Molecular Mechanisms Of MAPK Signaling Pathway And HSP70 Involved In High Temperature Adaptation Of Neoseiulus Barkeri

Phytoseiid mites(Acari: Phytoseiidae)are effective natural enemies and important biological control agents for spider mites.Phytoseiid mites,due to their advantages of small size,rapid development,large reproduction and strong predatory ability,can effectively control a variety of small suction-sucking pests such as spider mites,thrips and whiteflies.Changing or fluctuating environments provoke a range of stresses in arthropods which adversely affect most aspects of organismal biology and ecology.As ectothermic organisms,phytoseiid mites experience a variety of environmental stress stimuli under field conditions and seldom live in their optimal living environment,resulting in suppressed potential and low control efficiency on small insect and mite pests.Feasible biological control strategies should be utilized to enhance the adaptability of predatory mites in field conditions under global warming and climate change.In our previous study,a high-temperature adapted strain(HTAS)of the predatory mite Neoseiulus barkeri(Hughes)was screened from a conventional strain(CS)by long-term thermal acclimation(35°C)and regular thermal hardenings(45°C)(exposed for 2 h every fourth week for many generations).Surprisingly,long-term heat acclimation confers HTAS N.barkeri the thermotolerance a hefty boost.In this study,to understand the molecular basis of heat acclimation,‘omics’ analyses were performed to compare the differences between HTAS female adults to CS at transcriptional and translational levels.Based on the ‘omics’ analyses datasets,four HSP70 genes and eight MAPK pathway genes were cloned and characterized.By means of real-time quantitative PCR(q RT-PCR),RNA interference(RNAi),Western Blot and prokaryotic expression system were subsequently utilized to explore the functions of these genes in thermotolerance of HTAS N.barkeri.Finally,the phosphoproteomic was utilized to explore the role of phosphorylation modification in high temperature adaptation of HTAS N.barkeri.The main results in this thesis are as follows:1 Comparative transcriptome and proteome analysis between two strains of N.barkeriApproximately a total of 66.14 Gb transcriptome data and 39,623 unigenes were obtained from six samples of N.barkeri.After heat acclimation,a total of 2,571 and 2,803 up-and down-regulated transcripts were differentially expressed in HTAS.It’s conserved that some processes,such as high expression of heat shock protein(HSP)genes,involved in heat tolerance of transcriptome analyses,while many protective enzymes including glutathione S-transferase,superoxide dismutase,peroxidase and cytochrome P450 displayed down-regulated expression.In KEGG analysis,many pathways including ribosome,RNA transport,MAPK signaling pathway,citrate cycle,glyoxylate and dicarboxylate metabolism and fructose mannose metabolism were enriched.A total of 26,223 peptides and 5,082 trusted proteins were identified by i TRAQ proteomic sequencing.A total of 500 differentially expressed proteins were identified in HTAS in comparison with CS,with 225 proteins up-regulated and 275 down-regulated after heat acclimation.Proteins upregulated in HTAS included isocitrate dehydrogenase,chymotrypsin B,methyltransferase and serpin B10,while the significantly down-regulated proteins were involved in nutrient metabolism,signal transduction,detoxification and antioxidant defense.Correlation between DEGs and DEPs showed119 genes/proteins related to heat acclimation.Correlation analysis of differentially expressed proteins and genes displayed that a subset of genes and proteins were expressed with the same trend related to heat acclimation.These genes including HSP70,HSP90,isocitrate dehydrogenase,vitellogenin and ATP-dependent DNA helicase were up-regulated,while cytochrome P450,cuticle protein,choline dehydrogenase,endochitinase were among the genes down-regulated at both the transcriptional and translational levels.2 Functional analysis of MAPK pathway genes in thermotolerance and differential analysis of phosphoproteomic study of N.barkeriBased on the transcriptome data of N.barkeri,eight MAPK pathway genes were cloned and characterized.The sequence analysis showed these MAPK pathway genes all had their own conserved domains and characteristic sequences,such as S＿TKC motif and ATP binding site for the signal transduction reception.Phylogenetic tree analysis showed eight MAPK pathway genes were divided into individual cluster indicating the important roles of conserved structure on specific gene functions.The expression patterns of MAPK pathway genes in two strains of N.barkeri at different developmental stages and under 42°C stress were analyzed.Most of the MAPK pathway genes expressed low in egg and larva stages,but high in adult stages,indicating these genes may regulate the growth and development of N.barkeri.Besides,the expression difference of MAPK pathway genes at different development stages of HTAS was more obvious than that of CS,suggesting the MAPK pathway gene may be involved in the growth and reproduction of HTAS during long-term high temperature acclimation.High temperature stress also significantly induced