research article transcriptome sequencing of gynostemma...

Research ArticleTranscriptome Sequencing of Gynostemmapentaphyllum to Identify Genes and EnzymesInvolved in Triterpenoid Biosynthesis

Qicong Chen Chengtong Ma Jieying Qian Xiuwan Lan Naixia ChaoJian Sun and Yaosheng Wu

Key Laboratory of Biological Molecular Medicine Research of Guangxi Higher EducationDepartment of Biochemistry and Molecular Biology Guangxi Medical University Nanning Guangxi China

Correspondence should be addressed to Yaosheng Wu wuyaosheng03sinacom

Received 21 July 2016 Revised 29 September 2016 Accepted 7 November 2016

Academic Editor Sylvia Hagemann

Copyright copy 2016 Qicong Chen et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

G pentaphyllum (Gynostemma pentaphyllum) a creeping herbaceous perennial with many important medicinal properties iswidely distributed inAsia Gypenosides (triterpenoid saponins) themain effective components ofG pentaphyllum arewell studiedFPS (farnesyl pyrophosphate synthase) SS (squalene synthase) and SE (squalene epoxidase) are the main enzymes involved in thesynthesis of triterpenoid saponins Considering the important medicinal functions ofG pentaphyllum it is necessary to investigatethe transcriptomic information of G pentaphyllum to facilitate future studies of transcriptional regulation After sequencing Gpentaphyllum we obtained 50654708 unigenes Next we usedRPKM(reads per kilobases permillion reads) to calculate expressionof the unigenes and we performed comparison of our data to that contained in five common databases to annotate different aspectsof the unigenes Finally we noticed that FPS SS and SE showed differential expression of enzymes in DESeq Leaves showed thehighest expression of FPS SS and SE relative to the other two tissues Our research provides transcriptomic information of Gpentaphyllum in its natural environment and we found consistency in unigene expression enzymes expression (FPS SS and SE)and the distribution of gypenosides content in G pentaphyllum Our results will enable future related studies of G pentaphyllum

1 Introduction

Gynostemma pentaphyllum (Thunb) Makino is a kind ofcreeping herbaceous perennial that is distributed in AsiaGynostemma pentaphyllum (G pentaphyllum) grows in manyplaces of China including Guangxi Guangdong FujianGuizhou Yunnan Hubei Anhui Hebei Jiangsu HenanShandong Sichuan and Shanxi and in Taiwan G pentaphyl-lum also grows in neighboring countries such as BangladeshIndia Indonesia Japan Republic of Korea and Malaysia(data was obtained from the Checklist of South ChinaBotanical Garden) [1] Gypenosides (triterpenoid saponins)the major effective components of G pentaphyllum havevarious bioactivities that explain the extensive application ofG pentaphyllum in naturalmedicines [2ndash13] For instance thegypenosides exhibit a hypoglycemic effect by increasing thesecretion of insulin [11ndash13] Other functions like anticancer

function anti-inflammatory function antianxiety functionblood fat-reducing liver cells-protecting neuroprotectionand immunoprotection have also been reported [2ndash10]

Gypenosides are secondary metabolites in the synthesispathway of triterpenoids Mevalonate or isoprenoid are theprecursors in the beginning of the pathway of triterpenoidsynthesis which we refer to as the MVA (mevalonic acid) orMEP (methylerythritol phosphate) pathway (Figure 1) [14ndash16]The synthesis pathway of the triterpenoids can be decom-posed into three parts (1) the synthesis of IPP (isopentenylpyrophosphate) or DMAPP (dimethylallyl pyrophosphate)(2) the synthesis and cyclization of the squalene and (3) thefunctionalization reaction that proceeds with complexity ofthe squalene (Figure 1) FPS (farnesyl pyrophosphate syn-thase) SS (squalene synthase) and SE (squalene epoxidase)were previously identified as the main enzymes involvedin the synthesis of triterpenoid saponins [17ndash20] FPS SS

Hindawi Publishing CorporationInternational Journal of GenomicsVolume 2016 Article ID 7840914 10 pageshttpdxdoiorg10115520167840914

2 International Journal of Genomics

G3P PyruvateAcetyl-CoAAcetoacetyl-CoAAACT

EC 2319

EC 2217EC 23310 DXPS

DXP

DXR

HMG-CoA

EC 111267HMGR

HMGS

EC 11134

EC 11188MEPMVA CDP-ME2P

CMK

MCTEC 27760

CDP-ME

EC 46112EC 271148MKEC 27136

MVAP MECPP

HDSEC 11771EC 11773MPK

MDS

EC 2742

MVA2P HMBPP

EC 41133 MDCHDR

HDREC 11712

IPPIDI

DMAPPEC 5332

+IPPEC 2511 GPPS EC 2511

GPP

FPSEC 25110GGPPS

GGPPFPPEC 25129

+FPP

Monoterpenoids

DiterpenoidsSesquiterpenoids

EC 25121 SS

SESqualene TriterpenoidsEC 1141417 23-Oxidosqualene

GPPS + IPP

Figure 1 Triterpenoid synthesis pathway Note AACT acetyl-CoA C-acetyltransferase HMGS hydroxymethylglutaryl-CoA synthaseHMG-CoA hydroxymethylglutaryl-CoA HMGR hydroxymethylglutaryl-CoA reductase MVA mevalonate MK mevalonate kinaseMVAP mevalonate phosphate MPK mevalonate phosphate kinase MVAPP mevalonate diphosphate MDC mevalonate diphosphatedecarboxylase G3P D-glyceraldehyde-3-phosphate acetaldehydetransferase DXPS 1-deoxy-D-xylulose-5-phosphate synthase DXP 1-deoxy-D-xylulose-5-phosphate DXR 1-deoxy-D-xylulose-5-phosphate reductoisomerase MEP 2-C-methyl-D-erythritol 4-phosphateMCT 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase CDP-ME 4-(cytidine 51015840-diphospho)-2-C-methyl-D-erythritol CDP-ME2P 2-phospho-4-(cytidine 51015840-diphospho)-2-C-methyl-D-erythritol MECPP 2-C-methyl-D-erythritol 24-cyclodiphosphate HDS l-hydroxy-2-methyl-butenyl-4-diphosphate synthase HMBPP l-hydroxy-2-methyl-2-butenyl-4-diphosphate HDR 4-hydroxy-3-methylbut-2-enyl diphosphate reductase IPP isopentenyl-PP IDI isopentenyl-diphosphate delta-isomerase DMAPP dimethylallyl-PP GPS geranyl-diphosphate synthase GPS geranyl-diphosphate synthase GPPS geranylgeranyl diphosphate synthase GPP geranylgeranyl diphosphateFPS farnesyl diphosphate synthase GGPPS geranylgeranyl diphosphate synthase GGPP geranylgeranyl diphosphate SS squalene synthaseand SE squalene epoxidase

and SE are required for the synthesis and cyclization ofthe squalene that combines two sesquiterpenoids into onetriterpenoid (C15 + C15 = C30) [16 21] After this steptriterpenoids can be transformed into many isoform typeslike protosteryl type (chair-chair-chair-boat conformations)dammarenyl type (chair-chair-chair-boat conformations)cadinyl type (chair-chair-chair-boat conformations) and

hopene and tetrahymanol (chair-chair-chair-chair conforma-tions or chair-chair-chair-boat conformations) [21]

