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(英文)
cDNA CLONING AND EXPRESSION CHARACTERIZATION OF LYSOZYME GENE IN TWO FRESHWATER PRAWN
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摘要: 分别提取罗氏沼虾和日本沼虾血细胞总RNA,RT-PCR扩增获得特异性cDNA片段,纯化后克隆到T载体上。序列测定表明所克隆的两种沼虾溶菌酶基因的开放阅读框(ORF)为477bp,共编码158个氨基酸,包括溶菌酶成熟肽140个氨基酸残基和信号肽18个氨基酸残基。同源性分析表明,罗氏沼虾和日本沼虾溶菌酶基因的碱基序列及推测氨基酸序列高度同源,分别为99.4%和98.1%。两种沼虾溶菌酶基因的碱基序列和推测氨基酸序列与Gen-Bank上其他对虾溶菌酶的同源性达83.0%和80.0%以上。两种沼虾溶菌酶都具有c-型溶菌酶典型的两个酶活性位点(Glu51)和(Asp68),以及8个保守结构氨基酸残基Cys,且在101、106和107位上缺少Asp,因而推测本实验所克隆的两种沼虾溶菌酶基因属c-型溶菌酶基因的非钙结合亚型。以PCR法制备罗氏沼虾溶菌酶基因的生物素标记探针,斑点杂交检测感染弧菌后溶菌酶基因mRNA在各组织中的转录水平,结果表明受感染6h后在眼、肌肉、鳃、肝胰腺、肠管中的表达量均有升高,其中在肝胰腺中的表达量最高,约为对照组的560%。在不同感染时间里,肝胰腺中该基因表达量有较大的变化:感染后3h表达量最低,24h后表达量升至最高,大约为对照组的430%,48h时的表达量又有所下降,但仍明显高于对照组(约为330%)。受弧菌感染后罗氏沼虾溶菌酶基因转录的上调证明溶菌酶基因在非特异性免疫中的直接作用,同时表明肝胰腺可能在沼虾的免疫防御过程起重要作用。Abstract: 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.
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Keywords:
- Macrobrachium rosenbergii /
- M. nipponense /
- Lysozyme /
- Gene cloning /
- Expression pattern
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