Ko Chi-Fong, Chiou Tzu-Ting, Vaseeharan Baskaralingam, Lu Jenn-Kan, Chen Jiann-Chu
Department of Aquaculture, College of Life Sciences, National Taiwan Ocean University, Keelung, Taiwan 202, ROC.
Dev Comp Immunol. 2007;31(1):12-22. doi: 10.1016/j.dci.2006.05.002. Epub 2006 Jun 12.
A prophenoloxidase (proPO) cDNA was cloned from the haemocytes of mud crab Scylla serrata using oligonucleotide primers and RT-PCR. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA end (RACE) method. Analysis of the nucleotide sequence revealed that the cDNA clone has a full length of 2663bp, with an open reading frame of 2019bp, a 124-bp 5'-untranslated region, and a 520-bp 3'-untranslated region containing a poly A signal. It encodes a protein of 673 amino acids with a predicted molecular weight of 77.5kDa and with an estimated pI of 5.96. It contains two putative tyrosinase copper-binding motifs with six histidine residues (copper A, 185, 189, 211, and copper B, 346, 350, 386). The proPO has thiol-ester-like motif (GCGWPQHM), which showed similar structural features of proPOs from other decapod crustaceans. It also contains five possible glycosylation sites, and a conserved C-terminal region common to all known proPOs. Sequence comparison showed that the proPO-deduced amino acid of mud crab S. serrata has an overall similarity of 78%, 57%, 56%, 51-55%, 54%, 53%, 52%, 52%, and 52% to that of Dungeness crab Cancer magister, American lobster Homarus americanus, European lobster Homarus gammarus, kuruma prawn Marsupenaeus japonicus, crayfish Pacifastacus leniusculus, white shrimp Litopenaeus vannamei, tiger shrimp Penaeus monodon, green tiger shrimp Penaeus semisulcatus, and giant freshwater prawn Macrobrachium rosenbergii, respectively. The proPO was strongly expressed in haemocytes, but not in heart, eyestalk, gill, muscle, ovary, hepatopancreas, stomach, and intestine. The proPO transcript of mud crab S. serrata increased significantly in 12 and 24h post-lipopolysaccharide (LPS) injection, but returned to the original values in 72h post injection.
利用寡核苷酸引物和逆转录-聚合酶链反应（RT-PCR）从锯缘青蟹血细胞中克隆了一种酚氧化酶原（proPO）cDNA。通过cDNA末端快速扩增（RACE）方法分离出3'和5'区域。核苷酸序列分析表明，该cDNA克隆全长2663bp，开放阅读框为2019bp，5'非翻译区为124bp，3'非翻译区为520bp，包含一个多聚腺苷酸信号。它编码一个由673个氨基酸组成的蛋白质，预测分子量为77.5kDa，估计pI为5.96。它包含两个假定的酪氨酸酶铜结合基序，带有六个组氨酸残基（铜A，185、189、211，以及铜B，346、350、386）。该proPO具有硫酯样基序（GCGWPQHM），显示出与其他十足目甲壳类动物的proPO相似的结构特征。它还包含五个可能的糖基化位点，以及所有已知proPO共有的保守C末端区域。序列比较表明，锯缘青蟹proPO推导的氨基酸与珍宝蟹Cancer magister、美洲龙虾Homarus americanus、欧洲龙虾Homarus gammarus、日本对虾Marsupenaeus japonicus、小龙虾Pacifastacus leniusculus、凡纳滨对虾Litopenaeus vannamei、斑节对虾Penaeus monodon、墨吉对虾Penaeus semisulcatus和罗氏沼虾Macrobrachium rosenbergii的相应氨基酸总体相似度分别为78%、57%、56%、51 - 55%、54%、53%、52%、52%和52%。该proPO在血细胞中强烈表达，但在心脏、眼柄、鳃、肌肉、卵巢、肝胰腺、胃和肠道中不表达。锯缘青蟹的proPO转录本在注射脂多糖（LPS）后12小时和24小时显著增加，但在注射后72小时恢复到原始值。
