| Soybean is one of the world's important food crops, also China's major oil crops. Soybean which was moderately salt-tolerant plants was significantly affected by saline conditions, yield in saline conditions decreased significantly. China was one of the salinization widespread countries, and because improper irrigation, vegetation damage and water intrusion and other factors, resulting in the area of salinized land is constantly increasing every year. Therefore, breeding tolerant varieties is particularly urgent. Suppression subtractive hybridization (SSH) technology is effective ways in study of genes expression differences, and in most plants have been successful. However, in the library will be screening a large number of EST sequences to obtain the effective number of genes, and the screening was less efficient. Although the mechanism of salt-related information with understanding more in-depth, the basic theory of the mechanism of salt tolerance is not enough depth. To better understand the soybean molecular mechanism of response the salt stress and clone of a number of important salt tolerance gene.In this study, we constructed of soybean genotypes tolerant of salt induced suppression subtraction library (SSH1) by Wenfeng 7 (salt-tolerant varieties) and salt-sensitive soybean genotypes with the expression of salt-induced suppression subtractive library (SSH2) by Union (salt-sensitive varieties). Used the secondary PCR products of SSH1 and SSH2 library, constructed the third library (SSH3) after once again by suppression subtractive hybridization. In three libraries were randomly selected 600 positive clones, after sequencing, 1,500 high-quality EST sequences were obtained, 513 from the SSH1, 453 were from SSH2, 534 were from SSH3. Submitted to GenBank's dbEST database, and obtained accession number for the HO759947-HO761446.After sequence analysis, we selection 16 potential genes associated with salt tolerance. Real-time PCR used to analyze gene's expression in different soybean varieties after salt treatment. The results show that the abundance of these genes were differences in the expression after salt treatment in Wenfeng 7 and Union.Dot blot hybridization method was used to detect the same and similar EST sequences in the SSH1 and SSH2 library. Hybridization results showed that the library SSH3 can eliminated the same and similar EST sequences from the SSH1 and SSH2 library. Analysis of the EST sequences from SSH1 and SSH3 library by GO annotation and COG functional classification. The results show that the library SSH3 can eliminated the gene of growth and defense mechanisms. Therefore, the library SSH3 really enriched salt-related genes and also improved the efficiency of screening.We selected two candidate genes from highly salt tolerance relevant EST sequences. Compared to EST databases of the NCBI, predicted and cloned two genes. Bioinformatics analysis revealed that they were with the Thioredoxin and Resistance to sodium, lithium ion gene highly homology and were named GmTrx and GmSLT. Previous studies also found that two genes related to improve plant salt tolerance. We constructed GmSLT gene root transformation of sense, missing and antisense expression vector. In order to use the soybean root system to investigative these genes function. The results showed that, over-expression of GmSLT gene can increased the transgenic hairy roots of soybean and soybean complex plants salt tolerance. |
