Expression and Functional Characterization of Three Squalene Synthase Genes Associated with Saponin Biosynthesis in Panax ginseng
Tae-Dong Kim1,3,Jung-Yeon Han1,3,Gyung Hye Huh2and Yong-Eui Choi1,*
1Division of Forest Resources,College of Forest and Environmental Sciences,Kangwon National University,Chunchon200-701, Republic of Korea
2College of General Education,In Je University,Gimhae621-749,Republic of Korea
3These authors contributed equally to this work
*Corresponding author:E-mail,yechoi@kangwon.ac.kr;Fax,+82-33-252-8310
(Received August18,2010;Accepted November12,2010)
Squalene synthase(SQS)catalyzes the biosynthesis of squa-lene by condensing two molecules of farnesyl pyrophosphate (FPP),a key precursor in sterol and triterpene biosynthesis. Previously,we reported that PgSS1overexpression results in the enhanced biosynthesis of both phytosterols and tri-terpene saponins in Panax ginseng.Here,cDNAs encoding two new SQS homologs(PgSS2and PgSS3)from a P.ginseng expressed sequence tag(EST)library are described. Functional complementation analysis revealed that ectopic expression of PgSS1,PgSS2and PgSS3in the yeast erg9 mutant strain2C1lacking SQS activity restored ergosterol prototrophy.The recombinant mutant yeast produced squalene,squalene epoxide and ergosterol.PgSS1(mRNA) was highly transcribed in all organs,whereas PgSS2and PgSS3(mRNAs)were only transcribed in speci?c organs. All three genes were activated positively by an elicitor (methyl jasmonate),but their transcriptional patterns were different.In situ hybridization analysis revealed that both PgSS1and PgSS3transcripts were preferentially accu-mulated near conducting tissue in the petiole.The PgSS1 and PgSS3promoters were isolated,and the tissue-and organ-speci?c regulation of PgSS genes was examined. Transgenic ginseng was constructed by introducing PgSS1 and PgSS3promoters fused to the b-glucuronidase(GUS) gene.GUS expression driven by the PgSS1promoter was found in both roots and shoots,but PgSS3-driven GUS was only found in shoots.These results suggest that the three SQS genes are differently expressed and that all three SQS enzymes are involved in squalene production in P.ginseng.
Keywords:Functional complementation Panax ginseng Squalene synthase Yeast erg9mutant.
Abbreviations:ER,endoplasmic reticulum;EST,expressed sequence tag;FPP,farnesyl pyrophosphate;GC/MS,gas chromatography–mass spectrometry;GUS,b-glucuronidase; MeJA,methyl jasmonate;ORF,open reading frame;RT–PCR,reverse transcription–PCR;SQS,squalene synthase; UTR,untranslated region.
Introduction
Sterols and triterpenes are widely distributed isoprenoids,and they constitute one of the most important classes of natural products.Sterols are essential for all eukaryotic organisms to function(Bloch1992).The sterol biosynthetic pathway is a main target for pharmacological intervention,and sterol syn-thesis inhibitors are used in cholesterol-lowering therapies (Baxter et al.1992,Bergstrom et al.1993,Jones et al.1997). Unlike animal and fungal cells,higher plants synthesize a mix-ture of phytosterols,including campesterol,stigmasterol and sitosterol(Hartmann and Benveniste1987).Most plant triter-penoid compounds are in the form of saponin glycosides,which refers to the attachment of various sugar molecules to the triterpene unit.Triterpenoids have a wide range of structural diversity and biological activity,and saponins are of economical importance for drugs,detergents,sweeteners and cosmetics (Hostettmann and Marston1995).
