Study On The Innate Immune Function Of Genes In Toll And HMGB Family Of Portunus Trituberculatus

The swimming crab,Portunus trituberculatus is a kind of important Marine economic animals that distributed across China,Korea,Japan and other areas,particularly widespread in Bohai,Yellow Sea and East China sea of China.With the expansion of the marine farming,disease has become a major bottleneck of the development of aquaculture.Vibrio alginolyticus,white spot syndrome virus(WSSV)and Vibrio Parahemolyticus can cause pathological changes in P.trituberculatus.In addition,as a kind of important environment factor,water salinity has great influence on the immunologic function of P.trituberculatus.As P.trituberculatus does not have acquired immune system,it can largely rely on the innate immunity to induce rapid and effective immune responses to clear the intruding pathogens.In this study,we used V.alginolytica to infect P.trituberculatus and extracted total RNA from the hemocytes to establish a transcriptome library and selected the immune-related genes from this library.In addition,Using V.parahaemolyticus and WSSV infected P.trituberculatus and experimented in low salinity.we researched the function of the innate immune function of genes in Toll and HMGB Family of P.trituberculatus under V.parahaemolyticus,WSSV and low salinity stresses.This study will improve the theory of innate immune defense mechanism and provid the strategy about better disease management effectively.The main research contents and results are as follows:1.Transcriptomic analysis of P.trituberculatus hemocytes upon V.alginolytica infection using RNA-SeqV.alginolytica is one of the pathogen can caused diseases in the crustacean farming industry,and would proliferate in the host body,that can damage the cells and tissues of the crustacean.In this study,we used V.alginolytica to infect P.trituberculatus and extracted total RNA in 0h and 12 h from the hemocytes to establish a transcriptome library.A total of 75576 unigenes were generated.Of these unigenes,63515 unigenes were annotated in at least one database.Gene ontology(GO)analysis revealed diverse biological functions including metabolic process,cellular process,single-organism process,biological regulation,and immune system process.KEGG analysis of unigenes identified putative members of biological pathways related to signal transduction,Endocrine system,Translation,Transport and catabolism,and immunity.Comparing the infection group to the control group,a total of 791 significantly differentially expressed genes(DEGs)were identified,including 453 up-regulated DEGs and 338 down-regulated DEGs.The Toll-like receptor gene and the HMGB family gene were identified as the immune gene of this study by analyzing GO,KEGG and DEGs.In addition,After high-throughput sequencing,clustering,splicing,and annotation,we harvested plenty of c DNA sequences of P.trituberculatus.It could help to further explore its pathogenic mechanism and provide the basic data for the disease defense and histological analysis of P.trituberculatus.2.Cloning of Tolls in P.trituberculatus and its expression in responding to pathogenic infection and low salinity stressAs an economically important food species in China,Portunus trituberculatus lacks adaptive immunity but can induce effective immune responses to resist pathogen relying on innate immunity.Toll-like receptor is the greatest found of the innate immune system.As a pattern recognition receptors(PRRs),it plays a crucial role in innate immune responses.Two novel Toll genes were found in P.trituberculatus by rapid amplification of c DNA ends(RACE)technique,which named as Pt Toll4 and Pt Toll5,respectively.The full-length c DNA of Pt Toll4 was 4276 bp in length,which contains an open reading frame(ORF)of 2685 bp,a 3' untranslated region(UTR)of 1252 bp and a 5' UTR of 339 bp.The ORF encoded a polypeptide of 895 amino acids with a predicted molecular weight of 102.5 k Da.The full-length c DNA of Pt Toll5 was 4914 bp in length,which contains an open reading frame(ORF)of 