凡纳滨对虾酚氧化酶原激活因子基因的克隆及表达分析
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广西壮族自治区水产科学研究院,广西壮族自治区水产科学研究院,广西壮族自治区水产科学研究院,广西壮族自治区水产科学研究院,广西壮族自治区水产科学研究院,广西壮族自治区水产科学研究院,广西壮族自治区水产科学研究院,广西壮族自治区水产科学研究院

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广西自然科学基金项目(2012GXNSFDA053015);现代农业产业技术体系建设专项(CARS-47);国家现代农业产业技术体系广西凡纳滨对虾产业创新团队项目(nycytxgxcxtd-03-14)


Cloning and analysis of full length cDNA of prophenoloxidase activating factor in Litopenaeus vannamei
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Guangxi Academy of Fishery Sciences,Guangxi Academy of Fishery Sciences,,,,,,Guangxi Academy of Fishery Sciences

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    摘要:

    为研究酚氧化酶原激活因子(prophenoloxidase-activating factor,PPAF)在凡纳滨对虾感染传染性皮下及造血组织坏死病毒(IHHNV)过程中所起的免疫作用,实验采用逆转录聚合酶链式反应和cDNA末端快速扩增技术克隆了凡纳滨对虾PPAF基因的全长cDNA序列,并通过实时荧光定量PCR技术分析了该基因在正常凡纳滨对虾和感染IHHNV凡纳滨对虾不同组织的表达情况。结果显示,克隆获得了1 986 bp凡纳滨对虾PPAF基因cDNA全长序列(GenBank登录号JQ684529),其中含有一个1 629 bp开放阅读框(ORF),两翼分别存在21 bp(5’端)和336 bp(3’端)的非翻译区。该基因的开放阅读框共编码542个氨基酸,氨基酸序列269~517处存在功能结构域——丝氨酸蛋白酶结构域,氨基酸序列494~502区域存在一个ALPHA-2巨球型免疫蛋白功能位点,316~321区域有一个组氨酸酶活性位点;定量PCR分析发现,该基因无论在正常对虾还是感染IHHNV对虾的心脏、肝胰腺、肠、胃和肌肉中的表达量均较低,在血液和鳃腺中的表达量明显高于其他组织,但感染IHHNV对虾不同组织中PPAF基因的表达量均低于正常对虾相应组织中的表达量。定量检测PPAF基因在对虾鳃腺组织中的表达情况显示,对虾感染IHHNV后,PPAF基因的表达量急剧降低,3 h时至最低,之后表达量逐渐上升,48h时达最高值。研究表明,IHHNV可抑制PPAF基因的表达,因而抑制酚氧化酶原免疫系统的有效激活,或是其感染对虾并在对虾组织内增殖的原因之一。

    Abstract:

    Prophenoloxidase-activating factor(PPAF)plays a key role in prophenoloxidase activation system of crustacean's humoral immune system.In this study, the full-length cDNA sequence was cloned by reverse transcriptase polymerase chain reaction(RT-PCR)and rapid-amplification of cDNA ends(RACE), the expression patterns of PPAF gene in various tissues of normal shrimp and IHHNV-infected shrimp were determined by Quantitative real-time PCR.The results showed that the full-length cDNA sequence of PPAF was 1 986 bp, which contained a 21 bp 5'-UTR(untranslated region), 336 bp 3'-UTR(untranslated region), and the 1 629 bp open reading frame(ORF)encoded 542 amino acid.There was a serine protease domain between 269-517 amino acids residues of Litopenaeus vannamei PPAF, an ALPHA-2 macroglobulin functional site and a histamine enzyme active site were found within the 494-502 and 316-321 amino acids residues of Litopenaeus vannamei PPAF respectively.The expression of the gene in various tissues was analyzed by Real-time quantitative PCR, the result showed that the expression of the gene was higher in blood and gill than in heart, hepatopancreas, intestines, stomach and muscle of the normal shrimp or IHHNV-infected shrimp, but the expression of PPAF in various tissues of IHHNV-infected shrimp was lower than in same tissues of normal shrimp.The expression of PPAF in gill of Litopenaeus vannamei drastically reduced after infected with IHHNV, the expression levels reached a minimum within 3 hours, which was about 1/8 times as compared to the control group.After 3 hours shrimp infected with IHHNV, the expression of PPAF in gill increased gradually, and up to highest level after 48 hours.The results suggested IHHNV can protect them from the immunity of Prophenoloxidase by decreasing the expression of PPAF of shrimp when they infect shrimp.

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赵永贞,陈秀荔,曾地刚,杨春玲,彭敏,何苹萍,李咏梅,彭金霞,韦嫔媛,陈晓汉.凡纳滨对虾酚氧化酶原激活因子基因的克隆及表达分析[J].水产学报,2014,38(8):1068~1078

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  • 收稿日期:2013-11-13
  • 最后修改日期:2014-05-09
  • 录用日期:2014-06-20
  • 在线发布日期: 2014-08-21
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