Like the ginsenosides gypenosides (triterpenoids) in Gpentaphyllum have various and vital applications in medicineand health [22] However gypenosides showed much higherheterogeneity when compared with ginsenosides and morethan 169 kinds of gypenosides were found inG pentaphyllum

International Journal of Genomics 3

[23ndash28] In other words more than five times the number oftriterpenoid saponins was found in G pentaphyllum relativeto Panax ginseng (P ginseng) It was interesting that Gpentaphyllum has such diversity in triterpenoids compared toother plants [26] This is likely related to different expressionof genes and enzymes involved in the synthesis pathway oftriterpenoids Nowadays transcriptomic sequencing (RNAsequencing) is a more and more popular tool to exploretranscriptomic process [29ndash37] because RNA sequencinghas several advantages relative to DNA sequencing likelower fee higher efficiency more advanced features andso forth [36ndash39] In 2011 Sathiyamoorthy Subramaniyamanalyzed the transcriptome of G pentaphyllum related tothe synthesis pathway of triterpenoids However this articlehad two important limitations First the samples used forsequencing only included two tissues (leaves and roots)and G pentaphyllum that was sampled and sequenced wasplanted in water and not in its natural environment [32]This is important because G pentaphyllum exhibits greatphenotypic diversity in different environments because of itsstrong adaptability [33 40ndash44] Additionally although theauthor showed sequencing data in the paper no associationanalysis among unigenes expression enzyme expression orthe distribution of gypenosides content of G pentaphyllumwas determined In 2015 Zhao et al identified EST-SSRmakers by analyzing the sequencing data of two species ofGynostemma (Cucurbitaceae) [33] In that article the tissueswere natural and complete but the three tissues (youngleaves flowers and immature seeds) from each kind ofG pentaphyllum were mixed up together to extract RNAfor constructing cDNA to sequence In other words thesequencing data and related information in that article werea mixed result and these results could not be classified bytissues Therefore to address this deficiency of knowledgewe collected G pentaphyllum in natural environment andsequenced its transcriptome separately by Illuminarsquos NextSeq500

2 Materials and Methods

21 Sample Collection and Preparation G pentaphyllum usedfor sequencing was planted in the Medicinal Plant Gardenof Guangxi Traditional Chinese Medical University NanningCity Guangxi Autonomous Region China In July 2015we harvested G pentaphyllum after identification by MrYilin Zhu (Guangxi Traditional ChineseMedical University)Fibrous roots leaves and stems were separately collectedand cleaned and removed of impurities like soil (biolog-ical repeat of collections of each tissue was three times)(Figure S1ndashS5 in Supplementary Material available online athttpdxdoiorg10115520167840914) Finally the sampleswere saved in cryotubes and submerged in liquid nitrogenimmediately

22 Illumina Sequencing The plant tissue sample of G pen-taphyllum was sent to Personalbio Company (Shanghai CityChina) for transcriptome sequencing using Next-GenerationSequencing (NGS) technology based on the sequencingplatform of Illuminarsquos NextSeq 500 First the mRNA was

cleaved into little segments after treatment with chemicalreagents and high temperature Next the segments were usedto construct a cDNA library that was sequenced by paired end(PE) reads

23 Unigene Assembly Trinity (r20140717 k-mer 25 bp) pro-fessional software was used to assemble the RNA sequence[45] First high-quality sequences were constructed into ashort-sequence library with length of k-mer Next primarycontig sequences were obtained by the extension of the short-sequence library using overlaps with a k-mer-1 length Nextprimary contig sequences were categorized by their overlapsand categorical contigs were constructed into the De Bruijngraph Based on the recognition rate of reads in each categorytranscript sequences were restored by the contigs Afterassemblage by Trinity BLAST (version 2230+) was used tocompare the assembled sequences with reference sequencesin NCBI (National Center for Biotechnology Information)nonredundant protein (NR) sequences to determine thebest comparison results Finally sequences with the same ginumber were classified as the same unigene and the longestsequence was regarded as the representative sequence of thatunigene [46]

24 Analysis of Unigene Expression RPKM (reads per kilo-bases per million reads) was used to calculated unigeneexpression of G pentaphyllum and the calculation methodof RPKM is described below [47] Before we calculated theunigene expression we need to process the read count ofunigenes with Bowtie 2 (224 default setting) [48] TheRPKM density distribution generally reflected the pattern ofgene expression Typically unigenes withmedium expressioncover the majority of the area under the curve (AUC) in thedensity distribution of RPKM (as drawn with the densityfunction of software R) Oppositely unigenes with higheror lower expression occupy the minority of AUC DESeq(version 1180) software was used to analyze the differentialexpression of unigenes in our study [49] The expression ofunigenes was compared by the fold change (fold change gt2) and its significance (119901 value lt 005) The final result wasdisplayed by Venn diagram

RPKM = total exon reads(mapped reads (millions) lowast exon length (KB)) (1)

25 Functional Annotation After categorization unigeneswere annotated for functions using five databases NCBInonredundant protein (NR) sequences GeneOntology (GO)[50 51] Kyoto Encyclopedia of Genes and Genome (KEGG)[52 53] evolutionary genealogy of genes NonsupervisedOrthologousGroups (eggNOG) [54] and Swiss-Prot [55 56]

26 Analysis of the Distribution of Gypenosides Content inG pentaphyllum First dried powder of the sample (about15mg) was mixed with 10mL of extraction solvent (ethanolcontaining 50 pure water) and was processed by a con-tinued supersonic treatment for 30 minutes Second themixed solvent was evaporated to dryness using a rotaryevaporator The dry gypenosides were redissolved in 50mL


Table 1 Overview of the sequencing and assembly

Categories Description Number

Total reads Total number of reads (RAW) 356311342Number of clean reads 352999296

Contigs

Total length (bp) 249488543Sequence number 1119964Max length (bp) 13870Mean length (bp) 223

