Cloning And Functional Analysis Of Drought And Salinity Tolerance Of Soybean LEA Genes

Late embryogenesis abundant (LEA) proteins are associated with desiccation tolerance in many plant species, bacteria, fungi and in nematode. A number of genes have been identified and their functions of the LEA proteins have been variously proposed. However, the supporting evidence has been lacking and their exact function is still not clear. In this study, six different groups of LEA genes were cloned from DAF 40-45d soybean (Bainong 6)seeds. Three of which, PM11(LEA1),PM30(LEA3), ZLDE-2(LEA2 )were choosed for further research. ZLDE-2 was a new member of soybean LEA gene family, and the GenBank accession number was AY351918. They were expressed in pET-28a(+) system, respectively. Moreover , PMll(LEAl) was introduced into tobacco. Both the recombinant plasmids in E.coli and the transgenic plants were treated by several osmotic stresses for further research.PM11 gene is composed of 357nt. It was deduced that it contains an open reading frame coding for 105 amino acid residues. The results of analysis by Biology Software were that the deduced protein was extremely hydrophilic. The gene products were confirmed by SDS-PAGE and mass spectrometry. The expressed PM11 protein was soluble and heat stability. The E.coli cells containing the recombinant plasmids showed an improved tolerance to various osmotic stresses.PM30 gene is composed of 507nt. It was deduced that it contains an open reading frame coding for 140 amino acid residues. The results of analysis by Biology Software were that the deduced protein was extremely hydrophilic. The gene products were confirmed by SDS-PAGE and mass spectrometry. The expressed PM30 protein was soluble and heat stability. The E.coli cells containing the recombinant plasmids showed an improved tolerance under high salt stress or low temperature stress conditions. Moreover, compared to control cells or other cells containing recombinant plasmids, PM30-expressing cells display the highest enhanced growth in high KC1 medium.ZLDE-2 gene is composed of 749nt. It was deduced that it contains anopen reading frame coding for 242 amino acid residues. The results of analysis by Biology Software were that the deduced protein was extremely hydrophilic. It was a new number of LEA gene family. The gene products were confirmed by SDS-PAGE and mass spectrometry. The expressed ZLDE-2 protein was soluble and heat stability. Whereas the E.coli cells expressing ZLDE-2 did not show the obvious growth improvement in various stresses conditions except high NaCl medium. These data demonstrated for the first time that that E.coli expression system would be a simple useful method on study the functions of LEA genes. It also revealed that PM11 and PM30 proteins both have protective functions in E.coli under several osmotic stress conditions.Plant expression vector Y46-121 were constructed and introduced into tobacco in order to study the function of PM11. Compared to control, transgenic tobacco improved tolerance under 1.5% NaCl medium. These give the experimental evidence on study salinity tolerance functional of PM11 gene.