Article

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: 4.04). 03/2012; 13:126. 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.

Download full-text

Full-text

Available from: Russiachand Heikham, Aug 28, 2015
1 Follower
 · 
282 Views
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    Journal of Plant Biochemistry and Biotechnology 06/2015; DOI:10.1007/s13562-015-0317-7 · 0.81 Impact Factor
    • "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. "
    Journal of Plant Biochemistry and Biotechnology 05/2015; · 0.81 Impact Factor
  • Source
    • "Functional annotation and DGE analysis showed that 5032 transcripts were differentially expressed between the two transcriptomes and that 33 metabolic pathways changed significantly in response to salt stress. A method that can rapidly identify salt tolerance genes from the massive amount of RNA-seq data is crucial, because DGE results may be misleading in some circumstances, as previously reported [1] [2] [3]. Here, we identified fragments of 18 genes that are responsive to salt stress in R. trigyna using differential-display reverse transcription PCR (DDRT-PCR) followed by local BLAST searches against the RNA-seq data to identify the same or longer nucleotide sequences. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Next generation sequencing (NGS) technologies have been used to generate huge amounts of sequencing data from many organisms. However, the correct choice of candidate genes and prevention of false-positive results computed from digital gene expression (DGE) of RNA-seq data are vital when using these genetic resources. We indirectly identified 18 salt-stress-induced Reaumuria trigyna transcripts from the transcriptome sequencing data using differential-display reverse transcription PCR (DDRTPCR) combined with local BLAST searches. Highly consistent with the DGE results, the quantitative real-time PCR expression patterns of these transcripts showed strong upregulation by salt stress, suggesting that these genes may play important roles in R. trigyna’s survival under high-salt environments. The method presented here successfully identified responsive genes from the massive amount of RNA-seq data. Thus, we suggest that DDRT-PCR could be employed to mine NGS data in a wide range of applications in transcriptomic studies. In addition, the genes identified in the present study are promising candidates for further elucidation of the salt tolerance mechanisms in R. trigyna.
    International Journal of Plant Genomics 11/2014; 2014:381501. DOI:10.1155/2014/381501
Show more