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Cloning of a Near Complete Isochorismate synthase (ICS) cDNA from Morinda citrifolia L.

Tan Sia Hong and Hairul Azman Roslan

Pertanika Journal of Tropical Agricultural Science, Volume 31, Issue 1, February 2008

Keywords: Isochorismate synthase, Rubiaceae, 'mengkudu', anthraquinones, PCR, 3'RACE

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Isochorismate synthase (ICS) is a key enzyme that catalyses the conversion of chorismate to isochorismate which is then channelled to other secondary product such as the anthraquinones. A near complete cDNA was isolated through RT-PCR technique. Characterization of this gene is important in characterising its role in the production of anthraquinones in the Rubiaceae plant family. Anthraquinones are known for their medicinal properties and can be found in Rubiaceae especially in roots. In this study, total RNA was extracted from roots of 'mengkudu' using modified CTAB method. The total RNA was subjected to first strand synthesis using oligo-dT18 primer and M-MuLV reverse transcriptase. Subsequently, PCR technique using primers designed from conserved ICS domains from other plants were used to isolate an internal conservative region of 426 bp. The cDNA was subsequently sequenced and verified using BLASTn program through the NCBI Genebank database, which showed a high sequence identity (72%) to the ICS from Catharanthus roseus. Based on this sequence, 3'RACE was performed to obtain the 3'-end of the gene and a 1036 bp 3'-fragment was generated. Apart from that, another PCR managed to generate a fragment of 491 bp upstream of the cDNA. Both fragments were sequenced and verified. Contig analyses and assembly of the partial cDNAs generated showed a near complete cDNA of 1872 bp. Sequence analysis of this partial cDNA showed a high degree of identity with ICS cDNA from other plants with the highest identity of 72% with ICS from C. roseus. Deduced amino acid showed a high similarity with Rubia cardifolia ICS of 85%.

ISSN 1511-3701

e-ISSN 2231-8542

Article ID

JTAS-0015-2007

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