| Insect body cavity was immersed in hemolymph,which is not only the central place of nourishment and metabolism,but also the main place to eliminate the necrotic tissue,cell debris and foreign invader.As a classic lepidopteran model and an important economical insect,silkworm is a good model to study the function of hemocytes and hematopoiesis in insect.According to the traditional morphologic classification,silkworm hemocytes was divided into five different types,namely prohemocytes,granulocytes,plasmatocytes,oenocytoid,and spherulocyte.Lacking of the research tool,the research on the development and regulation mechanism of silkworm hemocytes is still at the early stage of exploration.In the present study,RNA-seq technology was used to analyze the gene expression difference in different hematopoietic lineages,and hemocyte-specific molecular markers were screened and confirmed.To explore the function of Integrin ?2 and Integrin ?3 in silkworm hemcytes,CRISP/Cas9 genome-editing technology were used in this study.Furthermore,hemocyte-specific genes were screened,and their promoter activities in vivo were detected by transgenic technology.Finally,the roles of MAPK and PI3 K signaling pathways in silkworm hematopoiesis were investigated using activators and inhibitors of corresponding signal pathways.The main results are as follows: 1.Expression Difference of larva silkworm granulocytes and plasmatocytesSilkworm larval granulocytes and plasmatocytes were sucessfully sorted,and the expression differences were analysed through transcriptome sequencing.A total of 10126 unigenes were identified,and there were have 831 differentially expressed genes between larva granulocytes and plasmatocytes.Among of them,there were 420 and 411 genes highly expressed in granulocytes and plasmatocytes,respectively.GO analysis showed that these genes mainly involved in binding,catalytic activity,metabolic process,single-organism process,cellular process,membrane composition as well as response to stimulus and biological regulation.KEGG analysis showed that the differentially expressed genes mainly related to the formation of the dorsal ventral axis,alpha linolenic acid metabolism,cell ECM receptor interaction,triglyceride metabolism,nicotinic acid and nicotinamide metabolism etc.Analysis of the metabolic pathways revealed that granuocytes and the plasmatocytes have different metabolic patterns.The metabolism of amino acids and fats were more vigorous in granuocytes,while the metabolism of nucleic acids and carbohydrates is stronger in plasmatocytes.Analysis of the immune related genes found that the plasmatocytes have higher activities in antibacterial peptides,serine protease cascade melanization,peroxidase,chitinase and superoxide dismutase.While the granunocytes have higher levels of cysteine protease and scavenger receptor.These results indicated that the plasmatocytes and granunocytes have different roles in the immune response2.Screening and identification of specific molecular markers in silkworm hemocytesThe quantitative real-time PCR were used to verify the results from the transcriptome analysis.Some of differentially expressed genes were chosen in the following studies.Our results suggested that Integrin ?2 and Integrin ?3 were mainly expressed in silkworm larva plasmatocytes,BmSCRB8 and BmSR-C were mainly expressed in granulocytes.Integrin ?1 was expressed in both hemocyte typies,and it seems that the expression level in plasmatocytes was a little higher.Subsequently,these candidate genes were cloned successfully.Through prokaryotic expression and protein purification,recombinant proteins were acquired and antibodies were obtained by immunizing animals.Immunofluorescence showed that anti-Integrin ?1 could label all detected hemocyte types,anti-Integrin ?2 and anti-Integrin ?3 could specific label plasmatocytes,anti-BmSCRB8 could label the granulocytes and oenocytoid.BmSR-C was expressed in oenocytoid and part of granulocytes.3.Molecular markers and the source of silkworm plasmatocytes(1)plasmatocyte-specific gene sequence analysisFull-length cDNA of Integrin ?2 and Integrin ?3 were acquired by PCR and RACE technology.These two genes are clustered on nscaf2847,which is located on chromosome 4,and the transcription directions of those gnees are opposite.The genomic DNA total length of Integrin ?2 is 6311 bp,composed of 25 exons and 24 introns.The full-length cDNA is 2895 bp,which contian a 118 bp,72 bp,and 2244 bp of 5 ' UTR,3' UTR,and CDS,respectively.Integrin ?2 enconding a 747 aa protein,the predicted molecular weight is 84.42 kDa,and isoelectric point is 5.349.The DNA total full-length of Integrin ?3 is 10086 bp,composed of 7 exons and 6 introns.The full-length of cNDA is 2653 bp,which contian a 122 bp,410 bp,and 2172 bp of 5 ' UTR,3' UTR,and CDS,respectively.Integrin ?2 enconding 723 aa,molecular weight is 81.79 kDa,and isoelectric point is 5.22.Both of those two integrin ? subunits own conserved structural features of integrin family,all of them contain a a long extracellular domains,a single transmembrane domain and a short cytoplasmic domain.