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 having a different linker. After hybridization, the DNA mixture was treated with T4 DNA polymerase and S1 nuclease so that only double-stranded tester/tester DNAs were amplified in subsequent polymerase chain reactions (PCR). After repeated rounds of subtraetive hybridization, the enriched DNAs were analyzed by sequencing and database search. Of the enriched sequences examined, several putative
defense-related genes have been identified. These include cytochrome P450, glutnthione S-transferase, pathogenesisrelated 1a protein, and a lipase homolog genes. Reverse transcription-PCR showed that these genes are induced by salicylic acid and/or wounding.
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