Both plant sterols(also called phytosterols)and triterpenoid saponins are synthesized from the same precursors,squalene and2,3-oxidosqualene(Haralampidis et al.2002).Squalene synthase(SQS)(farnesyl diphosphate:farnesyl diphosphate farnesyltransferase,EC2.5.1.21)catalyzes the?rst enzymatic step in sterol,brassinosteroid and triterpenoid biosynthesis from the central isoprenoid pathway(Abe et al.1993).SQS is a membrane-bound enzyme that condenses two FPP molecules into squalene.The SQS enzyme is also considered to play an important regulatory role in the sterol biosynthetic pathway (Devarenne et al.2002).SQS regulatory roles in sterol biosyn-thesis were discovered using SQS mutants(Karst and Lacroute 1977,Tozawa et al.1999),fungal elicitors(Threlfall and Whitehead1988,Vo¨geli and Chappell1988,Devarenne et al. 1998),speci?c inhibitors of SQS(Baxter et al.1992,Bergstrom
Plant Cell Physiol.52(1):125–137(2011)doi:10.1093/pcp/pcq179,available online at https://www.doczj.com/doc/8a4223842.html, !The Author2010.Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved.For permissions,please email:journals.permissions@https://www.doczj.com/doc/8a4223842.html, Regular Paper by guest on February 15, 2012 https://www.doczj.com/doc/8a4223842.html,/ Downloaded from
et al.1993,Wentzinger et al.2002)and overexpression of SQS genes in Panax ginseng (Lee et al.2004,Seo et al.2005).
In yeast (Jennings et al.1991)and humans (Robinson et al.1993),the SQS gene is present as a single copy.In plants,one or two copies of SQS genes have been reported.A single SQS gene has been found in Euphorbia tirucalli (Uchida et al.2009),Taxus cuspidate (Huang et al.2007)and Oryza sativa (Hata et al.1997).Two SQS genes have been found in tobacco (Devarenne et al.1998),Arabidopsis thaliana (Busquets et al.2008)and Glycyrrhiza glabra (Hayashi et al.1999).The genome of A.thaliana contains two SQS -annotated sequences,At4g34640(SQS1)and At4g34650(SQS2),organized in tandem array (Kribii et al.1997).SQS1is the only functional SQS in A.thaliana (Busquets et al.2008).
Panax ginseng is a famous and widely used medicinal plant.It is generally believed that ginsenosides and tetracyclic triterpenoids are responsible for the majority of ginseng’s pharmacological activities (Vogler et al.1999,Shibata 2001).Ginsenosides exert many pharmacological effects (Briskin 2000,Shibata 2001).Recently,hydrolyzed ginsenosides have been developed as anti-cancer drugs called ‘Shen-Yi Capsule’in China (Shibata 2001,Yue et al.2006).
We previously reported that overexpression of PgSS1en-hances P.ginseng sterol and triterpene saponin biosynthesis .We recently isolated two new P.ginseng SQS genes (PgSS2and PgSS3).In this work,we investigated the expression pattern of the three P.ginseng SQS genes (PgSS1,PgSS2and PgSS3)and functionally analyzed (through complementation)these genes in the yeast erg9mutant strain 2C1,which lacks SQS activity.
Results
PgSS2and PgSS3isolation and sequence analysis
We previously reported that overexpression of PgSS1results in enhanced biosynthesis of both phytosterols and triterpene sap-onins (Lee et al.2004).Recently,two other SQS full-length cDNA clones (PgSS2,GQ468527;and PgSS3,AB115496)were isolated from 4,226expressed sequence tags (ESTs)obtained from an in vitro cultured adventitious root cDNA library.We found that the PgSS2cDNA is 1,335bp long and encodes a protein of 415amino acid residues with a predicted molecular mass of 47kDa (Fig.1).The PgSS3cDNA is 1,329bp long and encodes a protein of 415amino acid residues with a predicted molecular mass of 47kDa.The PgSS2deduced amino acid se-quence is highly homologous (98%)to the PgSS3deduced amino acid sequence.However,the PgSS1amino acid sequence is only 92and 91%similar to the PgSS2and PgSS3sequences,respectively.Based on phylogeny,the three P.ginseng SS genes were subgrouped into two clades (Fig.2).PgSS2and PgSS3share high amino acid similarity with P.quinquefolium SQS proteins (AM182456and AM182457),whereas PgSS1shares similarity with the P.notoginseng SQS protein (DQ186630).SQS proteins contain three conserved regions (domains A–C)involved in catalysis,and speci?c amino acids within