GC 48

Transcriptome


GC 46

Unigenes


GC 44

Table 2 Overview of annotation

Annotation in database Unigene number Percentage ()NR 67068 100GO 40623 61KO 5884 9eggNOG 64768 97Swiss-Prot 55429 83In all databases 5031 75

hot water (50∘C) Third this solution was applied to achromatography column containing D101 macroporous resinand allowed to stand for 20 minutes Fourth pure water wasused to rinse unbound material from the macroporous resinwhile extraction solvent (ethanol containing 50 pure water)was used to remove the gypenosides form column Fifththe solvent of gypenosides was brought up to 50mL andprocessed with a color reaction by vanillin We then detectedthe absorbance of the solvent (after color reaction) by UV-Vis Spectrophotometer at a wavelength of 584 nm Finally weused Panaxadiol (C30H52O3) as a standard sample to calculatethe actual content of gypenosides in samples by the standardcurve method

3 Result

31 Overview of the Sequencing and Assembly ThemRNA ofG pentaphyllum was cut into many small segments to con-struct a cDNA library After sequencing the cDNA librarywe obtained 352999296 original reads However this setof original reads also contained a lot of adapters and low-quality sequences so 103500643 reads were filtered outleaving 249488643 clean reads of high qualityNext 1119964contigs with a total length of 249488543 bp were assembledby the overlaps of the original reads and we used these

contigs to restore the transcriptome sequences In the nextstep we harvested 159858904 transcriptome sequences andused BLAST (Basic Local Alignment Search Tool) for alltranscriptome sequences in the NR database The transcrip-tome sequence with the highest score in BLAST was savedand the transcriptome sequences with same gi number werecategorized as coming from the same unigene Finally weobtained 50654708 unigenes with a mean length of 755 bpThe overview of sequences is presented in Table 1

32 Result of Annotation We used five databases NR GOKEGG eggNOG and Swiss-Port to annotate unigenes forfunctions (Figure S6ndashS9)The overview of annotation is listedin Table 2 The result of each annotation is provided in thesupport file Generally speaking eggNOG showed an identi-fication rate of 9657 and KEGG displayed the lowest iden-tification rate of 877 when comparing unigenes with thereference sequences Based on GO annotation the unigeneswere categorized into different categories based on differentfunctions andGO Slim displayed general characteristic aboutthe distribution of the unigenes Then we used the eggNOGdatabase to explore the biological function of protein inmoredetail because eggNOG classifies different protein sequencesinto a more detailed directory Similarly Swiss-Port was alsoused for annotation of the protein sequences and Swiss-Portbuilds on eggNOG and provided more detailed structuralinformation about the protein Finally KEGG is the last butmost important database we used to annotate enzymes sincethe KEGG pathway annotation showed us the network ofthe intermolecular reaction This allows determination ofthe enzymes that are located in the synthesis pathway oftriterpenoid saponins

33 Expression ofUnigenes RPKMis a normalizationmethodto calculate gene expression and we used the density dis-tribution of the RPKM to show the expression of unigenesThe map of the density distribution showed that our uni-genes expression conformed to standards because unigeneswith mid-range expression occupied the majority of AUC(area under the curve) and unigenes with lower or higherexpression were the minority of AUC (Figure 2) In the den-sity distribution unigenes in fibrous roots showed muchhigher expression than in the stems and leaves Althoughstems and leaves showed similar unigene expression stemshad slightly higher unigene expression than the leaves Weanalyzed the result of expression of the unigenes of FPS SSSE and 120573-AS (beta-amyrin synthase) in Table 3 Noticeablythe unigenes that encoded FPS SS SE and 120573-AS showedthe highest expression in leaves and the lowest expression infibrous roots In the unigene expression of 120573-AS the leavesshowed almost 125 times higher expression than in the fibrousroots The unigenes of FPS SS SE and 120573-AS showed higherexpression in the stems than in the fibrous roots Based onthis sequencing data we detected the differential expressionof unigenes using the software DESeq We obtained theupregulated and downregulated unigenes in the pairwisecomparison among the data from the fibrous roots stemsand leaves (Table 4) We also determined the unigenes thatshowed differential expression in all samples We used Venn


Table 3 RPKM of unigenes (FPS SS SE and 120573-AS) in samples

Enzyme Unigene ID RPKM of sample 1 RPKM of sample 2 RPKM of sample 3Fibrous roots Stems Leaves Fibrous roots Stems Leaves Fibrous roots Stems Leaves

FPS c118250 g1 i1 835 793 1981 761 911 1953 844 384 97SS c136108 g1 i1 244 391 2703 239 436 2518 426 306 612SE-1 c127030 g2 i1 5923 16195 59115 5305 2111 72675 6754 7484 45422SE-2 c113536 g2 i1 079 095 566 026 094 287 06 052 27120573-AS c70785 g1 i1 649 8698 40379 55 6295 47136 166 1579 20631Note FPS farnesyl pyrophosphate synthase SS squalene synthase SE squalene epoxidase 120573-AS beta-amyrin synthase

Table 4 Overview of the upregulated and downregulated unigenes

Case Control Upregulated unigenes Downregulated unigenes Total DE unigenesNumber Number Number

GPG GPJ 3476 518 1759 262 5235 781GPG GPY 5590 833 3089 461 8679 1294GPJ GPY 2938 438 1890 282 4828 72Note GPG fibrous roots of G pentaphyllum GPJ stems of G pentaphyllum GPY leaves of G pentaphyllum DE differential expression

GPG1

GPG2

GPG3

GPY1

GPY2

GPY3

06

04

02

00

minus2 20 4

Den

sity

GPJ1

GPJ2

GPJ3

log10(TPM)

Figure 2 Density distribution of RPKM Note GPG fibrous rootsGPJ stems GPY leaves RPKM reads per kilobases per millionreads

diagram function in software R to describe the general dis-tribution of unigenes with differential expression (Figure 3)Combining the data in Table 4 and Figure 3 we concludedthat 10832 unigenes showed differential expression Addi-tionally 699 unigenes displayed differential expression in allsamples while 6512 unigenes showed differential expressionin the pairwise comparison of samples

34 Content Distribution of Gypenosides in G pentaphyllumA UV-Vis Spectrophotometer was used to detect the content

3736305 1963

699

2281495

1353

Fibrous roots versus leaves

Fibrous roots versus stems

Stems versus leaves

Figure 3 Venn diagram of differential expression of unigenes

distribution of gypenosides in G pentaphyllum Leaves hadthe highest content (3189) of gypenosides of all samples(Table 5) and the content of gypenosides in stems (0365)or fibrous roots (0172) was much lower than the leavesA correlation coefficient (1198772) of 0996 in the standard curveindicates that our result was accurate and reliable (FigureS10)


