Comparative Transcriptomics Of Panax Notoginseng In Different Cultivation Modes Provides Insights Into Ginsenoside Biosynthesis

Panax notoginseng is a valued medicinal plant.Together with P.ginseng,American ginseng(P.quinquefolius L.),it belongs to Panax,Araliaceace.The dried root of this plant,known as "Sanchi",is a famous haemostic top class tonic for more than 2,000 years in the folk of China.It was used to promote blood circulation to remove blood stasis.In pharmacology,P.notoginseng possesses anti-thrombotic,anti-hypertensive,anti-atherosclerotic,neuroprotective and hepatoprotective activities.Sanchi is best known for its uses in the treatment of cardiovascular diseases and valuable hemostatic effect.It is also a principal component of Yunnan Bai Yao and Xue Sai Tong,which are famous medicinal products for bleed diseases.The ginsenosides are considered to be the principal bioactive components in Sanchi,which are mainly derived from nature products.Secondary metabolic engineering is an important way to efficiently produce a large amount of secondary metabolites,which may ensure ginsenoside production.Up to date,we still lack an overall knowledge of of ginsenoside biosynthesis in P.notoginseng.In this study,the transcriptome of P.notoginseng was sequenced and analyzed using single molecular real time(SMRT)sequencing and Illumina(NGS)sequencing technologies.We characterized the expression profiling of genes involved in ginsenoside biosynthesis.Besides,we analyzed the genes associated with photoperiodic regulatory flowering pathway,disease resistance,transcript factors and the chloroplast genome of P.notoginseng.Main results of this study were summarized as follows.In order to obtain the high-quality reference transcriptome of P.notoginseng,we performed SMRT and Illumina sequencing technologies.The circular-consensus(CCS)reads via self correction and the correction with Illumina sequencing reads resulted in a high-quality reference transcriptome.The contig N50 was 2,967 bp,and the longest length reach 20,087 bp.Comparisons of NGS and SMRT sequenced transcriptomes show that the quality of SMRT sequencing transcriptome is good enough to be used as a reference of P.notoginseng.Of 51,404 unigenes,we identified 23 gene families involved in the ginsenoside biosynthetic pathway.Considering that plant genes are transcribed and expressed of genes show the spatial and temporal specificity,we performed RNA sequencing of the five different tissues(root,stem,leaf,flower and rhizome)of P.notoginseng using Illumina sequencing technology.We mapped these NGS reads to SMRT-based reference transcriptome to investigate specific expression patterns of genes related to ginsenoside biosynthesis.Our results showed that these 23 metabolite-related gene families exhibited considerably distinct patterns of gene expression among tissues,of which 13 gene families(ACAT,HMGS,HMGR,MK,MDD,DXS,DXR,GGR-GGPS,SS,SE,FPS,DDS and AS)were most highly expressed in the flowers.This result reveals that ginsenosides may be actively synthesized in flowers.Phylogenetic analyses of 23 genes families from P.notoginseng and 9 plant species showed that P.notoginseng experienced independent lineage-specific duplications of 14 gene families,including AACT,AS,DDS,DXR,DXS,FPS,HDR,SE,MK,HDS,HMGS,HMGR,SS and GGPS_GGR,The functional divergence of these duplicated genes,known as neofunctionalization,may enhance the evolution of a specified ginsenoside biosynthesis in P.notoginseng.To uncover the biosynthesis and accumulation of ginsenosides in P.notoginseng,we further collected RNA-seq datasets from 11 tissues of the one-year,two-year and three-year old plants,respectively,and three biological replicates were then set for each sample.Analyses of expression patterns of the ginsenoside biosynthesis-related genes revealed that at least 10 gene families(ACAT,HMGS,HMGR,FPS,DXS,DXR,GGR-GGPS,IPI,SS and SE)were most highly expressed in the flowers,further supporting the above-mentioned results.In addition,we analyzed the tissue-specific expression patterns of CYP450 and GT gene families in P.notoginseng that are functionally known to encode enzymes that synthesize protopanaxadiol-type Rd(CYP716A47,UGT94B1,UGT74A1)and protopanaxatriol-type Rgl(CYP716A531v2,UGTPg101),respectively.Comparisons of gene expression patterns of one-year old plant with two-and three-year old plants of P.notoginseng still suggest that massive biosynthesis of ginsenosides occurred in flowers.To determine the distribution of some important ginsenosides we performed using high-performance liquid chromatography analysis(HPLC)of the same tissues that we generated RNA-seq datasets.Our results showed that these ginsenosides are largely found in the root,and the contents of ginsenosides gradually increase with the growth of the P.notoginseng plants.Taken together,our results show that ginsenosides may be actively synthesized in flowers/leaves besides roots and then accumulated in roots.Based on reference transcriptome dataset of P.notoginseng,we identified the genes associated with photoperiodic regulatory flowering pathway,disease resistance and transcript factors.The results showed that the flowering time may be regulated by the photoperiod in P.notoginseng:the genes that encode photoreceptor proteins exhibited quick expansions,which may help to collect and utilize enough light under the habitats in lack of sunshine.