(2)The expression of integrin ?2 and integrin ?3 in silkwormThe expression of integrin ?2 and integrin ?3 were analysed by qRT-PCR.Results suggested that those two ? integrin subunits were highly expressed in hematopoietic system,lincluding in circulating hemocytes and hematopoietic organ,and no detection in other detected tissues.The expression of integrin ?2 and integrin ?3 in different hemocytes stages were also detected by qRT-PCR,and those two genes have a similar tendency.From the L4 M stage,expression levels of those two genes were gradually increased,until the L5D6 its expression levels reached the highest,then gradually decreased and up-regulated at PP2 stage.Through prokaryotic expression and protein purification,recombinant protein of integrin ?2 and integrin ?3 were obtained,and polyclonal antibodies were generated by animal immunization.Western blot results shown that those antibodies could specific recognition corresponding antigens.Immunofluorescence assay showed that anti-Integrin?2 and anti-integrin ?3 both can specifically recognize the plasmatocytes,and the fluorescence signal mainly distributed on the cell membrane.Hemocytes from A3-EGFP transgene strain was used to further confirm the results,and it was found that the signal of anti-Integrin?2 was coincidence with the EGFP signal.(3)The source of plasmatocytesQRT-PCR and western blot were used to detected the expression of Integrin ?2 and Integrin ?3 during the period of embryo development.Results shown that those two plasmatocyte-specific markers expressed from 7th day of embryo stage and maintained high expression levels untill the end of embryo stage.It indicated that plasmatocytes began to emerge from 7th day of embryo stage and maintained at a a certain number during the end of embryo stage.Using specific molecular markers and EDU proliferation marker,the development of larva plasmatocytes was studied.Statistics shown the proportion of plasmatocytes reached to the minimum peak,and gradually increased during the 5th instar larva.The proportion of plasmatocytes up to 30% at L5D6,and then gradually decreased.At the 4th molting and metamorphosis development stages,proliferation ability of the total hemocytes remained at a low level,and in the food supplement period,hemocytes has a high proliferation rate.Similar changes in proliferation of plasmatocytes was also found in different larval stages.These results indicated that the proliferative ability of hemocytes,including plasmatocytes,may be related to nutritional status.Hematopoietic organ removal in vivo and HPO cultured in vitro experiment suggested that the circulating plasmatocytes was mainly produced by hematopoietic organ.More importantly,our results shown that the newly discharged plasmatocytes released from HPO has a very strong proliferative ability,but decreased quickly with the release to the circulatory system.EDU label retaining indicated that the plasmatocytes could be markered long time,it confirmed that the proliferative capacity of circulating plasmatocytes is limited.4.Function study of Integrin ?2 and Integrin ?3 in hemocytesWhen the health silkworm larval were injected with different typies of microbial pathogens and pathogen associated molecular patterns,the expression of Integrin ?2 and Integrin ?3 were upregulated significantly,especially when treated with Staphylococcus aureus and PGN.Recombinant protein of Integrin ?2 and Integrin ?3 have strong agglutination activities to S.aureus in the presence of calcium in vitro.Furthermore,our results suggested that recombinant protein of Integrin ?2 and Integrin ?3 could bind to S.aureus,but not to E.coli and P.aeruginosa.ELISA results showed that both of those two recombinant protein could combine with PGN and LPS,but show stronger ability in combination with the former.The CRISP/Cas9 gene editing technology was used in the present study.Two transgenic strains,which could stable expression of Cas9 protein and guide RNA(gRNA)sequence,respectively,were acquired.Mutation strain of integrin ?2 was obtained after hybridization.Through sequencing screening and self-cross pdigree method,the homozygous mutant strain of integrin ?2 was acquired.Western blot confirmed that mutation strain could not detection the expression of integrin ?2 in protein level.Unfortunately,no obvious phenotypic changes were observed.The expression changes of all ? integrin subunits were investigated by qRT-PCR,we found that the expression levels of integrin ?1,integrin ?3 and integrin ?3 were significantly increased significantly in integrin ?2 mutation strain.Western blot and immunofluorescence assay confirmed that the expression level of integrin ?3 was increased.It was speculated that the hemocytes maybe upregulate the expression level of other integrin ? subunits,which have similar structure and function with integrin ?2,to maintain normal function of the hemocytes in silkworm.5.Identification and characterization of hemocyte-specific promoters(1)Identification and characterization of hemocyte-specific genes in silkwormSilkworm hemocyte-specific genes were analyzed using microarray data from SilkDB database.To demonstrate whether the genes obtained were hemocyte specific,expression profiles were further investigated in different tissues with qRT-PCR.Finally,four novel hemocyte-specific genes were obtained and conformed,namely,Integrin ?2,Integrin ?3,Cathepsin O,and sw04862,respectively.Integrin ?2 and Integrin ?3 belong to integrin family,which is a group of cell surface glycoprotein,it mediate cell to extracellular matrix(ECM)and cell-to-cell interactions as well as transduce the bidirectional transmembrane signal.Cathepsin O belong to cathpsin family,which are widely expressed in almost all organisms,function in intracellular protein degradation/turnover via catalyzation of protein hydrolysis,and play key roles in various physiological process.Sw04862 is a novel gene in silkworm,no homologous genes found in GeneBank.(2)Promoter cloning and activity analysisPromoters of the candidate genes were successful cloned and insert into modified recombinant baculovirus system.The recombinant virus was obtained by transfection of silkworm BmE celline,and then used to infect the normal silkworm larval.EGFP reporter gene expression patterns in hemocytes,silk gland and fat body were detected,our results shown that promoters of Integrin ?2,Integrin ?3,and Cathpsin O Could drive EGFP specifically expressed in hemocytes.However,promoter activity of sw04862 were detected in all detected tissues.(3)Construction of transgenic vectors and obtaining of positive individualsPromoter sequences of Integrin ?2 and Integrin ?3 were successful cloned into silkworm transgenic expression system,positive transgenic individuals were obtained after embryo microinjection and fluorescence screening.In these two transgenic strains,we found that the EGFP reporter gene were both expressed in hemopoietic system,including circulating hemocytes and hemopoietic organ,but not in other detected tissues.Furthermore,experimental evidences shown that EGFP only expressed in plasmatocytes,but not other hemocytes typies.In summary,the results of this study revealed that we haveidentified two novel plasmatocyte-specific promoters in silkworm that will not only great significance for better understanding of hemocyte-specific gene,but also has potential applications in insect hematopoiesis and innate immunity research.6.Study on the mechanism of hematopoietic regulation in silkwormHemopoietic organ could released hemocytes when cultured in vitro,and bovine insulin significantly promotes this process,and shown a certain concentration gradient dependent effect.Western blot results showed that bovine insulin can significantly activate Akt and Erk phosphorylation.Through inhibitor of MAPK and PI3 K signaling pathway,which are belong to the downstream regulatory pathways of insulin pathway,to study roles in silkworm hematopoiesis of those pathway.LY294002,which is a inhibitor of PI3 K pathway,could significant inhibitor silkworm hematopoiesis.However,MAPK pathway inhibitor U0126 significantly promoted the release of hemocytes.Furthermore,using U0126 to inhibit the activity of MAPK when activation of insulin pathway with bovine insulin,a significant hematopoietic promoting effect was observed.However,LY294002 could greatly inhibited the regulatory function of bovine insulin.These results suggest that MAPK and PI3 K pathways may play different roles in silkworm hematopoiesis.Further experiments shown that changes of relative activitities of MAPK and PI3 K pathways could dynamically regulate hemocytes release from hemopoietic organ.In conclusion,we hypothesized that insulin pathway,which is an important factor in silkworm hematopoiesis,regulate the activities of MAPK and PI3 K signal pathways to control silkworm hematopoiesis.Activation of PI3 K signal pathway could promote hematopoiesis,in order to prevent excessive hematopoiesis,insulin activates MAPK pathway simultaneously,which have a negative regulatory capacity of silkworm hematopoiesis. |
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