Study On The Mechanisms Of Phosphine Resistance In Tribolium Castaneum (Herbst) Based On Protective Enzyme And Cytochrome P450 Enzyme

Phosphine?PH₃?is the most commonly used stored grain pest fumigant in the world.It has the advantages of strong penetrability,good diffusion,low cost,convenient use and less residue.However,due to long-term,single use and some irregular operations during use,the stored grain pests have serious resistance to phosphine fumigants.In view of the fact that the phosphine resistance of stored grain pests is becoming more and more serious,seriously jeopardizing the safety of stored grain,and the real problem of causing huge economic losses,the research on the resistance to phosphine by using the stored grain pests,Tribolium castaneum.The resistance level of the stored grain pests to phosphine and the empty warehouse fumigant?dichlorvos?from different regions of China was determined.The relationship between superoxide dismutase,catalase and cytochrome P450 and phosphine resistance was studied by enzyme activity assay.Finally,using the gene quantification technique to study the molecular mechanism of two P450 genes?CYP345A1 and CYP345A2?mediated by phosphatase,The results of the study are as follows:1.Determination and analysis of phosphine and dichlorvos resistance levels in different regions of ChinaThe phosphine resistance test method recommended by the Food and Agriculture Organization of the United Nations?FAO?was used to determine the level of phosphine resistance of Tribolium castaneum from different regions.The results showed that the phosphine resistance of the 16 strains of Tribolium castaneum was 1.7-44.4.Combined with the data of the phosphine resistance in the laboratory,the research and analysis were carried out from two regions and different places.The results showed that highly resistant strains mostly occurred in southern China.Analysis of the level of resistance to phosphine in the strains of Tribolium castaneum in different places shows that highly resistant strains occur mostly in processing plants and grain depots.In comparison,the amount of resistance found in the processing plant is higher than the quantity in the grain depot.Determination of the level of dichlorvos resistance in the strains of Tribolium castaneum by the method of drug film.The results showed that the LC₅₀ values of the16 strains ranged from 0.044 to 0.110 g/L,and the relative resistance coefficient was within 2.50 times,indicating that these strains had little difference in resistance to dichlorvos.2.Determination and analysis of superoxide dismutase and catalase activities of Tribolium castaneumThe activity of superoxide dismutase?SOD?in different strains was determined by NBT photoreduction method.The results showed that the activity range of 25 strains superoxide dismutase was 4439.13 U/?g.-9098.67 U/?g.The results of data analysis showed that there was no significant correlation between SOD activity and phosphine resistance resistance level?P>0.05?.After phosphine stress,the activity of SOD increased with the prolongation of stress time.After 20 h of stress,the activity of SOD in the six strains increased by 0.90-2.08 times.The activity of catalase?CAT?in different strains was determined by ammonium molybdate colorimetry.The results showed that the activity of CAT enzyme ranged from 45.51 U/?g to 416.35 U/?g.Data analysis showed that there was no significant correlation between CAT enzyme activity and phosphine resistance level?P>0.05?.After phosphine stress,the activity of CAT enzyme increased with the stress time.After20 h of stress,the activity of CAT enzyme in the six lines decreased to 0.09-0.42 times.3.Determination and analysis of the specific activity of cytochrome P450The specific activity of P450 of 16 strains ranged from 0.81 EU of P450/?g-2.41EU of P450/?g,and the specific activity of P450 of resistant strains was significantly higher than that of sensitive strains?P<0.05?.Combined with the existing P450 specific activity data of the laboratory,the results show that,from the overall trend,with the increase of phosphine resistance,the specific activity of P450 is also increased,and the P450 ratio of resistant strains is improved.The viability was significantly higher than that of the sensitive strains?P<0.05?.It is speculated that the overexpression of P450protein may lead to the production of resistant strains.4.Determination of the expression level of two P450 genes and their correlation with phosphine resistanceThe expression levels of CYP345A1 and CYP345A2 genes of different phosphine-resistant Phytosterols were determined by real-time quantitative PCR.The results showed that the expression level of CYP345A1 gene ranged from 1.00 to 16.60,and the expression level of CYP345A2 gene ranged from 1.00 to 12.05,and with the increase in phosphine resistance,the amount of gene expression also increased.