A secondary suppression subtractive hybridization method for isolation and identification of some salt-induced genes in soybean (Glycine max L. Merr)

The yield of soybean (Glycine max L. Merr.) has been seriously challenged by increased salinization of the land. Continuous efforts are needed to understand the salt tolerance mechanism in soybean using suitable biotechnological tools. Although the suppression subtractive hybridization (SSH) method was a powerful technique and has been successful in some cases, it may retain some less effective genes which make screening difficult. In order to enrich the target sequences and improve screening efficiency, a secondary subtraction was implemented. In this study, three SSH cDNA libraries were constructed and 1500 clean EST sequences were obtained from forward SSH libraries for further analysis. Using real-time PCR, the expression of 16 representative candidate genes was examined and their differential expressions in NaCl treated Wenfeng7 (salt-tolerant) and Union (salt-sensitive) was confirmed. Dot blot hybridization analysis showed that the retention of common EST sequences were significantly reduced or eliminated in the SSH3 library. GO (gene ontology) annotation and COG (cluster of orthologous group) functional classification revealed that development-related genes and defense mechanism genes were removed from the SSH3 library and salt-tolerance related EST sequences were further enriched, suggesting that the secondary subtraction is a simple and effective way to enrich response-specific genes and improve screening efficiency.