Alterations in Gene Expression during Exposure of Bermudagrass to Low, Non-lethal Temperatures

Abstract

Temperature is a limiting factor for plant growth. Warm-season turfgrasses can experience winter-kill when grown in the “transition zone.” On the other hand, when properly cold-acclimated, these same plants can withstand otherwise lethal temperatures. As part of our investigations into the biochemistry and molecular biology of cold acclimation in bermudagrass, total RNA from crowns (rhizome buds) isolated at different timepoints before and after chilling temperature exposure, was isolated by salt-buffer/phenol extraction, followed by LiCl precipitation and DNAse treatment. Differential display reverse transcriptase polymerase chain reaction (DD-RT-PCR) was performed using specific-(dT11NN) or variable-(dT12VN) anchor primers (where V = dA, dG and dC and N = dA, dG, dC or dT) for first strand cDNA synthesis by RT. The ss-cDNAs were converted to double stranded molecules and PCR amplified using a randomly chosen 10-mer primer paired with the same anchor primer used for cDNA synthesis. The dCTP32 labeled cDNAs were fractionated on non-denaturing polyacrylamide gels. Individual bands exhibiting differential expression between treated and nontreated samples were identified for reamplification, cloning, sequencing and further characterization of the differential nature of their expression by reverse northern hybridization and RT-PCR. Only those excised bands able to be reamplified using the anchor:10-mer pair were selected for cloning. To date, 90 variable-anchor:10-mer or specific-anchor:10-mer pairs have been screened. Of these, ≈27 have exhibited possible differential expression with one or more bands. Nucleotide (and deduced amino acid) sequence information was used to search on-line databases for similarity/homology with previously reported gene or protein sequences.