MSc

The study entitled “Differential expression of genes involved in anthocyanin pigmentation in Red banana and Green-red clones” was conducted at the Department of Plant Biotechnology, Vellayani, Thiruvananthapuram during the period from 2004 to 2006 with an objective of contrasting the expression of genes (chalcone synthase, dihydroflavonol 4-reductase and UDP: glucose 3-oxy-glucosyl transferase) involved in anthocyanin pigmentation in Red banana and Green-red clones. Experiments were also conducted to differentiate the clones based on total sugar concentration and protein profile.

Reverse transcription-polymerase chain reaction was carried out to study the expression of genes. Heterologous primers designed based on the gene sequences of Oryza sativa and Vitis vinifera were used for RT-PCR. Total RNA from tissue culture shoot, leaves and fruit from three different stages of Red banana and Green-red clones were isolated using hot phenol method which gave an yield of 80 - 200 µg g -1 of the tissue and a A260/A280 ratio ranging between 1.6 –2.0. Messenger RNA was purified from the total RNA using the mRNA purification kit from GENEI (Bangalore). The RT-PCR amplified products of the two clones, representing chalcone synthase (CHS), dihydroflavonol 4- reductase (DFR), and UDP: glucose flavonoid 3-oxy-glucosyl transferase (UFGT) were eluted and purified. The cDNA fragments were cloned to pCRII vector (TA Cloning Kit, Invitrogen. Inc., USA) and sequenced.

The nucleotide to nucleotide BLAST of cDNA clone of 183 bp from Red banana showed similarity with the chalcone synthase mRNA sequence of rice (Acc. No. X89859). cDNA from Green-red clone showed no significant similarity to any other chalcone synthase gene during homology search. The translated query vs. protein database (blastx) search exhibited similarity with shikimate 3- dehydrogenase, an enzyme in the biochemical pathway for aromatic amino acids. The cDNAs synthesized from Red banana and Green-red clone with gene specific primers for dihydroflavonol 4- reductase were having 354 bp and 325 bp length respectively. The homology search (blastn) revealed no similarities with any of the nucleotide sequences specific for dihydroflavonol 4- reductase. The cDNAs amplified from Red banana and Green-red clone with UDP: glucose 3-oxy-glucosyltransferase specific primers were of 361 bp and 345 bp, respectively. The homology search using blastn showed similarity with mRNA sequence for UDP: glucose 3-oxy-glucosyltransferase in grapes. In Red banana the blastx search revealed similarity with a glucosyltransferase from Synechocystis sp. There was no similarity for UDP: glucose 3-oxy-glucosyltransferase cDNA of Green-red clone in the NCBI database.

SDS-PAGE analysis showed no difference in the protein profile of Red banana and Green-red clone. Total sugar content in the peel, pulp, peel together with the pulp of red banana and Green-red clone did not showed any significant difference.

The expression analysis of the key genes of the general pathway showed no significant difference in both the clones. All the three genes selected for the study were present in both Red and Green-red clones. The genes isolated were not totally identical in the two banana clones. Further studies are needed to get a better insight into the cause of colour change.