Identification of elicitor-induced cytochrome P450s of soybean (Glycine max L.) using differential display of mRNA.
Elicitor-inducible glyceollin biosynthesis in soybean depends on five presumably transcriptionally regulated cytochrome P450-dependent enzymes (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 conditions. The differential display of mRNA (DD-RT-PCR) technique was performed with upstream primers based on the conserved heme-binding region of P450s, and ten different 3'-terminal partial P450 sequences were isolated. They were subsequently used to isolate nine different full-length cDNA clones from a cDNA library. As shown by Northern blot analysis, eight of the clones represented P450s, which were activated under glyceollin-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 expression in yeast identified one cDNA clone coding for cinnamate 4-hydroxylase. Thus, at least one of the isolated clones definitively encodes a P450 of the glyceollin pathway. Consequently, this approach offers a straightforward alternative to classical P450 isolation strategies via protein purification and should prove especially useful for isolating P450s that are expressed at a low level.
Schopfer
C R
CR
Botanisches Institut, Ludwig-Maximilians-Universität München, Germany. schopfer@botanik.biologie.uni-muenchen.de
Ebel
J
J
D001578:Benzopyrans / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D018076:DNA, Complementary / Q000302:isolation & purification / Q000378:metabolism; D018744:DNA, Plant / Q000302:isolation & purification / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010449:Peptide Mapping; D016133:Polymerase Chain Reaction / Q000379:methods; D036343:Pterocarpans; D012333:RNA, Messenger / Q000378:metabolism*; D018749:RNA, Plant / Q000378:metabolism; D013025:Glycine max / Q000201:enzymology*; D050564:Trans-Cinnamate 4-Monooxygenase
D001578:Benzopyrans; D018076:DNA, Complementary; D018744:DNA, Plant; D036343:Pterocarpans; D012333:RNA, Messenger; D018749:RNA, Plant; C017343:glyceollin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; C071072:vestitone reductase; D050564:Trans-Cinnamate 4-Monooxygenase; D044925:Oxidoreductases Acting on CH-CH Group Donors