these domains are essential for the two half-reactions catalyzed by the enzyme (Gu et al.1998).These residues were found in the corresponding PgSS1,PgSS2and PgSS3domains,with the only exception of a serine residue found in PgSS2(Fig.1).SQS proteins are membrane bound and localize to the endo-plasmic reticulum (ER;Favre and Ryder 1996).Amino acids within the C-terminal region were the most variable compared with other regions of the proteins (Fig.1).Hydrophobic se-quences within the C-terminus have a role in anchoring the enzyme to the ER membrane (Robinson et al.1993),which allows it to channel squalene through the yeast sterol pathway (Kribii et al.1997).Most membrane proteins have hydrophobic regions that span the hydrophobic core of the membrane bi-layer,with hydrophilic regions located on either side of the membrane.Membrane-spanning amino acids derived from PgSS1,PgSS2and PgSS3were predicted using TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/)(Krogh et al.2001),a program that predicts transmembrane helices based on a hidden Markov model.PgSS1,PgSS2and PgSS3not only had C-terminal hydrophobic sequences,but they also had add-itional membrane-spanning helices between Ile281and Asn303near domain C (Supplementary Fig.S1).Arabidopsis thaliana ,tobacco,human and yeast SQS all have a single predicted membrane-spanning helix,found in their C-terminal hydropho-bic sequences (Jennings et al.1991,Robinson et al.1993,Kribii et al.1997).Interestingly,the additional membrane-spanning helices near domain C are seen in saponin-rich plant species such as Glycine max (GenBank accession No.BAA22559),G.glabra (GenBank accession No.BAA13084)and Aralia elata (GenBank accession No.ADC32654).
Complementation of the erg9yeast mutant
Expression of the complete SQS proteins from humans and A.thaliana (SQS1)did not convert yeast erg9mutant cells to ergosterol prototrophy,even though both enzymes are catalyt-ically active (Robinson et al.1993,Kribii et al.1997).In both cases,the ergosterol requirement caused by the erg9mutation reverted only when chimeric SQS proteins,in which the C-terminal region of the human and plant SQS proteins was replaced with the equivalent region of the yeast enzyme,were expressed (Robinson et al.1993,Kribii et al.1997).Interestingly,all yeast erg9mutant strain 2C1cultures expressing the com-plete PgSS1,PgSS2or PgSS3gene had restored growth on ergosterol-free media (Fig.3).Yeast harboring the pYES2.1plasmid (negative control)did not have restored growth (Fig.3),except in media containing an ergosterol supplement (Fig.3A ).
Gas chromatography–mass spectrometry (GC/MS)analysis of FPP,squalene,squalene epoxide,and ergosterol
To demonstrate ergosterol prototrophy,extracts from erg9mutant yeast expressing PgSS1,PgSS2or PgSS3were analyzed by GC/MS.Peak retention times for authentic GC/MS
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standards were 9.6,28.9,30.4and 33.7min for farnesol,squa-lene,squalene epoxide and ergosterol,respectively.Most of the farnesol,the hydrolyzed product of FPP,is detected in the GC chromatogram.Farnesol is detected as a major FPP-derived product in GC analysis.Farnesol,squalene,squalene epoxide and ergosterol were all identi?ed in yeast carrying PgSS1,PgSS2or PgSS3(Fig.4).The amounts of farnesol,squalene,squalene epoxide and ergosterol are listed in Table 1.Squalene and squalene epoxide were not detected in control yeast carrying the empty vector,except for when ergosterol was fed to yeast to allow growth (Fig.4).The MS fragmentation pattern of
squalene produced in yeast harboring PgSS1was the same as that of authentic squalene (Supplementary Fig.S2).The same squalene fraction pattern was observed in yeast harboring PgSS2and PgSS3(data not shown).This result indicates that all three P.ginseng SQS genes are functionally expressed and restore ergosterol prototrophy in yeast erg9mutants.
PgSS1,PgSS2and PgSS3mRNA accumulation
Densitometric analysis revealed that PgSS2mRNA accumulated preferentially in the leaf,petiole and main root body.PgSS3mRNA accumulated preferentially in the leaf only,
indicating
Fig.1Alignment of the deduced amino acid sequences of three SQS polypeptides:P.ginseng (PgSS1,AB122078;PgSS2,FJ393274;PgSS3,GU183406),tobacco (TSS ,U60057)and Arabidopsis (SQS1,D29017).Identical residues are boxed in black and similar residues in gray.The sky-blue box (A)in amino acid sequences indicates domain A.The red box (B)indicates domain B.The green box (C)indicates domain C.The dark-blue box (D)indicates the high sequence variation region in the C-terminal region of the enzyme.
