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Submitted to: Mid Atlantic Plant and Molecular Biology Proceedings
Publication Type: Abstract Only Publication Acceptance Date: 7/20/1998 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: Plant cytochrome P450 monooxygenases are heme-containing enzymes that participate in the synthesis of secondary products, some of which are shown to inhibit insects, pathogens and animal herbivores. Using RT-PCR of poly(A)+ RNA from N. plumbaginifolia containing the potato inhibitor wound- inducible promoter-isopentenyl transferase gene construct (PI-II-ipt), two full length clones of P450, designated as pNp11 and pNp12, were isolated and sequenced. pNp11 has an open reading frame of 1524 nucleotides and its deduced amino acid sequence has 44% identity to Catharanthus roseus P450 (CYP72). pNp12 is similar to pNp11 except for 81 nucleotides deletion and an internal stop codon, and so possibly represents a pseudogene. When in vitro transcribed and translated, two 35S-methionine labeled polypeptides with molecular masses of 56 and 34 kDa were synthesized corresponding to the products of pNp11 and pNp12, respectively. The complete coding region of pNp11 was amplified by PCR and used to estimate the copy number of P450 genes and to study the expression of P450 in PI-II-ipt-transformed and normal N. plumbaginifolia. Southern blot hybridization of genomic DNA indicated that P450 exists as multiple copies of the same gene. Northern blot analysis revealed that transcript accumulation was maximum during the day but was lowest at night. When infested with tomato hornworm (Manduca sexta) larvae or mechanically wounded, this rhythm was disrupted resulting in an elevated level of expression at night in the wounded leaves. The level of induction was 4.5- to 6-fold higher in PI-II-ipt-transformed leaves after 6 to 12 h of mechanical wounding in comparison to a 2- to 3.5 fold induction from wounded but untransformed leaves. The response to feeding insect larvae was systemic. |
