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Complementary DNA cloning, sequencing and expression of an unusual dehydrin from blueberry floral buds
Author(s) -
Levi Am,
Panta Ganesh R.,
Parmentier Cecile M.,
Muthalif Mubarack M.,
Arora Rajeev,
Shanker Savita,
Rowland Lisa J.
Publication year - 1999
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1034/j.1399-3054.1999.100114.x
Subject(s) - cloning (programming) , biology , dna sequencing , dna , botany , computational biology , genetics , computer science , programming language
Levels of three major dehydrins of 65, 60, and 14 kDa have been observed to increase in blueberry ( Vaccinium smpp.) floral buds during chill unit accumulation and cold acclimation and decrease during deacclimation and resumption of growth. Indeed, levels of the 65‐, 60‐, and 14‐kDa dehydrins increase such that they become the most predominant proteins visible on sodium dodecyl sulfate (SDS)‐polyacrylamide gels. The peptide sequence information from the 65‐ and 60‐kDa dehydrins was used to synthesize degenerate DNA primers for amplification of a part of the gene(s) encoding the dehydrins. One pair 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 floral buds) and resulted in the isolation of a clone with a 2.0‐kb insert. The cDNA was sequenced and found to be a full‐length clone encoding a K 5 ‐type dehydrin (5 K boxes). Five high‐confidence peptide sequences, ranging from 9 to 25 amino acids long, obtained from the 60‐kDa dehydrin exactly matched sequences encoded within the cDNA clone. Furthermore, amino acid composition of the 60‐kDa dehydrin agreed well with the expected amino acid composition based on the cDNA sequence. However, the DNA sequence and coupled in vitro transcription/translation reactions of the cDNA clone indicated that it encodes a dehydrin with a native molecular mass of ∼40 kDa instead of 60 kDa. Experiments to determine if the dehydrins undergo post‐translational modifications revealed that the 65‐ and 60‐kDa dehydrins are glycosylated. 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 the 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.