Flavonoid pathway gene discoveries in Boesenbergia rotunda through RNA-SEQ transcriptome profiling of cell suspension cultures in response to phenylalanine / Noor Diyana Md Mustafa

Noor Diyana , Md Mustafa (2017) Flavonoid pathway gene discoveries in Boesenbergia rotunda through RNA-SEQ transcriptome profiling of cell suspension cultures in response to phenylalanine / Noor Diyana Md Mustafa. PhD thesis, University of Malaya.

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Abstract

Panduratin A extracted from Boesenbergia rotunda is a flavonoid reported to possess a range of medicinal properties which include anti-dengue, anti-HIV, anti-cancer, antioxidant and anti-inflammatory activities. B. rotunda is a plant from the Zingiberaceae family commonly used as a food ingredient and traditional medicine in Southeast Asia and China. Reports on the health benefits of secondary metabolites extracted from B. rotunda over the last few years have increased demands for panduratin A. However, large scale extraction has been hindered by low abundance of the compound in nature and limited knowledge of its biosynthetic pathway. Experiments showed an increase in panduratin A production after 14 days post treatment with exogenous phenylalanine, an aromatic amino acid derived from the shikimic acid pathway. Transcriptome sequencing and digital gene expression (DGE) analysis of untreated and phenylalanine treated B. rotunda cell suspension cultures were carried out to elucidate the key genes differentially expressed in the panduratin A biosynthetic pathway. Total RNA of untreated and 14 days post-phenylalanine treated cell suspension cultures were extracted and sequenced using next generation sequencing technology employing an Illumina-Solexa platform. The transcriptome data generated 101,043 unigenes with 50,932 (50.41%) successfully annotated in the public protein databases; including 49.93% (50,447) in the non-redundant (NR) database, 34.63% (34,989) in Swiss-Prot, 24.07% (24,316) in Kyoto Encyclopedia of Genes and Genomes (KEGG) and 16.26% (16,426) in Clusters of Orthologous Groups (COG). Through DGE analysis, it was found that 14,644 unigenes were up-regulated and 14,379 unigenes down-regulated in response to exogenous phenylalanine treatment. In the flavonoid pathway leading to the proposed panduratin A production, 2 unigenes encoded for phenylalanine ammonia-lyase (PAL), 3 for 4-coumaroyl:coenzyme A ligase (4CL) and 1 for chalcone synthase (CHS) were found up-regulated. In this thesis, a flavonoid pathway leading to panduratin A biosynthesis was proposed. In addition, two enzymes, namely flavonoid O-methyltransferase and prenyltransferase, were suggested to be key enzymes for panduratin A biosynthesis. In the transcriptome data, Unigene891_All, Unigene21507_All were predicted to encode flavonoid O-methyltransferase whereas, Unigene31983_All was predicted to encode prenyltransferase. At the gene regulatory level, transcripts for MYB transcription factors in the transcriptome database were further analysed by transcriptome-wide R2R3 MYB transcription factor analysis. As a result, transcripts for three MYB transcription factors namely BrMYB1, BrMYB2 and BrMYB3 were successfully sequenced and characterized. To date, this is the first report of B. rotunda de novo transcriptome data that could serve as a reference for gene or enzyme functional studies in the Zingiberaceae family. Although enzymes that are directly involved in the panduratin A biosynthetic pathway were not completely elucidated, the data provide an overall picture of gene regulation patterns leading to panduratin A production.

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