| Diamondback moth(DBM),Plutella xylostella,is a destructive cosmopolitan pest of cruciferous crops and is thought to be an important model pest for studying the resistance level to insecticides.In order to reveal the relationship of the fitness between the chlorpyrifos-resistant(R)and-susceptible(S)DBM under heat stress and the reason why high temperature influences genotype frequency at Gly227Ala mutation of ace1 in R DBM population,effects of heat stress on expression levels of mitochondrial apoptosis-related genes and Hsp family genes in R and S DBM(larvae,pupae and adults)were studied.Apoptosome(Cytochrome c-Apaf-1-procaspase-9)plays a critical role in the mitochondria-associated apoptotic pathway.Cytochrome c,Apaf-1 and caspase-9 were identified from DBM.The full length cDNA of cytochrome c(GenBank:KC507801)was 873 bp including a 327 bp ORF encoding a putative protein of 108 amino acids.The calculated molecular mass and isoelectric point of deduced protein were 11.74 kDa and 9.62,respectively.Two full length cDNAs of Apaf-1,Apaf-1a and Apaf-1b,were identified.The full length cDNA of Apaf-1a(GenBank:KC588901)was 5054 bp including a 4647 bp ORF encoding a putative protein of 1548 amino acids.The calculated molecular mass and isoelectric point of deduced protein were 175.96 kDa and 6.85,respectively.The full length cDNA of Apaf-1b(GenBank:KF318722)was 4981 bp including a 4647 bp ORF encoding a putative protein of 1548 amino acids.The calculated molecular mass and isoelectric point of deduced protein were 175.96 kDa and 6.85,respectively.Apaf-1a shared the same region of ORF and 5'-UTR(5'-untranslated region)(172 bp)of cDNA with Apaf-1b,but 73 bp longer than Apaf-lb in the region of 3'-UTR of cDNA.The ORP of Apaf1la and Apaf-1b shared 100%identity.The ORF of caspase-9(GenBank:K.F365914)was 1356 bp encoding a putative protein of 451 amino acids.The calculated molecular mass and isoelectric point of deduced protein were 51.33 kDa and 7.56,respectively.In this study,three cDNA sequences of hsp70,i.e.,hsp72-1a,hsp72-1b and hsc70,were identified from DBM.hsp72-1a and hsp72-1b were the type of stress-inducible hsp70,while hsc70 was the type of constitutive hsp70.The ORF of hsp72-1a(GenBank:JQ693015.1)was 1992 bp encoding a putative protein of 663 amino acids with the calculated molecular mass of 72.16 kDa and isoelectric point of 5.27.The ORF of hsp72-1b(GenBank:JQ711194.1)was 1992 bp encoding a putative protein of 663 amino acids with the calculated molecular mass of 72.13 kDa and isoelectric point of 5.34.The ORF of hsc70(GenBank:JN676213.1)was 1953 bp encoding a putative protein of 650 amino acids with the calculated molecular mass of 71.18 kDa and isoelectric point of 5.26.Three highly conserved Hsp70 family signatures,a putative ATP/GTP binding site and putative nuclear localization signal motifs were found in the amino acid sequences of hsp72-1a,hsp72-1b and hsc70.Up-regulated expressions of mitochondrial apoptosis-related genes(cytochrome c,Apaf-1,caspase-9 and caspase-7)were found in DBM larvae and adults treated with 37 and 42? for 3 h,and the extent of up-regulated expression of these genes was higher in R DBM than that in S DBM,in general.As compared to 25? treatment,38? for 48 h,40? for 8 or 16 h,42? for 4 or 8 h and 44? for 1 h resulted in up-regulated expression of mitochondrial apoptosis-related genes in DBM pupae,and significantly higher up-regulated expression of caspase-9 and caspase-7 was observed in R DBM than in S DBM.High temperatures(33.5-50? for 3 h)could induce up-regulated expression of Apaf-1,caspase-9 and caspase-7 in DBM adults,and with the increase of temperature,the expressions of these genes increased and reached at a plateau at 42 or 47?and then decreased.40?(24,48 and 72 h)could result in up-regulated expression of Apaf-1,caspase-9 and caspase-7 in DBM adults,and with the increase of the treatment time period,the expressions of these genes presented a tendency of increased at the first and then declined(except for caspase-7 whose expression declined gradually).In addition,the extent of up-regulated expression of caspase-9 and caspase-7 was higher in R DBM than in S DBM under 40?conditions.Heat stress could induce apoptosis in both R.and S DBM,and the degree of apoptosis in R DBM was higher than that in S DBM.The basic expression level(at 25?)of hsc70 was high,but no significant up-regulated expression of hsc70 was found in DBM under heat stress.The basic expression levels of inducible hsp70s,hsp90 and hsp20 were lower,but high temperature could result in significantly up-regulated expression of these genes.In DBM larvae and adults,the expressions of inducible hsp70s,hsp90 and hsp20 were up-regulated under 37 and 42? for 3 h,and significantly higher expression levels of these genes were found at 42? than 37?.In addition,the extent of up-regulated expression of these genes was higher in S DBM than that in R DBM under heat stress.In DBM pupae,as compared to 25? treatment,38? for 48 h,40? for 8 or 16 h,42? for 4 or 8 h and 44? for 1 h resulted in significantly up-regulated expression of inducible hsp70s,hsp90 and hsp20.The expression levels of these genes in R DBM were higher than those in S DBM at 25?,while under high temperatures,these genes expression levels in R DBM were lower than those in S DBM.High temperatures(33.5-50°C for 3 h)could induce up-regulated expression of inducible hsp70s,hsp90 and hsp20 in DBM adults,and with the increase of temperature,the expressions of these genes increased and reached at a plateau at 42 or 47? and then decreased.40?(24,48 and 72 h)could result in significantly up-regulated expression of inducible hsp70s,hsp90 and hsp20 in DBM adults,and with the increase of the treatment time period,the expressions of these genes presented a tendency of increased at the first and then declined or declined gradually.In general,heat stress could induce inducible hsp70s,hsp90 and hsp20 up-regulated expression,and the extent of up-regulated expression of these genes was higher in S DBM than that in R DBM.In the absence of insecticides,frequency of resistant homozygous genotype(RR)at Gly227Ala mutation of acel decreased gradually with the increasing generations in R DBM population,while high temperature could accelerate the sharp decrease of the frequency of RR genotype.In addition,the frequency of RR genotype in summer declined sharply,and was significantly lower than that in spring and autumn. |
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