the up-regulation of expression of MAPK pathway genes in HTAS.The expression of MAPK pathway genes in CS decreased significantly after a long time of high temperature treatment,while the gene expression in HTAS was maintained at a high level or the same as the control level,indicating the high temperature adaptability of HTAS may be related to a large number of modifications of MAPK signaling pathway at the transcriptional process.The analysis of phosphoproteomics with two strains 3,156 proteins and 10,641 protein phosphorylation modification sites were determined,of which 217 phosphorylation protein phosphorylation levels up-regulated,144 phosphorylation protein phosphorylation levels downregulated,containing 310 phosphorylation increased loci and 198 phosphorylation decreased loci.Motif phosphorylation analysis revealed Ser/Thr kinase MAPKs and CDKs related to stress signal transduction were significant enriched.Besides,multiple MAPK pathway genes and the genes associated with heat shock protein binding were up-regulated at phosphorylation levels in HTAS of N.barkeri.3 Identification and functional analysis of HSP70 genes in thermotolerance of N.barkeriFour HSP70 genes were cloned and characterized from N.barkeri.The sequence analysis showed four HSP70 genes had three classical HSP70 protein signatures,one ATP/GTP-binding site and two deduced bipartite nuclear localization signal(NLS).Phylogenetic tree of HSP70 s was divided into two main branches: HSP70 in Arachnida and HSP70 in Insecta.All four Nb HSP70 s were all clustered into the Arachnida group.The m RNA expression levels of four HSP70 genes under developmental expression patterns showed the most expression in adults and least in eggs and larvae.Besides,the expression levels between two strains showed significant difference.Most of the HSP70 s genes showed more dramatic changes in m RNA expression at different developmental stages in HTAS.At42°C,four HSP70 genes in two strains increased rapidly upon exposure to high temperature within a short time;meanwhile the expression of Nb HSP70-1 and Nb HSP70-2 in CS sharply decreased after 4h displaying a distinct contrast with the remained elevated expression in HTAS.Furthermore,HSP70 suppression by RNAi knockdown had a greater influence on the survival of HTAS,causing a higher mortality under high temperature than CS.In the Western blot analysis,the Nb HSP70-1 was significantly induced in HTAS by heat stress and increased with exposure time.The protein expression levels of CS N.barkeri were dramatically elevated at 0.5 h and down-regulated at 6 h,while in HTAS N.barkeri,Nb HSP70-1 protein was increased and peaked at 6 h.4 Prokaryotic expression and characterization of recombinant HSP70 s and MAPK pathway genes of N.barkeriBased on prokaryotic expression method,the recombinant expression vectors of three HSP70 genes and two key genes of MAPK pathway of N.barkeri were successfully constructed by introducing Bam HI and Nhe I double restriction sites,and subsequently transferred to E.coli BL21 to induce the expression of the fusion proteins.The molecular masses of the fusion proteins were estimated by comparisons with a molecular mass the marker to be approximately 71.33,70.03,69.04,49.07 and 40.93 k Da for Nb HSP70-1,Nb HSP70-3,Nb HSP70-4,Nb JNK and Nbp38,respectively.The SDS-PAGE and Western-blot showed the five recombinant proteins were successfully induced.The growth curve of E.coli with recombinant proteins under high temperature stress was measured.The results showed when treated with heat shock at 50°C,the growth rate of recombinant proteins cells increased significantly,surpassing even the growth rate of recombinant proteins cells without heat treatment,whereas the growth rate of control cells was arrested,which indicated the expression of recombinant proteins could greatly improve the heat resistance of E.coli.The cell viability of E.coli treated by the high temperature of 50 °C was determined through counting the number of colony forming units after heat shock.After 30 min,the survival rate of E.coli with empty p ET28 a vectors decreased to 20%,approximately 43% lower than for that containing Nb HSP70-1.The viability of Nb HSP70-1 cells was reduced to 18% while control cells were almost unable to provide any protection with 5% viability for 90 min.These results indicate the capability of Nb HSP70-1 expression in providing thermotolerance capability for cells in vivo.In conclusion,the comparisons of transcriptomics and proteomics were conducted to analyze the differences at transcriptome and protein translation levels between CS and HTAS strains of N.barkeri,which made clear the high temperature adaptability strategy of HTAS.Furthermore,the key genes and pathways including heat shock protein 70 and MAPK signaling pathway genes were identified and their thermal functions were analyzed.The results of this study illustrated the mechanism of high temperature adaptability in HTAS of N.barkeri under high temperature at the molecular level,providing a theoretical basis for the adaptation and improvement of predatory mite products under environment stress and technical supports for the efficient utilization of predatory mites to control high-temperature living pests(mites).