cDNA cloning and 3-D protein structure prediction of intracellular copper/zinc superoxide dismutase （ic-Cu/Zn-SOD） in Cristaria plicata | 褶纹冠蚌胞内Cu/Zn-SOD的cDNA克隆及蛋白质三维结构预测

Chinese. 摘　　要：运用兼并引物结合RT-PCR方法从褶纹冠蚌血细胞中克隆出一种细胞内铜锌超氧化物歧化酶（cpSOD）的cDNA,用半定量PCR方法研究了cpSOD的mRNA在褶纹冠蚌不同组织的表达情况。结果表明：cpSOD cDNA全长为891 bp,包含1个468 bp的开放阅读框,编码含155个氨基酸的蛋白。预测蛋白质分子质量为15.8 ku,等电点为5.8。含4个与铜离子结合的保守的氨基酸残基（H46,H48,H63和H120）和4个与锌离子结合的保守的氨基酸残基（H63,H71,H80和D83）及1个保守的分子内二硫键（Cys57-Cys146）。氨基酸序列同源性比较显示cpSOD与海洋贝类太平洋牡蛎Crassostrea gigas及栉孔扇贝Chlamys farreri的胞内Cu/Zn-SOD相似性分别为70%和69%;半定量PCR分析显示在褶纹冠蚌血液、外套膜、鳃、闭壳肌和肝胰腺5种组织中均能检测到cpSOD的基因表达,且在鳃和肝胰腺中表达量较大。嗜水气单胞菌刺激后cpSOD基因在褶纹冠蚌血细胞中表达上调,12 h后达到最大值。由此推断cpSOD可能在机体的免疫防御中起着重要的作用。

English. An intracellular copper/zinc superoxide dismutase（cpSOD） cDNA was cloned from the haemocytes of freshwater mussel Cristaria plicata using reverse transcription-polymerase chain reaction（RT-PCR） by degenerate primers.Further studies were conducted on cpSOD mRNA expression in various tissues of C.plicata by semi-quantitative PCR method.The results revealed that the cpSOD cDNA clone consists of 891 bp with an open reading frame of 468 bp and with a protein of 155 amino acids.The calculated molecular mass is 15.8 ku with an estimated pI of 5.8.Four conserved amino acids responsible for binding copper（H46,H48,H63 and H120）,four conserved amino acids responsible for binding zinc（H63,H71,H80 and D83） and a conserved intramolecular disulfide bond were observed.Sequence comparison showed that the deduced amino acid sequence of cpSOD presented 70% similarity to that of saltwater shellfish Crassostrea gigas ic-Cu/Zn-SOD and 69% to Chlamys farreri ic-Cu/Zn-SOD,respectively.Semi-quantitative RT-PCR analyses showed that cpSOD genes were expressed in haemocytes,mantle,gill,adductor muscle and hepatopancreas of C.plicata.Stimulating C.plicata by injection with Aeromonas hydrophila significantly up-regulated the expression of cpSOD in haemocytes,indicating that cpSOD is involved in the immune responses against A.hydrophila.