Isolation of the detoxification enzyme EgP450 from an oil palm EST library.

Amornrat Phongdara, Alisa Nakkaew, Sureeporn Nualkaew

Index: Pharm. Biol. 50(1) , 120-7, (2012)

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Abstract

Sequencing of cDNA clones from plant tissue to generate expressed sequence tags (ESTs) is an effective tool for gene discovery. Together with powerful bioinformatics tools, EST sequences allow the prediction of functions of putative bioactive compounds that can later be confirmed.To isolate a detoxification enzyme from an EST library from the oil palm (Elaeis guineensis Jacq. Arecaceae).In total, 750 clones from an oil palm cDNA library were randomly sequenced and analyzed. A clone homologous to cytochrome P450 monooxygenases (P450) was selected from the list of highly expressed genes. The full-length cDNA of P450 from E. guineensis (EgP450) was generated and transformed into a bacterial host to produce recombinant protein. A 3D model of EgP450 was generated and used in a molecular docking analysis to screen for target herbicide substrates. Finally, the detoxification activity of EgP450 was confirmed by an herbicide tolerance test with rice seedlings.The full-length EgP450 has an open reading frame (ORF) of 1515 bp that encodes a protein of 505 amino acids. Docking analysis showed that EgP450 bound to phenylurea-like herbicides such as isoproturon, chlortoluron and fluometuron. The herbicide tolerance test demonstrated that the presence of EgP450 protected the rice seedlings from the killing action of the phytotoxic agent isoproturon.The gene EgP450 was detected in the roots and stems of oil palm tissues, and its recombinant product was shown to protect rice seedlings from exogenous herbicides of the phenylurea family.