EcNH Gene Enhances Alkali Tolerance Of Transgenic Soybean

Jilin Province is known as"the homeland of soybeans"for its large planting area and high yield.However,the growth,distribution and yield of soybean were limited by the large area of soda salt soil in the central and western regions of Jilin province.How to improve the alkali tolerance of soybean and grow in salt soil is an important research topic in soybean breeding.Due to the lack of effective alkali resistant germplasm resources and long breeding years,the traditional breeding methods have made slow progress.In this study,the alkali resistant gene EcNH from Escherichia coli was introduced into the soybean cultivar Williams 82 through Agrobacterium mediated high-efficiency genetic transformation of soybean cotyledon nodes and the alkali tolerance soybean plants were obtained.The main results are as follows:?1?The EcNH gene,originated from Escherichia coli,is Na⁺/H⁺antiporter gene.Bioinformatics analysis of the EcNH gene showed that the total length of the gene is 1179 bp and encode 388 amino acids.The molecular weight of the protein is 41 k Da,and the protein has 11 transmembrane helices and a typical hydrophilic region at 200-300 amino acids.?2?The plant expression vector pTF101.1-EcNH was constructed by using Xba I enzyme and Eco RI restriction enzyme.In this study,total 5320 soybean explants were infected by using Agrobacterium strains,EHA101,and 360 transformed plants were obtained,and the plant regeneration rate was about 6.8%.?3?The obtained transformed plants were detected by PCR,RT-PCR,Southern blot and Quick PAT/bar strip detection methods.A total of 125 positive transgenic plants were obtained with a plant conversion rate of about 2.3%.?4?Alkali tolerance identification of genetic soybeans.T3 transgenic soybean and non-transgenic soybean were treated with 60 mmol·L^-1 Na HCO₃ stress.The photosynthetic parameters including chlorophyll content,intercellular CO₂?Ci?,transpiration rate?E?,stomatal conductance?Gs?,net rate of photosynthesis?Pn?were observed,and the physiological parameters malondialdehyde content?MDA?,proline content?PRO?,soluble protein content,soluble sugar content,superoxide dismutase?SOD?,peroxidase?POD?and catalase?CAT?activities were detected.The results showed that under alkali stress,the leaves of transgenic plants maintained significantly higher photosynthetic efficiency than non-transgenic plants by enhancing the absorption and utilization of weak light.The osmoregulation substances accumulated in leaf and roots,especially proline and soluble protein in roots,of transgenic plants were significantly higher than that in non-transgenic plants.SOD,POD and CAT enzyme activities of the transgenic plants increased significantly than non-transgenic plants,and therefore MDA content decreased significantly.In this study,the transgenic plants were resistant to 60 mmol·L^-1 Na HCO₃.Compared with the control plants,the alkali resistance of the transgenic plants was significantly improved.