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作 者 ：吴 凡1，余 梅1，鲁茂龙2，李 娟2，王荣富2，王先磊1

期 刊 ：西北植物学报 2012年 10期 页码：1957~1964

关键词：盐角草；MnSOD；Cu/ZnSOD；克隆；耐盐性；

Keywords:Salicornia europaea, MnSOD, Cu/ZnSOD, cloning, salt tolerance,

摘 要 ：为了探究盐生植物的耐盐机制，比较不同盐生植物超氧化物歧化酶（SOD）基因耐盐性的大小，采用RACE技术克隆了盐角草锰和铜/锌两种SOD的cDNA全长。序列分析表明，盐角草MnSOD基因（SeMSD，GenBank登录号为JQ061158）含有699 bp的开放阅读框，编码一个长233个氨基酸的多肽，其预测相对分子量为25.7 kD。Cu/ZnSOD基因（SeCSD，GenBank登录号为JQ061160）开放阅读框长为684 bp，并编码228个氨基酸的多肽，相对分子量为23.3 kD。根据已获得的这两个cDNA序列和GenBank公布的盐芥MnSOD基因序列（ThMSD，EF140719），构建了3个原核表达载体pET30a-SeMSD、pET30a-SeCSD和pET30a-ThMSD，并将它们转化大肠杆菌BL21，成功表达出了目的蛋白。通过优化IPTG的诱导浓度，在6.5%和7.5%盐度下对这3个SOD基因的耐盐能力验证结果显示，相比对照菌BL21（pET30a），转化了pET30a-SeMSD和pET30a-ThMSD载体的重组菌具有更高的耐盐性,而转化pET30a-SeCSD载体的重组菌没有表现出耐盐性的提高。

Abstract:In order to study the halophyte salt-tolerance mechanism and compare the salt tolerance of superoxide dismutase (SOD) from different halophytes,we cloned the full-length cDNAs of manganese (Mn) and Cu/Zn SOD from Salicornia europaea by RACE method.Sequence analysis indicated that the S.europaea MnSOD gene (SeMSD) (GenBank accession number:JQ061158) comprised an open reading frame of 699 bp,encoding a polypeptide of 233 amino acids with a predicted molecular mass of 25.7 kD.Correspondingly,the Cu/ZnSOD gene (SeCSD,GenBank accession number:JQ061160) consisted of an open reading frame of 684 bp which encoded a protein of 228 amino acids with a predicted molecular mass of 23.3 kD.According to these two obtained cDNA sequences and Thellungiella halophila MnSOD gene (ThMSD) published on GenBank (EF140719),the prokaryotic expression vectors pET30a-SeMSD,pET30a-SeCSD and pET30a-ThMSD were constructed,and the target proteins were expressed successfully in BL21 Escherichia coli.Through optimizating the concentration of Isopropyl β-D-Thiogalactopyranoside (IPTG),the salt tolerance of these bacteria with the three superoxide dismutases were tested under 6.5% and 7.5% NaCl.The results demonstrated that the recombinants BL21 (pET30a-SeMSD) and BL21 (pET30a-ThMSD) strains showed better tolerance to salinity stress in comparison with the control stains BL21 (pET30a),but the recombinant BL21 (pET30a-SeCSD) strains displayed no salt tolerance increase.

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