Expression and functional characterization of three ginseng squalene synthase genes
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that PgSS2and PgSS3have different expression patterns.PgSS1mRNA accumulated ubiquitously in various organs of P.gin-seng ,such as roots (branch and tap roots),petioles,leaves and ?owers (Fig.5),similar to what was found in previous experiments (Lee et al.2004).
Methyl jasmonate (MeJA)treatment stimulates the biosyn-thesis of many secondary metabolites (Gundlach et al.1992),especially ginseng saponin in P.ginseng (Han et al.2006b).The accumulation of PgSS1,PgSS2and PgSS3transcripts in ad-ventitious roots was enhanced after MeJA treatment for 24h,although PgSS1mRNA had the highest accumulation (Fig.6).
In situ hybridization of PgSS1and PgSS3
In situ hybridization was performed to examine PgSS1and PgSS3tissue-speci?c expression patterns in the petiole.Tissue
sections were probed with gene-speci?c fragments from the 50-untranslated region (UTR),including the non-conserved 50coding regions,to discriminate between PgSS1and PgSS3cDNAs.Both PgSS1and PgSS3transcripts preferentially accu-mulated in vascular bundle tissues (phloem cells and parenchy-matous cells near xylem)and resin ducts in petioles (Fig.7A–C,E–G ).No signal was detected in tissue hybridized with a sense probe (Fig.7D,H ).
GUS expression regulated by PgSS1and PgSS3promoters in transgenic ginseng plants
The 754bp 50-?anking sequence (GenBank accession No.GU323919)from the PgSS1cDNA promoter site and the 1,789bp 50-?anking sequence (GenBank accession No.GU323920)from the PgSS3cDNA promoter site were obtained.
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Fig.2Phylogenetic tree containing the deduced amino acid sequences from PgSS1,PgSS2and PgSS3along with other SS proteins.The DDBJ/GenBank/EMBL accession numbers of the sequences are AB122078(P.ginseng ,PgSS1),FJ393274(P.ginseng ,PgSS2),GU183406(P.ginseng ,PgSS3),DQ386734(P.notoginseng ),DQ186630(P.notoginseng ),AM182456(P.quinquefolium ),AM182457(P.quinquefolium ),D29017(Arabidopsis thaliana ),AB007503(Glycine max ),AB007501(Oryza sativa )and U60057(Nicotiana tabacum ).Phylogenetic tree SS distances from plants between each clone and group were calculated using the program CLUSTAL W (Thompson et al.1994)and viewed using the Neighbor–Joining method in the TreeView software (Page 1996).The scale bar (number of substitutions/site)corresponds to the relative branch length.
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The 50-UTR from both PgSS1and PgSS3cDNAs contained one intron region,at positions à121to à16(106bp)and à137to à18(120bp),respectively.
Putative cis -elements in the PgSS1and PgSS3promoters were investigated using the software program PLACE (Higo et al.1999).A number of potential regulatory motifs corres-ponding to known eukaryotic cis -elements were found.The potential regulatory elements were associated with light-,hor-mone-and stress-related responses (data not shown).However,the position,frequency and kinds of the cis -elements were quite different between the two promoters.
To investigate the PgSS1and PgSS3organ-and tissue-speci?c transcriptional regulation,transgenic P.ginseng plants harboring the GUS reporter gene fused to each promoter were constructed.GUS expression driven by the PgSS1promoter was strong in all parts of the plant (leaves,petiole and roots),but GUS expression driven by the PgSS3promoter was very weak in roots (Figs.8,9).In older roots,GUS was preferentially expressed in the inside of vascular bundles from both pro-moters (Figs.8B,C ,9B,C ).However,in newly developed lateral roots,GUS expression driven by the PgSS1promoter was very strong in all tissues of the root.GUS expression driven by the PgSS3promoter was faint and restricted to root tips (Figs.8D ,9D ).
