| Insect immune response is related to its development alstages,andinsectshave different ability to resist pathogen infection in different developmental stages.In order to explore the interation between immune response and developmentin insect pests,in this study,the transcriptomes of different developmental stagesfrom Plutella xylostella,including 4th larva,pupae,and adults,were sequenced,andbioinformatics used to analyze the differentially expressed genes and alternative splicing at different development stages in P.xyllostella,the mutual regulators and differential expressed genes between immunity and development of P.xylostella were identified,the results provided a molecular network to explore the interaction between pest immune response and development.C-type lectins(CTLs),a kind of important pattern recognition receptors in insects immune system,mediated the signal pathway by binding pathogenic microorganisms specifically,and play an important role in the immune system and development.The full-length sequence of Px CTL5 gene was cloned by RT-PCR and RACE.Based on this,the prokaryotic expression of PxCTL5 was carried out in vitro and the polyclonal antibody was prepared.At mRN A and protein level,the expression patterns of PxCTL5 were identified by q RT-PCR and western blot after different bacteria induced,respectively.With the exits of calcium ion,the agglutination of rPxC TL5 on different bacteria was observed.The results of this study provide a theoreticalmolecular basis for studying on the mutual regulation of immunity and development in pests.In order to investigate the interation between immune response and development,nine transcriptomes were obtained from three developmentalstagesof P.xylostella by high throughput sequencing,including 4th instar larva,pupaeand adults.For each developmental stage of P.xylostella,the average clean reads were 16,266,300,16,597,631 and 16,275,979,the average bases were 2,033,287,500 nt,2,094,632,500 nt and 2,034,497,417 nt,respectively,and total bases were around 2G.From each developmental stage of P.xylostella respectively,the new transcripts were 1131,1474 and 1361,and the number of alternative splicing were 8854,14075 and 11793,and the SNP site were 105197,160897 and 133363.The transcriptomes of the three developmental stages of P.xylostella were compared between each every two groups and the differential expressed genes were identified by qualitative analysis.Comparing the DGEs(digital gene expression)from 4th instar larvae to adult,456 differential genes were up-regulated,223 differential genes were down-regulated.Frompupa to adult,449 differential genes were up-regulated,334 differential genes were down-regulated.From pupa to 4th instar larvae,160 differential genes were up-regulated,365 differential genes were down-regulated.Among these differential expressed genes,there are 42 immune related genes,including the pattern recognition receptor,thegenesfrom Toll and Imd pathway and the downstream effect factors,there are 40 genes developmentrelated,including hormone genes,P450,caspase and bHLH(basic helix-loop-helix Per/Arnt/Sim).Based on bioinformatics analysis of transcriptomics database from P.xylostella,a full-length cDNA of C-type lectin gene(designated as Px CTL5,Genbank No.: KY807084)was obtained by RT-PCR and RAC E technique.The full length c DNA sequence of Px CTL5 is 1243 bp,and the complete open reading frame is 672 bp,which encoding 223 amino acid,with a 19 amino acid signal peptide and 204 mature peptide,and the predicated molecular weight is 23.66 kDa,the isoelectric point is 6.24.PxC TL5 owns a 172 aa CRD domain between 34 and 205 aa which is specific to C-type lectin families,which containing a QPD(Glu-Pro-Asp)site.QRT-PCR analysis indicated that Px CTL5 was expressed in all developmental stages,with the highest expression level in the adult stage.The expression of Px CTL5 gene in hemocyte,epidermis and midgut could be induced by Gram-positive bacteria Bacillus thuringiensis at mRNA level,and reached the peak at 42 h,18h and 24 h respectively.The expression of Px CTL5 gene was significantly up-regulated after stimulation with Gram-negative bacteria Escherichia coli in hemocyte,epidermis and malpighian tubule,and reached the peak at 30 h,24 h and 18 h,respectively.In order to further elucidate the function of PxCTL5 in P.xylostella,the prokaryotic expression vector p GEX-4T-1-PxCTL5 was constructed in vitro and the soluble fusion protein with 49 k Da was expressed by the induction of IPTG and purified by affinity column chromatography,and the polyclonal antibody(anti-PxCTL5)was successfully prepared.The hemolymph of 4th larvae of P.xylostella treated by different bacteria were collected and detected by anti-PxCTL5,the results showed that the expression of PxCTL5 is significantly induced by B.thuringiensis and E.coli,and the expression patterns induced by different bacteria are defirrent.The agglutination assay showed that purified fusion protein rPxC TL5 had high activity of agglutination on gram-negative and positive bacteria,in comparition,agglutination effect on B.thuringiensis is stronger.In the present study,through the transcriptome sequencing,the mutual regulation between immune response and development of P.xylostella were preliminaryly studied,and the immune and development related genes were also identified.The full-length c DNA encoding Px CTL5 was obtained by RT-PCR,and the spatial and temporal expression was analyzed by q RT-PCR in P.xylostella after gram-negative and gram-positive bacteria induction.The agglutination of PxCTL5 on bacteria was confirmed in the presence of calcium ion.The results of this study provide a molecular basis for elucidating the interation between the immune system and development of P.xylostella,and also provide a theoretical basis for clarifying the function of PxC TL5 in pest immune defense against pathogenic bacterial infection. |
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