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 CYP450s and five UDPGs 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 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.

Figure 1

Putative mogrosides biosynthesis pathway in Siraitia grosvenorii. AACT: acetyl-CoA acetyltransferase, EC:2.3.1.9; HMGS: hydroxymethylglutaryl-CoA synthase, EC:2.3.3.10; HMGR: 3-hydroxy-3-methylglutaryl-coenzyme A reductase, EC:1.1.1.34; MK: mevalonate kinase, EC:2.7.1.36; PMK: phosphomevalonate kinase, EC:2.7.4.2; MVD: diphosphomevalonate decarboxylase, EC:4.1.1.33; DXS: 1-deoxy-D-xylulose-5-phosphate synthase, EC:2.2.1.7; DXR: 1-deoxy-D-xylulose-5-phosphate reductoisomerase, EC:1.1.1.267; MCT: 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, EC:2.7.7.60; CMK: 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, EC:2.7.1.148; MCS: 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, EC:4.6.1.12; HDS: 4-hydroxy-3-methylbut-2-enyl diphosphate synthase, EC:1.17.7.1; IDS: 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (isopentenyl/dimethylallyl diphosphate synthase), EC:1.17.1.2; IPI: isopentenyl-diphosphate delta-isomerase, EC:5.3.3.2; GPS: geranyl diphosphate synthase, EC:2.5.1.1; FPS: farnesyl diphosphate synthase/farnesyl pyrophosphate synthetase, EC:2.5.1.10; SQS: squalene synthetase; CAS: cycloartenol synthase, EC:2.5.1.21; SQE: squalene epoxidase, EC:1.14.99.7; CS: cucurbitadienol synthase, EC:5.4.99.8; P450: cytochrome P450, EC:1.14.-.-; and UDPG: UDP-glucosyltransferase, EC:2.4.1.-.

Figure 2

Histogram of gene ontology classification. The results are summarized in three main categories: biological process, cellular component and molecular function. The right y-axis indicates the number of genes in a category. The left y-axis indicates the percentage of a specific category of genes in that main category.

Figure 3

Changes in gene expression profile among the different developmental stages. The number of up-regulated and down-regulated genes between 3 DAF and 50 DAF; 3 DAF and 70 DAF; 50 DAF and 70 DAF are summarized. DEGs: Differentially Expressed Genes.

Figure 4

Expression pattern of genes involved in mogrosides biosynthesis at different fruit developmental stages by DGE. 21 genes expression in 3 DAF, 50 DAF and 70 DAF were analyzed by DGE.

Figure 5

Clustering of CYP450 genes expression profiles at three different fruit developmental stages. Hierarchical clustering of expression data for 17 candidate CYP450 genes using CS and SQE as reference profiles. Expression ratios are expressed as Log 2 values.

Figure 6

Clustering of UDPG genes expression profiles at three different fruit developmental stages. Hierarchical clustering of expression data for 16 candidate UDPG genes using CS and SQE as reference profiles. Expression ratios are expressed as Log 2 values.