Histological analysis revealed that the overall GUS expres-sion patterns were similar for both promoters,except in newly
developed roots.GUS expression was evenly distributed in leaves (Figs.8E ,9E ).In the petiole,GUS expression preferen-tially accumulated near vascular strands (Figs.8F ,9F ).In aged roots,GUS expression using either promoter was abundant near vascular tissues situated in the inside of the casparian strip (Figs.8G ,9G ).In newly formed roots,GUS expression driven by the PgSS1promoter was strong,whereas GUS expres-sion driven by the PgSS3promoter was restricted to root caps (Figs.8H ,9H ).
Discussion
PgSS1,PgSS2and PgSS3mRNA isolation
Until now,only one or two copies of SQS genes have been reported in plant genomes.A single SQS gene was reported in E.tirucalli (Uchida et al.2009),T.cuspidate (Huang et al.2007)and O.sativa (Hata et al.1997).Two SQS genes were reported in tobacco (Devarenne et al.1998), A.thaliana (Busquets et al.2008)and G.glabra (Hayashi et al.1999).Unlike the organisms mentioned above,P.ginseng has more than three SQS genes.The PgSS1amino acid sequence is iden-tical to that of the previously registered SQS gene (BAA24289),but there were differences in the 50-and 30-UTR nucleotide sequences.It is believed that P.ginseng is tetraploid,like several other Panax species (Wen and Zimmer 1996).Thus,
the
A B Fig.3Functional complementation analysis of S.cerevisiae erg9mutants through ectopic expression of PgSS1,PgSS2and PgSS3.(A,C)erg9mutant strains expressing PgSS1,PgSS2and PgSS3were cultured on solid (A)and liquid (C)YPG media supplemented with ergosterol for 5d.(B,D)erg9mutant strains expressing PgSS1,PgSS2and PgSS3were cultured on solid (B)and in liquid (D)YPG medium without ergosterol for 5d.
Expression and functional characterization of three ginseng squalene synthase genes
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existence of multiple P.ginseng SQS homologs may have re-sulted from evolution following chromosome duplication.
Functional complementation
Expression of the complete SQS protein from humans and A.thaliana (SQS1)did not convert yeast erg9mutant cells to ergosterol prototrophy,even though both enzymes are
catalytically active (Robinson et al.1993,Kribii et al.1997).In both cases,the ergosterol requirement caused by the erg9mu-tation reverted only when chimeric SQS proteins,in which the C-terminal region of human and plant SQS proteins were replaced with the equivalent region of the yeast enzyme,were expressed (Robinson et al.1993,Kribii et al.1997).Interestingly,ectopic expression of the three full-length
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Fig.4GC/MS chromatograms of P.ginseng SQS products from erg9yeast mutants.(A)GC chromatogram of erg9yeast cell extracts containing the empty vector as a control.(B–D)GC chromatograms of erg9yeast cell extracts containing PgSS1(B),PgSS2(C)and PgSS3(D),respectively.(E)GC chromatogram of FPP,squalene,squalene epoxide and ergosterol standards.
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P.ginseng SQS genes restored growth of yeast erg9mutants in media lacking ergosterol.Further experiments are needed to explain why the P.ginseng SQS genes could confer ergosterol prototrophy in yeast erg9mutant cells.
SQS appears to be anchored to the membrane by a short C-terminal membrane-spanning domain,with its large N-terminal catalytic domain facing the cytosol.SQS genes from Arabidopsis,tobacco,human and yeast all have a single predicted membrane-spanning helix found in the C-terminal hydrophobic sequences identi?ed by the TMHMM program.Interestingly,all three P.ginseng SQS genes (PgSS1,PgSS2and PgSS3)not only have a membrane-spanning helix within the C-terminal hydrophobic sequences,but they also have
additional membrane-spanning helices near domain C.Kribii et al.(1997)questioned the possibility that the lack of comple-mentation is due to the inability of plant SQS to be targeted to the yeast ER membrane.If the P.ginseng SQS C-terminal membrane-spanning helices are not appropriately targeted to the yeast ER membrane,additional membrane-spanning helices might be advantageous in localizing SQS to the ER membrane.Interestingly,well-known saponin-rich species such as G.max ,G.glabra and A.elata contain additional membrane-spanning helices near domain C,similar to P.ginseng .
