Identification and analysis of genes associated with the synthesis of bioactive constituents in Dendrobium officinale using RNA-Seq

Abstract

Dendrobium officinale L. is an important traditional herb with high commercial value in China. Several bioactive constituents, including polysaccharides and alkaloids, reportedly make major contributions toward the excellent medicinal effect of D. officinale. In this study, the contents of polysaccharides and alkaloids in various organs of D. officinale were measured and compared. We took advantage of transcriptomes from four organs to explore biological mechanisms in the organ-specific distribution of active ingredients in D. officinale. Based on Kyoto Encyclopedia of Genes and Genomes pathways, unigenes related to the enzymes involved in fructose and mannose metabolism and unigenes associated with putative upstream elements of the alkaloid biosynthetic pathway were identified. A large number of candidates, including 35 full-length glycosyltransferase genes and 49 full-length P450 genes, were also identified based on the transcriptome data, and the organ-specific expression pattern of these genes was determined. Furthermore, differential expression of all candidate genes was analyzed in two Dendrobium species, D. nobile L. and D. officinale. The data will supply important clues to exploit useful genes involved in polysaccharide and alkaloid synthesis.

Figure 1

Determination of polysaccharide and alkaloid contents in four different organs of D. officinale L. (a) Samples from different organs of D. officinale L. (b) Determination of polysaccharide contents in four organs, including flower, leaf, stem and root, of D. officinale L. (c) Determination of alkaloid contents in four organs, including flower, leaf, stem and root, of D. officinale L.

Figure 2

MeV cluster analysis of organ-differentially expressed genes. (a) Heat map illustrating the expression profiles of the organ-differentially expressed genes. (b) Cluster analysis by the K-means method from the gene expression profiles. Red circles indicated the organ-specific expressed genes.

Figure 3

Overview of the differentially expressed unigenes (DEGs) among four different organs in D. officinale. (a) Volcanoplots of the DEGs in different comparisons, including S vs R, S vs F and S vs L. (b) VennDiagrams of the DEGs in different comparisons. Gene Ontology (GO) classification of DEGs in the S vs R comparison (c), S vs F comparison (d) and S vs L comparison (e).

Figure 4

Expression patterns of the putative fructose and mannose metabolism unigenes in D. officinale. (a) The pathway of fructose and mannose metabolism is based on KEGG analysis. The full names of enzymes by EC IDs are provided in Table S5. The average expression level of the enzyme encoding unigenes in various organs is indicated by a heat map. The grids with different colors from green to red show the relative expression levels to maximum RPKM values, from 0 to 100%, respectively. (b) Significance analysis of glycosyltransferase unigenes in three stem contained comparisons, including S vs R, S vs F and S vs L. The grids with different shades of blue show the different significant values, from 0 to 0.01.

Figure 5

Expression pattern of the unigenes associated with putative alkaloid biosynthesis in D. officinale. (a) Expression pattern of the unigenes associated with putative upstream elements of alkaloid biosynthetic pathway. Full names of enzymes represented by their abbreviated names were showed in Table S7. The average expression level of the enzyme encoding unigenes in various organs is indicated by a heat map. The grids with different colors from green to red show the relative expression levels to maximum RPKM values, from 0 to 100%, respectively. (b) Phylogenetic analysis of all P450 genes with full-length cDNA from D. officinale. Branches in different background colors indicated different subfamilies. (c) Significance analysis of P450 genes in three leaf contained comparisons, including L vs R, L vs S and L vs F. The grids with different shades of blue show the different significant values, from 0 to 0.01.

Figure 6

Polysaccharide contents determination and expression analysis of key polysaccharide biosynthetic genes in two different dendrobium species. (a) The polysaccharide contents were measured in four organs of two different dendrobium species (D. nobile L. and D. officinale L.). The significantly changes in polysaccharide contents between D. nobile L. and D. officinale L. were indicated by “*”. (b) The related expression levels of eight D. officinale L. highly expressed genes. The significantly changes in expression levels of polysaccharide genes between D. nobile L. and D. officinale L. were indicated by “*”.

Figure 7

Alkaloid contents determination and expression analysis of key alkaloid biosynthetic genes in two different dendrobium species. (a) The alkaloid contents were measured in four organs of two different dendrobium species (D. nobile L. and D. officinale L.). The significantly changes in polysaccharide contents between D. nobile L. and D. officinale L. were indicated by “*”. (b) The related expression levels of seven D. nobile L. highly expressed genes. The significantly changes in expression levels of polysaccharide genes between D. nobile L. and D. officinale L. were indicated by “*”.