Identification of elicitor-induced cytochrome P450s of soybean (Glycine max L.) using differential display of mRNA

Mol Gen Genet. 1998 May;258(4):315-22. doi: 10.1007/s004380050736.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Benzopyrans / metabolism
  • Cytochrome P-450 Enzyme System / biosynthesis
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / isolation & purification*
  • DNA, Complementary / isolation & purification
  • DNA, Complementary / metabolism
  • DNA, Plant / isolation & purification
  • DNA, Plant / metabolism
  • Gene Expression Regulation, Enzymologic
  • Kinetics
  • Mixed Function Oxygenases / biosynthesis
  • Mixed Function Oxygenases / genetics
  • Molecular Sequence Data
  • Oxidoreductases / biosynthesis
  • Oxidoreductases / genetics
  • Oxidoreductases Acting on CH-CH Group Donors*
  • Peptide Mapping
  • Polymerase Chain Reaction / methods
  • Pterocarpans
  • RNA, Messenger / metabolism*
  • RNA, Plant / metabolism
  • Soybeans / enzymology*
  • Trans-Cinnamate 4-Monooxygenase

Substances

  • Benzopyrans
  • DNA, Complementary
  • DNA, Plant
  • Pterocarpans
  • RNA, Messenger
  • RNA, Plant
  • glyceollin
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • Oxidoreductases
  • vestitone reductase
  • Trans-Cinnamate 4-Monooxygenase
  • Oxidoreductases Acting on CH-CH Group Donors

Associated data

  • GENBANK/X92437
  • GENBANK/Y10489
  • GENBANK/Y10490
  • GENBANK/Y10491
  • GENBANK/Y10492
  • GENBANK/Y10493
  • GENBANK/Y10982
  • GENBANK/Y10983