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De novo sequencing and characterization of Picrorhiza kurroa transcriptome at two temperatures showed major transcriptome adjustments

Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Council of Scientific and Industrial Research, PO Box No 6, Palampur 176 061, Himachal Pradesh, India.
BMC Genomics (Impact Factor: 3.99). 03/2012; 13(1):126. DOI: 10.1186/1471-2164-13-126
Source: PubMed

ABSTRACT

Picrorhiza kurrooa Royle ex Benth. is an endangered plant species of medicinal importance. The medicinal property is attributed to monoterpenoids picroside I and II, which are modulated by temperature. The transcriptome information of this species is limited with the availability of few hundreds of expressed sequence tags (ESTs) in the public databases. In order to gain insight into temperature mediated molecular changes, high throughput de novo transcriptome sequencing and analyses were carried out at 15 °C and 25 °C, the temperatures known to modulate picrosides content.
Using paired-end (PE) Illumina sequencing technology, a total of 20,593,412 and 44,229,272 PE reads were obtained after quality filtering for 15 °C and 25 °C, respectively. Available (e.g., De-Bruijn/Eulerian graph) and in-house developed bioinformatics tools were used for assembly and annotation of transcriptome. A total of 74,336 assembled transcript sequences were obtained, with an average coverage of 76.6 and average length of 439.5. Guanine-cytosine (GC) content was observed to be 44.6%, while the transcriptome exhibited abundance of trinucleotide simple sequence repeat (SSR; 45.63%) markers.Large scale expression profiling through "read per exon kilobase per million (RPKM)", showed changes in several biological processes and metabolic pathways including cytochrome P450s (CYPs), UDP-glycosyltransferases (UGTs) and those associated with picrosides biosynthesis. RPKM data were validated by reverse transcriptase-polymerase chain reaction using a set of 19 genes, wherein 11 genes behaved in accordance with the two expression methods.
Study generated transcriptome of P. kurrooa at two different temperatures. Large scale expression profiling through RPKM showed major transcriptome changes in response to temperature reflecting alterations in major biological processes and metabolic pathways, and provided insight of GC content and SSR markers. Analysis also identified putative CYPs and UGTs that could help in discovering the hitherto unknown genes associated with picrosides biosynthesis.

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    • "For example, the expression level of the HMGR gene in the MVA pathway and that of the HDS gene in the MEP pathway were relatively lower than those of other genes in each pathway in Picrorhiza kurrooa Royle ex Benth. leaf (Gahlan et al., 2012). In contrast, the expression level of the HMGR gene in the MVA pathway was highest among those of other genes in the MVA pathway in Euphorbia fischeriana root and Chlorophytum borivilianum leaf and root similar to P. tricornutum (Barrero et al., 2011;Kalra et al., 2013). "
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    ABSTRACT: The marine diatom Phaeodactylum tricornutum is expected to be a source of hydrocarbons and carotenoids that are synthesized via isoprenoid precursor biosynthesis pathways such as the mevalonate (MVA) and the 2-C-methyl-D-erythritol phosphate (MEP) pathways, because the molecular biotechnological techniques for metabolic engineering have been established. In this study, we investigated the expression profiles of the genes, including those in the MVA and MEP pathways, under various culture conditions using RNA-seq analysis to obtain information useful for the metabolic engineering and development of endogenous promoters that can highly drive the expression of transgenes in P. tricornutum. The expression levels of the genes in the MVA pathway, except for the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) gene, were low under all the conditions tested, and the expression levels of the 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK) and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS) genes in the MEP pathway were relatively low. Eleven genes with high expression levels, including the V-type proton ATPase subunit C-like gene, which are potential sources of endogenous promoters in P. tricornutum, were selected. These results are expected to provide useful information for the metabolic engineering of P. tricornutum.
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    • "Henkes et al. (2001) also showed that precursors of primary metabolism colimit flux into the shikimate pathway and phenylpropanoid metabolism. The connection of phenylpropanoid metabolism to picroside biosynthesis was previously documented by various studies (Gahlan et al. 2012; Kumar et al. 2013). This may imply the role of HK in picroside biosynthesis. "
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    ABSTRACT: Picrosides, the terpenoids synthesized by Picrorhiza kurroa, have ample usage in medicine. Identification of the regulatory enzymes involved in picroside biosynthesis needs to be explored for improving the level of these secondary metabolites. Current efforts are based on the analysis of secondary metabolism in picroside biosynthesis but its interpretation is limited by the lack of information on the involvement of primary metabolic pathways. The present study investigated the connection of primary metabolic enzymes with the picrosides levels in P. kurroa. The results showed changes in the catalytic activities as well as in the gene expression profiles of hexokinase, pyruvate kinase, isocitrate dehydrogenase, malate dehydrogenase, and NADP+-malic enzyme in congruence with picroside-I content under different conditions of P. kurroa growth, which indicates the role of these enzymes in the accumulation of picrosides. The significant correlation coefficients (p<0.05) observed between gene expression and enzyme activity underline the role of integrative studies for a better understanding of connecting links between metabolic pathways leading to picroside biosynthesis. This is apparently the first report on the involvement of glycolytic and TCA cycle enzymes in the accumulation of picrosides in P. kurroa.
    Full-text · Article · Jun 2015 · Journal of Plant Biochemistry and Biotechnology
    • "Previous studies have shown that regulatory genes in terpenoids biosynthesis are hmgr and dxs, which encode 3-hydroxy-3-methylglutaryl coenzyme A reductase and 1-deoxy-D-xylulose-5-phosphate synthase, respectively (Kawoosa et al. 2010). Recently, the involvement of phenylpropanoid pathway in the biosynthesis of picrosides has been reported (Gahlan et al. 2012; Kumar et al. 2013) which provides broader class of targets which could be triggered for the enhancement of picrosides biosynthesis. Secondary metabolic pathways are dependant on primary metabolic pathways for the supply of precursors. "

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