斜带石斑鱼L-氨基酸氧化酶基因克隆及表达分析

鱼类的L-氨基酸氧化酶(L-amino acid oxidase, LAAO)具有广泛的抑菌杀虫效果, 为了解斜带石斑鱼(Epinephelus coioides)LAAO基因序列特征及其在刺激隐核虫(Cryptocaryon irritans)感染后的表达变化, 该试验克隆得到2个石斑鱼LAAO基因: EcLAAO-1和EcLAAO-2, 它们的ORF长度分别为1536和1569 bp, 编码511和522个氨基酸, 均含有氨基酸氧化酶(Amino_oxidase)结构域以及LAAO保守序列: DBM和GG motif。多重序列比对显示石斑鱼LAAO与其他鱼类LAAO具有较高的相似性。系统进化树分析表明, 斜带石斑鱼的LAAO与硬骨鱼类亲缘关系较近。实时荧光定量PCR检测结果显示EcLAAO-1和EcLAAO-2在石斑鱼各组织均有表达, 其中皮肤、鳃、胸腺、肝脏和肌肉中含量较丰富; 在感染刺激隐核虫后, 鳃和脾脏EcLAAO-1, EcLAAO-2表达量显著升高(P<0.05), 这些结果暗示了石斑鱼LAAO参与先天性免疫, 并在抗御刺激隐核虫感染中发挥重要作用。

MOLECULAR CLONING AND EXPRESSION ANALYSIS OF L-AMINO ACID OXIDASE IN GROUPER (EPINEPHELUS COIOIDES)

Fish L-amino acid oxidase (LAAO) has a strong killing effect on bacteria and parasites. In order to study the gene sequence characteristics and expression pattern of LAAO gene in grouper (Epinephelus coioides) post Cryptocaryon irritans infection, two LAAO cDNA sequences were identified and designated as EcLAAO-1 and EcLAAO-2. The ORF of EcLAAO-1 and EcLAAO-2 were 1536 bp and 1569 bp, coding 511 and 522 amino acids respectively. They both have Amino_oxidase domain and LAAO conserved sequences of DBM and GG motif. Multiple sequence alignment showed that EcLAAO-1 and EcLAAO-2 protein shared high sequence identity with LAAO of other teleost. EcLAAO-1 and EcLAAO-2 were clustered into the same subgroup with other teleost in the phylogenetic tree. EcLAAO-1 and EcLAAO-2 were widely distributed in various tissues, especially in skin, gill, thymus, liver and muscle. EcLAAO-1 and EcLAAO-2 were up-regulated in the gill and spleen post C. irritans infection. Together, these results suggest that LAAO might be involved in host’s innate immunity and play an important role in the defense against C. irritans infection.