Monoterpenoids


Triterpenoids

FPS

GGPPSGGPPFPP

EC 25129+FPP

SS

SESqualene

EC 1141417

IPPIDI

DMAPPEC 5332

EC 2511+IPPEC 2511

EC 25110

EC 25121

GPPS

GPP

Mevalonatepathway

MEPDOXPpathway

23-Oxidosqualene

GPPS + IPP

Figure 4 FPS SS and SE in the synthesis pathway of triterpenoids Note IPP isopentenyl-PP IDI isopentenyl-diphosphate delta-isomerase DMAPP dimethylallyl-PP GPS geranyl-diphosphate synthase GPPS geranylgeranyl diphosphate synthase GPP geranylgeranyldiphosphate FPS farnesyl diphosphate synthase GGPPS geranylgeranyl diphosphate synthase GGPP geranylgeranyl diphosphate SSsqualene synthase and SE squalene epoxidase

Table 5 Content distribution of gypenosides in G pentaphyllum

Sample Content of gypenosidesNumber 1 NUmber 2 Number 3 Average

Fibrous roots 01725 01731 01731 01729Stems 03645 03657 03682 03662Leaves 31887 31912 31931 31910

35 Expression of Enzymes in the Synthesis Pathway of Triter-penoids Based on the KEGG Pathway a more detailed resultabout the enzymes expression in the triterpenoids synthesiswas obtained We categorized related enzymes into threeparts according to the synthesis pathway of the triterpenoids(1) enzymes involved in the synthesis of IPP or DMAPP (2)enzymes involved in the synthesis and cyclization of squaleneand (3) enzymes in the squalene functionalization reaction(Figure 1) We focused on FPS SS and SE because thesethree enzymes play an important role in the synthesis andcyclization of triterpenoids (Figure 4) [17ndash19] The results areshown in Table 6 and Figure 5 We found three noticeableresults First in the comparison of FPS only one comparisonbetween the fibrous roots and leaves was found Leavesshowed higher expression of FPS compared to the fibrousroots Second in the comparison of SS two comparisonswerefound and the result showed that leaves had higher expressionthan the fibrous roots and stems Third in the comparisonof SE leaves showed higher expression than the stems andfibrous roots The fibrous roots showed higher expression ofSE than the stems

4 Discussion

G pentaphyllum is a creeping herbaceous perennial withmedicinal properties used in traditional Chinese medicineTriterpenoid saponins the main effective components of Gpentaphyllum have been widely studied [2ndash13 23 24] In thisstudy we obtained transcriptome information using RNAsequencing a technique that exhibits higher efficiency andis less expensive than DNA sequencing [38] We analyzedthe associations of unigene expression enzyme (the outputof the unigenes) expression and the content distributionof gypenosides (enzyme output) after functional annotation(Table 2) RPKM calculating (Table 3) and measurement ofgypenosides content (Table 5)

We found that the expressions of unigenes and enzymeswere positively associated with the distribution of gypeno-sides content Generally speaking unigenes and enzymeexpression (FPS SS and SE) in the samples (fibrous rootsstems and leaves) determined to the distribution of gypeno-sides content Higher expression of unigenes and enzymes(encoded by unigenes) caused the higher content of enzymesrsquoproduction (gypenosides) and the lower expression of uni-genes and enzymes caused less enzyme productionThis con-sistent result could facilitate future studies of other secondarymetabolites in G pentaphyllum Since G pentaphyllum haswide applications for health and medicine it is essential toidentify the secondary metabolites with various and vitalmedicinal functions

One interesting finding was the observed differencesbetween general expression and individual expression ofunigenes (Figure 2 and Table 3) A group of specific unigenes


Table 6 Enzyme expression of FPS SS SE and 120573-AS

Full name Unigene ID Short name EC number G versus J G versus Y J versus YFarnesyl diphosphate synthase c118250 g1 i1 FPS EC 251125110 GPY upSqualene synthase c136108 g1 i1 SS EC 25121 GPY up GPY up

Squalene epoxidase c127030 g2 i1 SE EC 11413132 GPJ up GPY up GPY upGPY up GPY up

120573-amyrin synthase c70785 g1 i1 120573-AS EC 549939 GPJ up GPY up GPY upNote GPG or G fibrous roots of G pentaphyllum GPJ or J stems of G pentaphyllum GPY or Y leaves of G pentaphyllum

FPS RPKM25

20

15

10

5

0

GPG1 rpkmGPJ1 rpkmGPY1 rpkmGPG2 rpkmGPJ2 rpkm

GPY2 rpkmGPG3 rpkmGPJ3 rpkmGPY3 rpkm

(a)

25

20

15

10

5

0

30SS RPKM



(b)

SE-1RPKM800

700

600

500

400

300

200

100

0



(c)

120573-AS RPKM500

450

400

350

300

250

200

150

100

50

0



(d)

Figure 5 RPKM of FPS SS SE and 120573-AS Note (a) RPKM of FPS (b) RPKM of SS (c) RPKM of SE-1 (d) RPKM of 120573-AS GPG fibrousroots GPJ stems GPY leaves RPKM reads per kilobases per million reads


(FPS SS and SE) located in a special pathway like triter-penoids synthesis could increase their expression to a muchhigher level as required for a special physiological activity liketriterpenoid synthesis This obvious difference in expressionmay result from changes in the regulation of transcriptionIt is currently a hot topic to study the differential expressionof the transcriptome and the regulation of transcription ofplants in response to stresses of a special environment orother genetic factors [57ndash63] Another interesting observa-tion was that the transcriptome sequences ofG pentaphyllumdetermined in our study showed high similarity to thetranscriptome sequences related to bitterness in cucumberas reported previously [64] We downloaded the availablemRNA sequences of related enzymes from that article andblasted them against the unigene sequences of G penta-phyllum The BLAST result was surprising as all thirteenavailable sequences related to bitterness in that article showeda high degree of similarity to the specific unigene sequencesof G pentaphyllum This high similarity predicted that genesrelated to the biosynthesis regulation and domesticationof bitterness in cucumber may also be present in G pen-taphyllum G pentaphyllum also has two tastes (sweet andbitter) and this difference of taste may be caused as incucumber The taste of G pentaphyllum from bitter to sweetpredicts that G pentaphyllum may change in response todomestication and contain a similar mutation A furthergenetic exploration of the domestication of G pentaphyllummay provide understanding of its changed taste