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Fig.6RT–PCR analysis of PgSS1,PgSS2and PgSS3mRNAs in cultured adventitious roots following 24h MeJA treatment.(A)Accumulation of PgSS1,PgSS2and PgSS3mRNAs in adventitious roots following treatment with various concentrations of MeJA for 24h.(B)Densitometric analysis of PgSS1
,PgSS2and PgSS3mRNAs in adventi-tious roots.Densitometric analysis represents the mean ±SE from three independent experiments,each done in triplicate.All of the results were normalized to b -actin.
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Fig.5RT–PCR analysis of PgSS1,PgSS2and PgSS3mRNAs from dif-ferent P.ginseng plant organs.(A)Accumulation of PgSS1,PgSS2and PgSS3mRNAs in different organs.(B)Densitometric analysis of PgSS1,PgSS2and PgSS3mRNAs in plant organs.Densitometric analysis rep-resents the mean ±SE of three independent experiments,each done in triplicate.All results were normalized to b -actin.
Table 1Fanesol,squalene,squalene epoxide and ergosterol content in erg9mutant yeast containing empty vector with PgSS1,PgSS2and PgSS3
Empty vector
Ectopic expression of SQS in yeast (k g g DW à1)PgSS1PgSS2PgSS3Fanesol 212.2±36.7
969.1±112.31,123±198.8755.4±96.8Squalene – 2.9±0.35 3.8±0.534.9±2.9Squalene epoxide – 1.6±0.13 2.3±0.3 1.7±0.23Ergosterol
8,314.7±278.5
139.5±54.3
145.5±27.5
116.4±30.7
The presence of ergosterol in erg9mutants containing empty vector results from ergosterol in the culture medium.
Expression and functional characterization of three ginseng squalene synthase genes
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Ganoderma lucidum is a basidiomycete white rot fungus (medicinal mushroom)that produces triterpenes (ganoderic acids).Ectopic expression of G.lucidum SQS in yeast erg9mu-tants results in ergosterol prototrophy on media lacking ergos-terol (Zhao et al.2007).These results reveal that full-length SQS proteins from some organisms are functional in yeast erg9mutants.
There are two SQS -annotated sequences in A.thaliana ,At4g34640(SQS1)and At4g34650(SQS2).Busquets et al.(2008)demonstrated that SQS1is the only functional SQS ,as SQS2has no SQS activity.In P.ginseng ,all three SQS genes were functionally expressed in yeast https://www.doczj.com/doc/8a4223842.html,pared with Arabidopsis and tobacco,P.ginseng is a saponin-rich plant,and the amount of saponin is >4%in dried roots and much higher in leaves (Chang 1998).Having multiple functional P.ginseng SQS genes may be advantageous for saponin biosynthesis.
Reverse transcription–PCR (RT–PCR)analysis revealed that the PgSS1mRNA is actively transcribed in every organ,but the PgSS2and PgSS3mRNAs are only transcribed in speci?c organs.If SQS expression directly affects phytosterol and triterpene biosynthesis,the organ-speci?c transcription of PgSS2and PgSS3mRNA could contribute to the organ-speci?c accumula-tion of phytosterols and triterpenes in P.ginseng plants.Saponin accumulation is higher in leaves than in roots of P.ginseng (Chang 1998).The high level of expression of PgSS2and PgSS3in leaves could be related to the high level of accu-mulation of saponin in leaves.
