ISOLATION AND CHARACTERIZATION OF THE LYSOZYME-ENCODING GENE FROM THE BLACK TIGER SHRIMP,PENAEUS MONODON

Lysozyme is an enzyme which acts as a hydrolase to break down the Beta-(1,-4)linkages in the peptidoglycan layer of bacterial cell walls.Bacteria is either destroyed directly or is opsonized,enabling its destruction through phagocytosis.Researches showed that lysozyme is also an important immune factor in shrimps.Lysozyme activity has been considered as one of the basic biochemical characters to estimate the immune state of shrimps.In order to study the immune function of lysozyme in shrimps,it is necessary to isolation the shrimp lysozyme gene.Lysozyme gene of Penaeus monodon was cloned by RT-PCR (reverse transcription PCR).The primer was designed referring to the lysozyme gene and lysozyme-like gene sequences of shrimps and other species.High quality total RNA was isolated from hemocytes of Penaeus monodon. The first strand cDNAs encoding lysozyme were obtained by using total RNA as templates and then amplified.A specific band about 700bp in length in PCR products was showed by gel electrophoresis.After purified the amplified cDNA fragments were inserted into pGEM-T Easy vector and sequenced.Sequencing result showed that the total length of the cDNA is 658bp,containing an open reading frame (ORF) of 477bp and a 181bp 3′-untranslated region.Of the 477bp ORF sequence,421bp encode 140 amino acid residues of the mature peptide and 54bp for 18 amino acid residues of the signal peptide.The estimated molecular mass of the mature peptide (140 amino acids)is 16,32kD and pI is 8.78.Alignment analyses showed that the similarities of nucleotide sequences and deduced amino acid sequences of lysozyme between P.monodon and Litopenaeus vannamei are 89.5%and 93.5%,and that are 84.0%and 91.0%between P. monodon’s and Marsupenaeus japonicus’s.Further analysis showed that the lysozyme from P. monodon possesses high homology with that of chicken-type lysozyme of other species.The lysozyme from P.monodon has the conserved Glu51 Asp68 catalytic sites,as well as the eight structural cysteine residues,which are highly conserved across many species of chicken-type lysozymes.In addition,the sequences around the catalytic site are quite conserved too.Therefore the cloned P. monodon lysozyme is presumed to be chichen-type lysozyme.