Cloning And Functional Analysis Of Superoxide Dismutase (SOD) Genes From Suaeda Salsa And Salicornia Europaea

Soil salinity, which has serious effect on crop growth and yield, is one of the major factors limiting agricultural production. China is one of the countries which have large area of saline-alkali soil, the saline-alkali soil is widespread, and its area comes to34.6million hm2. So how to develop the saline-alkali soil resource and increase the crop production have become an urgent problem. At present, isolation salt-tolerance genes and overexpression in transgenic plants to breed new varieties through biotechnology method have become an effective approach to improve saline-alkali soil. Superoxide dismutases (SOD) are metalloenzymes which exist widely in plant、animal and microorganism, and important antioxidant enzymes of active oxygen removal system. Many studies found that the activity of SOD is closely related to plant stress tolerance, and the overexpression of SOD genes can enhance the plant resistance against salt stress obviously. In this study, we characterized the Mn-SOD and Cu/Zn-SOD gene from Salicornia europaea and Suaeda salsa by RACE technology; the prokaryotic expression vectors were constructed and transferred to E coli BL21(DE3) for protein expression; through cultivating the recombinants in LB medium with different NaCl concentrations, we detected the salt tolerance of these5SOD genes. The main results are as follow:1. we characterized the Mn-SOD and Cu/Zn-SOD gene from Salicomia europaea and Suaeda salsa by degenerate primers amplification and RACE technology. The full-length SsMSD cDNA (GenBank number:JQ061157) is1050bp long containing a699bp open reading frame (ORF), the ORF encodes a233-amino acid polypeptide with a predicted molecular mass of25.9KDa. The putative amino acid sequences of SsMSD shows84%identify with the Gossypium hirsutum and Hevea brasiliensis sequences. Transit peptide and subcellular location analysis indicate the Suaeda salsa Mn-SOD is a mitochondrial SOD gene. The full-length SsCSD cNDA (GenBank number:JQ061159) is843bp long including a459bp ORF, the ORF encoded a153-amino acid polypeptide with a predicted molecular mass of15.1KDa. The predicted SsCSD protein exhibited94%identify with the Spinacia oleracea sequence and92%with the Mesembryanthemum crystallinum cytoplasmic Cu/Zn-SOD sequence. The full-length SeMSD cDNA (GenBank number: JQ061158) is1053bp long and contains a699bp open reading frame encoding233amino acids, and the amino acid polypeptide has a predicted molecular mass of25.7kDa. The deduced SeMSD shows highest identity with the enzymes from Gossypium hirsutum (83%) and Tamarix androssowii (81%) at the amino acid level. Transit peptide and subcellular location analysis imply it may be mitochondrial SOD gene. The full-length SeCSD cDNA (GenBank number:JQ061160) is985bp long and includes a684bp open reading frame encoding228amino acids with a predicted molecular mass of23.2KDa. The putative Cu/ZnSOD protein exhibits81%identity with the Spinacia oleracea Cu/ZnSOD, and transit peptide analysis indicates it is a chloroplast SOD gene.2. According to the obtained cDNA sequences and ThMSD cDNA (GenBank accession number:EF140719), the primers with Nedl and EcoRV sites were used to amplify the entire ORF. We constructed three prokaryotic expression vectors pET30a/SsCSD pET30a/SsMSD、pET30a/SeCSD、pET30a/SeMSD and pET30a/ThMSD, and transformed to E. coli BL21(DE3). SDS-PAGE analysis indicated that the molecular mass of these5expressed proteins were consistent with the predicted molecular weight.3. Considering the growth inhibition derived from the excess IPTG, we optimized the IPTG concentration and confirmed that the SOD proteins expressed well, the growth can not be affected adversely simultaneously. We chose0.025mM as the optimal IPTG concentration. The same amount of recombinants and control strains were inoculated into the liquid LB (0.025mM IPTG,50mgL-1Kan) with6.5%,7%,7.5%,8%NaCl concentrations, and detected the OD600. The result showed that the recombinants BL21(pET30a/SsMSD)、BL21(pET30a/SeMSD) and BL21(pET30a/ThMSD) displayed better tolerance to salinity stress in comparison with the control stain, which implied that the SsMSD、SeMSD and ThMSD genes may play an important role in plant defense against salt stress.