| Sea cucumber,also known as Apositichopus japonicas,belongs to Echinodermata,Holothuroidea.It is one of the most important breeding species in Northern Huanghai and Bohai area.However,due to the deterioration of the marine environment,sea cucumber is very susceptible to some bacteria and viruses.For example,bacterial infection causes sea cucumber's skin rot syndrome,mouth swelling,autolysis and even death.A large number of economic losses have been caused to the farmers,which seriously restricts the healthy development of the aquaculture industry.As invertebrates,sea cucumber is lack of acquired immune system and relies on innate immunity against pathogens.Therefore,studies on immune related genes and immune defense mechanism of sea cucumber are very important for the prevention and healthy breeding of sea cucumber.In the present study,A.japonicas was used as the experimental material,AjFucolectin-1(AjFL-1)and AjCystain genes were used to clone gene,expression and immunological function verification by using the techniques of molecular biology,bioinformatics,and immunology.Firstly,a fucolectin was identified from sea cucumber A.japonicus(designated AjFL-1).The open reading frame(ORF)of AjFL-1 was of 546 bp,encoding a polypeptide of 181 amino acids with a predicted molecular mass of about 20 kDa.The deduced amino acid sequence of Aj FL-1 shared 30%-40% similarity with those fucolectins from vertebrates and invertebrates.There were a typical F-type lectin domain(FLD)(residues 39-180)and a signal peptide(residues 1-24)in AjFL-1.The mRNA transcript of Aj FL-1 could be detected by qRT-PCR in various tissues,such as intestinum,coelomocytes,respiratory tree,tentacle,and body wall,while undetectable in the gonads and longitudinal muscle.The mRNA expression level of AjFL-1 in coelomocytes was significantly up-regulated(47.06-fold to that in control group,p < 0.05)at 12 h after Vibrio splendidus challenge.Immunofluorescence assay showed that AjFL-1 protein was mainly distributed on the membrane of coelomocytes in sea cucumber.The recombinant AjFL-1(r AjFL-1)could bind Lipopolysaccharide(LPS),Peptidoglycan(PGN),Mannan(MAN)and Fucose(FUC),and exhibited a broader binding activities towards Gram-negative bacterium Escherichia coli,Gram-positive bacterium Micrococcus luteus,as well fungi Pichia pastoris.In addition,rAjFL-1 could strongly agglutinate fungi P.pastoris.These results indicated that AjFL-1 was a novel member of fucose-binding lectin family,which functioned as a pattern recongnization receptor with broad spectrum of microbial recognition,and involved in innate immune response of sea cucumber.Secondly,the AjCystain was cloned and analyzed in A.japonicus.The ORF of AjCystain is 348 bp in length and encodes a protein of 115 amino acids with a predicted molecular mass of about 14 kDa.There is a high homology with the Family1 of cystatin,and has conserved functional site with Cystain.Under normal physiological conditions,the mRNA transcripts of AjCystain could be detected in the tissues of intestinum,coelomocytes,respiratory tree,tentacle,body wall,with the highest level in body wall the lowest level in intestinum.The mRNA expression level of AjCystain in body wall was significantly up-regulated(30.20-fold to that in control group,p < 0.05)at 12 h after V.splendidus challenge.The immunohistochemistry showed that the Aj Cystain protein was mainly distributed in body wall and intestinum.The above results showed that AjCystain may regulate the response of sea cucumber to the immune response. |
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