An efficient approach to finding Siraitia grosvenorii triterpene biosynthetic genes by RNA-seq and digital gene expression analysis

Abstract

Background

Siraitia grosvenorii (Luohanguo) is an herbaceous perennial plant native to southern China and most prevalent in Guilin city. Its fruit contains a sweet, fleshy, edible pulp that is widely used in traditional Chinese medicine. The major bioactive constituents in the fruit extract are the cucurbitane-type triterpene saponins known as mogrosides. Among them, mogroside V is nearly 300 times sweeter than sucrose. However, little is known about mogrosides biosynthesis in S. grosvenorii, especially the late steps of the pathway.

Results

In this study, a cDNA library generated from of equal amount of RNA taken from S. grosvenorii fruit at 50 days after flowering (DAF) and 70 DAF were sequenced using Illumina/Solexa platform. More than 48,755,516 high-quality reads from a cDNA library were generated that was assembled into 43,891 unigenes. De novo assembly and gap-filling generated 43,891 unigenes with an average sequence length of 668 base pairs. A total of 26,308 (59.9%) unique sequences were annotated and 11,476 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. cDNA sequences for all of the known enzymes involved in mogrosides backbone synthesis were identified from our library. Additionally, a total of eighty-five cytochrome P450 (CYP450) and ninety UDP-glucosyltransferase (UDPG) unigenes were identified, some of which appear to encode enzymes responsible for the conversion of the mogroside backbone into the various mogrosides. Digital gene expression profile (DGE) analysis using Solexa sequencing was performed on three important stages of fruit development, and based on their expression pattern, seven CYP450 s and five UDPG s were selected as the candidates most likely to be involved in mogrosides biosynthesis.

Conclusion

A combination of RNA-seq and DGE analysis based on the next generation sequencing technology was shown to be a powerful method for identifying candidate genes encoding enzymes responsible for the biosynthesis of novel secondary metabolites in a non-model plant. Seven CYP450 s and five UDPG s were selected as potential candidates involved in mogrosides biosynthesis. The transcriptome data from this study provides an important resource for understanding the formation of major bioactive constituents in the fruit extract from S. grosvenorii.

Background

Siraitia grosvenorii, which belongs to the Cucurbitaceae family, has long been used in traditional Chinese medicine as a natural sweetener and as a folk medicine for the treatment of lung congestion, colds and sore throats. In recent years, important pharmacological characteristics, such as anti-cancer and anti-hyperglycemic effects and inhibition of oxidative modification of low-density lipoprotein, have been reported [1–4]. Many cucurbitane-type triterpene glycosides have been isolated and characterized from the fruits [5–10]. The mixed mogrosides have been estimated to be about 300 times as sweet as sucrose so that an 80% extract was nearly 250 times sweeter than sugar [7]. Among them, mogroside V which accounts to 20% of mogrosides is extremely sweet. The purified, sweet principle, mogroside V, has been approved as a high-intensity sweetening agent in Japan [11] and the non-caloric sweet taste extract is a generally recognized as safe (GRAS) non-nutritive sweetener, flavor enhancer, and food ingredient in the USA [33], American ginseng[34], Glycyrrhiza uralensis[35], Medicago truncatula[24, 5, 6 and 7.

Evaluation of DGE libraries

A statistical analysis of the frequency of each tag in the different cDNA libraries was performed to compare gene-expression in different developmental stages. Statistical comparison was performed using the method described by Audic et al[22]. FDR (false discovery rate) was used to determine the threshold of P-value in multiple test and analysis. We used FDR < 0.001 as the threshold to judge the significance of gene expression difference. For pathway enrichment analysis, we mapped all differentially expressed genes to terms in the KEGG database and looked for significantly enriched KEGG terms compared to the genome background.

Clustering of CYP450 and UDPG gene expression profiles

Hierarchical clustering of log-transformed expression data was carried out using the Cluster 3.0 and Treeview programs [46]. Correlations between gene clusters were determined using Pearson's correlation. Heat maps were constructed using the University of Toronto BAR Heatmapper tool http://www.bar.utoronto.ca/ntools/cgi-bin/ntools_heatmapper.cgi.