| Soybean is one of the most important crops in China, which serves as the primary source of plant proteins for both human consumption and as fodder. Cultivated soybean is middle sensitive to saline. Its salt-sensitivity threshhold is 5.0ds/m. Soybean plants will be die if the electric conductivity of irrigated water is over 6.7ds/m. The salt tolerance capability of soybean limited its growing area especially in saline land and arid and semiarid areas. Little progress has been achieved in generating cultivars with enhanced tolerance to stresses and good adaptability by traditional breeding. Soybean genetic transformation has been projected as an option to realize this goal. However, low efficiency of soybean transformation imposed great restriction on its application in gene function investigation and soybean improvement. Because of its rapidity and technical simplicity, Agrobacterium rhizogenes-mediated transformation has become a powerful tool to study gene function and root biology in plants. This transformation is particularly suitable for those plants including legumes whose transformation using Agrobacterium tumefaciens has been challenging for many laboratories.In this reseach, three plant overexpression vectors GFPGUSPlus-GmNHX1, GFPGUSPlus-GmCLC1 and GFPGUSPlus-GmPAP3, respectively harboring GmNHX1, GmCLC1 and GmPAP3 gene was constructed. To enhance salt tolerance of soybean, the transformation of GmNHX1 gene which encodes soybean Na+/H+ antiporter protein, GmCLC1 gene which encodes soybean Cl-/H+ antiporter protein, purple acid phosphatase GmPAP3 gene, into soybean roots was conducted with the mediation of Agrobacterium rhizogenes in the present study. Hpt screening, GUS and RT-PCR analysis confirmed that GmNHX1, GmCLC1, GmPAP3 gene were successfully integrated into the soybean hairy roots. Studying the capability of salt tolerance of the transgenic hairy roots under different salt stress environment, it provided a theoretical and applied foundation for evaluating the breeding utility value of candidate gene in objective crops. Analysis of salt tolerance of transgenic hairy roots showed that the length and weight increments of in vitro transgenic hairy roots on solid or liquid culture medium were superior to that of the non-transgenic control at 100, 150 and 200 mmol·L-1 NaCl. Both cotyledons and composite plants which produced hairy roots had stronger capacity to survive than the control on solid culture medium with salinity stress. The results further proved that the overexpression of GmNHX1, GmCLC1and GmPAP3 gene could enhance the salt tolerant of soybean roots, which provides a confident evidence for improving soybean salt tolerance by the use of GmNHX1, GmCLC1and GmPAP3 gene.To confirm the relations between the GmCLC1 gene and the plant salt endurance further, the GmCLC1 gene was introduced into Lotus corniculatus cultivar Superroot by Agrobacterium rhizogenes-mediated transformation system highly effective. The Gm CLC1 gene has been successfully conformed to the transgenic Lotus corniculatus after Hpt screening, PCR identification and GUS transient expression assay. Studying on salt tolerance of transgenic plants on solid culture medium, the results showed that overexpression the GmCLC1 gene had increased the 150mmol/L NaC1 salt endurance of transgenic Lotus corniculatus adult plants, thus had proven this conversion system might study the platform as the gene function the feasibility.Our results suggested that GmNHX1, GmCLC1, GmPAP3 gene have function on soybean and can improve its capability of salt resistance. This also implies that gene function analysis can be done only in one tissue or in one organ. If soybean shoot can be induced from the hairy root, then the efficiency of soybean transformation must be improved greatly. And it will be of great significance for soybean molecular breeding.
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