cDNA CLONING AND EXPRESSION CHARACTERIZATION OF LYSOZYME GENE IN TWO FRESHWATER PRAWN
两种沼虾溶菌酶基因ORF的克隆和罗氏沼虾溶菌酶基因的组织表达

Total RNAs were isolated from haemocytes of M.rosenbergii and M.nipponense.The cDNAs encoding lysozyme were amplified by RT-PCR.The amplified cDNA fragments were inserted into pGEM-T vector.Sequence analysis revealed that both of the two freshwater prawn lysozyme cDNAs contain an open reading frame(ORF) of 477nt,which encodes 158 amino acid residues,including 140 residues of mature peptide and 18 residues of signal peptide.The nucleotide and amino acid sequence identity between the two prawn cDNAs is 99.4% and 98.1%,respectively.The two prawn cDNAs possess high identity with marine shrimp cDNAs,too.Their nucleotide and amino acid sequence identity is above 83.0% and 80.0%,respectively.The prawn lysozymes are presumed to be the non-calcium binding family of chicken-type lysozyme because they have two conserved catalytic sites Glu~(51) and Asp~(68),as well as eight structural Cys residues,which are highly conserved among the species of chicken-type lysozymes but lack of three Asp residues at the site 101,106 and 107,which are conserved sites in calcium-binding chicken-type lysozyme.The expression pattern of lysozyme gene of M.rosenbergii infected with Vibrio spp was analyzed by Northern Dot Blot with biotin-labeled probe produced by PCR.The result showed that 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 the lysozyme mRNA levels in hepatopancreas varied at different infection time: The lowest level was at 3h post-infection,the highest was at 24h post-infection,which was about 430% of that of the control group.At 48h post-infection,the mRNA level decreased slightly,but still higher than that of the control group(about 330%).The up-regulation of lysozyme gene expression observed in Vibrio infected M.rosenbergii suggested that freshwater prawn lysozyme gene is undoubtedly related to the non-specific immune defense and hepatopancreas may also play an important role in prawn immune mechanism.