Ginseng Genome Research And Multi-omics Data Elaboration On The Ginsenosides Biosynthesis

Panax ginseng C.A.Meyer is a precious herbal medicine and has been clinically used in East Asia for several millennia.Despite its importance for human uses,the genetic background of ginseng is still largely unknown and major details of ginsenoside biosynthetic enzymes have remained unresolved.Although many reports have described the dynamics distribution and accumulation of ginsenosides in plants,molecular mechanism for their biosynthesis and regulatory remain to be characterized.In this paper,we firstly completed and assembled the genome sequence of ginseng based on the research strategy of herbgenomics.Then,mass spectrometry images were applied to precisely locate the spatial distribution of ginsenosides in ginseng roots.Transcriptome and proteome were combined in parsing the molecular expression pattern of ginseng root at the tissue level.An integration analysis of the distribution of ginsenosides and molecular expression characteristics were conducted to screen the potential key genes responsible for ginsenosides synthesis.Thus,we aimed to elucidate the biosynthetic pathway and regulatory mechanisms.The main findings and results are as follows:1.Whole genome sequence of Panax ginseng and analysis of biological genetic backgroundThe whole genome sequence of P.ginseng was assembled using next-generation sequencing.Ginseng has 3.4Gb nucleotide sequence and more than 62%was predicted to be repeats.The ginseng genome contained 42,006 predicted protein-coding genes models,including 488 cytochrome P450 genes,2,556 transcription factors,and 3,745 transporters.These annotated genes may be associated with secondary metabolism.Thirty-one genes were identified to be involved in the mevalonicacid pathway(MVA)and 33 in the methylerythritol phosphate pathway(MEP).Many of these enzymes displayed multiple copies and isoforms.We speculate that the multicopy phenomenon of ginseng MVA/MEP enzymes may be associated with the flexible production and diverse regulatory control of triterpenoid or steroid biosynthesis in plants.In addition,225 UDP-glycosyltransferases(UGTs)were identified,accounting for one of the largest gene families in ginseng.Information on the genome structure and the evolutionary analysis showed that tandem repeats contributed to the duplication and divergence of UGTs.A large number of UGTs were enriched in the ginseng genome during its evolution and partially accounted for the diversity of ginsenosides.These results will facilitate the ginseng genome research and enhance our understanding the biosynthesis of triterpenes.2.Spatial distribution of ginsenosides in ginseng root sections through mass spectrometric imagingDesorption electrospray ionization mass spectrometry(DESI-MS)imaging was utilized to determine the spatial distribution of ginsenosides in ginseng root sections.Ginsenosides Rgl/Rf were found to be highly concentrated in the periderm and internal stele of the root.The ginsenosides Re,Rbl,Rs1/Rs2,Ra1/Ra2,and pseudoginsenoside Rcl were mainly distributed at high concentrations in the periderm and at low concentrations in the center of root.The ginsenosides Ra3 and Rd displayed a diffuse distribution within the root section.In particular,three main roots of ginseng were dissected into periderm,cortex,and stele,respectively.High-performance liquid chromatography(HPLC)was used to quantify the ginsenosides Re,Rf,Rgl,Rg2,Rb1,Rc,Rb2,and Rd within these three tissues.We found that the contents of these eight ginsenosides in the periderm were significantly higher than in the cortex and stele(P<0.001).This confirms that ginsenosides were distributed unevenly in ginseng root,which might be related to plant defense and the differential expression of the biosynthesis genes of triterpenes.These results provide the basis for further analysis of molecular transcription and translation.3.The ginseng transcriptome revealed gene expression pattern in root tissueAccording to the spatial distribution of ginsenosides,we focused on the transcriptome aspects of the three tissues that were previously analyzed with HPLC,especially the periderm.A total of 32,144 expressed genes were detected from the transcriptome data,and 83.3%were shared among the three tissues.Approximately 659 to 797 genes showed tissue specific expression,which reflected the consistency and difference of gene transcription in ginseng root at the tissue level.More than 21,000 expressed gene were positively correlated with the content of ginsenosides,suggesting that complex mechanisms are involved in ginsenosides synthesis and regulation.The gene expression patterns were compared among three tissues,in which we screened for 2,116 differentially expressed genes,which mainly involved in metabolism,cell process,single-organism,localization,response to stimuli,and biological regulation within the biological processes category.Catalytic and protein binding were the most abundant GO terms within the molecular function category.Cell and cell part were the most highly represented terms in the cellular component category.On the whole,the expression profiles of the genes involved in ginsenosides synthesis were varied among the three tissues.Three 3-hydroxy-3-methylglutaryl-CoA reductases(HMGRs),two 3-hydroxy-3-methylglutaryl-CoA reductases(FPSs),two dammarendiol synthases(DDSs),and 60 UGTs were highly expressed in the periderm,which may be responsible for the unevenly accumulation of ginsenosides.In addition,differential splicing was found in 19 ginsenoside synthesis genes,indicating that the splicing events played an important regulatory role in the biosynthesis of triterpenes.4.Ginseng proteome uncovered the protein expression pattern in root tissueLabel-free technology was applied to explore the protein expression pattern for the three tissues that were previously analyzed with HPLC and transcriptome methods.A total of 2,719 expressed proteins were detected from the proteome data,and 56.4%were shared among the three tissues.Approximately 172 to 500 proteins had tissue specific expression,reflecting the consistency and difference of protein expression in ginseng root at the tissue level.When compared among the three tissues,the protein expression pattern was screened for 839 differentially expressed proteins,which were mainly involved in amino acid biosynthesis,carbon metabolism,starch and sugar metabolism,and glycolysis/gluconeogenesis.The periderm was compared with the cortex and stele,showing that 90%of the resistance-proteins were highly expressed in the periderm,and 22 of them were significantly up-regulated,which reflects the molecular mechanisms of periderm defense.In addition,a total of 28 proteins were identified to be involved in the biosynthesis of ginsenosides,including nine enzymes involved in the MVA pathway,seven enzymes in the the MEP pathway,and 12 UGTs.The expression profiles of the proteins involved in the synthesis of ginsenosides varied among the three tissues,which may be contribute to the unevenly enrichment of ginsenosides.5.Combined analysis for transcriptome and proteomeA weak correlation was found between RNA-seq and label-free data through a combined analysis.Specifically,62,98,and 18 molecules displayed a common significant differential expression in the periderm compared with the cortex,the periderm compared with the stele,and the cortex compared with the stele,respectively.Most of these molecules were highly expressed in the periderm,and were mainly related to metabolic process,single-organism process,cellular process,and response to stimuli in the biological processes category and catalytic activity and binding in the molecular function category.Among these molecules,the genes involved in metabolic pathways are the most numerous,suggesting that these molecules play an important regulatory role in the growth and development of ginseng roots.More than 88%of molecules were differentially expressed at the mRNA level,but the corresponding proteins,including 31 genes associated with ginsenosides synthesis,were not detected in this study,Among molecules differentially expressed at the protein level but not at the mRNA level,more than 99%were detected via gene transcription,including seven ginsenosides synthesis related enzymes.This data suggests that some molecules may be affected by post-translational regulation or transportation.This work presents the draft genome of P.ginseng and a comprehensive characterization of molecular expression patterns in ginseng root at the tissue level.This study provides a powerful resource for furthering research on ginseng and will help to improve the interpretation of complex mechanisms of the biosynthesis,accumulation,and transportation of ginsenosides.