Spatio-Temporal Expression Pattern of Six Novel Candidate Genes in Ginsenoside Biosynthesis from Panax ginseng C. A. Meyer

Abstract: To explore the mode of the spatio-temporal expression of six newly discovered ginsenoside biosynthesis candidate gene transcripts, both Northern blotting and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) were used to elucidate the mRNA expression levels of the transcripts in various tissues and organs of Panax ginseng C. A. Meyer during different growth development stages. The six gene transcripts were all differentially expressed in cultured callus, root, stem, leaf, and seed. The mRNA expression levels were significantly higher in four-year-old roots than in one-year-old roots, and results of semi-quantitative RT-PCR assays were in accordance with those of Northern blotting analyses. The results strongly suggest that all six genes were differentially expressed at root-specific developmental stages. In particular, when a quiescent early stage culture suspension of P. ginseng cells was exposed to the ginsenoside biosynthesis-promoting elicitor Aspergillus niger polysaccharide, the GBR6 gene transcript response showed time-dependent increments and was parallel with ginsenoside productivity (P < 0.01). Overexpressionof the GBR6 gene is likely to play a critically important role in the biosynthesis of ginsenosides. The results of the present study provided a background for the further elucidation of the structure and physiological function of these six candidate genes.

Advances in Transcriptomic Studies and Ginsenoside Biosynthesis of American Ginseng

American ginseng（Panax quinquefolius L.）, belonging to the Araliaceae family, is one of the most widely used traditional herbs in the world. Its major bioactive constituents are triterpene saponins known as ginsenosides. Up to date, it is still a big challenge to sequence and assemble the large and repeat-enriched genome of tetraploid American ginseng, using whole genome shotgun（WGS） sequencing strategy. The lack of American ginseng genome information has significantly impeded its genetic and functional genomic studies. With the development of next-generation sequencing（NGS） technologies, sequencing and analysis of transcriptomes have become powerful tools for the discovery of novel genes and elucidation of specific biosynthetic pathways of secondary metabolites. Here we summarized the recent advances in the transcriptomic studies of American ginseng, including high-throughput transcriptome sequencing, assembly, and functional gene annotation and classification. Based on the results of transcriptomic data mining and co-expression analyses, many candidate genes possibly involved in the biosynthetic pathway of ginsenosides have been found, thereby providing an unparalleled opportunity to fully understand the mechanism of ginsenoside biosynthesis and its regulations in American ginseng. Advances in transcriptomic studies will contribute to the molecular breeding and planting management of American ginseng and to the development of novel ginsenoside-type drugs.

Integrative SMRT sequencing and ginsenoside profiling analysis provide insights into the biosynthesis of ginsenoside in Panax quinquefolium

Panax quinquefolium is one of the most common medicinal plants worldwide.Ginsenosides are the major pharmaceutical components in P.quinquefolium.The biosynthesis of ginsenosides in different tissues of P.quinquefolium remained largely unknown.In the current study,an integrative method of transcriptome and metabolome analysis was used to elucidate the ginsenosides biosynthesis pathways in different tissues of P.quinquefolium.Herein,22 ginsenosides in roots,leaves,and flower buds showed uneven distribution patterns.A comprehensive P.quinquefolium transcriptome was generated through single molecular real-time(SMRT)and second-generation sequencing(NGS)technologies,which revealed the ginsenoside pathway genes and UDP-glycosyltransferases(UGT)family genes explicitly expressed in roots,leaves,and flower buds.The weighted gene co-expression network analysis(WGCNA)of ginsenoside biosynthesis genes,UGT genes and ginsenoside contents indicated that three UGT genes were positively correlated to pseudoginsenoside F11,notoginsenoside R1,notoginsenoside R2 and pseudoginsenoside RT5.These results provide insights into ginsenoside biosynthesis in different tissues ofP.quinquefolium.

Penicillium sp.YJM-2013 induces ginsenosides biosynthesis in Panax ginseng adventitious roots by inducing plant resistance responses

Objective:Fusarium oxysporum is a common pathogenic fungus in ginseng cultivation.Both pathogens and antagonistic fungi have been reported to induce plant resistance responses,thereby promoting the accumulation of secondary metabolites.The purpose of this experiment is to compare the advantages of one of the two fungi,in order to screen out more effective elicitors.The mechanism of fungal elicitor-induced plant resistance response is supplemented.Methods:A gradient dilution and the dural culture were carried out to screen strains.The test strain was identified by morphology and 18 s rDNA.The effect of different concentrations(0,50,100,200,400 mg/L)ofPenicillium sp.YJM-2013 and F.oxysporum on fresh weight and ginsenosides accumulation were tested.Signal molecules transduction,expression of transcription factors and functional genes were investigated to study the induction mechanism of fungal elicitors.Results:Antagonistic fungi ofF.oxysporum was identified as Penicillium sp.YJM-2013,which reduced root biomass.The total ginsenosides content of Panax ginseng adventitious roots reached the maximum(48.95±0.97 mg/g)treated with Penicillium sp.YJM-2013 at 200 mg/L,higher than control by 2.59-fold,in which protopanoxadiol-type ginsenosides(PPD)were increased by 4.57 times.Moreover,Penicillium sp.YJM-2013 activated defense signaling molecules,up-regulated the expression of PgWRKY 1,2,3,5,7,9 and functional genes in ginsenosides synthesis.Conclusion:Compared with the pathogenic fungi F.oxysporum,antagonistic fungi Penicillium sp.YJM-2013 was more conducive to the accumulation of ginsenosides in P.ginseng adventitious roots.Penicillium sp.YJM-2013 promoted the accumulation of ginsenosides by intensifying the generation of signal molecules,activating the expression of transcription factors and functional genes.

De novo assembly and comparative analysis of root transcriptomes from different varieties of Panax ginseng C. A. Meyer grown in different environments

Panax ginseng C. A. Meyer is an important traditional herb in eastern Asia. It contains ginsenosides, which are primary bioactive compounds with medicinal properties. Although ginseng has been cultivated since at least the Ming dynasty to increase production, cultivated ginseng has lower quantities of ginsenosides and lower disease resistance than ginseng grown under natural conditions. We extracted root RNA from six varieties of fifth-year P. ginseng cultivars representing four different growth conditions, and performed Illumina paired-end sequencing. In total, 163,165,706 raw reads were obtained and used to generate a de novo transcriptome that consisted of 151,763 contigs(76,336 unigenes), of which 100,648 contigs(66.3%) were successfully annotated. Differential expression analysis revealed that most differentially expressed genes(DEGs) were upregulated(246 out of 258, 95.3%) in ginseng grown under natural conditions compared with that grown under artificial conditions. These DEGs were enriched in gene ontology(GO) terms including response to stimuli and localization. In particular, some key ginsenoside biosynthesis-related genes, including HMG-Co A synthase(HMGS), mevalonate kinase(MVK), and squalene epoxidase(SE), were upregulated in wild-grown ginseng. Moreover, a high proportion of disease resistance-related genes were upregulated in wild-grown ginseng. This study is the first transcriptome analysis to compare wild-grown and cultivated ginseng, and identifies genes that may produce higher ginsenoside content and better disease resistance in the wild; these genes may have the potential to improve cultivated ginseng grown in artificial environments.