Effect Of Cytochrome P450 On Cryptolestes Ferrugineus’s Resistance To Phosphine

Cryptodestes ferrugineus（Stephens）is one of the world’s stored grain pest and one of the main stored grain pests in China.At present,the most commonly used prevention and control method C.ferrugineus is phosphine fumigation.In recent years,the resistance of C.ferrugineus to phosphine has become more and more serious,that has become the most difficult problem for pest control in stored grain.At present,the resistance mechanism of C.ferrugineus to phosphine is still not clear.In order to effectively prevent and control the damage caused by C.ferrugineus in grain storage,it is urgent to study the resistance mechanism of these pests.Cytochrome P450 plays an important role in insect defense against insecticides by metabolizing and detoxifying exogenous substances.The overexpression of cytochrome P450 gene is one of the main mechanisms for the high level of insecticide resistance in many insects.In this study,resistance detection and screening were carried out on several geographical populations in China,and on this basis,resistant strains with different resistance levels were constructed,including extremely high resistance strains,high resistance strains,medium resistance strains,etc.On the one hand,the activity of P450enzyme in the sensitive and resistant strains of C.ferrugineus was measured.On the other hand,the transcriptome sequencing analysis of the resistant and susceptible strains of R.ruticosa was carried out by using high-throughput sequencing technology.Bioinformatics analysis was conducted on C.ferrugineus at the RNA level.The species,expression pattern and amino acid sequence of the screened P450 gene were investigated and analyzed.The results of this study lay a molecular basis for the in-depth study of the resistance mechanism of C.ferrugineus to phosphine,and have important significance for the control and resistance management of C.ferrugineus.The main research results are as follows:1.Determination of Phosphine Resistance of Different Strains of Rusty Raspberry Raspberry.Through eight different geographical populations of C.ferrugineus valley steal the FAO recommended method is used for bioassay test,the result shows:The LC₅₀（95%confidence interval）of the sensitive strain SS was 0.024 mg/L.Seven strains were resistant,including Kaiyuan LNKY from Liaoning Province,Sanya HNSY from Hainan Province,Xiaoshan ZJXS from Zhejiang Province,Shenqiu HNSQ from Henan Province,BJZL from Beijing Cofco,GDZS from Guangdong Province and SXLL from Luliang,Shanxi Province.Among them,LNKY,HNSY,ZJXS,HNSQ and BJZL were all highly resistant strains,and their resistance ratios were 206,236,473,511 and 942 times higher than those of sensitive strains,respectively.The resistance ratio of GDZS was 114 times that of the sensitive strain,and it was highly resistant strain.The resistance ratio of SXLL was 17 times that of the sensitive strain,indicating that SXLL was medium resistant strain2.Determination of cytochrome P450 enzyme activity of different strains of C.ferrugineusCytochrome P450 enzyme activity in different strains of C.ferrugineus was determined by using SS sensitive strains,SXLL moderate resistant strains,GDZS high resistant strains and HNSQ extremely high resistant strains.The results showed that the P450 activity of HNSQ,GDZS and SXLL strains was 2.07,1.53 and 1.04 times of the sensitive SS strain,respectively.The activity of P450 enzyme in HNSQ strain increased at 24 h and reached the highest value of 15.06（Eu of P450）/μg at 48 h after different time of LC₃₀ stress with phosphine sublethal dose.The P450 activity of GDZS strain increased with the prolonging of treatment time,and the highest P450 activity reached 10.27（Eu of P450）/μg.The highest enzyme activity of SXLL strain was 9.20（Eu of P450）/μg.The results showed that the P450enzyme activity of the resistant strains was higher than sensitive strains.The P450 enzyme activity of the extremely high resistance strains was higher than the high resistance strains,medium resistance strains,and sensitive strains,indicating that the resistance of C.ferrugineus to phosphide was related to the increase of P450 enzyme activity.3.Screening of genes related to phosphide resistance in C.ferrugineusThrough transcriptome sequencing analysis of resistant and susceptible strains of C.ferrugineus,the differentially expressed genes between susceptibility and resistance were screened and analyzed.A total of 7464 differentially expressed genes were screened between the two groups of SS-VS-RS,including 6324 up-regulated genes and 1140 down-regulated genes.Through comparison and analysis of differentially expressed genes by NR annotation,there are 38 genes of P450 differentially expressed up-regulated and 4 genes of P450differentially expressed down-regulated.The phylogenetic tree of the 38 up-regulated genes was constructed and analyzed,and 15 of these P450 genes were accurately classified into 4Clan.In Cyp2 clan,Cyp3 clan,Cyp4clan and P450 Mitochondrion clan.4.Relative expression of resistance-related P450 geneIn the resistant and susceptible strains,the genes,expression differential multiple Log Fc value was greater than 1.5.The expression level of 7 P450 genes were further verified by qPCR.The results showed that,the expression levels of DN10621,DN4282,DN30828,DN1575 and DN828 in resistant strain HNSQ were 10.33±1.07,15.82±0.27,25.05±1.53,4.69±1.11,5.48±0.97 times of those in sensitive strain SS,respectively.The expression level of DN27063 in different strains ranged from 0.897 to 1.149 times.The expression level of DN1830 in different strains was low,ranging from 0.198 to 0.768 times.After analysis of the expression levels of different differentially expressed genes,it was found that DN10621,DN4282,DN30828,and DN828 were all overexpressed in Cryptolestes ferrugineus resistant strains,while DN1575 was only overexpressed in the highly resistant strains,and the expression levels were significantly higher than those in the moderately resistant and sensitive strains.After the sublethal concentration of phosphine LC₃₀ stress treatment,the five P450 genes were further tested and verified.The results showed that the expression levels of DN30828,DN828,DN4282 and other three genes were up-regulated to different degrees.Among them,DN828 and DN4282 showed a general trend of decreasing first and then increasing in the process of 48 h of phosphate stress,and reached the peak value at 2 h and 12 h of phosphate stress,respectively,and then slightly decreased,and the expression levels of resistant strains were higher than those of sensitive strains.Therefore,The overexpression of cytochrome P450 gene may be related to the resistance to phosphine in C ferrugineus.5.Cloning and Sequence Analysis of DN4282 and DN828 Genes of C ferrugineusAccording to transcriptome sequencing sequences designed the primers,gene sequences of DN4282 and DN828 were amplified and named as CYP4XU1 and CYP4XV1,respectively.The ORF of DN4282 was 1494 bp and encoded 497 amino acids.The ORF of DN828 was 1506 bp,encoding 501 amino acids.The protein molecular formula encoded by DN4282 gene is C₂₆₃₉H₄₁₅₆N₆₉₀O₇₂₆S₁₉,the theoretical isoelectric point is 8.74,and the molecular weight of the protein is 57.78k Da,.The protein molecular formula encoded by DN828 gene is C₂₆₅₇H₄₂₁₅N₆₉₁O₇₂₈S₂₁,the theoretical isoelectric point is 8.96,and the molecular weight of the protein is 58.16 k Da.Phylogenetic tree analysis shows that the homology of C ferrugineus is relatively high with T.castaneum,Laodelphax striatellus,and Tenebrio molitor.