Complementary DNA cloning, sequencing and expression of an unusual dehydrin from blueberry floral buds

Levels of three major dehydrins of 65, 60, and 14 kDa have been observed to increase in blueberry (Vaccinium spp,) floral buds during chill unit accum ulation and cold acclimation and decrease during deacclimation and resumpti on of growth, Indeed, levels of the 65-, 60-, and 14-kDa dehydrins increase such that they become the most predominant proteins visible on sodium dode cyl sulfate (SDS)-polyacrylamide gels. The peptide sequence information fro m the 65- and 60-kDa dehydrins was used to synthesize degenerate DNA primer s for amplification of a part of the gene(s) encoding the dehydrins. One pa ir of primers amplified a 174-bp fragment, The 174-bp fragment was used to screen a cDNA library (prepared from RNA from cold-acclimated blueberry flo ral buds) and resulted in the isolation of a clone with a 2.0-kb insert. Th e cDNA was sequenced and found to be a full-length clone encoding a K-5-typ e dehydrin (5 K boxes). Five high-confidence peptide sequences, ranging fro m 9 to 25 amino acids long, obtained from the 60-kDa dehydrin exactly match ed sequences encoded within the cDNA clone. Furthermore, amino acid composi tion of the 60-kDa dehydrin agreed well with the expected amino acid compos ition based an the cDNA sequence. However, the DIVA sequence and coupled in vitro transcription/translation reactions of the cDNA clone indicated that it encodes a dehydrin with a native molecular mass of similar to 40 kDa in stead of 60 kDa, Experiments to determine if the dehydrins undergo post-tra nslational modifications revealed that the 65- and 60-kDa dehydrins are gly cosylated. Thus, our results indicate that the 2.0-kb dehydrin cDNA encodes the native version of the 60-kDa dehydrin. The dehydrin cDNA hybridized on RNA blots to two chilling/cold-responsive messages of 2.0 and 0.5 kb. Both the 2.0- and 0.5-kb messages increased to higher levels more quickly in th e cold-hardy cultivar Bluecrop than in the less hardy cultivar Tifblue. In addition, the 0.5-kb message remained at a higher level longer in Bluecrop than in Tifblue.