| In previous works, it were proven that overexpression of GmDREB5 can enhance stress tolerance of transgenic plants. In order to study regulating mechanism of GmDREB5 protein, we cloned some genes encoding proteins interacting with GmDREB5 using yeast two hybrid. GmE2 was one of interacting proteins with GmDREB5. In this research, GmE2 was further studied. GmE2 was ubiquitin conjugating enzyme, which involves ubiquitin protein degradation system dependent on the ATP. In general, ubiquitin protein degradation system contains E1, E2 and E3 three key proteins and GmE2 belonged E2 enzyme. On other hand, a stress-related gene GmNAC2a was isolated from salt-tolerance soybean variety TieFeng 8 using NTT system (Nuclear Transportation Trap). GmNAC2a belongs to NAC transcription factor family which is unique in plants and plays an important role not only in regulation of growth and development of plants but also in responses to biotic or abiotic stresses. The characteristics and functions of GmE2 and GmNAC2a were showed as following:1. The GmE2 gene contains 462bp and encodes 153 amino acids. The 89th cystein residue was anticipated as active site. It has the UBC domain from 7th to 146th amino acids residue, and the 8th, 65th, 66th, 99th, 100th amino acid residues were anticipated as interaction sites with the E3 ubiquitin ligase.2.The results of subcellular localization and expression pattern analysis of GmE2 showed that GmE2 protein located in the plant nucleus and expression of GmE2 were induced by drought, low temperature, ABA, ethylene stress conditions, but not by the high temperature and high salt stress conditions.3. GmE2 was transformed into Arabidopsis and tobacco using Agro bacterium-mediated method. The functional analysis showed that tolerance to high temperature and high salt stresses between transgenic plants and wild type plants were similarly, whereas under drought stress condition, germination percentage of transgenic tobacco seeds was higher than that of wild type, which suggesting that overexpression of GmE2 can enhance drought tolerance of transgenic plants, whereas it didn't affect tolerance of transgenic plants to high temperature and high salt stresses.4. The GmNAC2a contains 900bp encoding 299 amino acids with a conserved NAM domain in N terminal of protein. The phylogenetic analysis showed that the GmNAC2a belongs to the ATAF subfamily in NAC family, and GmNAC2a had the highest homology with a member of NAC transcriptional factor family, AhNAC1 in Arachis hypogaea (76.47%).5. The results of subcellular localization and expression analysis of GmNAC2a showed that GmNAC2a located in the plant nucleus and expression of GmNAC2a were induced by drought, high temperature, low temperature, salt, ABA and ethylene stresses, and was most sensitively to the ethylene, which suggesting that GmNAC2a might be the crosstalking point and involved in both abiotic responsive pathway and biotic stress response pathways related with ethylene.6.The GmNAC2a was transformed into tobacco and it were proven by PCR or RT-PCR that GmNAC2a was inserted in plant genome and expressed in transgenic plants. The functional analysis showed that tolerance to high salt stress between transgenic plants and wild type plants were similarly, whereas under high salt stress condition, phenomenon of flower early emerged in transgenic plants and some tuft shoot grow up on top of stem. We suggested as published papers that GmNAC2a might involved in regulation of development in plant and induced growth of apical meristem.In this research, two important stress-related genes GmE2 and GmNAC2a were isolated from soybean. The results of expression analysis showed that those two genes involved in all kinds of stress responses in plants. The results of characteristics and functional analysis will be contributed to understand regulation mechanism of GmDREB5 protein. The functional analysis of GmNAC2a will be as basic to select novel candidate gene for crop stress improvement in the further. |
