Isolation of Salt Stress Tolerance Genes from Roots of Mangrove Plant, Rhizophora stylosa Griff., Using PCR-Based Suppression Subtractive Hybridization

Abstract

Salinity is a major abiotic stress that seriously limits plant growth and crop productivity. The halophytic Rhizophora stylosa is useful for the study of the molecular mechanisms behind salinity tolerance in mangrove trees. To isolate anti-salt stress genes from mangrove plants, a cDNA library of R. stylosa roots was constructed and screened for stress-related genes by polymerase chain reaction (PCR)-based suppressive subtractive hybridization (SSH). The mangrove seedlings were grown for 8 months under two conditions: exposure to water with 3% salt and exposure to fresh water. cDNA of seedlings exposed to water with 3% salt was used as a tester and cDNA for freshwater germination was used as a driver. We isolated and sequenced 240 up-regulated expressed sequence tags (ESTs) from the SSH library. Among these up-regulated ESTs, 48 unique clones were putatively identified and classified into ten functional categories, such as cell rescue and defense, secondary metabolism, protein synthesis, and metabolism. Fifteen genes from different categories were selected and their expression was studied by real-time RT-PCR. Significantly increased expression levels were confirmed for 13 of these 15 transcripts, which suggest that these genes contribute to the salt tolerance of this plant. Among them, two transcription factors and several genes involved in isoprenoid biosynthesis were identified from mangrove trees as salt tolerance genes for the first time. The physiological significance of the increased expression of these genes in the long-term adaptation of mangrove trees to salt stress is discussed.