RNA-Seq for gene identification and transcript profiling of three Stevia rebaudiana genotypes

Abstract

BackgroundStevia (Stevia rebaudiana) is an important medicinal plant that yields diterpenoid steviol glycosides (SGs). SGs are currently used in the preparation of medicines, food products and neutraceuticals because of its sweetening property (zero calories and about 300 times sweeter than sugar). Recently, some progress has been made in understanding the biosynthesis of SGs in Stevia, but little is known about the molecular mechanisms underlying this process. Additionally, the genomics of Stevia, a non-model species, remains uncharacterized. The recent advent of RNA-Seq, a next generation sequencing technology, provides an opportunity to expand the identification of Stevia genes through in-depth transcript profiling.ResultsWe present a comprehensive landscape of the transcriptome profiles of three genotypes of Stevia with divergent SG compositions characterized using RNA-seq. 191,590,282 high-quality reads were generated and then assembled into 171,837 transcripts with an average sequence length of 969 base pairs. A total of 80,160 unigenes were annotated, and 14,211 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. Gene sequences of all enzymes known to be involved in SG synthesis were examined. A total of 143 UDP-glucosyltransferase (UGT) unigenes were identified, some of which might be involved in SG biosynthesis. The expression patterns of eight of these genes were further confirmed by RT-QPCR.ConclusionRNA-seq analysis identified candidate genes encoding enzymes responsible for the biosynthesis of SGs in Stevia, a non-model plant without a reference genome. The transcriptome data from this study yielded new insights into the process of SG accumulation in Stevia. Our results demonstrate that RNA-Seq can be successfully used for gene identification and transcript profiling in a non-model species.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-571) contains supplementary material, which is available to authorized users.

Transcriptome Analysis to Identify the Putative Biosynthesis and Transport Genes Associated with the Medicinal Components of Achyranthes bidentata Bl.

“…In addition, the cytochrome P450 CYP716A52v2 has been illustrated to engage in the formation of oleanane-type ginsenoside biosynthesis in P. ginseng (Han et al, 2012, 2013). Saponins are high-molecular-weight glycosides consisting of a sugar moiety linked to a triterpenoid or a steroid aglycone, and plant UDP-glycosyltransferases (UGTs) are a widely divergent group of enzymes that transfer a sugar residue from an activated donor to an acceptor molecule (Chen et al, 2014). Moreover, it has been previously reported that UGT73 family genes participate in saponin glycosylation in other plants (Thimmappa et al, 2014).…”

Comparative transcriptomics unravel biochemical specialization of leaf tissues of Stevia (Stevia rebaudiana) for diterpenoid production

“…As the Stevia genome has not yet been sequenced, only limited sources of gene information are available from randomly selected ESTs . Recently, Chen et al (2014) reported a RNA sequencing (RNA-seq) data set of three Stevia genotypes comparing expression patterns of previously reported genes involved in SG biosynthesis in these genotypes, but there were no additional new genes identified related to the MEP pathway, SG biosynthesis, or the synthesis of other terpenoids. Detailed or enriched transcriptome data sets from different tissues of terpenoid-producing nonmodel plants not only provide opportunities for the discovery of new genes and enzymes, but also explain the biosynthetic ability of specialized terpene metabolism that may be spatially restricted to organs, tissues, or cell types (Zerbe et al, 2013;Jin et al, 2014;Zerbe and Bohlmann, 2015).…”

Biotechnological approaches in Stevia rebaudiana and its Therapeutic Applications

“…RNA-Seq, the next generation sequencing technology can be successfully used for gene identification and transcript profiling in a non-model species like Stevia. A comprehensive landscape of the transcriptome profiles of three genotypes of Stevia with divergent steviol glycosides compositions characterized using RNA-seq [52]. Kim et al (2015) integrated metabolome and transcriptome analyses of Stevia to explore the biosynthetic capacity of leaf tissues for diterpenoid metabolism.…”