| Aleurolyphus ovatus(Trouperau),belonging to Acari,Acaridae,is a worldwide distributed stored grain mite that is harmful to grain and affects its quality.Temperature is an important environmental factor in controlling stored grain pests.Temperature can cause stress to mites and prompte them to response to and resist this stress.The insulin signaling pathway plays an important role in regulating the metabolism,growth,and reproduction of the mites,and can be used as an important target for pest control.There has no report on the control of A.ovatus by insulin signaling pathway genes and RNA interference.In this study,we analyzed the transcriptome under different temperature stresses.The cDNA sequences of six key genes in the insulin signaling pathway were cloned using RACE and RT-PCR techniques,and their bioinformatics analysis was performed.RT-qPCR was used to explore the expression levels of six genes at different developmental stages and under different temperature stresses.After silencing AoInR by RNA interference technology,we explored its impact on six key genes of the insulin signaling pathway and provided a new way to control the A.ovatus.The main results are as follows:1.Analysis of differentially expressed genes in the transcriptome and the insulin signaling pathway of A.ovatus under temperature stress.Transcriptome data of A.ovatus under half-lethal time stress at different temperatures(4,8,12,28,39,42,and 45℃)were obtained through transcriptome sequencing.A total of 56379 Unigenes were obtained from 7 groups of samples,of which 44980 Unigene were annotated.Compared with the control group,there were 2151,6515,and 3420 differentially expressed genes in the hyperthermia group(39,42,and 45℃),with 674,3664,and 1988 up-regulated genes and 1477,2851,and 1432 down-regulated genes,respectively.In the hypothermia group(4,8,and 12℃),there were 695,4380,and 3116 differentially expressed genes,with 165,1524,and 1246 upregulated genes and 530,2856,and 1870 down-regulated genes,respectively.Enrichment of differential gene KEGG pathways revealed that compared to the control group,the significantly enriched pathway in the high-temperature treatment group was the highest at 39℃ and lowest at 45℃,while in the low-temperature group,the highest was 12℃ and the lowest was 4℃.Regardless of high or low-temperature stress,the enriched KEGG pathways are mainly metabolic pathways,involving fatty acid metabolism,steroid hormone biosynthesis,and lysosomes.The differentially expressed genes involved in these pathways may play an important role in temperature stress in A.ovatus.Further analysis revealed that the number of differentially expressed genes annotated to the insulin signaling pathway was higher in the hyperthermia group than in the downregulation group,while the number of differentially expressed genes downregulated was higher in the hypothermia group than in the upregulation group.2.Cloning and sequence analysis of six genes involved in insulin signaling pathwayBased on transcriptome data,six key genes of the insulin signaling pathway were cloned:AoInR,AoIRS1,AoPI3Kc,AoPDK1,AoAkt1,and AoAkt2.The amplified cDNA sequences were 3975,2985,1623,2017,1281,and 1299 bp in length,encoding 1324,994,448,476,362,and 371 amino acids,respectively.Six genes are hydrophilic proteins that have no signal peptides or phosphorylation sites,among which AoPI3Kc and AoPDK1 have no glycosylation sites.Except for AoInR,which has a transmembrane region and is a transmembrane protein,the other five genes have no transmembrane region and are non-transmembrane proteins.There are varying degrees of differences in the tertiary structure of proteins’ spiral,folding,and irregular curling.Phylogenetic tree analysis showed that the amino acid sequences of the six genes in the insulin signaling pathway of A.ovatus and the homologous sequences of mites clustered into one branch.3.mRNA expression characteristics of six genes in insulin signaling pathwayThe six genes of the insulin signaling pathway in A.ovatus can be expressed at different developmental stages.The expression levels of the AoInR,AoIRS1,AoPDK1,and AoAkt2 genes were the highest in the egg stage,significantly higher than those in other developmental stages(P<0.05).The expression levels of the AoPI3Kc gene were the highest in the adult mite stage,significantly higher than those in other developmental stages(P<0.05).The expression levels of AoAkt1 gene were significantly higher in the egg and tritonymph developmental stage(P<0.05).The six genes of the insulin signaling pathway in A.ovatus can be expressed at different temperatures.Compared with the control group,the expression of AoInR was significantly increased at 39℃(P<0.05),while there was no significant difference between the 42 and 45℃ groups(P>0.05).The expression level at 39℃ was higher,16.9 times that of the control group.The expression level at 4℃ was significantly decreased(P<0.05),while the expression level at 8℃ was significantly increased(P<0.05).The expression level at 8℃ was 2.60 times that of the control group.The expression level of AoIRS1 at 39℃ was significantly higher than that of the control group(P<0.05),13.17 times higher than that of the control group.There was no significant difference between the expression levels at 42 and 45℃(P>0.05).The expression level was significantly increased at 12℃(P<0.05),2.81 times higher than that of the control group,and significantly decreased at 4 and 8℃(P<0.05).The expression of AoPI3Kc at 39 and 45℃ was significantly higher than that of the control group(P<0.05),32.02 and 3.99 times higher than that of the control group,respectively.There was no significant difference in the expression level at 42℃(P>0.05).The expression level was significantly increased at 4,8,and 12℃(P<0.05).The expression levels of AoPDK1,AoAkt1,and AoAkt2 genes under high and low-temperature stress were significantly higher than those in the control group(P<0.05).4.Effects of RNAi with the AoInR gene on the expression levels of 6 genes of insulin signaling pathway.Using the immersion method to introduce dsAoInR into the body of A.ovatus,Compared to the control group,with the prolongation of the interference time,the mortality rate significantly increased(P<0.05),with a mortality rate of 19.72%at 24 h and 51.67%at 48 h.Compared with the control group dsGFP,the expression of AoInR,AoIRS1,and AoAkt2 decreased after 24 h of interference,with a significant decrease in AoAkt2(P<0.05);The expression levels of AoPI3Kc,AoPDK1,and AoAkt1 increased,with the expression level ofAoAkt1 significantly increasing(P<0.05),8.64 times higher than that of dsGFP;after 48 h of treatment,the expression of AoInR and AoAkt2 decreased significantly(P<0.05),the expression of AoIRS1,AoPI3Kc,AoPDK1,and AoAkt1 was significantly increasing(P<0.05),with the highest expression of AoPI3Kc,4.69 times higher than dsGFP.In conclusion,this study constructed a transcriptome library of A.ovatus under different temperature stresses for the first time,annotated the differentially expressed genes in the insulin signaling pathway,and found that six key genes of insulin signaling pathway can be expressed at different development stages and after different temperature treatments.After RNAi silences the AoInR gene,the mortality rate increases,indicating that AoInR can be a potential biological control target gene which provides a theoretical basis for further control of A.ovatus. |
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