Expressed Sequence Tags Analysis And Immune-Related Genes Cloning,Expression Pattern In Chinese Mitten Crab (Eriocheir Sinensis)

The freshwater Chinese mitten crab,Eriocheir sinensis,is an economically important species cultured in China in the last few decades.With the development of intensive aquaculture,various diseases caused by bacteria,viruses and rickettsia-like organisms have emerged,and started to threaten the sustainability of the aquacultured populations of the mitten crab,severely affecting its production.In the present study, we presented the methodology and results of EST analysis,targeting immune-related genes in the Chinese mitten crab and conducted experiments to further clone the immune-related gene by reverse transcript-polymerase chain reaction(RT-PCR)and rapid amplification of cDNA ends(RACE)methods from haemocytes of Chinese mitten crab,compare its sequence with other related proteins.Based on the expression of the immune-related gene in various tissues,we evaluated the immune response by Real-time quantitative reverse transcription-PCR(qRT-PCR)when the E.sinensis were injected with a gram-negative bacterium,Aeromonas hydrophila.The identification and isolation of immune-related genes in crab will improve our understanding of crustacean immunity and lead to the development of new therapeutics or bacterin.1 To identify distinctive genes associated with immunity in the Chinese mitten crab(E.sinensis),an expressed sequence tag(EST)library was constructed from haemocytes of this economically important species.3,118 clones were unidirectionally sequenced and analyzed by homology searches against sequences in the GenBank,KEGG and Uniprot.Significant homology to known genes was found in 488 of the 1,049 unique sequences.The automatic functional classification based on KEGG and Gene Ontology revealed 26 putative immune-related genes.These genes coded for enzymes and proteins in the clotting and prophenoloxidase-activating system,antioxidative enzymes,antimicrobial peptides,and pattern recognition molecules.The existence of these molecular processes in the activation of cellular defense in crab has not been previously reported.According to EST abundance,the major immune-related genes were the Kazal-type proteinase inhibitor and the C-type lectin.The EST sequences of mitten crab haemocytes provide important information for understanding the evolution of the immune system among crustaceans in general.2 The lipopolysaccharide andβ-1,3-glucan binding protein(LGBP)is a pattern recognition protein which is fundamental for the innate immune response of crustaceans.The full-length cDNA of LGBP gene cloned from E.sinensis LGBP gene was 1,236 bases long and was capable of encoding a polypeptide of 362 amino acids showing significant similarity to homologous genes in shrimp.The crab LGBP deduced amino acid sequence carrying conserved features of this gene family including a potential recognition motif forβ-1,3 linkages of polysaccharides and putative RGD(Arg-Gly-Asp)cell adhesion sites.LGBP gene mRNA expression was detected in haemocytes,hepatopancreas,muscles,gills,stomachs,and intestines with the highest level in haemocytes and the lowest in the stomach.The LGBP gene expression is up-regulated upon bacterial infection and the binding of lipopolysaccharide andβ-1,3-glucan to LGBP could induce a series of immune reactions.The temporal expression of the LGBP gene after having bacterial challenge was up-regulated at 1.5 h post-injection of bacteria followed by a step by step recovery at 12 and 24 h.Our data suggest that the crab LGBP is an inducible acute-phase protein that could be critical in crab immunity.3 The proteolytic activation of prophenoloxidase(proPO)played a critical role of resisting pathogen invading in crustaceans.Analysis of the nucleotide sequence of E.sinensis PPAF genes c DNA revealed that the PPAF gene cDNA consists of 1386 bp with an open reading frame(ORF)of 1,134 bp,a 154 bp 5'-untranslated region, and a 95 bp 3'-untranslated region containing a poly A signal.The open reading frame encoded a protein of 378 amino acids with 16 residues signal sequence.The E. sinensis PPAF amino acids sequence contained putative functional domains including a signal peptide region,cysteine residues forming the clip