Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds

Abstract

BackgroundTea is one of the most popular non-alcoholic beverages worldwide. However, the tea plant, Camellia sinensis, is difficult to culture in vitro, to transform, and has a large genome, rendering little genomic information available. Recent advances in large-scale RNA sequencing (RNA-seq) provide a fast, cost-effective, and reliable approach to generate large expression datasets for functional genomic analysis, which is especially suitable for non-model species with un-sequenced genomes.ResultsUsing high-throughput Illumina RNA-seq, the transcriptome from poly (A)+ RNA of C. sinensis was analyzed at an unprecedented depth (2.59 gigabase pairs). Approximate 34.5 million reads were obtained, trimmed, and assembled into 127,094 unigenes, with an average length of 355 bp and an N50 of 506 bp, which consisted of 788 contig clusters and 126,306 singletons. This number of unigenes was 10-fold higher than existing C. sinensis sequences deposited in GenBank (as of August 2010). Sequence similarity analyses against six public databases (Uniprot, NR and COGs at NCBI, Pfam, InterPro and KEGG) found 55,088 unigenes that could be annotated with gene descriptions, conserved protein domains, or gene ontology terms. Some of the unigenes were assigned to putative metabolic pathways. Targeted searches using these annotations identified the majority of genes associated with several primary metabolic pathways and natural product pathways that are important to tea quality, such as flavonoid, theanine and caffeine biosynthesis pathways. Novel candidate genes of these secondary pathways were discovered. Comparisons with four previously prepared cDNA libraries revealed that this transcriptome dataset has both a high degree of consistency with previous EST data and an approximate 20 times increase in coverage. Thirteen unigenes related to theanine and flavonoid synthesis were validated. Their expression patterns in different organs of the tea plant were analyzed by RT-PCR and quantitative real time PCR (qRT-PCR).ConclusionsAn extensive transcriptome dataset has been obtained from the deep sequencing of tea plant. The coverage of the transcriptome is comprehensive enough to discover all known genes of several major metabolic pathways. This transcriptome dataset can serve as an important public information platform for gene expression, genomics, and functional genomic studies in C. sinensis.

Comparative transcriptome of rhizome and leaf in Ligusticum Chuanxiong

“…More than 61 % (67,373 of 109,486) unigenes were annotated by sequence similarity search in public databases. The percentages of the annotated unigenes were similar to those in Chinese fir (Huang et al 2012), parsley (Li et al 2014a, b) and tea (Shi et al 2011).…”

The de novo Transcriptome and Its Analysis in the Worldwide Vegetable Pest, Delia antiqua (Diptera: Anthomyiidae)

“…Altogether, BLAST searches against Nr, Nt, Swiss-Prot, GO, COG, and KEGG databases showed that a total of 21,605 (72.8% of 29,659) identified unigenes could be annotated with known biological functions. After Nr database annotation, the E-value distribution, identity distribution, and species distribution were then analyzed (Shi et al 2011) (Figure 1). For the E-value distribution of the predicted proteins, the top hits indicated that 41.2% of the mapped sequences had a significant similarity with a stringent threshold of less than 1e245, and 58.8% of the similar sequences ranged from 1e25 to 1e245 ( Figure 1A).…”

Functional Characterization of Proanthocyanidin Pathway Enzymes from Tea and Their Application for Metabolic Engineering

“…Zhao et al (2005) sequenced 4,320 clones from a tea library and analyzed the sequences of a total of 2,963 ESTs. Recently, the transcriptome of tea was analyzed using a high-throughput Illumina RNAseq technique (Shi et al, 2011); however, no detailed functional characterization of gene products was attempted beyond preliminary biochemical assays (Singh et al, 2009;Zhang et al, 2012). Biochemical studies using leaf protein extracts revealed high activities of ANR in tea leaves and linked this to the formation of E and EGC ; the combined enzymatic reactions of dihydroflavonol 4-reductase (DFR) and LAR confirmed that the formation of catechin and GC proceeded from leucoanthocyanidins.…”