Study On The Mechanisms Of Phosphine Resistance In Tribolium Castaneum(Herbst) Based On CYP346 Family

Tribolium castaneum is a worldwide storage pest with important economic significance.By far,the most important control method is phosphine fumigation.Because of long-term large and unreasonable use,the resistance of Tribolium castaneum to phosphine increased gradually.To clear the red flour beetle of the resistance mechanism of phosphine with reasonable prevention and control of strategy and the development of effective fumigant plays an important role.Cytochrome P450is one of the three major detoxification and metabolic enzymes in insects,and it is the only one that can mediate the resistance of many insecticides in insects.In order to investigate the effect of cytochrome P450 on the resistance to phosphine,the resistance folds of the cytochrome P450 to phosphine were determined by taking the red flour beetle from different areas as the research object.On the basis of this study,the relationship between cytochrome P450 and phosphine resistance were studied.The main results were as follows:?1?The phosphine test by FAO was used to determine the resistance of two strains from different regions.The results showed that Shenzhen?SZ?strain belonged to a highly resistant strain with LC₅₀ of 7763.9?g/L and resistance fold of 862.7;Yunnan?YN?strain was a sensitive strain with resistance fold of 3.0.The specific activity of resistant strain?SZ?P450 was significantly higher than that of the sensitive strain?YN??P<0.05?.After treatment with PBO which has a specific inhibition on cytochrome P450,the activity of P450 was significantly inhibited in both strains.After treatment for 6 hours,the inhibition rate of enzyme activity reached the maximum,and the inhibition rate in high resistant strain?SZ??88.5%?was significantly higher than that in sensitive strain?YN??50.9%??P<0.05?.When the activity of P450 was inhibited,the susceptibility of strains to phosphine were increased;and the synergistic effect of PBO on resistant strain was the most significant,with a synergistic effect of 172.0%.Based on this,it could be preliminarily judged that the resistance to phosphine was related to cytochrome P450?P<0.05?.?2?The full-length sequences of 5 genes CYP346 family were obtained based on the information of the transcriptome database.The length of open reading frame of these five genes ranged from 1482 bp to 1512 bp,the relative molecular weight and isoelectric point were 56.54 kDa to 58.08 kDa and 7.20 to 9.06,respectively.The protein composition,transmembrane region,nucleotide and amino acid homology of CYP346 gene family were compared and predicted by TMHMM,ProParam and other softwares.The results showed that the homology of amino acid coded by five genes ranged from 48.31%to 84.49%,and the homology of nucleotide sequence ranged from 56.75%to 83.47%,and all of them contained transmembrane fragments and conserved regions.?3?The expression patterns of five genes of CYP346 gene family in different strains?10 strains of Tribolium castaneum?and under phosphine stress were studied by qPCR.The results showed that the expression of CYP346B subfamily genes?CYP346B1,CYP346B2 and CYP346B3?in resistant strains was significantly higher than that in susceptible strains?P<0.05?,and the expression of CYP346B subfamily genes in SZ strain were 11.97±4.55?5.03±0.27?9.00±1.89 times higher than that in JX strain,respectively.However,there was no significant difference in the expression of CYP346A1 and CYP346A2 among different strains or there was no direct regularity between the expression level and the resistance level?P>0.05?.After phosphine induction,the expression of CYP346B subfamily genes increased in YN and SZ strains in varying degrees and had similar variation patterns.In SZ strain,the expression of CYP346B1 was positively correlated with induction time,and was 5.0times higher than that of non-induction at 20 h.In sensitive strain?YN?,the expression of CYP346B subfamily genes reached its maximum after 2 h treatment,which was 5.6 times higher than that of non-induction.After phosphine induction,the gene expression of SZ strain was higher than that of YN strain.After phosphine induction,the gene changes were similar.This further indicated that the CYP346B subfamily gene might be involved in the formation of phosphine resistance.?4?Functional validation of 5 genes of CYP346 family by RNAi.The results showed that after injection of dsRNA?300 ng/head?of five genes of CYP346 gene family,5 genes of sensitive strain?YN?and resistant strain?SZ?were successfully silenced.The silencing efficiency was 50.2%-76.9%and 42.5%-62.8%,respectively.After sublethal dosage treatment,the results showed that,after inhibiting the expression of CYP346B subfamily genes,the mortality of YN and SZ strain increased by 32%-42%and 20%-31%,respectively.However,while inhibiting the expression of CYP346A subfamily genes,the sensitivity of YN and SZ strain to phosphine did not change significantly?P>0.05?.These results indicated that all three CYP346B subfamily genes participated in the formation of phosphine resistance of Tribolium castaneum,while the two CYP346A subfamily genes were not participated in the formation of phosphine resistance.