Abstract
BackgroundSiraitia 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.ResultsIn 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 CYP450s and five UDPGs were selected as the candidates most likely to be involved in mogrosides biosynthesis.ConclusionA 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 CYP450s and five UDPGs 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.
Highlights
Siraitia grosvenorii (Luohanguo) is an herbaceous perennial plant native to southern China and most prevalent in Guilin city
Digital gene expression profile (DGE) of S. grosvenorii at 3, 50 and 70 days after flowering (DAF) identified seven cytochrome P450 (CYP450) and five UDPGs out of a total of eighty-five CYP450 and ninety UDPG unigenes, as potential candidate genes responsible for mogroside backbone modifications. These results demonstrated the powerful ability of high-throughput sequencing to identify candidate genes involved in novel metabolic pathways in non-model plant systems
Illumina Sequencing and de novo assembly To obtain an overview of the S. grosvenorii fruit transcriptome, a cDNA library was generated from an equal mixture of RNA isolated from 50 DAF and 70 DAF fruits, and pair end sequenced using the Illumina platform
Summary
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. Mogroside V is nearly 300 times sweeter than sucrose. 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. Many cucurbitane-type triterpene glycosides have been isolated and characterized from the fruits [5,6,7,8,9,10]. 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]. 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 [12]
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