Identification of genes induced by salt stress from Medicago truncatula L. seedlings

Abstract

In order to identify genes induced during the salt stress response in barrel medic (Medicago truncatula L) seedlings, a cDNA library by salt stress was constructed  by suppression subtractive hybridization (SSH). Total RNA from 15-day-old seedlings was used as a ‘driver’, and total RNA from seedlings induced by salt was used as a ‘tester’. One hundred and sixty nine clones identified as positive clones by reverse northern dot-blotting resulted in 75 uni-ESTs that comprised of 13 contigs  and 62 singletons. Basic Local Alignment Search Tool (BLAST) analysis of deduced protein sequences revealed that 35 expressed sequence tags (ESTs) had identity similar to proteins with known function, while 27 could not be annotated at all. Most of the known function sequences were homologous to genes involved in abiotic stress in plants. Among these protein, citrate synthase, ribulose- 1,5-bisphosphate carboxylase, chloroplast protein, phosphoenolpyruvate carboxylase and  chloroplast outer envelope protein are related to photosynthesis; DNA binding/transcription factor, putative AP2/EREBP transcription factor, Cab9 gene, photosystem II polypeptide and calcium-dependent protein kinase play a significant role in signal transduction and transcription regulation; and aldolase and sucrose synthase are interrelated to osmolyte synthesis. Moreover, 5 of the ESTs, similar to genes from other plant species and closely involved in salt stress were isolated from M. truncatula L. They are superoxide dimutase (SOD)-1, gene for copper/zinc superoxide dismutase, cysteine protease, Na+/H+ antiporter and salt overly sensitive 2 (SOS2). To further assess the expression level of salt-induced ESTs, real-time polymerase chain reaction (PCR) analysis was employed, and the result showed that these genes have significantly increased expression and probably play an important role in the response of plants to salt stress.

Key words: Barrel medic (Medicago truncatula L.), suppression subtraction hybridization (SSH), reverse northern dot-blotting, salt stress, real-time polymerase chain reaction (PCR).