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盐角草Cu/Zn-SOD基因的克隆及耐盐性分析
臧洁1,余梅2,王先磊2,王荣富2,李娟2,吉虎2,徐凯2,鲁茂龙3
1. 安徽农业大学
2.
3. 昆山科腾生物科技有限公司
Cloning and Salt-tolerance Analysis of the Salicornia europaea Gene Cu/Zn-SOD
1, 1, 1, 1, 1, 1, 1,
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摘要 盐角草(Salicornia europaea)是一种典型的耐盐植物,为了研究盐角草Cu/Zn-SOD基因在的盐胁迫耐受中的机制,本研究利用已知植物Cu/Zn-SOD基因的保守序列设计简并引物,采用RACE技术的方法从盐角草中扩增获得Cu/Zn-SOD基因。使用生物学软件分析其氨基酸序列,并进行同源性比对。构建原核表达载体,转化大肠杆菌(Escherichia coli),使目的蛋白在重组菌中表达,并分析了不同盐浓度并含有抗生素的液体LB培养基中菌的生长情况,IPTG诱导表达,通过测定OD600值来分析Cu/Zn-SOD基因的耐盐功能。结果通过简并引物PCR扩增和RACE技术,克隆出盐角草Cu/Zn-SOD基因。盐角草Cu/Zn-SOD基因(GenBank登录号: JQ074238.2)全长为660 bp,开放阅读框长为459 bp,推测编码152个氨基酸,蛋白分子量约为15.1 kD,其氨基酸序列与碱蓬(Suaeda salsa)的序列相似性为96%,与黄灯笼辣椒(Capsicum chinense)的序列相似性为88%。生物学软件分析表明Cu/Zn-SOD蛋白可能存在于细胞质。构建原核表达载体pET-Cu/Zn-SOD和对照pETDuet-1,转化大肠杆菌BL21中。蛋白经IPTG诱导表达。经SDS-PAGE蛋白电泳检测发现表达蛋白条带大小与预期一致,说明目的蛋白成功表达。耐盐性分析表明重组菌BL21(pET-Cu/Zn-SOD)在高盐度培养基中的生长明显优于对照菌BL21(pETDuet-1),说明盐角草Cu/Zn-SOD基因可能在盐胁迫逆境中起到耐受性作用。
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臧洁
余梅
王先磊
王荣富
李娟
吉虎
徐凯
鲁茂龙
关键词 盐角草超氧化物歧化酶基因克隆耐盐性    
Abstract:In order to study the salt-tolerant mechanism of the Salicornia europaea gene Cu/Zn-SOD, conserved sequences of Cu/Zn-SOD from the known plants were designed for degenerate primers in this research. The coding region of Cu/Zn-SOD was amplified by RACE technology. Amino acid sequences and multiple sequences alignment were analyzed by biological software. The gene carried by prokaryotic expression vector pet-duet1 was transferred to Escherichia coli BL21 and expressed after IPTG induction. The status of recombinants cultivated in LB medium with different NaCl concentrations were observed. The salt-tolerant function of Cu/Zn-SOD was assessed by OD600 value. As a result, the Salicornia europaea gene Cu/Zn-SOD was amplified by degenerate primers and RACE technology. The full-length of Cu/Zn-SOD cDNA(GenBank number: JQ074238.2) was 660 bp, containing a 459 bp open reading frame (ORF). The ORF encoded a 152-amino acid polypeptide with a predicted molecular mass of 15.1 kD. The predicted amino acid sequences showed 96% similarity with the corresponding Suaeda salsa gene sequences and 88% similarity with the Capsicum chinense gene sequences. Biological software analysis implied that the Cu/Zn-SOD protein existed in cytoplasm. We constructed a prokaryotic expression vector pETCu/Zn-SOD, contrasted by control pETDuet-1, and transformed them into Escherichia coli BL21. Protein was expressed by IPTG induction. The target protein was successfully expressed, as the molecular mass of it accorded with theoretical value by SDS-PAGE electrophoresis. Salt-tolerance analysis showed that recombinant bacteria BL21 (pET-Cu/Zn-SOD) grew significantly better in the high salinity mediums than that of the control bacteria BL21 (pETDuet-1). It indicates that the Salicornia europaea gene Cu/Zn-SOD may play an important role in the salt stress.
Key wordsSalicornia europaea    Superoxide dismutase(SOD)    Gene cloning    Salt tolerance
收稿日期: 2013-04-15     
ZTFLH:  Q785  
通讯作者: 鲁茂龙   
引用本文:   
臧洁1,余梅2,王先磊2,王荣富2,李娟2,吉虎2,徐凯2,鲁茂龙3. 盐角草Cu/Zn-SOD基因的克隆及耐盐性分析[J]. , 2013, 21(7): 847-854.
1, 1, 1, 1, 1, 1, 1,. Cloning and Salt-tolerance Analysis of the Salicornia europaea Gene Cu/Zn-SOD. , 2013, 21(7): 847-854.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2013/V21/I7/847
 
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