Preliminary Study On Drug Resistance-related Gene CA?

Insecticide/drug resistance is organisms' resistance and tolerance to insecticides/drugs.The emergence of resistance makes the insecticides/drugs fail to work under normal dose or even no effect at all.Insecticides/drug resistance can be inherited to the offspring and is related to the species' evolution,survival and reproduction.At present,resistance is becoming a more and more serious challenge to human beings,whether in entomology,bacteriology or oncology research.Elucidating the mechanism of resistance is a major scientific problem that is urgently needed in the field of biomedicine.In our laboratory,we has long been engaging in insect resistance mechanism research,identifying and cloning a number of new insect resistance genes,such as cytochrome P450 monooxygenases(P450s),glutathione-S-transferases(GSTs),trypsin,ribosomal proteins and so on.Based on the interdisciplinary literature reading of entomology,bacteriology and oncology,and further comparative analysis and inductive analysis,we found that some insect resistance genes are also tumor and bacterial resistance related genes.Therefore,we put forward a scientific hypothesis that insect resistance,bacterial resistance and tumor cells resistance may have similar mechanisms,which deserves further study.Currently,the research in oncology has found that carbonic anhydrase?(CA?)is tumor cell drug-resistant gene.Previously,we undertook a large-scale transcription profiling study using high-throughput transcriptome sequencing in susceptible and resistant strains of Culex pipiens pallens,from which we found that CA ? was differentially expressed.Based on the above experimental reports and scientific hypothesis,we detected the relative expression levels of CA ? in insect-susceptible and resistant strains by real-time quantitative PCR,determined the sensitivity of mosquitoes to deltamethrin by CDC Bottle Bioassay method after knocking down the expression of CA ? with dsRNA injection.We also observed the changes in P450s,GSTs and choline/carboxylesterases(CCEs)activities before and after dsRNA injection.The results showed that the expression level of CA ? in susceptible strain was 3.35 times higher than that in the resistant strain(P<0.001).At the same time,the mortality rate of mosquitoes exposed to deltamethrin was decreased after knocking down the expression of CA ?.These results suggest that CA ? was a new mosquito resistant gene.The results of metabolic enzymes activities tests showed that the activities of P450s,GSTs and CCEs respectively increased by 32%,31%and 43%after knocking down the expression of CA ?(P<0.05).This suggests that CA ? may be involved in mosquito insecticide resistance by affecting metabolic enzymes activities.We also cloned the ORF sequences of CA? from cDNA library of Culex pipiens pallens.Gene sequence analysis showed that the ORF region was 1041 bp in length and encoded 346 amino acids.It shared the highest homology with Culex pipiens quinquefasciatus(94%identity).The results of analysis of its coding protein domain showed that it belongs to the a-CA family and exists one protein domain.The expression level of CA in Escherichia coli showed that the 2 kinds of CA were 2.77 times and 4.56 times higher in susceptible strain than in resistant strain repectively(P<0.05).This suggests that CA may also be a bacteria resistance related gene,which needs further study.Our results suggest that CA? is a novel mosquito insecticide-resistant gene,which may be involved in the resistance by affecting the activities of P450s,GSTs and CCEs.The study also reports that the two kinds of CA in Escherichia coli may also be drug-resistant genes.The literature shows that CA ? is a tumor cell resistance gene.Therefore,this study demonstrates that CA is a common resistance gene for insects,bacteria and tumor cells,and the three may have similar mechanisms.These results provides preliminary evidence and research direction for exploring the inherent laws and relationships of the three.