斑节对虾溶菌酶基因克隆及序列分析

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

参考对虾溶菌酶基因和类溶菌酶基因及其他多种生物的溶菌酶基因序列 ,设计并合成引物。运用RT PCR技术 ,从斑节对虾血细胞总RNA中扩增获得特异性片段。所获片段回收纯化后克隆到pGEM TEasyVector系统的T载体上。重组子的序列分析表明 ,所克隆的斑节对虾溶菌酶基因片段长 6 5 8bp ,包括溶菌酶基因开放阅读框 (ORF)4 77bp和 3′端非编码区的 181bp。 4 77bpORF共编码 15 8个氨基酸 ,包括溶菌酶成熟肽 14 0个氨基酸残基和信号肽 18个氨基酸残基。斑节对虾溶菌酶成熟肽推测分子量为 16 ,32kd ,等电点为 8 78。与南美白对虾溶菌酶基因的碱基序列及推测氨基酸序列比较 ,同源性分别为 89 5 %和 93 0 % ;与日本对虾类溶菌酶基因的同源性分别为 84 0 %和91 0 %。进一步的序列分析表明 ,斑节对虾溶菌酶氨基酸序列与多种类群生物的c 型溶菌酶氨基酸序列具有较高的同源性 ,并具有与c 型溶菌酶相同的活性位点氨基酸残基Glu51和Asp68,且与活性位点相邻的序列高度保守。斑节对虾溶菌酶氨基酸序列还具有与c 型溶菌酶相同的结构氨基酸——— 8个半胱氨酸残基。因而可认为所克隆的斑节对虾溶菌酶基因属c 型溶菌酶基因。

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 Glu 51 Asp 68 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.