Isolation and characterisation of cDNA encoding dihydroflavonol 4- reductase gene from Orchid Dendrobium variety Sonia17

Abstract

The thesis entitled “Isolation and characterization of cDNA encoding dihydroflavonol 4-reductase gene from Dendrobium orchid variety Sonia 17” was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2006-2008 with an objective of characterizing one of the key genes involved in anthocyanin biosynthesis, viz., dihydroflavonol 4-reductase from flower buds of Dendrobium Sonia 17 orchid. Reverse Transcription-PCR technique was used in the present study for the amplification of cDNA encoding dfr gene. Heterologous forward and reverse primers were designed based on the gene sequences of Cymbidium orchid and Oryza sativa using Primer 3 software. Total RNA isolated from the flower buds using TRIZOL reagent yielded good quality intact RNA, with no genomic DNA contamination and showed an A260/A280 ratio of 1.6-2.0. The cDNA synthesized from purified mRNA was amplified using gene specific primers designed. All the primers included in the study yielded reproducible amplification. The primer sets A (Af and Ar) and C (Cf and Cr) which were designed based on the dfr gene sequence of Cymbidium orchid yielded amplified fragments of size approximately 400-500 bp and 100 bp respectively. Primer set B (Bf and Br) synthesized from Oryza sativa dfr gene sequences yielded an amplified product of size nearly 300 bp. The cDNA amplified with primer set A was cloned in pGEM-T Easy Vector and multiplied in competent E. Coli strain DH5α for sequencing. The transformed E. Coli colonies were grown on L.B agar plates and selected by blue – white screening. Three white colonies were randomly selected and plasmid DNA was isolated from transformed clones. The presence of insert was checked by PCR analysis of the plasmid DNA with gene specific primer (primer set A). The agarose gel electrophoresis showed the presence of amplified fragment. Sequencing of two clones at Chromous Biotech Pvt. Ltd. using T7 and SP6 primer failed to amplify the plasmid DNA. This work was an initial step towards isolation and characterization of dihydroflavonol 4-reductase gene in Dendrobium variety Sonia 17. The primers designed in this study could successfully amplify the cDNA encoding dfr. PCR reactions gave good results but the sequencing was not satisfactory. For the better understanding of the dfr sequences, the sequencing reaction needs to be repeated with other clones.