| The desert beetle,Microdera punctipennis(Coleoptera:Tenebrionidae),is an endemic species in the Gurbantonggut Desert in Xinjiang.The climate in this region is harsh,and the temperature difference between day and nights,and the seasonal change varies greatly.M.punctipennis has evolved a range of physiological and molecular adaptations to survive the extreme temperature variation in the desert environment.Its mechanism in cold hardiness is representative in the desert insects in northern Xinjiang.Therefore,the study of the cold tolerance of M.punctipennis helps to understand the adaptive mechanism of insects to the extreme desert environment.Under low temperature conditions,insects not only cope with the stress of low temperature,but also face the stress of microbial pathogens due to reduced cellular metabolism.Insect success at low temperatures is shaped in part by interactions with biotic stressors,such as pathogens,thus it is important to understand how and why immunity might be activated by cold.At present,research on this related aspect is limited.M.punctipennis adults survived for nearly 5 months in winter,and the transcriptome sequencing analysis showed that cold exposure activated the expression of various immune response genes in M.punctipennis.However,the functional significance of the relationship between cold and immunity is unclear.The aim of this study was to answer from the transcriptional level which genes of the immune system of M.punctipennis are activated,and whether those components differ among different kinds of cold exposure,ie how the cold activates immunity in M.punctipennis.In this dissertation,we analyzed the transcriptome data sets of short term low temperature stressed and overwintering M.punctipennis adults,and annotated the immune-related genes of M.punctipennis.According to the screening results of immune genes,preliminary drew the M.punctipennis immune pathway.We compared the differences of immune gene expression levels in response to low temperatures in laboratory conditions and the natural environment,including pattern recognition receptors,immune signal transduction and regulatory factors and immune effectors.In addition,In addition,the gene Mpattacin,which is significantly up-regulated after cold induction,was cloned and expressed.The main conclusions were as follows:(1)Immune gene discovery in the low temperature transcriptome of M.punctipennis:M.punctipennis transcriptome library was successfully constructed,which contain 198206unigenes.A total of 49034 genes were annotated,accounting for 24.74%of the total unigenes of the transcriptome.1,750 homologous unigenes of 107 immunerelated genes were obtained from the M.punctipennis transcriptome by annotation information and manual screening.Through the analysis of immune related genes,identified the main pattern recognition receptors,signal transduction factors and immune effect factors exist in insects.Based on 1:1orthologous relationships in most cases,the Toll,MAPK-JNK-p38 and JAK-STAT pathways were thought to intact and operative in the desert beetle M.punctipennis,as are most of the regulatory mechanisms.Similarly,cellular processes such as autophagy,apoptosis and RNA interference probably function in similar ways,because their modulators are mostly conserved in this species.A large number of genes were associated with immune effect factors,including genes that encode antimicrobial peptide proteins and heat shock proteins,and enzymes such as lysozyme,chitinase,phenoloxidase and antioxidase.But IMD,FADD and domeless gene were not identified in M.punctipennis.According to the screening results of immune genes,preliminary drew the M.punctipennis immune pathway.Such information provided a genomic perspective of the intricate signaling system in a non-drosophiline insect,and provided a basis for the subsequent analysis of the effects of cold on the immune response of M.punctipennis.(2)Transcriptional analysis of the effects of cold on the immune response of M.punctipennis:This study compared the transcriptional changes of pattern recognition receptors,immune signal transduction and regulatory factors,and immune effectors in individuals between the overwintering adults and the adults that were kept in laboratory at4?and-4?.The results showed that the beetle's immune system is differently modulated by cold induced in laboratory settings than that which occurs in natural conditions.Overwinter caused complex transcriptional activation of innate immunity potential:The pattern recognition receptors,modulators,Toll pathway and JNK pathway and autophagy were activated,IMD