Characterization of phenylalanine ammonia lyase and revealing flavonoid biosynthesis in Gymnema sylvestre R. Br through transcriptomic approach

Gymnema sylvestre R.Br. is famous medicinal plant among diabetics for its gymnemic acid content. It also contains flavonoids, which are an essential component in various other products. Though some molecular information on the biosynthesis of gymnemic acid, polyoxypregnane, micro RNAs and photosynthetic efficiency is available, there is no gene level information available on the biosynthesis of flavonoids in this plant. RNA was extracted from winter-collected Gymnema sylvestre leaves and cDNA libraries were prepared and used for next generation sequencing. De novo transcriptome assembly were prepared and Coding DNA Sequences (CDS) of 13 major genes involved in flavonoids biosynthesis were identified from transcriptome data. Phenylalanine ammonia lyase gene containing full-length CDS was employed for in silico protein modelling and subsequent quality assessment. These models were then compared against publicly available databases. To confirm the identification of these genes, a similarity search was conducted using the NCBI BLAST tool. Therefore, in the present study, an effort has been made to provide molecular insights into flavonoid biosynthesis pathway by examining the expressed transcripts in G.sylvestre. Gene sequences of total thirteen major genes viz., phenylalanine ammonia lyase, 4-coumarate CoA ligase, cinnamic acid 4-hydroxylase, shikimate O-hydroxycinnamoyl transferase, coumaroyl quinate (coumaroyl shikimate) 3′-monooxygenase, caffeoyl-CoA O-methyltransferase, chalcone synthase, chalcone isomerase, naringenin 3-dioxygenase, flavanol synthase, flavonoid 3′-monooxygenase, Flavanone 7-O-glucoside 2″-O-beta-L-rhyamnosyltransferase and leucoanthocyanidin dioxygenase were identified and a putative pathway of flavonoids biosynthesis has been illustrated based on transcriptome data. This transcriptome study has contributed gene-level insights into the biosynthesis of flavonoids in plants as a whole and represents the first report within a non-model plant, Gymnema sylvestre perticullarly.