Isolation And Funcational Identification Of Salt Tolerance Related-genes GmA3 And GmA5 From Soybean

There are 95 hundred million hm2 salt in the world. It is about 10% of the terrene, and it is increases by 1.0-1.5 million hm2 every year. 6.7 million hm2 salt lands are soiled lands, and it is about 7% of the whole soiled lands. A lot of lands are becoming hypo-salt lands because of unsuitable irrigation, the destruction of vegetation, and the intrusion of seawater. The more content of salt in the soil will affect growth of plants, even leads to their withering and death. It will reduce the producties of crops and destroy the enviroment. So scientists emphasize on both soil improvement and increasing plant's salt tolerance through molecular breeding.Salt tolerance of plant are complex traits controlled by many genes and involved by many metabolize approaches. In recent years, there are some advances in mechanism of plant resistance to salt stress. Isolation of salt tolerance genes is one of the most difficult and innovative subjects in this field. It is important for us to isolate salt tolerance genes, especially new genes, and elucidate genes and their products reciprocity. It is also a important part anti-stress functional genomic. These works contribute to academic and practical researches.Though functional screening, 28 transformants are isolated from plates containing high concentration salt and identified by PCR when transforming E.coli by cDNA library from immature embryo of anti-salt soybean. The plasmids extracted from recombinants and transform host E.coli again. 5 transformants are confirmed the anti-salt clones. We choose two clones they growth better and name them transformantsXLOLR/pBK-GmA3 and XLOLR/pBK-GmA5 to deeply research.Transformant XLOLR/pBK-GmA3 could survive on plate with HOOmM NaCl but on that control could not survive. Growth curve of transformant XLOLR/GmA3 is distinctly better than that of control in LB broth with lOOOmM NaCl, 700mM KC1, 180mM MgCl2. It has shorter lag phase and more growth. GmA3 of soybean has 369bp OFR coding 122 amino acids. Its ammo acids sequence has 61% homology with HEN4 and KH domain protein of Arabidopsis thaliana. So it is a novel gene. Amino acids sequence of soybean GmA3 contains a KH domain found in many species. It could bind RNA or DNA and contribute to regulation of cell cycle. It is speculated that KH domain is functional part in soybean GmA3 and contributes to salt tolerance of transformant in osmotic stress.Transformant XLOLR/pBK-GmA5 could survive on plate with HOOmM NaCl. GmAS of soybean has 756bp OFR coding 251 amino acids. Its amino acids sequence has high homology with soybean Sali3-2 protein that induced by aluminum in root. It is a reported gene with unknown function. Subclone BL21/pET-GmA5 was constructed with high expression. Growth curve of this subclone has is distinctly better than that of control in LB broth with 800mM NaCl, 800mM KC1, 220mM MgCl2 with shorter lag phase and more growth. Amino acids sequence of soybean. GmA5 contain a BURP domain found in many species. Many proteins with BURP could response to stress or found in high stage of development. It is speculated that BURP domain is functional part in soybean GmAS and contributes to salt tolerance of transformant in osmotic stress.