日本沼虾酚氧化酶原基因cDNA的全长克隆及表达分析

cDNA cloning and characterisation of prophenoloxidase from Macrobrachium nipponense

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(5.0×109/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2013bp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相似性为50%～53%。该基因表达具有组织特异性,血细胞最高,肝胰腺有较弱表达,肌肉、鳃、肠、大颚器官和卵巢不表达;血细胞proPO基因的表达量在蜕皮前期(D0/1)最高;嗜水气单胞菌刺激后6 h血细胞proPO mRNA水平及血清中PO活力均显著增加。结论与其他甲壳动物相似,日本沼虾proPO基因具有2个保守的铜离子结合位点;其表达呈组织特异性,最高出现在血细胞;在蜕皮周期的前期(D0/1)血细胞表达量最高;嗜水气单胞菌可诱导proPO基因的表达以及PO活力,提示该基因是参与机体免疫防御反应的一种重要分子。

Objective To clone the prophenoloxidase （proPO）gene from Maerobrachium 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 eDNA 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 x 109/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 eDNA 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. Comparisonof amino acid sequences showed that the proPO-deduced amino acid of Macrobrachittm 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 haemocytcs, weakly in the hepatopancreas, and negative in the muscle, gill, intestine, mandibular organ and ovarian. Its transcript derived from haemocytes was highest in stage D0zI 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 D0/1. 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 hydrophila.