Preliminary Research Based On Ran And Insecticide Stress Response

It is well known that the unreasonable use of insecticides not only pollutes the ecological environment,but also exacerbates the evolution of resistance of various pests under high selective pressure.Even more frightening is that these insecticides can indirectly threaten human health through the enrichment of the food chain.Finding a balance point in the game of protection has become a worldwide problem.With the rapid development of molecular biology and genomics,the study of the molecular mechanism of insect resistance has brought new ideas.At present,the main views on drug-resistance are related gene amplification,differential expression and structural changes.Many countries have reported and cloned a number of target genes related to resistance.The mechanism of resistance to insects is very complicated.Research and discovery of drug-resistant genes can provide a solid theoretical basis for further elucidating the molecular mechanisms of insect resistance and formulating effective pest control efforts.In our lab,we found that Ran can obviously respond to the resistance of Drosophila Kc cells to DM stress through two-dimensional electrophoresis(2-DE).Subsequently,it was demonstrated by the cell transfection technique that the gene Ran is a gene involved in the formation of DM resistance in cells.This led to the next question:whether the Ran gene responds to insecticide stimulation has a certain representative and broad spectrum?Studies on the relationship between Ran and other insecticides have not been reported.In order to further explore the relationship between Ran and various insecticides,this study still used Drosophila melanogaster Kc cells as the research object,using fluorescence quantification,western blotting,cell transfection,RNAi,flow cytometry and other experimental techniques.To investigate the response of Kc cells to Ran under the stress of various insecticides(DDT,parathion,carbaryl and spinosad)and the relationship between the expression level of Ran and tolerance to insecticide stress.Get the following main experimental results:1.We stimulated the Kc cells with different concentrations of four insecticides.The results of quantitative real-time PCR shown that Ran were both up-regulated in Kc cells after stimulation.Then we selected a suitable concentration of these insecticides to stimulate the Kc cells in different time,we also found that Ran were both overexpressed.This suggests that Ran may play an important role in the formation of a variety of insecticide tolerance.2.Amplification of Ran gene ORF sequences and exogenous transfection into cells.After the detection of transfection,different insecticides were applied.The results showed that the expression of Ran was significantly increased in the cells transfected with the recombinant plasmid for 24 h.After 24 h exposure to insecticide,the expression level of Ran was increased by 3.15 and 2.46 times compared with the control in the experimental group with exogenous transfection of Ran.Exogenous addition of Ran increases cell tolerance to both insecticides.3.Preparation of Ran dsRNA and exogenous transfection into cells.After the detection of transfection,different insecticides were applied.RNAi experiments showed that the expression level of Ran was significantly down-regulated after 24 h transfection of exogenous dsRNA.However,when the insecticides stress the cells for 24 h,the expression level of Ran can be gradually restored.Compared to the control group,the expression of Ran was increased by 1.61 and 1.15 times in the experimental group transfected with dsRNA,respectively.The results showed that reducing the expression level of Ran can reduce the tolerance level of the cells to these two insecticides.4.FCM detected apoptosis in each treatment group to analyze the effect of Ran on insecticide induction.The results showed that Kc cells apoptosis was the lowest when the insecticide treatment dose was 20 mg/L DDT and 40 mg/L parathion.Transfection of the recombinant plasmid significantly reduced apoptosis;in contrast,the mortality of transfected dsRNA was significantly increased.The combination of 20 mg/L DDT and 40 mg/L parathion is also the optimal response dose for Ran.We speculate that the level of expression of Ran can affect the tolerance of cells to insecticides.The results showed that the exogenous addition of Ran can enhance the cell survival rate for two insecticides.The results of the above studies indicate that upregulating the expression level of the gene Ran can increase the resistance of the cells to insecticide stress,thus having a certain support and protection for the cells.Based on the above experimental results,we believe that Ran is a tolerant gene associated with various insecticides.However,the relevant mechanisms still need to be further explored.