Cloning genes differentially expressed in freezing tolerant orchids
Genes responsible for differences in gene regulation and expression in normal cells and freezing tolerant cells were identified using two related wintergreen orchid species, Aplectrum hyemale and Tipularia discolor. Changes in gene expression observed in field-collected tissues obtained from different seasons were compared as were changes observed in plants subjected to cold shock in a laboratory environmental chamber. In order to clone these differentially expressed genes which may confer photosynthesis cold tolerance, the recently developed technique, mRNA differential display was employed. Using this process, mRNA was isolated from the tissue and reverse transcribed to cDNAs, which were amplified using specific anchored 3' primers and various random 5' primers. The 50-100 bands resulting from specific primers were compared on denaturing polyacrylamide gels. Bands differently expressed were excised from the gel and purified. In the future, if partial sequence analysis indicates they may code important regulatory proteins, they will be used as probes to obtain full-length genes from a cDNA library for further characterization. This study provides an opportunity not only to obtain important regulatory genes in plants, but also to understand more about temperature regulated gene expression in orchids.