仿刺参铜锌超氧化物歧化酶基因全长cDNA的克隆及表达分析

Cloning,Characterization and Expression of Full-length cDNA of a Copper/Zinc Superoxide Dismutase Gene in Sea Cucumber(A postichopus japonicus)

采用cDNA末端快速克隆技术首次克隆了仿刺参铜锌超氧化物歧化酶基因的全长cDNA序列，该基因cDNA全长1500 bp ，其中包含5′-非翻译区长129 bp ，3′-UTR长912 bp ，开放阅读框459 bp ，编码152个氨基酸，预测蛋白分子量为15．47 ku。仿刺参铜锌超氧化物歧化酶基因 cDNA推导的氨基酸序列具有保守的铜锌超氧化物歧化酶蛋白家族标签序列42 GFHIHQFGDNT52及136 GNAGGRAACGVI147，4个Cu2＋结合位点（His-44，-46，-61，-118）和4个Zn2＋结合位点（His-61，-69，-78，Asp-81），一对二硫键（Cys-55，-144）。氨基酸序列比对结果显示，仿刺参铜锌超氧化物歧化酶与匙胸瘿蜂和烟粉虱相似度最高，为71％。系统进化分析显示仿刺参处于无脊椎动物分支中，与紫海胆位于同一小支上。实时定量 PCR结果显示，铜锌超氧化物歧化酶基因 mRNA在仿刺参肠、体壁、肌肉、呼吸树、体腔细胞和管足中均有表达，在管足中表达量最高。在细菌脂多糖刺激后4～12 h ，体腔细胞中铜锌超氧化物歧化酶基因 mRNA表达量显著下降，表明仿刺参的铜锌超氧化物歧化酶基因对细菌脂多糖刺激有免疫应答。

In this study ,the full‐length cDNA of Cu/Zn‐SOD gene as an immune‐related gene was cloned and characterized in sea cucumber （A postichopus japonicus） .It was found that the Cu/Zn‐SOD gene had full‐length of 1 500 bp including a 129 bp 5’‐untranslated region （UTR） ,459 bp ORF encoding 152 amino acids ,and a 912 bp 3’‐UTR with predicted molecular weight of 17 .47 ku .Four copper binding sites （His‐44 ,‐46 ,‐61 ,and‐118） and four Zn binding sites （His‐61 ,‐69 ,‐78 ,and Asp‐81） ,a conserved disulfide bond （Cys‐55 ,‐144 ） , and two conserved Cu/Zn‐SOD signatures were identified in the deduced amino acid sequence in the Cu/Zn‐SOD .The deduced amino acid sequence showed 71% amino acid sequence identity to the Cu/Zn‐SOD in Leptopilinaboulardi and Bemisia tabaci .Phylogenetic analysis revealed that the Cu/Zn‐SOD gene in the sea cucumber was close to that in purple sea urchin Strongylocentrotus purpuratus in invertebrate cluster .Quantitative real‐time PCR analysis showed that the Cu/Zn‐SOD gene was expressed in all the tested tissues including body wall ,feet ,coelomocytes ,respiratory tree ,muscle ,and intestine , the maximal expression level in feet . The expression of the Cu/Zn‐SOD mRNA was down‐regulated significantly in the coelomocytes 4—12 h after bacterial lipopolysaccharide （LPS） challenge ,indicating that the Cu/Zn‐SOD gene would be one of the immune‐related factor .