Although this study was not the first report of the tran-scriptome information for G pentaphyllum using sequenc-ing we corrected the limitations of the previous studyand enriched the analysis of triterpenoid synthesis of Gpentaphyllum G pentaphyllum used for analysis in our studywas planted in a natural environment and three commonkinds of plant tissues (fibrous roots stems and leaves) wereused to provide tissue samples for sequencing Togetherwith the sequencing an exploration of the distribution ofgypenosides content was performed to confirm the analysisof enzymes and unigenes We found a positive association ofunigene expression enzyme expression and the distributionof gypenosides Our study will facilitate more genetic studiesexamining the regulation of transcription and the change ofbitterness in G pentaphyllum

5 Conclusions

To provide more complete and high-quality transcriptionalinformation of natural G pentaphyllum we used RNAsequencing technology to sequence the transcriptome ofG pentaphyllum We found a positive association of uni-gene expression enzyme expression and the distribution ofgypenosides Our results will enable future related studies ofG pentaphyllum

Abbreviations

G pentaphyllum Gynostemma pentaphyllum (Thunb)Makino

FPS Farnesyl pyrophosphate synthase

SS Squalene synthaseSE Squalene epoxidaseRPKM Reads per kilobases per million readsNR Nonredundant protein sequencesGO Gene OntologyeggNOG Evolutionary genealogy of genes

Nonsupervised Orthologous GroupsKEGG Kyoto Encyclopedia of Genes and GenomeMVA Mevalonic acidMEP Methylerythritol phosphateIPP Isopentenyl pyrophosphateDMAPP Dimethylallyl pyrophosphateP ginseng Panax ginsengBLAST Basic Local Alignment Search ToolGO Slim Cut-down versions of the GO ontologiesAUC Area under the curve120573-AS Beta-amyrin synthaseUV-Vis Ultraviolet-visibleNGS Next-Generation SequencingPE Paired endNCBI National Center for Biotechnology

InformationCDS Coding sequenceRefSeq Reference sequencesPDB Protein databaseEMBL European Molecular Biology LaboratoryDDBJ DNA Data Bank of JapanKO KEGG Ortholog

Disclosure

Qicong Chen is first author

Data Access

The data and materials were listed in the supplementaryinformation

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Yaosheng Wu Chengtong Ma and Jieying Qian preparedthe sample of G pentaphyllum for sequencing in GuangxiTraditional Chinese Medical University All authors workedtogether to analyze the sequencing data but Qicong Chenwas responsible for the specific analysis of sequencing dataYaosheng Wu provided the guidelines to detect the distri-bution of gypenosides content in G pentaphyllum and theguidelines to write this report Qicong Chen detected thedistribution of gypenosides content in G pentaphyllum andwrote this manuscript All authors read and approved thefinal manuscript


Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (no 31260069) The authors thank MrYilin Zhu (Guangxi Traditional Chinese Medical University)for the identification of samples

References

[1] Checklist of South China Botanical Garden httpwwweflorasorgflorataxonaspxflora id=610amptaxon id=200022642

[2] X Piao Q Wu J Yang S Y Park D Chen and HLiu ldquoDammarane-type saponins from heat-processed gynos-temma pentaphyllum show fortified activity against A549 cellsrdquoArchives of Pharmacal Research vol 36 no 7 pp 874ndash879 2013

[3] X-L Piao S-F Xing C-X Lou and D-J Chen ldquoNoveldammarane saponins from Gynostemma pentaphyllum andtheir cytotoxic activities against HepG2 cellsrdquo Bioorganic andMedicinal Chemistry Letters vol 24 no 20 pp 4831ndash4833 2014

[4] F Yang H Shi X Zhang and L Yu ldquoTwo novel anti-inflammatory 21-nordammarane saponins from tetraploidjiaogulan (Gynostemmapentaphyllum)rdquo Journal of Agriculturaland Food Chemistry vol 61 no 51 pp 12646ndash12652 2013

[5] F Yang H Shi X Zhang H Yang Q Zhou and L L YuldquoTwo new saponins from tetraploid jiaogulan (Gynostemmapentaphyllum) and their anti-inflammatory and 120572-glucosidaseinhibitory activitiesrdquo Food Chemistry vol 141 no 4 pp 3606ndash3613 2013

[6] C Muller A Gardemann G Keilhoff D Peter I Wiswedeland L Schild ldquoPrevention of free fatty acid-induced lipidaccumulation oxidative stress and cell death in primaryhepatocyte cultures by a Gynostemma pentaphyllum extractrdquoPhytomedicine vol 19 no 5 pp 395ndash401 2012

[7] M Wang F Wang Y Wang et al ldquoMetabonomics study ofthe therapeutic mechanism of Gynostemma pentaphyllum andatorvastatin for hyperlipidemia in ratsrdquo PLoS ONE vol 8 no11 Article ID e78731 2013

[8] H S Choi M S Park S H Kim B Y Hwang C K Lee andM K Lee ldquoNeuroprotective effects of herbal ethanol extractsfrom gynostemma pentaphyllum in the 6-hydroxydopamine-lesioned rat model of parkinsonrsquos diseaserdquoMolecules vol 15 no4 pp 2814ndash2824 2010

[9] H S Choi T T Zhao K S Shin et al ldquoAnxiolytic effects ofherbal ethanol extract from gynostemma pentaphyllum inmiceafter exposure to chronic stressrdquo Molecules vol 18 no 4 pp4342ndash4356 2013

[10] S-A Im H S Choi S O Choi et al ldquoRestoration of elec-tric footshock-induced immunosuppression in mice by gynos-temma pentaphyllum componentsrdquoMolecules vol 17 no 7 pp7695ndash7708 2012

[11] X-S Zhang X-L Bi X Wan et al ldquoProtein tyrosine phos-phatase 1B inhibitory effect by dammarane-type triterpenesfrom hydrolyzate of total Gynostemma pentaphyllum saponinsrdquoBioorganic ampMedicinal Chemistry Letters vol 23 no 1 pp 297ndash300 2013

[12] D Gao M Zhao X Qi et al ldquoHypoglycemic effect of Gynos-temma pentaphyllum saponins by enhancing theNrf2 signalingpathway in STZ-inducing diabetic ratsrdquo Archives of PharmacalResearch vol 39 no 2 pp 221ndash230 2016

[13] E F Lokman H F Gu W N Wan Mohamud and C-GOstenson ldquoEvaluation of antidiabetic effects of the traditional

medicinal plant Gynostemma pentaphyllum and the possiblemechanisms of insulin releaserdquo Evidence-Based Complementaryand Alternative Medicine vol 2015 Article ID 120572 7 pages2015

