Please use this identifier to cite or link to this item: https://hdl.handle.net/11499/10283
Title: Cloning and expression of a CYP720B orthologue involved in the biosynthesis of diterpene resin acids in Pinus brutia
Authors: Semiz, Aslı
Sen, A.
Keywords: Cloning
CYP720B
Cytochrome P450
Diterpene resin acids
Expression
Pinus brutia
diterpene
oxygenase
protein CYP720B
protein CYP720B1
protein CYP720B4
unclassified drug
codon
cytochrome P450
Article
biosynthesis
CYP720B gene
gene sequence
genetic transfection
loblolly pine
molecular cloning
nonhuman
open reading frame
pine
plant gene
polymerase chain reaction
protein expression
protein motif
Saccharomyces cerevisiae
sequence analysis
sitka spruce
Western blotting
amino acid sequence
biology
chemistry
gene expression
genetics
metabolism
methylation
molecular genetics
phosphorylation
sequence alignment
Amino Acid Sequence
Cloning, Molecular
Codon
Computational Biology
Cytochrome P-450 Enzyme System
Diterpenes
Gene Expression
Methylation
Molecular Sequence Data
Phosphorylation
Pinus
Sequence Alignment
Publisher: Kluwer Academic Publishers
Abstract: Cytochrome P450 monooxygenases mediate a broad range of oxidative reactions involved in the biosynthesis of both primary and secondary metabolites in plants. Until now, only two P450 genes, CYP720B1 from Pinus taeda and CYP720B4 from Picea sitchensis, have been functionally characterised and described in the literature. The purpose of this study was to describe the cloning and expression of CYP720B from Pinus brutia due to its suggested role in the synthesis of bioactive compounds used for chemical defence against insects. A PCR product of the P. brutia CYP720B gene was cloned into the pCR8/GW/TOPO cloning vector. After optimising the sequence for codon usage in yeast, it was transferred into the inducible expression vector pYES-DEST52 and transfected into the S. cerevisiae INVSc1 strain. Sequence analysis showed that the P. brutia CYP720B gene contains an open reading frame of 1,464 nucleotides, which encodes a 53,570 Da putative protein of 487 amino acid residues. The putative protein contains the classic heme-binding sequence motif that is conserved in all P450 enzymes. It shares 99 and 61 % identity with the deduced amino acid sequences of CYP720B1 from Pinus taeda and CYP720B4 from Picea sitchensis, respectively. Recombinant CYP720B protein expression was confirmed using western blot analysis. Furthermore, recombinant CYP720B was functionally active, showing a Soret peak at approximately 448 nm in the reduced CO difference spectra. These data suggest that the cloned gene is an orthologue of CYP720B in P. brutia and might be involved in DRA biosynthesis. © 2014, Springer Science+Business Media Dordrecht.
URI: https://hdl.handle.net/11499/10283
https://doi.org/10.1007/s11033-014-3822-1
ISSN: 0301-4851
Appears in Collections:Fen-Edebiyat Fakültesi Koleksiyonu
PubMed İndeksli Yayınlar Koleksiyonu / PubMed Indexed Publications Collection
Scopus İndeksli Yayınlar Koleksiyonu / Scopus Indexed Publications Collection
WoS İndeksli Yayınlar Koleksiyonu / WoS Indexed Publications Collection

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