Transcriptional activity of PgSS1,PgSS2and PgSS3mRNA
MeJA treatment strongly activates the biosynthesis of triterpene saponins in many species,including P.ginseng (Gundlach et al.1992,Suzuki et al.2002,Han et al.2006b).Accumulation of SQS,squalene epoxidase and dammarenediol synthase mRNAs,genes involved in P.ginseng saponin biosyn-thesis,is enhanced after MeJA treatment (Lee et al.2004,Han et al.2006b,Han et al.2010).Enhancement of PgSS1,PgSS2and PgSS3mRNA accumulation following MeJA treatment suggests that the activity of all the three P.ginseng SQS genes is positively involved in saponin biosynthesis.However,the squalene epox-idase proteins PgSQE1and PgSQE2,which are involved in the step following SQS in saponin biosynthesis (Han et al.2010),respond differently to exogenous stimuli (elicitor and precursor).MeJA treatment enhances PgSQE1expression but suppresses PgSQE2expression.Han et al.(2010)postulated that PgSQE1has a regulatory role in triterpene biosynthesis,whereas PgSQE2has a regulatory role in phytosterol biosynthesis.
In situ hybridization of PgSS1and PgSS3
In situ hybridization revealed that both PgSS1and PgSS3tran-scripts preferentially accumulate in vascular bundle tissues (phloem cells and parenchymatous cells near xylem)and resin ducts in petioles.A similar result has been revealed by in situ hybridization analysis of squalene epoxidase (PgSQE1and PgSQE2),where these mRNAs mainly accumulate near vascular bundles and resin ducts.In situ hybridization using an EtSE antisense probe has revealed prominent EtSE expression in a parenchyma cell adjacent to primary laticifers located in the inner region of the cortex (Uchida et al.2009).
Madey et al.(2002)reported that phloem isolated from canola (Brassica napus )stems contains phospholipids,diacyl-glycerol,triacylglycerol,steryl and wax esters,and comparative-ly high concentrations of unesteri E ed fatty acids.Resin ducts are specialized tissues that conduct and secrete hydrocarbons in many plants,particularly in coniferous trees.Phloem-and resin duct-speci E c accumulation of SQS and SQE mRNAs in P.ginseng petioles indicates that phloem and resin ducts are both metabolically active sites for sterol and saponin biosyn-thesis and play a role in conducting squalene oil.
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Fig.7In situ hybridization of PgSS1and PgSS3mRNA in petiole cross-sections.(A–D)Sections probed with PgSS1antisense (A–C)and sense probes (D).(E–H)Sections probed with PgSS3antisense (E–G)and sense probes (H).Pf,phloem ?ber;Ph,phloem;Xy,xylem;Rd,resin duct.
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A
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Fig.8Histochemical analysis of GUS activity in transgenic P.ginseng plants harboring chimeric PgSS1promoter::GUS.(A)Leaf.(B and C)Aged root segments.(D)Newly formed roots (left,dark culture;right,light illumination culture).(E–H)Histological analysis of P.ginseng plants:leaf (E),petiole (F),aged roots (G)and newly formed root tip portion (H).
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Fig.9Histochemical analysis of GUS activity in transgenic P.ginseng plants harboring chimeric PgSS3promoter::GUS.(A)Leaf.(B and C)Aged root segments.(D)Newly formed roots (left,dark culture;right,light illumination culture).(E–H)Histological analysis of P.ginseng plants:leaf (E),petiole (F),aged roots (G)and newly formed root tip portion (H).
Expression and functional characterization of three ginseng squalene synthase genes
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Promoter analysis
Analysis of the PgSS1and PgSS3promoters using GUS staining of transgenic plants revealed that the PgSS1promoter was strongly expressed in roots and the PgSS3promoter was weakly expressed in roots.The different promoter activities in tissues and organs of plants might reveal the different responses to environmental stimuli.In fact,the DNA sequences of these two promoters are very different.In Arabidopsis,the SQS1pro-moter directs widespread expression of GUS activity (Busquets et al.2008).In contrast,transgenic Arabidopsis harboring SQS2::GUS exhibits narrow expression patterns of GUS,primar-ily in vascular tissues.The different expression patterns of Arabidopsis SQS promoters are in agreement with those of P.ginseng .
Materials and Methods
Isolation of cDNA clones for PgSS2and PgSS3
Total RNA was isolated from in vitro cultured adventitious roots.Poly(A)+RNA was isolated using a Poly (A)quick mRNA isolation kit (Stratagene).The SMARTTM cDNA library construction kit (Clontech)was used to synthesize cDNA from poly(A)+RNA.Size-selected cDNA was ligated to TriplEx2vector arms and packaged into phage particles using Gigapack III Gold packaging extract (Stratagene).
