Construction Of SSH CDNA Library And Cloning, Expression Of Immune-related Gene Of Grass Carp Intestine

Construction of SSH cDNA library and Cloning,Expression of immune-related Gene of Grass carp intestineFish lives in water environment comparing with the others livings on land,large areas of mucosa of fish are the first barrier to pathogen infection.Therefore,the mucosal immune response plays an important role in the process of immune response of fish.A subtractive cDNA library of grass carp(Ctenopharyngodon idellus)was constructed by suppression subtractive hybridization(SSH)after perfusing Aeromonas hydrophila into grass carp cDNA clones were select form library randomly and partially sequenced.These sequences were analysed using BLASTX software and classified for the results.Moreover,two full-length cDNA of immune-related genes,major histocompatibility complexⅡ-α(MHCⅡ-α)and Alpha-1-microglobulin in/bulinin precursor protein(AMBP),were cloned and sequenced,and these sequences and expression of profile was analysed in different tissues of grass carp.The results provided a series of important information for further to research and explore immune mechanism of fish mucosa.The content of these studies and the main results are as followings:1.Subtracted cDNA library construction and sequence analysisA constructed subtracted cDNA library was constructed using suppression subtractive hybridization method between two different physiological states, bacterial-induced and non bacterial-induced group,from grass carp intestine.211 cDNA clones partially and single-pass sequenced and 147 ESTs were obtained.The result of Sequence homology search shows that there are 97 with significant homology to GenBank database sequences that were identified,of which 28 matched MHC type genes.There are 17 sequences matched hypothetical open reading frames of unknown function protein,while 33 clones did not match any sequences in the database and presumably represented unidentified cDNAs.According to Adams'method,All of these ESTs were classified into eight categories:protein/gene expression,signal transduction,cell structure and motility,metabolism,cell defense,cell division,miscellaneous and hypothetical protein.The percentage of cDNA sequence of cell defense is the highest in obtained ESTs,up to 27.2%;Signal transduction and metabolism were up to 12.9%,12.2%,respectively; The percentage of protein/gene expression was 11.6%;Cell structure and motility, cell division are quite low proportion.All of these shows that the expression of the factors of cell defense,signaling,metabolism,protein/gene expression,especially defense-related factor from intestinal tissue were up-regulated after stimulating by bacterial infection.2.Cloning and sequencing of Grass carp intestinal immune-related genes MHCⅡalpha gene full-length cDNABased on the sequence of MHCⅡαEST(GenBank Accession No:EW688194) obtained from subtractive library of grass carp.The full-length cDNA of MHC-Ⅱαgene was obtained using 5'-RACE and 3'-RACE(GenBank Accession No:EU186148). MHC-Ⅱalpha cDNA sequence was analyzed using bioinformatics,The length of MHC-Ⅱαgene cDNA is composed of 1007 bp,including a 717 bp open reading frame,which encodes 238 amino acids,flanked by a 34 bp of 5'untranslated region(UTR)and a 256 bp of 3'-UTR with poly(A)tail.A poly-adenylation signal of AATAAA is located within the 3'-UTR at nucleotides 962-967.The isoelectric point of the protein deduced from cDNA is 4.92 and the molecular weight is 26.4KDa. there are a 20 amino acid of signal peptide,and a transmembrane region between amino acid of 210-232.The results of homology analysis shows that grass carp and carp shared very high similarity.Phylogenetic tree of evolution reveals that evolutionary relationship between Grass carp and carp,zebrafish is very closely, while its evolutionary relationship is farther to nurse Shark and Africa cichlid.The expression profile of MHC-Ⅱαwas detected in heart,liver,spleen,intestine,kidney, gills and muscle through Semi-quantitative RT-PCR by infecting Aeromonas hydrophila.The results showed that MHC classⅡαwere highly expressed in spleen, head kidney,back intestine,whereas quite lowly in the liver,and even no expression in muscle.All of these suggest that the expression of MHC-Ⅱαis mainly concentrated in immune-related tissues.3.Cloning and sequencing of Grass carp intestinal immune-related gene, Alpha-1-microglobulling precursor(AMBP)full-length cDNAA pair of specific primers was designed according AMBP EST sequence (GenBank Accession No:EW688152)from the grass carp subtractive library.The full-length cDNA sequence of AMBP was obtained using 5'-and 3'-RACE RACE method(GenBank Accession No:EU186151).The length of AMBP cDNA is composed of 1230bp,including a 1047bp open reading frame which encoding 348 amino acids.Two instability signals ATTTA are located within the 3'-UTR at nucleotides 1103-1107 and 1146-1150,respectively.A poly-adenylation of AATAAA is located within the 3'-UTR at nucleotides 1166-1171.Bioinformatics analysis showed that the isoelectric point of AMBP protein deduced from cDNA is 6.27,and the molecular weight is 38.84KDa.There is a 18 amino acids of signal peptide,but no transmembrane region is found.there are two different space domains predicted by Swiss-Model,including trypsin inhibitor(bikunin)andα-1-microglobulin precursor which is classified into lipocalin family protein.Homology analysis via Clustal W shows that amino acid sequence of AMBP also highly conservative regions comparing with close species.Analysis of phylogenetic relationships reveals that Phylogenetic tree is divided into two large branchs, Phylogenetic relationship of AMBP of fishies is closely;and the others including human,rat,cow,pig,their Phylogenetic relative are similar,belong to another branch. Under infecting Aeromonas hydrophila,RT-PCR result shows that the expression level of grass carp AMBP is highest in the liver,and higher in back-kidney,head kidney and spleen,lower in heart and intestinal,whereas,no expression in gill and muscles.