IDENTIFICATION OF ELICITOR-INDUCED CYTOCHROME-P450S OF SOYBEAN (GLYCINE-MAX L) USING DIFFERENTIAL DISPLAY OF MESSENGER-RNA

Elicitor-inducible glyceollin biosynthesis in soybean depends on five presumably transcriptionally regulated cytochrome P450-dependent enzym es (P450s). In order to isolate corresponding cDNA clones, we devised a novel polymerase chain reaction (PCR)-based approach targeting P450s that are transcriptionally activated under glyceollin-inducing condit ions. The differential display of mRNA (DD-RT-PCR) technique was perfo rmed with upstream primers based on the conserved heme-binding region of P450s, and ten different 3'-terminal partial P450 sequences were is olated. They were subsequently used to isolate nine different full-len gth cDNA clones from a cDNA library. As shown by Northern blot analysi s, eight of the clones represented P450s, which were activated under g lyceollin-inducing conditions similar to two enzymes of the glyceollin biosynthesis pathway, CHS and IFR. Therefore, these eight clones are candidate cDNAs for the glyceollin-related P450s. Functional expressio n in yeast identified one cDNA clone coding for cinnamate 4-hydroxylas e. Thus, at least one of the isolated clones definitively encodes a P4 50 of the glyceollin pathway. Consequently, this approach offers a str aightforward alternative to classical P450 isolation strategies via pr otein purification and should prove especially useful for isolating P4 50s that are expressed at a low level.