pathway and JAK-STAT pathway and apoptosis were inhibited,and immune effector genes antibacterial peptide,lysozyme,phenol oxidase,heat shock protein,chitinase,and antioxidant enzyme were significantly up-regulated during winter.The different transcriptomic response to 4?low temperatures was instead dominated by upregulation of IMD pathway and the JAK-STAT pathway and RNAi,and downregulation of the phenol oxidase.Differences in response to low temperatures in laboratory conditions and the natural environment suggested that the simultaneous presence of other stress factors during winter such as desiccation and starvation may have a significant influence on the activity of beetle's immune system.The results provided a basis for further testing the effects of cold on the immune system of M.punctipennis from physiological and biochemical levels.(3)Identification and validation of reference genes for gene expression analysis in M.punctipennis using RT-qPCR:According to the results of M.punctipennis transcriptome data,elongation factor1?(EF1?),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),?-actin(ACTB),18S ribosomal RNA(18s rRNA)and?-TUBulin(TUB)were selected and their expression stability was detected under different treatment conditions(including development duration,tissues,and mixed samples under temperature stress),by using real-time fluorescence quantitative PCR.The stability of the 5 candidate genes was evaluated by RefFinder,BestKeeper,GeNorm,NormFinder and?C_t.EF1?,GAPDH and ACTB were highly stable in different development durations.EF1?was highly stable in different tissues.EF1?and TUB were relatively stable under temperature stress.EF1??TUB and ACTB were stable in mixed samples of M.punctipennis.EF1?and TUB were used as reference genes,the relative expression of 10 genes of beetle M.punctipennis in low temperature treated samples were detected by RT-qPCR.The results showed that the expression was consistent with the low temperature transcriptome data,and provided a reference for the selection of suitable reference genes in different experimental conditions,which is beneficial to obtain more reliable and accurate data in M.punctipennis gene expression research.(4)Cloning and expression of two cold stress related genes MpAtt1 and MpAtt2:Characterizing two attacins,MpAtt1 and MpAtt2,from the desert beetle M.punctipennis and investigating the expression profiles of the two genes in response to cold stress.These newly identified Mpattacins showed characteristics different from those previously reported in other insects,and they showed 28.1%identity at the amino acid level,and 38.3%identity at the nucleotide level.The secondary structure of the two proteins mainly included random coil and?-sheet.Tertiary structures were highly conservative with Attacin_C domain and Gloverin domain.It was predicted that the two proteins were soluble and extracellular protein,and there were amount of potential phosphorylation sites.These proteins belonged to Attacin-C superfamilies,and there was a signal peptide,respectively.Phylogenetic tree showed that the Mpattacins and the other Coleoptera attacins diverge earlier than Diptera and Lepidoptera attacins in evolution.The prokaryotic expression purified fusion proteins MpAtt1 and MpAtt2 showed antibacterial activity against Escherichia coli and Staphylococcus aureus,but no antibacterial activity against Candida albicans.The results of RT-qPCR showed that MpAtt1 and MpAtt2 are highly expressed in hindgut plus Malpighian tube,and MpAtt1 also highly expressed in fat body.The mRNA level of MpAtt1 and MpAtt2 were increased when the insects were treated at 4?and-4?,but the responsiveness to-4?was lower than to4?.From the aspect of antimicrobial peptide synthesis,the cold inducible expression of the attacin genes from the desert beetle M.punctipennis supports the hypothesis that the insect immune system is stimulated during cold exposure.In conclusion,the immune signaling pathway of M.punctipennis constructed was different from that of model organisms.Low temperature could affect the immune signaling pathway of M.punctipennis and induced the change of the immune system of M.punctipennis at transcriptional level.The transcriptional activation mechanism of the immune pathway of of M.punctipennis needed to be further verified at physiological level.Attacin protein,which was expressed in large quantities at low temperature,could play a role in the cold tolerance mechanism of M.punctipennis.The results may help to find some new multifunctional proteins of M.punctipennis. |
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