Article

The First Insight into the Tissue Specific Taxus Transcriptome via Illumina Second Generation Sequencing

Biotechnology Institute, Dalian Jiaotong University, Dalian, China.
PLoS ONE (Impact Factor: 3.23). 06/2011; 6(6):e21220. DOI: 10.1371/journal.pone.0021220
Source: PubMed

ABSTRACT

Illumina second generation sequencing is now an efficient route for generating enormous sequence collections that represent expressed genes and quantitate expression level. Taxus is a world-wide endangered gymnosperm genus and forms an important anti-cancer medicinal resource, but the large and complex genomes of Taxus have hindered the development of genomic resources. The research of its tissue-specific transcriptome is absent. There is also no study concerning the association between the plant transcriptome and metabolome with respect to the plant tissue type.
We performed the de novo assembly of Taxus mairei transcriptome using Illumina paired-end sequencing technology. In a single run, we produced 13,737,528 sequencing reads corresponding to 2.03 Gb total nucleotides. These reads were assembled into 36,493 unique sequences. Based on similarity search with known proteins, 23,515 Unigenes were identified to have the Blast hit with a cut-off E-value above 10⁻⁵. Furthermore, we investigated the transcriptome difference of three Taxus tissues using a tag-based digital gene expression system. We obtained a sequencing depth of over 3.15 million tags per sample and identified a large number of genes associated with tissue specific functions and taxane biosynthetic pathway. The expression of the taxane biosynthetic genes is significantly higher in the root than in the leaf and the stem, while high activity of taxane-producing pathway in the root was also revealed via metabolomic analyses. Moreover, many antisense transcripts and novel transcripts were found; clusters with similar differential expression patterns, enriched GO terms and enriched metabolic pathways with regard to the differentially expressed genes were revealed for the first time.
Our data provides the most comprehensive sequence resource available for Taxus study and will help define mechanisms of tissue specific functions and secondary metabolism in non-model plant organisms.

  • Source
    • "RNA-Seq has been leveraged with de novo transcriptome assembly to learn about plant innovations including parasitism4567and C4 photosynthesis[8], plant processes including fruit ripening[9], drought response[10], and flavonoid biosynthesis[11], chemical defenses[12], and the evolution of sex chromosomes[13]. The recent boom of RNA-Seq studies involving de novo assembly has motivated innovations in assemblers developed specifically for RNA-Seq data (Velvet[14,15], Oases[16,17](includes Velvet[18]), SOAPdenovo1920212223242526272829, SOAPdenovo- Trans[30], CLC[31], ABySS[32], Trinity[5,13,333435363738). Comparison of de novo transcriptome assembler performance is hindered by lack of widely used standard quality metrics[39]or rigorous evaluation of a comprehensive selection of assemblers with a transcriptome from a high quality reference genome. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Whereas de novo assemblies of RNA-Seq data are being published for a growing number of species across the tree of life, there are currently no broadly accepted methods for evaluating such assemblies. Here we present a detailed comparison of 99 transcriptome assemblies, generated with 6 de novo assemblers including CLC, Trinity, SOAP, Oases, ABySS and NextGENe. Controlled analyses of de novo assemblies for Arabidopsis thaliana and Oryza sativa transcriptomes provide new insights into the strengths and limitations of transcriptome assembly strategies. We find that the leading assemblers generate reassuringly accurate assemblies for the majority of transcripts. At the same time, we find a propensity for assemblers to fail to fully assemble highly expressed genes. Surprisingly, the instance of true chimeric assemblies is very low for all assemblers. Normalized libraries are reduced in highly abundant transcripts, but they also lack 1000s of low abundance transcripts. We conclude that the quality of de novo transcriptome assemblies is best assessed through consideration of a combination of metrics: 1) proportion of reads mapping to an assembly 2) recovery of conserved, widely expressed genes, 3) N50 length statistics, and 4) the total number of unigenes. We provide benchmark Illumina transcriptome data and introduce SCERNA, a broadly applicable modular protocol for de novo assembly improvement. Finally, our de novo assembly of the Arabidopsis leaf transcriptome revealed ~20 putative Arabidopsis genes lacking in the current annotation.
    Full-text · Article · Jan 2016 · PLoS ONE
  • Source
    • "Species Explant Profiling method Number of transcripts Reference T. chinensis MeJA elicited cells Illumina deep sequencing 46,581 unigenes Li et al. (2012a, 2012b) T. chinensis MeJA elicited cells Illumina deep sequencing 1,256,425 sRNAs Qiu et al. (2009) T. chinensis MeJA elicited cells Random Sanger sequencing of cDNA library 3563 unigenes Jennewein et al. (2004) T. cuspidata MeJA elicited cells Sanger sequencing of subtractive hybridization library 331 unigenes Lenka et al. (2012) T. cuspidata (Cambial) cells 454 deep sequencing 26,906 unigenes Lee et al. (2010) T. cuspidata Needles 454 deep sequencing 20,557 unigenes Wu et al. (2011) T. mairei Roots, leaves, stems Illumina deep sequencing 36,493 unigenes Hao et al. (2011a, 2011b "
    [Show abstract] [Hide abstract]
    ABSTRACT: Taxol is a complex diterpene alkaloid scarcely produced in nature and with a high anticancer activity. Biotechnological systems for taxol production based on cell cultures of Taxus spp. have been developed, but the growing commercial demand for taxol and its precursors requires the optimization of these procedures. In order to increase the biotechnological production of taxol and related taxanes in Taxus spp. cell cultures, it is necessary not only to take an empirical approach that strives to optimize in-put factors (cell line selection, culture conditions, elicitation, up-scaling, etc.) and out-put factors (growth, production, yields, etc.), but also to carry out molecular biological studies. The latter can provide valuable insight into how the enhancement of taxane biosynthesis and accumulation affects metabolic profiles and gene expression in Taxus spp. cell cultures.
    Full-text · Article · Nov 2014 · Biotechnology advances
  • Source
    • "e l s e v i e r . c o m / l o c a t e / g e n e (Hao et al., 2011; Li et al., 2011; Sandmann et al., 2011; Iseli et al., 1999). In the current study, we compared transcriptome sequences of above-and underground tissues of R. algida to obtain a profile of molecular mechanisms involved in synthesis of bioactive compounds . "
    [Show abstract] [Hide abstract]
    ABSTRACT: Transcriptome sequencing is a powerful tool for the assessment of gene expression and the identification and characterization of molecular markers in non-model organisms. Rhodiola algida L. (Crassulaceae), endemic to the Qinghai-Tibetan Plateau, has long been used in traditional Chinese medicine to prevent altitude sickness and eliminate fatigue. Illumina-based high-throughput transcriptome sequencing of aboveground and underground tissues of R. algida respectively yielded 5.40 million and 5.18 million clean reads. A total of 82,664 unigenes averaging 577bp in length were generated from the aboveground clean reads, with 86,237 unigenes of 502-bp mean length obtained from the underground tissues. Of 55,028 unigenes compared with sequences in UniProt databases, 20,413 unigenes had significant similarities with existing sequences in NR, NT, Swiss-Prot, GO, KEGG, and COG databases. Single nucleotide polymorphism (SNP) analysis identified 237,294 SNPs from 154,636 contigs of aboveground tissues and 197,540 SNPs from 144,963 underground-derived contigs. The information uncovered in this study should serve as a valuable resource for the characterization of important traits related to secondary metabolite formation and for the identification of associated molecular mechanisms.
    Full-text · Article · Oct 2014 · Gene
Show more