To gain a better understanding of differentially expressed sequence tags (ESTs) for aluminum (Al) tolerance and to investigate the molecular mechanisms of Al toxicity, cDNA subtraction libraries were generated from Al-stressed roots of alfalfa (Medicago sativa L.) compared with no Al-stressed ones, employing suppression subtractive hybridization. Using differential screening technique in which the probes were labeled with DIG, we identified 45 non-redundant ESTs in Al-stressed alfalfa root tips with significantly altered expression. Among the up-regulated ESTs, we have found genes encoding identified proteins, including malate dehydrogenase, 6-phosphogluconate dehydrogenase, peroxidase, and an ABC transporter, while the down-regulate genes included ATPase, secretory carrier membrane protein 2, pectinesterase inhibitor. In addition, two novel ESTs, EW678752 and EY976957, up- and down-regulated by Al stress were sequenced. Analyzed by real-time PCR, the expressions of EST EW678718, EW678739, EY976969 and EW678728, which encode for ABC transporter, malate dehydrogenase, peroxidase and 6-phosphogluconate dehydrogenase correspondingly, increased 1.64-, 2.75-, 3.27- and 6.54-folds, respectively, and the expression of EY976957 encoding for ATPase decreased 3.27 folds. The expression of EST EW678752 increased 34.54-fold, while that of EY976957 decreased 16.68 folds. It suggested that the two novel ESTs maybe play a significant role in the aluminum tolerance of alfalfa.
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