Expression and Function of a Highly Conserved Water Stress Protein

New Mexico agriculture depends upon irrigation to avoid water deficit stress in agricultural crops. Targeted manipulation of plant genomes for enhanced salinity and water deficit tolerance through genetic engineering will require a sophisticated understanding of plant water stress proteins and their genes.

Photosynthetic cyanobacteria, the free-living relatives of chloroplasts, share with higher plants at least one ubiquitous water stress protein, dehydrin. The biochemical function of dehydrin is still unknown. Our goal has been to clone and sequence the cyanodehydrin gene. Knock-out mutagenesis would then afford an opportunity to study dehydrin function during water stress. Ultimately, these studies should empower rational improvements in water and salt-stress tolerance.

The 40 kilo-Dalton (kD) cyanodehydrin protein was purified and the sequence of 16 N-terminal amino acids was obtained from an immuno-positive 20 kD CNBr peptide. An expression library was constructed in lambda gt11 and screened with the affinity-purified antibodies. Three immuno-positive clones were identified. One of these three contains a 2.2 kb fragment that hybridized with a degenerate oligonucleotide probe based on the amino acid sequence information. Preliminary DNA sequence has identified the coding region for the peptide sequence in a 700 bp EcoR1-Sca1 fragment, thus unambiguously identifying this fragment as containing a portion of the cyanodehydrin gene. Three short regions of similarity to barley dehydrin (dhn5) were identified in the derived amino acid sequence for the cyanodehydrin using the computer similarity program, BLAST. One of these includes the sequence IKQKLP, similar to one of the barley dhn5 K-segments. Southern hybridization with the 700 bp EcoR1-Sca1 fragment identified a series of unique fragments in Anabaena 7120 genomic DNA, suggesting there is only one copy of the dehydrin gene in this organism. The entire 2.2 kb fragment identifies multiple bands in genomic southern blots, suggesting that a segment of DNA downstream of the dehydrin gene itself is part of a small gene family. Preliminary sequence from the repeated region shows similarity in BLAST searches to bacterial insertion sequences. Six genomic clones of 12-16 kb have been isolated using the 2.2 kb fragment to probe a lambda L47.1 library of Anabaena 7120 DNA. These will be used to determine the sequence of the 5′ end of the dehydrin gene which is not present on the 4b insert.