[14] J D Newman and J Chappell ldquoIsoprenoid biosynthesis inplants carbon partitioning within the cytoplasmic pathwayrdquoCritical Reviews in Biochemistry and Molecular Biology vol 34no 2 pp 95ndash106 1999

[15] K Haralampidis M Trojanowska and A E Osbourn ldquoBiosyn-thesis of triterpenoid saponins in plantsrdquo Advances in Biochem-ical EngineeringBiotechnology vol 75 pp 31ndash49 2002

[16] Terpentoid backbone biosynthesis httpwwwgenomejpkegg-binshow pathway14607215648484ko00900args

[17] Y-K Kim Y B Kim M R Uddin S Lee S-U Kim and SU Park ldquoEnhanced triterpene accumulation in Panax ginsenghairy roots overexpressingmevalonate-5-pyrophosphate decar-boxylase and farnesyl pyrophosphate synthaserdquo ACS SyntheticBiology vol 3 no 10 pp 773ndash779 2014

[18] J W Seo J H Jeong C G Shin et al ldquoOverexpression ofsqualene synthase in Eleutherococcus senticosus increases phy-tosterol and triterpene accumulationrdquo Phytochemistry vol 66no 8 pp 869ndash877 2005

[19] J M Rasbery ldquoGenetic and biochemical characterization of thesqualene epoxidase gene family in Arabidopsis thaliana 2007rdquohttphdlhandlenet191120636

[20] F He Y Zhu M He and Y Zhang ldquoMolecular cloning andcharacterization of the gene encoding squalene epoxidase inPanax notoginsengrdquo DNA SequencemdashJournal of DNA Sequenc-ing and Mapping vol 19 no 3 pp 270ndash273 2008

[21] Sesquiterpenoid and Triterpenoid Biosynthesis httpwwwgenomejpkegg-binshow pathwayko00909

[22] Pharmacopoeia of Peoplersquos Republic of China Beijing ChemicalIndustry Press National Pharmacopoeia Committee 1997

[23] Y Hu F C F Ip G Fu H Pang W Ye and N Y IpldquoDammarane saponins from Gynostemma pentaphyllumrdquo Phy-tochemistry vol 71 no 10 pp 1149ndash1157 2010

[24] P T Ky P T Huong T KMy et al ldquoDammarane-type saponinsfromGynostemma pentaphyllumrdquo Phytochemistry vol 71 no 8-9 pp 994ndash1001 2010

[25] Z Zhang W Zhang Y-P Ji Y Zhao C-G Wang and J-F Hu ldquoGynostemosides A-E megastigmane glycosides fromGynostemma pentaphyllumrdquo Phytochemistry vol 71 no 5-6 pp693ndash700 2010

[26] J H Kim and Y N Han ldquoDammarane-type saponins fromGynostemma pentaphyllumrdquo Phytochemistry vol 72 no 11-12pp 1453ndash1459 2011

[27] L Shi J Q Cao S M Shi and Y Q Zhao ldquoTriterpenoid sap-onins from Gynostemma pentaphyllumrdquo Journal of AsianNatural Products Research vol 13 no 2 pp 168ndash177 2011

[28] X S Zhang J Q Cao C Zhao X D Wang X J Wuand Y Q Zhao ldquoNovel dammarane-type triterpenes isolatedfrom hydrolyzate of total Gynostemma pentaphyllum saponinsrdquoBioorganic amp Medicinal Chemistry Letters vol 25 no 16 pp3095ndash3099 2015

[29] J Blanca J Canizares C Roig P Ziarsolo F Nuez and BPico ldquoTranscriptome characterization and high throughputSSRs and SNPs discovery in Cucurbita pepo (Cucurbitaceae)rdquoBMC Genomics vol 12 article 104 2011

[30] T K Hyun Y Rim H Jang et al ldquoDe novo transcriptomesequencing of Momordica cochinchinensis to identify genesinvolved in the carotenoid biosynthesisrdquo Plant Molecular Biol-ogy vol 79 no 4-5 pp 413ndash427 2012


[31] R S Sangwan S Tripathi J Singh L K Narnoliya and NS Sangwan ldquoDe novo sequencing and assembly of Centellaasiatica leaf transcriptome for mapping of structural func-tional and regulatory genes with special reference to secondarymetabolismrdquo Gene vol 525 no 1 pp 58ndash76 2013

[32] S Subramaniyam R Mathiyalagan I J Gyo L Bum-SooL Sungyoung and Y D Chun ldquoTranscriptome profiling andinsilico analysis of Gynostemma pentaphyllum using a nextgeneration sequencerrdquo Plant Cell Reports vol 30 no 11 pp2075ndash2083 2011

[33] Y-M Zhao T Zhou Z-H Li and G-F Zhao ldquoCharac-terization of global transcriptome using illumina paired-endsequencing and development of EST-SSR markers in twospecies of Gynostemma (Cucurbitaceae)rdquoMolecules vol 20 no12 pp 21214ndash21231 2015

[34] X Zheng H Xu X Ma R Zhan and W Chen ldquoTriterpenoidsaponin biosynthetic pathway profiling and candidate genemining of the Ilex asprella root using RNA-Seqrdquo InternationalJournal of Molecular Sciences vol 15 no 4 pp 5970ndash5987 2014

[35] J A Martin and Z Wang ldquoNext-generation transcriptomeassemblyrdquo Nature Reviews Genetics vol 12 no 10 pp 671ndash822011

[36] U Nagalakshmi K Waern M Snyder et al ldquoRNA-Seq amethod for comprehensive transcriptome analysisrdquo in CurrentProtocols in Molecular Biology M A Frederick Ed chapter 42010

[37] Z Wang M Gerstein and M Snyder ldquoRNA-Seq a revolution-ary tool for transcriptomicsrdquo Nature Reviews Genetics vol 10no 1 pp 57ndash63 2009

[38] B Rabbani H Nakaoka S Akhondzadeh M Tekin and NMahdieh ldquoNext generation sequencing implications in per-sonalizedmedicine and pharmacogenomicsrdquoMolecular BioSys-tems vol 12 no 6 pp 1818ndash1830 2016

[39] Next Generation Sequencing (NGS) httpsenwikibooksorgwikiNext Generation Sequencing (NGS)

[40] C Wang H Zhang Z-Q Qian and G-F Zhao ldquoGenetic dif-ferentiation in endangeredGynostemma pentaphyllum (Thunb)Makino based on ISSR polymorphism and its implications forconservationrdquo Biochemical Systematics and Ecology vol 36 no9 pp 699ndash705 2008

