| Land resources in the world are increasingly suffering from drought, weathering, soil salinization and so on many kinds of stress. Our country has widely saline-alkali land. The area of saline-alkali land is about0.33billion hectares and it is still expanding. The salt stress problem is getting worse. The high salt concentration in the soil caused irreversible damage to many of the plants, restrained the growth of the plants, and even caused the death of plants. Countries around the world control and improve of saline-alkali land, mainly through such as irrigation agriculture, physical chemistry and biotechnology measures. At present, by studying the salt tolerance mechanism of plants, using biotechnology methods to clone salt tolerance gene form the salt-tolerant plants and cultivating salt tolerance of the new crop varieties, can effectively solve the negative influence of soil salinization. Salicornia europaea is belong to Chenopodiaceae plant, is annual low and herb plant. Salicornia europaea born in the plain desert area or on the moist saline soil of the lake. They are typical salt-tolerant plants. The principle of Salt-tolerance is concerned by more a growing number of researchers. Superoxide dismutases(SODs) are metalloenzymes which exist widely in organisms with catalyzing the dismutation of superoxide anion (O2-) to O2and H2O2to scavenge reactive oxygen species. Previous studies have found that SODs play important role in medicine, food, health care and cosmetics and have wide range of applications. SODs have important activity in the defense injury and connect with varieties of diseases. They are closely related to the resistance of plants.In this study, Salicornia europaea is selected as the salt-tolerant plants. Cu/Zn-SOD gene was cloned by RACE method from the Salicornia europaea, used of biological software to analysis the nucleotide and amino acid sequence and carried on the homologous comparison. The prokaryotic expression vectors were constructed and transferred to E.coli BL21for protein expression and analyzed through cultivating the recombinants in LB medium with different NaCl concentrations. The salt-tolerance function of Cu/Zn-SOD gene was analyzed by measuring the wavelength of OD600value. For future to study the application and in plant tolerance of the Cu/Zn-SOD gene from Salicornia europaea lay the foundation, such as cultivating new varieties of salt-tolerant crops. The main results of this experiment are as follows:1. The Cu/Zn-SOD gene from Salicornia europaea was coned by degenerate primers amplification and RACE technology. The full-length Cu/Zn-SOD cDNA (GenBank number:JQ074238.2) is660bp long, containing a459bp open reading frame (ORF). The ORF encodes a152-amino acid polypeptide with a predicted molecular mass of15.1KDa. The predicted amino acid sequences shows96%similarity with the Suaeda salsa sequences and88%similarity with the Capsicum chinense sequences. Biology software analysis indicates that the Cu/Zn-SOD protein may present in the cytoplasm.2. According to the obtained cDNA sequences of the Cu/Zn-SOD gene design primer with BamHI and NotI enzyme sites respectively to amplify the coding region. We constructed a prokaryotic expression vector pET-Cu/Zn-SOD and contrast pETDuet-1, transformed into BL21. Protein was induced by IPTG to express. By SDS-PAGE electrophoresis detecting protein band size in line with expectations showed that the target protein was successful expressed.3. The recombinant bacteria and control bacteria were inoculated into the liquid LB medium with1%,5%,6%,7%salt concentrations. After culturing bacteria for10hours detected the OD600every3hours. The result showed that the recombinant bacteria displayed better tolerance to salinity stress in comparison with the control bacteria and increased salt tolerance is about1-3times. This implies that the Cu/Zn-SOD gene from Salicornia europaea may play an important role in plant salt tolerance. |
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