Cloning And Differential Gene Expression Of TsMT In Thellungiella Salsuginea In Response To Salt Stress And The Study On Agrobacterium-mediated SsNHX1 Transformation For Soybean (Glycine Max L.)

The soil salination is a significant abiotic stress for agricultural production and ecological environment and the research on salt tolerance represents an important part for basic plant biology. Studying the salt-tolerance mechanism of euhalophytes and improving plant ability of salt tolerance by genetic engineering breeding has become one of the central issues.Thellungiella salsuginea is an euhalophyte that may have evolved the unique salt-tolerance mechanism and can complete its life history normally in soil containing 100mmol . L-1.NaCl.The results of many studies suggest that plant salt tolerance is usually correlated with a more efficient antioxidant system. Plant has evolved protection mechanism which contains enzymatic reaction system and no-enzymatic reaction system. There may have distinct difference on the scavenging of reactive oxygen species (ROS) between euhalophyte and glycophyte. So cloning those genes that encode proteins engaged in scavenging ROS from Thellungiella salsuginea may attribute to get insight into the mechanism of plant salt tolerance. Metallothionein(MT) is a salt-induced protein and a good scavenger of hydroxyl radical. Its ability of scavenging hydroxyl radical derives from reduced - SH group in MT. In the first part of this experiment, we isolated the TsMT gene ( GeneBank accession number :AF499726. 1 )from a A Zap-cDNA library constructed from a 200mmolL-1 NaCl-treated library of Thellungiella salsuginea and analyzed its sequence characterization, genomic organization and the differential expression in response to salt stress. TsMT encodes metallothionein and there is only one copy in Thellungiella salsuginea genome according to its Southern Blotting. The expression of TsMT was slightly induced by saltstress and the activity of MT increased gradually in response to salt stress .The result indicates that TsMT may play an important role in protecting Thellungiella salsuginea from the oxidation damage.The maladies caused by salt stress arise from the disruption of cellular aqueous and ionic equilibrium. Na+ accumulates excessively in the cytoplasm can be toxic to the metabolism and affects the regulation mechanism of the plant cell. To cope with salt stress, plants have developed a variety of adaptation mechanisms. The mechanisms of ion homeostasis of some halophytes include extrusing Na+, sequestering Na+ into the vacuolar and regulating the ratio of KV Na+, and Na+/H+antiporter plays an important role in these processes. Soybean is a generally acknowledged recalcitrant crop for transformation. The regeneration system of soybean cytoledon node and Agrobacteriunr mediated transformation method is the first selection at present. In the second part of this experiment, the expression vector pROK2 containing NPT II and SsNHX1 (Na+/H+ antiporter) gene from suaeda salsa was introduced into soybean cytoledon nodes by gene transformation mediated by Agrobacterium tumefaciens , and kanamycin resistant transgenic plants were obtained by screening in selective condition. The suitable concentration of the selective agent kanamycin is 50mg. L-1. The duration of infection and co-culture with Agrobacterium influences soybean transformation evidently. The suitable infection duration is about 15 minutes, and the appropriate co-culture duration is 3 days. Both the concentration of acetosyringane and pH value at stages of infection and co-culture influence the transformation frequency of soybean. The suitable pH value is 5. 2 and the optimum concentration of acetosyringane is 150 mol.L-1.