[41] HWMing and Z Z Cheng ldquoEffects of external support on theforaging behavior and reproductive strategies in Gynostemmapentaphylum populationsrdquo Acta Ecologica Sinica vol 21 no 1pp 47ndash50 2001

[42] J Zhou Y S Wu R Q Zhao et al ldquoMolecular authenticationof Gynostemma pentaphyllum through development and appli-cation of random amplification polymorphic DNA sequence-characterized amplified region markerrdquo Genetics and MolecularResearch vol 14 no 4 pp 16204ndash16214 2015

[43] G Xinfen S K Chen Z J Gu and J Z Zhao ldquoA chromosomalstudy on the genus Gynostemma (Cucurbitaceae)rdquo Acta Botan-ica Yunnanica vol 17 pp 312ndash316 1995

[44] Gynostemma pentaphyllum httpsenwikipediaorgwikiGynostemma pentaphyllum

[45] B J Haas A Papanicolaou M Yassour et al ldquoDe novotranscript sequence reconstruction from RNA-seq using theTrinity platform for reference generation and analysisrdquo NatureProtocols vol 8 no 8 pp 1494ndash1512 2013

[46] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[47] A Ammar Z Elouedi and P Lingras ldquoRPKM the roughpossibilistic K-modesrdquo in Foundations of Intelligent Systems20th International Symposium ISMIS 2012 Macau ChinaDecember 4ndash7 2012 Proceedings vol 7661 of Lecture Notes inComputer Science pp 81ndash86 Springer Berlin Germany 2012

[48] B Langmead and S L Salzberg ldquoFast gapped-read alignmentwith Bowtie 2rdquo Nature Methods vol 9 no 4 pp 357ndash359 2012

[49] S Anders and W Huber ldquoDifferential expression analysis forsequence count datardquo Genome Biology vol 11 no 10 articleR106 2010

[50] Gene Ontology httpgeneontologyorg[51] M Ashburner C Ball D Ja Botstein et al ldquoGene ontology tool

for the unification of biologyTheGene Ontology ConsortiumrdquoNature Genetics vol 25 no 1 pp 25ndash29 2015

[52] KMinoru G SusumuK Shuichi O Yasushi andHMasahiroldquoThe KEGG resource for deciphering the genomerdquo NucleicAcids Research vol 32 no 22 pp D277ndashD280 2004

[53] Kyoto Encyclopedia of Genes and Genome httpwwwkeggjp

[54] Evolutionary genealogy of genes Non-supervised OrthologousGroups httpeggnogdbembldeapphome

[55] S Powell K Forslund D Szklarczyk et al ldquoEggNOG v40nested orthology inference across 3686 organismsrdquo NucleicAcids Research vol 42 no 1 pp D231ndashD239 2014

[56] Swiss-Prot httpwebexpasyorgdocsswiss-prot guidelinehtml

[57] B Qi Y Yang Y Yin M Xu and H Li ldquoDe novo sequencingassembly and analysis of the Taxodium ldquoZhongshansardquo rootsand shoots transcriptome in response to short-term waterlog-gingrdquo BMC Plant Biology vol 14 article no 201 2014

[58] T Yang L Hao S Yao Y Zhao W Lu and K Xiao ldquoTabHLH1a bHLH-type transcription factor gene inwheat improves planttolerance to Pi and N deprivation via regulation of nutrienttransporter gene transcription and ROS homeostasisrdquo PlantPhysiology and Biochemistry vol 104 pp 99ndash113 2016

[59] W Jin H Wang M Li et al ldquoThe R2R3 MYB transcriptionfactor PavMYB101 involves in anthocyanin biosynthesis anddetermines fruit skin colour in sweet cherry (Prunus avium L)rdquoPlant Biotechnology Journal vol 14 no 11 pp 2120ndash2133 2016

[60] S Bai P A Tuan T Saito et al ldquoEpigenetic regulation ofMdMYB1 is associated with paper bagging-induced red pig-mentation of applesrdquo Planta vol 244 no 3 pp 573ndash586 2016

[61] Q Zhu B Li S Mu et al ldquoTTG2-regulated development isrelated to expression of putative AUXIN RESPONSE FACTORgenes in tobaccordquoBMCGenomics vol 14 no 1 article 806 2013

[62] F-Q Tan H Tu W-J Liang et al ldquoComparative metabolicand transcriptional analysis of a doubled diploid and its diploidcitrus rootstock (C junos cv Ziyang xiangcheng) suggests itspotential value for stress resistance improvementrdquo BMC PlantBiology vol 15 article 89 2015

[63] T Liu S Zhu Q Tang and S Tang ldquoGenome-wide transcrip-tomic profiling of ramie (Boehmeria nivea L Gaud) in responseto cadmium stressrdquo Gene vol 558 no 1 pp 131ndash137 2015

[64] Y Shang Y Ma Y Zhou et al ldquoBiosynthesis regulation anddomestication of bitterness in cucumberrdquo Science vol 346 no6213 pp 1084ndash1088 2014

