λ Exonuclease-Based Subtractive Hybridization Approach to Isolate Differentially Expressed Genes from Leaf Cultures of Paulownia kawakamii

Genes that are preferentially expressed in a particular developmental pathway can be isolated by subtractive hybridization (SH). We developed a PCR-based approach coupled with λ exonuclease digestion that allows for generating single-stranded tester and driver nucleic acids suitable for SH starting from cDNA libraries. An efficient subtraction strategy was developed to overcome some of the problems in the previously described SH protocols, such as the need for large amounts of experimental tissue, RNase contamination during solution hybridization, and postsubtraction recovery of nucleic acids. We used this method to obtain cDNA corresponding to genes expressed during adventitious shoot regeneration from excised leaf cultures of the fast-growing tree Paulownia kawakamii. Over 36 cDNA clones were isolated and 1 of the differentially expressed clones codes for a leucine zipper transcription factor. This clone showed about sixfold higher level of expression in the shoot-forming tissues (tester) compared to that in the callus-forming tissues (driver) of Paulownia, suggesting that differentially expressed genes can be efficiently isolated using this simple λ exonuclease-based subtractive hybridization method.