Isolation and characterization of a harvest-inducible gene hi11 and its promoter from alfalfa

The harvesting and storing of alfalfa is a routine practice in the agricultural industry worldwide. To investigate gene expression in harvested alfalfa, cDNA from non-harvested and harvested plants in the field was subjected to subtractive hybridization to identify, in particular, those genes that are induced by the harvesting treatment. One cDNA clone, named hi11, was isolated and analysed. The full length cDNA of the hi11 gene was cloned by RACE amplification. The hi11 gene, which has high homology to a putative protein of unknown function in Arabidopsis, was induced in alfalfa following harvesting, a 38°C heat shock and a wounding treatment. Northern blot analysis confirmed that the expression patterns of hi11 in alfalfa in response to harvesting, heat shock, and wounding. In addition, genomic walking was performed to isolate the 5′ flanking region of the hi11 gene. The promoter of the hi11 gene was fused to the GUS reporter gene and transferred to Medicago truncatula and tobacco. In all transgenic plants of M. truncatula and tobacco, GUS gene expression was observed in harvested tissue, especially in the transgenic tobacco plants, but not in the non-harvested control tissue.