Study On The Mechanisms Of Phosphine Resistance In Cryptolestes Ferrugineus (Stephens) Based On Mitochondrial Gene

The rusty grain beetle,Cryptolestes ferrugineus?Coleoptera:Laemophloeidae?,is an important grain storage pest.The resistance to the main fumigants-phosphine developed rapidly,which has become an important factor restricting the safety of grain storage.In this study,the resistance level of C.ferrugineus of different geographic populations was determined by FAO's recommended method.Based on the sequencing information of the transcriptome of Cryptolestes ferrugineus,the full-length sequences of the six cuticular protein genes of CPR family were obtained by reverse transcription polymerase chain reaction?RT-PCR?technology and rapid amplification cDNA?RACE?technology.The nucleotide sequence and protein sequence of the six cuticular protein genes of CPR family were analyzed by bioinformatics.After screening the stable reference genes of C.ferrugineus in different developmental stages and different strains stimulated with phosphins,the expression patterns of the four mitochondrial genes?nad3,atp6,cob and cox1?were studied to explore the mechanism of phosphine resistance of C.ferrugineus.The results in this study are as follows:1.The measurement of the lever of phosphine-resistance in Cryplotestes ferrugineusPhosphine-resistance in 3 populations of Cryplotestes ferrugineus?Stephens?collected from Zhang jiagang,Cheng du and Tai cang was detected through the method recommended by FAO.The results indicated that,all above the mentioned stored grain insect species have a varying degree of resistance and resistant varieties have reached 100%.The value of LCso of ZJGCF was 0.079 mg/L and the value of resistance factoris?Rf?was 7.18 times,so ZJGCF was low resistant.The value of LC50 of CDCF was 0.126 mg/L and the the value of resistance factoris?Rf?was 11.45 times,so ZJGCF was medium resistant.The value of LC50 of TCCF was 20.795 mg/L and the value of resistance factoris?Rf?was 1906.82 times,so TCCF was resistant.2.Cloning and Sequence Analysis of Cuticular Protein Gene in C.ferrugineusBased on the sequencing information of the transcriptome of Cryptolestes ferrugineus,the full-length sequences of the six epidermal protein genes of CPR family were obtained by reverse transcription polymerase chain reaction?RT-PCR?and rapid amplification cDNA?RACE?.The six cuticular genes of CPR family were named as C1,C2,C3,C4,C5 and C6.And then we analyzed the six cuticular genes structure of CPR family,sequence characteristics and the protein homology by bioinformatics methods.Showing as the Table 3.2,the length of each cuticular gene of CPR family was:C1=704 bp,C2=794 bp,C3=771 bp,C4=796 bp,C5=630 bp and C6=711 bp,respectively.Each cuticular gene of CPR family has a typical polyA structure.The length of each mature protein sequence of CPR family varied from 137 to 176 amino acids.The isoelectric point of each mature protein sequence of CPR family ranged from 4.56 to 7.10.The protein molecular of each mature protein sequence of CPR family weight ranged from 14.51 to 17.81 kDa.C1 has the typical R&R conserved domain of epidermal protein:CHIT_BIND_RR₂,and C2,C3,C4,C5 and C6 have typical R&R conserved domains of epidermal proteins:CHIT_BIND-RR₁ and CHIT_BIND_RR₂;each protein of CPR family has chitin binding domain:Chitin_bind₄;signal peptide sequence of each protein of CPR family was 16-18 amino acids.The protein signal peptide sequences of each mature protein sequence of CPR family were 16-18 amino acids.Homology analysis of each mature protein sequence of CPR family found that amino acids obtained have higher homology with epidermal proteins of other species.3.Selecting and validating the reference genes of Cryptolestes ferrugineus.In this study,four programs including geNorm,NormFinder,Bestkeeper and deltaCt were used to evaluate the expression of 9 candidate reference genes-?-Tubulin??-TUB?,Cytochrome oxidase?CO?,CyclinA?CycA?,Elongation factor 1??EF1??,?-Tubulin??-TUB?,Glycerladehyde-3-phosphate dehydrogenase?GAPDH?,Ribosomal protein S13a?RPL13a?,Ribosomal protein S13?RPS13?,Succinate dehydrogenase complex subunit A?SD?and Beta actin?ACTB?in different developmental stages and different strains stimulated with phosphins at 0.083 mg/L for 0 h,0.5 h,1 h,2 h,4 h,8 h,12 h and 48 h.The results showed that the most stable gene was RPS13+EF1?,and the most unstable was ?-TUB in different developmental stages.The most stable gene was RPS13+?-TUB,the most unstable gene were GAPDH and Cyc A in different strains.4.The expression pattern of the four mitochondrial genes of Cryptolestes ferrugineus in different strains under phosphine stressThe expression levels of the four mitochondrial genes-NADH dehydrogenase subunit 3?nad3?,ATP synthase FO subunit 6?ATPs?,cytochrome b?cob?,cytochrome oxidase subunit I?coxl?were detected by qRT-PCR method in different strains.Test results showed:under normal circumstances,the expression levels of nad3,atp6 and cob were significantly different between sensitive strain and resistant strain?P<0.05?.The expression level of nad3 in sensitive strains was 753.29 times as much as that in resistant strains.The expression level of atp6 in sensitive strains was 1479.01 times as much as that in resistant strains.The expression level of cob in susceptible strain was 5756.93 times of that in resistant strain.But coxl had no significant difference?P>0.05?.Under the induction of phosphine,the expression levels of nad3,atp6 and cob decreased gradually in the sensitive and resistant strains,while no significant change was observed in the expression level of coxl in the susceptible and resistant strains.Inconclution,the study indicates that mitochondrial genes?nad3,atp6,cob?may be involved in the formation of phosphine resistance of C,ferrugineus,while coxl might not be be involved in it.