ISOLATION OF SALICYLIC ACID-INDUCED GENES IRE BRASSICA-NAPUS BY SUBTRACTIVE HYBRIDIZATION

Subtractive hybridization has been performed to isolate salicylic acid -induced genes in Brassica napus, as a first step toward understanding plant defense mechanisms. In this procedure, tester cDNA was adapted with a linker and hybridized with a 10-fold excess of driver cDNA havi ng a different linker. After hybridization, the DNA mixture was treate d with T4 DNA polymerase and S1 nuclease so that only double-stranded tester/tester DNAs were amplified in subsequent polymerase chain react ions (PCR). After repeated rounds of subtractive hybridization, the en riched DNAs were analyzed by sequencing and database search. Of the en riched sequences examined, several putative defense-related genes have been identified. These include cytochrome P450, glutathione S-transfe rase, pathogenesis-related la protein, and a lipase homolog genes. Rev erse transcription-PCR showed that these genes are induced by salicyli c acid and/or wounding.