cDNA CLONING AND EXPRESSION CHARACTERIZATION OF LYSOZYME GENE IN TWO FRESHWATER PRAWN

Total RNAswere isolated from haemocytes of M. rosenbergii and M. nipponense. The cDNAs encoding lysozyme were am plified by RT -PCR. The amplified cDNA fragmentswere insertedinto pGEM-T vector. Sequence analysisrevealed that both of the twofreshwater prawn lysozyme cDNAs contain anopenreading frame (ORF) of 477nt, which encodes 158 amino acidresidues, including140 residues of mature peptide and 18 residues of signal peptide.The nucleotide and amino acid sequence identity between the twoprawn cDNAs is 9914% and 9811%, respectively. The two prawn cDNAs possess high identity with marine shrimp cDNAs, too.Their nucleotide and amino acid sequence identity is above 8310% and 8010%, respectively. The prawn lysozymes are presumedtobe the non-calcium binding family of chicken -type lysozyme because they have two conserved catalytic sites Glu51andAsp68, as wellaseight structural Cys residues, which are highly conserved among the species of chicken-type lysozymes but lack of three Aspresidues at the site 101,106 and 107,which are conserved sites in calcium -binding chicken type lysozyme.The expression pattern oflysozyme gene of M. rosenbergii infected with Vibrio spp was analyzed by Northern Dot Blot with biotin-labeled probe produced byPCR. The result showedthat the expression levels of lysozyme were up-regulated in eye, muscle, gill, hepatopancreas, intestine.Among them the lysozyme mRNA level in hepatopancreas was the highest which was 560% of that of the control group. And thelysozyme mRNA levels in hepatopancreas varied at different infection time: The lowest levelwas at 3h post infection,the highestwasat 24h post infection,which was about 430% of that of the controlgroup. At 48h post infection,the mRNA level decreased slightly,but still higher than that of the controlgroup ( about 330%).The up -regulationof lysozyme gene expressionobserved in Vibrio infect-ed M.rosenbergii suggested that freshwater prawn lysozyme gene is undoubtedly related to the non -specific immune defense and hep -atopancreas may also play an important role in prawn immune mechanism.