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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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