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


G3P PyruvateAcetyl-CoAAcetoacetyl-CoAAACT

EC 2319

EC 2217EC 23310 DXPS

DXP

DXR

HMG-CoA

EC 111267HMGR

HMGS

EC 11134

EC 11188MEPMVA CDP-ME2P

CMK

MCTEC 27760

CDP-ME

EC 46112EC 271148MKEC 27136

MVAP MECPP

HDSEC 11771EC 11773MPK

MDS

EC 2742

MVA2P HMBPP

EC 41133 MDCHDR

HDREC 11712

IPPIDI

DMAPPEC 5332

+IPPEC 2511 GPPS EC 2511

GPP

FPSEC 25110GGPPS

GGPPFPPEC 25129

+FPP

Monoterpenoids


EC 25121 SS

SESqualene TriterpenoidsEC 1141417 23-Oxidosqualene

GPPS + IPP

Figure 1 Triterpenoid synthesis pathway Note AACT acetyl-CoA C-acetyltransferase HMGS hydroxymethylglutaryl-CoA synthaseHMG-CoA hydroxymethylglutaryl-CoA HMGR hydroxymethylglutaryl-CoA reductase MVA mevalonate MK mevalonate kinaseMVAP mevalonate phosphate MPK mevalonate phosphate kinase MVAPP mevalonate diphosphate MDC mevalonate diphosphatedecarboxylase G3P D-glyceraldehyde-3-phosphate acetaldehydetransferase DXPS 1-deoxy-D-xylulose-5-phosphate synthase DXP 1-deoxy-D-xylulose-5-phosphate DXR 1-deoxy-D-xylulose-5-phosphate reductoisomerase MEP 2-C-methyl-D-erythritol 4-phosphateMCT 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase CDP-ME 4-(cytidine 51015840-diphospho)-2-C-methyl-D-erythritol CDP-ME2P 2-phospho-4-(cytidine 51015840-diphospho)-2-C-methyl-D-erythritol MECPP 2-C-methyl-D-erythritol 24-cyclodiphosphate HDS l-hydroxy-2-methyl-butenyl-4-diphosphate synthase HMBPP l-hydroxy-2-methyl-2-butenyl-4-diphosphate HDR 4-hydroxy-3-methylbut-2-enyl diphosphate reductase IPP isopentenyl-PP IDI isopentenyl-diphosphate delta-isomerase DMAPP dimethylallyl-PP GPS geranyl-diphosphate synthase GPS geranyl-diphosphate synthase GPPS geranylgeranyl diphosphate synthase GPP geranylgeranyl diphosphateFPS farnesyl diphosphate synthase GGPPS geranylgeranyl diphosphate synthase GGPP geranylgeranyl diphosphate SS squalene synthaseand SE squalene epoxidase

and SE are required for the synthesis and cyclization ofthe squalene that combines two sesquiterpenoids into onetriterpenoid (C15 + C15 = C30) [16 21] After this steptriterpenoids can be transformed into many isoform typeslike protosteryl type (chair-chair-chair-boat conformations)dammarenyl type (chair-chair-chair-boat conformations)cadinyl type (chair-chair-chair-boat conformations) and

hopene and tetrahymanol (chair-chair-chair-chair conforma-tions or chair-chair-chair-boat conformations) [21]

Like the ginsenosides gypenosides (triterpenoids) in Gpentaphyllum have various and vital applications in medicineand health [22] However gypenosides showed much higherheterogeneity when compared with ginsenosides and morethan 169 kinds of gypenosides were found inG pentaphyllum
















Contigs


GC 48

Transcriptome


GC 46

Unigenes


GC 44




3 Result












GPG1

GPG2

GPG3

GPY1

GPY2

GPY3

06

04

02

00

minus2 20 4

Den

sity

GPJ1

GPJ2

GPJ3

log10(TPM)




3736305 1963

699

2281495

1353



Stems versus leaves




Monoterpenoids


Triterpenoids

FPS

GGPPSGGPPFPP

EC 25129+FPP

SS

SESqualene

EC 1141417

IPPIDI

DMAPPEC 5332

EC 2511+IPPEC 2511

EC 25110

EC 25121

GPPS

GPP

Mevalonatepathway

MEPDOXPpathway

23-Oxidosqualene

GPPS + IPP






4 Discussion









FPS RPKM25

20

15

10

5

0



(a)

25

20

15

10

5

0

30SS RPKM



(b)

SE-1RPKM800

700

600

500

400

300

200

100

0



(c)

120573-AS RPKM500

450

400

350

300

250

200

150

100

50

0



(d)





5 Conclusions


Abbreviations






Disclosure


Data Access


Competing Interests





Acknowledgments


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















Contigs


GC 48

Transcriptome


GC 46

Unigenes


GC 44




3 Result












GPG1

GPG2

GPG3

GPY1

GPY2

GPY3

06

04

02

00

minus2 20 4

Den

sity

GPJ1

GPJ2

GPJ3

log10(TPM)




3736305 1963

699

2281495

1353



Stems versus leaves




Monoterpenoids


Triterpenoids

FPS

GGPPSGGPPFPP

EC 25129+FPP

SS

SESqualene

EC 1141417

IPPIDI

DMAPPEC 5332

EC 2511+IPPEC 2511

EC 25110

EC 25121

GPPS

GPP

Mevalonatepathway

MEPDOXPpathway

23-Oxidosqualene

GPPS + IPP






4 Discussion









FPS RPKM25

20

15

10

5

0



(a)

25

20

15

10

5

0

30SS RPKM



(b)

SE-1RPKM800

700

600

500

400

300

200

100

0



(c)

120573-AS RPKM500

450

400

350

300

250

200

150

100

50

0



(d)





5 Conclusions


Abbreviations






Disclosure


Data Access


Competing Interests





Acknowledgments


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








GPG1

GPG2

GPG3

GPY1

GPY2

GPY3

06

04

02

00

minus2 20 4

Den

sity

GPJ1

GPJ2

GPJ3

log10(TPM)




3736305 1963

699

2281495

1353



Stems versus leaves




Monoterpenoids


Triterpenoids

FPS

GGPPSGGPPFPP

EC 25129+FPP

SS

SESqualene

EC 1141417

IPPIDI

DMAPPEC 5332

EC 2511+IPPEC 2511

EC 25110

EC 25121

GPPS

GPP

Mevalonatepathway

MEPDOXPpathway

23-Oxidosqualene

GPPS + IPP






4 Discussion









FPS RPKM25

20

15

10

5

0



(a)

25

20

15

10

5

0

30SS RPKM



(b)

SE-1RPKM800

700

600

500

400

300

200

100

0



(c)

120573-AS RPKM500

450

400

350

300

250

200

150

100

50

0



(d)





5 Conclusions


Abbreviations






Disclosure


Data Access


Competing Interests





Acknowledgments


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Monoterpenoids


Triterpenoids

FPS

GGPPSGGPPFPP

EC 25129+FPP

SS

SESqualene

EC 1141417

IPPIDI

DMAPPEC 5332

EC 2511+IPPEC 2511

EC 25110

EC 25121

GPPS

GPP

Mevalonatepathway

MEPDOXPpathway

23-Oxidosqualene

GPPS + IPP






4 Discussion









FPS RPKM25

20

15

10

5

0



(a)

25

20

15

10

5

0

30SS RPKM



(b)

SE-1RPKM800

700

600

500

400

300

200

100

0



(c)

120573-AS RPKM500

450

400

350

300

250

200

150

100

50

0



(d)





5 Conclusions


Abbreviations






Disclosure


Data Access


Competing Interests





Acknowledgments


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






FPS RPKM25

20

15

10

5

0



(a)

25

20

15

10

5

0

30SS RPKM



(b)

SE-1RPKM800

700

600

500

400

300

200

100

0



(c)

120573-AS RPKM500

450

400

350

300

250

200

150

100

50

0



(d)





5 Conclusions


Abbreviations






Disclosure


Data Access


Competing Interests





Acknowledgments


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




5 Conclusions


Abbreviations






Disclosure


Data Access


Competing Interests





Acknowledgments


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Acknowledgments


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article transcriptome sequencing of gynostemma...

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