4083 bp,a 3' untranslated region(UTR)of 681 bp and a 5' UTR of 150 bp.The ORF encoded a polypeptide of 1361 amino acids with a predicted molecular weight of 151.6 k Da.PtToll4 and PtToll5 are transmembrane proteins that contain several common structural domains of TLRs,such as LRR,LRRCT,LRRNT motif in the extracellular domain and a TIR domain in the intracellular region.The BLAST analysis showed that Pt Toll4 shared the highest homology with Eriocheir sinensis Toll2,Pt Toll5 shared the highest homology with Macrobrachium rosenbergii Toll.Pt Toll4 and Pt Toll5 were widely expressed in many tissues,such us hemocyte,heart,gill,hepatopancreas,stomach,intestine,muscle,cuticle,eyes.Pt Toll4 mainly distributed in the hemocyte and cuticle,A weak expression was detected in the hepatopancreas.Pt Toll5 mainly distributed in the eyes,A weak expression was detected in the hemocyte.Quantitative Real-time PCR analysis showed that Pt Toll4 transcripts were modulated in hemocyte after WSSV inflection,with significant up-regulation at 6 h(P<0.05).However,there was no significant change after V.Parahemolyticus infection except 48 h.Pt Toll5 transcripts were significant up-regulation(P<0.05).Meanwhile,compared with the control group,Pt Toll4 and Pt Toll5 transcripts were decreased significantly in low salinity experimental groups.Our results suggested that Pt Toll4 and Pt Toll5 may play important roles in innate immune of P.trituberculatus and would provide useful information for the research of immunity regulation in crustaceans.3.Gene cloning and expression analysis of HMGB from P.trituberculatusV.Parahemolyticus and WSSV have caused great economic damage to P.trituberculatus aquaculture,and the sustainable development of the crab aquaculture industry is under serious threat.High mobility group box(HMGB)proteins are known to be involved in diverse functions in mammalian cells.In crustacean,very limited studies on HMGB proteins have been documented.To investigate the role of high mobility group box(HMGB)proteins in innate immune of Portunus trituberculatus,two HMGB homologue c DNAs from P.trituberculatus,named Pt HMGBa and Pt HMGBb,respectively,were first cloned using RACE technique in our study.The full-length c DNA of Pt HMGBa was 1030 bp in length,which contains an open reading frame(ORF)of 681 bp,a 255 bp 3' untranslated region and a 94 bp 5' untranslated region.The full-length c DNA of Pt HMGBb was 981 bp in length,which contains an open reading frame(ORF)of 618 bp,a 239 bp 3' untranslated region and a 61 bp 5' untranslated region.The ORF of Pt HMGBa encoded a polypeptide of 227 amino acids with a predicted molecular weight of 25.82 k Da.The ORF of Pt HMGBb encoded a polypeptide of 206 amino acids with a predicted molecular weight of 24.07 k Da.Their polypeptide containd two HMG boxes domain and a C-terminal acidic tail composed of 24 rich Asp/Glu residues.The BLAST analysis showed that Pt HMGBa and Pt HMGBb shared highest homology with Lv HMCBa and Lv HMCBb from Litopenaeus vannamei respectively.The expressions of Pt HMGBa and Pt HMGBb were mainly distributed in the hemocyte.A weak expression of Pt HMGBa and Pt HMGBb were detected in the eyestalk and intestines,respectively.Quantitative Real-time PCR analysis showed that Pt HMGBa transcripts were significantly up-regulated in hemocyte at 6 h post V.Parahemolyticus inflection(P<0.05).Meanwhile,Pt HMGBa transcripts were significantly up-regulated in hemocyte at 12 h post WSSV inflection(P<0.05).Pt HMGBb transcripts were significantly up-regulated in hemocyte at 24 h post V.Parahemolyticus inflection and transcripts were significantly up-regulated in hemocyte at 12 h post WSSV inflection(P<0.05).Meanwhile,compared with the control group,Pt HMGBa and Pt HMGBb transcripts were decreased significantly in low salinity experimental groups.Our results suggested that Pt HMGBa and Pt HMGBb may play important roles in innate immune of the swimming crab,and would provide useful information for the research of immunity regulation in P.trituberculatus and other crustaceans.