domain and conserved serine proteinase domain.Alignment of the deduced amino acid sequence to other species PPAF revealed that PPAF was highly similar to other PPAF from crustaceans with identities from 70%to 55%.The conserved domains and clip domains,and higher similarity with other PPAF or prophenoloxidase-activating protein(PAP) suggested that PPAF was a member of the serine proteinase homgones family.The mRNA transcripts of PPAF could be detected in all the examined tissues with the highest level in hepatopancreas and PPAF transcripts in haemocytes of E.sinensis increased significantly in 6,12 and 48 h post-Aeromonas hydrophila injection.4 In order to protect against oxidative stress,aerobic cells regulate excessive ROS by a group of antioxidant enzymes,such as superoxide dismutase(SOD), glutathione peroxidase(GPx),glutathione-S-transferase(GST).These three antioxidant enzymes were all cloned from the haemocytes of Chinese mitten crab.The full-length cDNA of SOD genes consists of 1,339 bp with a 867 bp open reading frame,encoding 288 amino acids.The deduced amino acid sequence contains a putative signature sequence of Mn-SOD(DVWHHAYY).Sequence comparison showed that the cMn-SOD of E.sinensis shares 90%,89%,87°%,84%and 81% identity with that of Litopenaeus vannamei,Penaeus monodon,Callinectes sapidus,Macrobrachium rosenbergii and Procambarus clarkia,respectively. cMn-SOD transcripts were detected in all the examined tissues with the highest level in haemocytes and the temporal expression of the SOD gene after bacterial challenge was up-regulated at 1.5 h post-injection of bacteria followed by a step by step recovery at 12 h,and increased the level same as the 1.5 h at 48 h post bacterial injection.cDNA encoding glutathione peroxidase(GPx)mRNA of E.sinensis was obtained from haemocytes.The l,101bp cDNA contained an open reading frame of 660 bp,a 94 bp 5'-untranslated region,and a 347 bp 3'-untranslated region containing the poly A tail.It contains no putative selenocysteine residue which is encoded by the unusual stop codon,TGA.The conserved GPx domain,AhpC domain and the signature of peroxidase catalytic center were also identified in the crab GPx.Comparison of the crab GPx gene amino acid sequences showed similarity of 81%to that of Scylla serrata,67%to that of Suberites domuncula and 66%to that of Hymeniacidon perlevis.The mRNA transcript of GPx could be detected in all the examined tissues with highest expression level in hepatopancreas.The expression level of GPx in haemocytes was down-regulated after bacterial challenge.As time progressed,the expression level began to increase but did not recover to the original level during the experiment.The full-length GST gene cDNA comprised of 958 bp,containing 85 bp in the 5'-UTR,651 bp in the ORF,222 bp in 3'-UTR with a poly(A)tail of 25 bp and a putative polyadenylation consensus signal(AATAAA)seventeen nucleotides upstream the polyA tail.The ORF encodes a polypeptide of 216 amino acids including a 17 amino acid signal peptide.Sequence comparison of the GST deduced amino acid showed similarity of 53%to that of Tribolium castaneum and Culex quinquefasciatus GST,52%to that of Anopheles dims and Culicoides variipennis GST,51%to that of Anopheles gambiae and Anopheles dirus GST.We also discovered that GST had a G-site(1-82 aa),which binds the tripeptide glutathione in the N-terminal region and an H-site(88-213 aa),which is a substrate binding site in the C-terminus.Additionally,a kinase C phosphorylation site(ITI)and one putative N-linked glycosylation sites(DNIT)for N-linked carbohydrate chains were identified suggesting that the GST is a glycoprotein.The mRNAs of GST was detected in haemocyte,hepatopancreas and muscle with different levels of expression while was not detected in gill,intestis and stomatch.mRNAs of the gene showed the highest expression levels in haemocytes.The temporal expression of the GST gene in the control and bacteria-challenged samples is shown that the expression of GST was up-regulated significantly(p＜0.05)at 6 h post-injection of bacteria,followed by a step by step recovery at 12 h.These results suggested antioxidant enzymes genes were involved in the response to bacterial infection and played functional importance role in the immune system of E.sinensis.