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

Isochorismate svnthase (rCS) 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-dT!8primer and M-MuLV reverse transcriptase. Subsequently, PCR technique using primers designed from conserved lCS 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 NCBr Genebank database, which showed a high sequence identity (72%) to the rcs 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 rcs 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%.