解剖学报

›› 2012, Vol. 43 ›› Issue (6): 744-750.doi: 10.3969/j.issn.0529-1356.2012.06.005

• 细胞和分子生物学 • 上一篇    下一篇

日本沼虾酚氧化酶原基因cDNA的全长克隆及表达分析 

王文锋1,2; 马建敏3; 杨洪1; 刘方1; 陈香丽1;宁黔冀1*    

  1. 1.河南师范大学生命科学学院，河南 新乡 453007; 2.新乡医学院生命科学技术学院，河南 新乡 453003; 3.新乡市疾病预防控制中心检验科，河南 新乡 453003
  • 收稿日期:2011-12-16 修回日期:2012-02-08 出版日期:2012-12-06
  • 通讯作者: 宁黔冀

cDNA cloning and characterisation of prophenoloxidase from EM>Macrobrachium nipponense/EM>

  1. 1. College of Life Sciences, He’nan Normal University, He’nan Xinxiang 453007, China; 2. Department of Life Science and Technology, Xinxiang Medical University, He’nan Xinxiang 453003, China; 3. Department of Clinical Laboratory, Xinxiang Center for Disease Control and Prevention, He’nan Xinxiang 453003, China
  • Received:2011-12-16 Revised:2012-02-08 Online:2012-12-06
  • Contact: NING Qian-ji

摘要： 目的 克隆日本沼虾酚氧化酶原（proPO）基因，进行生物信息学及时空表达分析。方法 利用RT-PCR和快速扩增cDNA末端（RACE）技术从血细胞中克隆proPO基因cDNA全长序列，用生物软件对其序列进行生物信息学分析； proPO基因时空表达分析采用RT-PCR和Real-time PCR方法；腹部肌肉注射嗜水气单胞菌（5.0×10SUP>9/SUP>/L）诱导酚氧化酶（PO），20μl/只，注射后3、6、12、24h取其血淋巴，分别测定血细胞 proPO mRNA水平及血清酚氧化酶（PO）活力。结果 日本沼虾proPO基因cDNA全长2428bp，包含71 bp的 5’UTR、344 bp的 3’UTR 和2013 bp的开放阅读框（ORF）。ORF编码 671 个氨基酸，预测蛋白分子量为76.5kD，理论等电点（pI）约为7.31；含有2个保守的铜离子结合位点和6个组氨酸残基、1个蛋白酶水解位点和硫酯样的基序(GCGWPRHM)；含有3个血蓝蛋白结构域；与罗氏沼虾proPO的相似性最高，为93%，与其他甲壳动物proPO相似

关键词: 酚氧化酶原, 时空表达, 快速扩增cDNA末端技术, 实时荧光定量聚合酶链反应, 日本沼虾

Abstract: Objective To clone the prophenoloxidase（proPO）gene from Macrobrachium nipponense, and to analyze the bioinformatics and spatial and temporal expression of the gene. Methods The proPO gene was cloned from haemocytes of Macrobrachium nipponense using RT-PCR and rapid amplification of cDNA ends (RACE) method, and its sequence was analyzed with a biological software. Spatial and temporal expressions of the gene were detected by RT-PCR and Real-time PCR. The immune challenge test was carried out by injecting with 20μl of a Aeromonas hydrophila suspension (5.0×10SUP>9/SUP>/L) into the ventral sinus of prawns. After 3 hours, 6 hours, 12 hours and 24 hours of injection, haemolymph were collected and used for the proPO mRNA expressions of haemocytes and the phenoloxidase (PO) activity of serum. Results The full-length cDNA of hemocyanin was 2428bp and contains a 71bp of 5’-untranslated region (UTR), a 344bp of 3’UTR and a 2013bp of open reading frame (ORF) that encoded a protein of 671 amino acids with a calculated molecular mass of 76.5 kD and pI at 7.31. It was predicted to possess all the expected features of proPO members, including two putative tyrosinase copper-binding motifs with six histidine residues,a proteolytic activation site and a thiol ester-like motif (GCGWPRHM). Deduced amino acid sequence contained three hemocyanin domains. Comparison of amino acid sequences showed that the proPO-deduced amino acid of Macrobrachium nipponense exhibited higher similarity to that of Macrobrachium rosenbergii(93%) and 50% -53% to that of other crustaceans. The gene was expressed highly in the haemocytes, weakly in the hepatopancreas, and negative in the muscle, gill, intestine, mandibular organ and ovarian. Its transcript derived from haemocytes was highest in stage DSUB>0/1/SUB> among the molt cycle. Injection of pathogenic bacteria A. hydrophila resulted in the significant increase of proPO gene expressions and PO activity 6hours after treatment. Conclusion Like other crustaceans, the proPO gene of Macrobrachium nipponense contains two conserved copper-binding sites, and is highly expressed in haemocytes. The levels of proPO transcripts in haemocytes are the highest in molt stage DSUB>0/1/SUB>. Injection of Aeromonas hydrophila results in increase of proPO gene expressions and PO activity, indicating that proPO may act as an important molecule involved in immune defense against Aeromonas hydrop

Key words: Prophenoloxidase（proPO）, Spatial and temporal expression, Rapid amplification of cDNA ends, Real-time RT-PCR, Macrobrachium nipponense

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