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| Cloning and Salt-tolerance Analysis of the Salicornia europaea Gene Cu/Zn-SOD |
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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.
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Received: 15 April 2013
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