.With the phylogenetic development,the ferritin gene did not change significantly at all stages of development of Culex pipiens pallens,and the expression level of the ferritin gene was higher in susceptible strains than in susceptible strains.There was no significant difference in the expression of the iron regulatory protein gene at each developmental stage of Culex pipiens pallens.The expression level of the iron regulatory protein gene was lower in the resistant strain Culex pipiens pallens than in the sensitive strain.CCK-8 was used to detect the viability of C6/36 cells transfected with recombinant and empty plasmids.It was found that the viability of C6/36 cells transfected with plasmids carrying ferritin gene fragment was higher than that of C6/36 cells transfected with empty plasmids.The viability of C6/36 cells transfected with the plasmid containing the iron regulatory protein gene fragment was lower than that of C6/36 cells transfected with the empty plasmid.Conclusion The expression levels of ferritin and iron regulatory protein genes were different in sensitive and cypermethrin resistant strains of Culex pipiens pallens.The expression level of ferritin gene in resistant strain Culex pipiens pallens was significantly higher than that of susceptible strain Culex pipiens pallens;iron regulatory protein gene In the resistant strain Culex pipiens pallens,the expression level decreased,suggesting that the ferritin gene and the iron regulatory protein gene may be related to the resistance of mosquitoes;at different developmental stages,the expressions of ferritin and iron regulatory protein genes in Culex pipiens pallens did not show significant changes,and the development Stage ferritin expression resistance strains were higher than susceptible strains,and iron resistance protein expression levels in all developmental stages were lower than those of susceptible strains;the target gene was transfected into Aedes albopictus C6/36 cells and observed.In the cypermethrin environment,the survival rate of the ferritin gene-transfected recombinant plasmid was higher than that of the plasmid transfected with the empty plasmid.The cell survival rate of the recombinant plasmid transfected with the iron regulatory protein gene was lower than that of the plasmid transfected with the empty plasmid.