Abstract
Natural indigo is the most important blue dye for textile dyeing and valuable secondary metabolite biosynthesized in Indigofera tinctoria and Polygonum tinctorium plants. Present investigation is made to generation of gene resource for pathway enrichment and to understand possible gene expression involved in indigo biosynthesis. The data about raw reads and the transcriptome assembly project has been deposited at GenBank under the accessions SRA180766 and SRX692542 for I. tinctoria and P. tinctorium, respectively.
Keywords: Unigenes, Indican, Transcriptome dataset
| Specifications | |
|---|---|
| Organism/cell line/tissue | Indigofera tinctoria and Polygonum tinctorium |
| Sequencer or array type | Illumina HiSeq™ 2000-RNA sequencing |
| Data format | Raw and processed |
| Experimental factors | Laboratory grown different species yielding indigo dye |
| Experimental features | RNA-seq dataset for gene expression profiling in young leaves |
| Sample source location | India (Indigofera tinctoria) and Japan (Polygonum tinctorium) |
1. Direct link to deposited data
Transcriptome assembly project and raw data have been deposited at GenBank under the accessions SRA180766 (http://www.ncbi.nlm.nih.gov/sra/?term=SRA180766) and SRX692542 (http://www.ncbi.nlm.nih.gov/sra/?term=SRX692542) for Indigofera tinctoria and Polygonum tinctorium, respectively.
2. Experimental design, materials and methods
2.1. Plant materials
In the mature plants of P. tinctorium, younger leaves (especially first and second leaves) contain larger amounts of indican compared to older ones [1], similar observation was made in the case of I. tinctoria (data not shown). The seeds of I. tinctoria and P. tinctorium were germinated in pots containing soil and sand (1:1). Plants were grown in a plant growth chamber under 28 ± 2 °C and ~ 70% humidity.
2.2. Total RNA extraction and quality control
To maximize the number of indigo biosynthetic pathway genes, fresh leaves from two month old plant were sampled for total RNA extraction. Quantity and purity of extracted total RNA were determined using NanoDrop (Thermo Fisher Scientific Inc., USA) and Agilent 2100 bioanalyzer (Agilent Technologies, USA), respectively.
2.3. Transcriptome sequencing, de novo assembly, annotation and classification
These RNA samples were used for mRNA enrichment, fragmentation and cDNA library construction. The quality check was carried out by Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System. The cDNA library construction and sequencing with an Illumina sequencing platform were performed at the Beijing Genomics Institute (BGI), China. Transcriptome de novo assembly is carried out with short reads assembling program — Trinity (release-20130225) [2] (Table 1). Further, BLASTX search was performed with obtained unigene sequences against several databases, including databases as NCBI Nr (http://www.ncbi.nlm.nih.gov/), Swissprot (http://www.expasy.ch/sprot/), KEGG (http://www.genome.jp/kegg/) and COG (http://www.ncbi.nlm.nih.gov/cog/), using a cut-off E-value of 10− 5 (Table 2). To classify the unigenes, the Blast2GO program was used to get GO annotation based on molecular function, biological process and cellular component [3].
Table 1.
Statistics of sequencing and assembly of the I. tinctoria and P. tinctorium transcriptome.
| Attributes |
Value |
|
|---|---|---|
| I. tinctoria | P. tinctorium | |
| Total raw reads | 56,681,030 | 58,664,978 |
| Total clean reads | 53,337,778 | 54,784,512 |
| Number of contigs | 102,459 | 155,283 |
| Number of unigenes | 60,395 | 96,913 |
| Q20 percentage | 98.27% | 98.10% |
| GC percentage | 45.28% | 49.73% |
Table 2.
Statistics of functional annotation of the I. tinctoria and P. tinctorium unigenes.
| Databases | NR | NT | Swiss-Prot | KEGG | COG | GO | ALL |
|---|---|---|---|---|---|---|---|
| Number of unigenes | 89,333 | 74,698 | 57,053 | 51,348 | 31,460 | 68,956 | 92,731 |
3. Conclusion
RNA-seq analysis of I. tinctoria and P. tinctorium from leaf tissues using Illumina HiSeq™ 2000 platform has led to the identification of transcripts of interest and may be useful in defining the role towards indigo biosynthesis, also several transcription factors. Further, the gene prediction and dataset development in non-model plants by Illumina platform are a much faster and cost-effective approach to enrich the gene resource for molecular and functional genomics of the blue dye producing species.
Conflict of interest
All the authors have approved submission and there are no conflicts of interest.
Acknowledgments
This work was funded by DST-JSPS grant number (Grant ID DST/INT/JSPS/P-163/2013) from DST, Govt. of India — JSPS, Japan. Authors wish to gratefully acknowledge Director, NEERI, Nagpur, and Council of Scientific and Industrial Research (CSIR), New Delhi, India and Okayama University of Science, Japan for providing facilities and encouragement.
References
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