Single-pass partial sequences were determined with an auto-mated DNA sequencer (model ABI Prism 3700,Applied Biosystems).A total of 4,226high-quality ESTs were obtained from the library of in vitro cultured adventitious ginseng roots (NCBI GenBank dbEST accession Nos.HS076062–HS080287).A putative full-length SQS cDNA was sequenced,and its nucleo-tide and predicted amino acid sequences were analyzed using the DNASIS program (Hitachi Software Engineering Co.).Two full-length SQS cDNA clones (PgSS2,GQ468527;and PgSS3,AB115496)were isolated from 4,236ESTs obtained from an in vitro cultured adventitious root cDNA library.
Comparison of SS protein sequences
SS protein sequences were obtained from EMBL,GenBank and DDBJ sequence data,and the accession numbers of se-quences used in this analysis were as follows:Panax notoginseng (DQ186630),Panax quinquefolium (AM182456and AM182457),Nicotiana tabacum (U60057),Arabidopsis thaliana (D29017),Glycine max (AB007503)and Oryza sativa (AB007501).PgSS1,PgSS2and PgSS3amino acid sequences were analyzed using the BLAST program and aligned with ClustalW (https://www.doczj.com/doc/8a4223842.html,/clustalw/)and the DNASIS program (Hitachi).
Functional complementation in yeast erg9mutants
To construct an expression plasmid for yeast,PgSS1,PgSS2and PgSS3open reading frames (ORFs)were ampli?ed from cDNA
by PCR (25cycles of 40s at 94 C,40s at 50 C and 2min at 72 C)using Pfu DNA polymerase (Stratagene)and were cloned into pYES2.1using the TOPO TA expression kit (Invitrogen).Primer pairs used to isolate the cDNAs were 50-ATG GGA AGT TTG GGG GCA ATT CT-30and 50-GTT CTC ACT GTT TGT TCA GTA GTA GGT T-30for PgSS1;50-ATG GGA AGT TTG GGG GCA ATT CT-30and 50-TGT GGG ATT TGC AAA ACC CAA TCA-30for PgSS2;and 50-ATG GGA AGT TTG GGG GCA ATT CT-30and 50-TGT GGG ATT TGC AAA ACC CAA TCA-30for PgSS3.The PCR products were ligated into pYES2.1/V5-His-TOPO and transformed into Escherichia coli .The ORFs were ligated to the GAL1promoter in the sense orientation.The nucleotide sequence of the inserted DNA was con?rmed by sequencing.The erg9mutant strain 2C1(Mat a erg9::his3,aux32,ura3,trp1,leu2)was kindly provided by F.Karst,Colmar,France (Karst and Lacroute et al.1977).The erg9-de?cient yeast was transformed with pYES2.1/V5-His-TOPO encoding PgSS1,PgSS2or PgSS3by electroporation.Transformants were selected on minimal media lacking uracil supplemented with casein hydrolysate (7.5g l à1)and,after 5d of growth,subcultured on YPG medium with or without ergosterol to test for ergosterol pro-totrophy (Kribii et al.1997).
GC/MS analysis of squalene,squalene epoxide and ergosterol in yeast
Quantitative analysis of farnesol,squalene,squalene epoxide and ergosterol in erg9-de?cient yeast harboring PgSS1,PgSS2or PgSS3was performed by GC (Agilent 7890A)linked to an MSD system (Agilent 5975C).Freeze-dried yeast (1g)were ex-tracted by sonication with 15ml of 20%KOH containing 50%ethanol for 10min.After extraction with the same volume of hexane,the solvent was evaporated,and the residue was dis-solved in chloroform (1ml)and then centrifuged for 10min at 6,000r.p.m.to remove the suspended particles.A 2m l aliquot of the solution was analyzed by GC/MS (Agilent)equipped with a Agilent 5975C mass detector (EI,70eV)and the HP-5MS capil-lary column (30m ?0.25mm,?lm thickness 0.25m m),carrier gas:He (4ml min à1),oven temperature:150–300 C (20 C min à1).The farnesol,squalene,squalene epoxide and ergosterol contents were calculated from the ratios of the peak areas of the relevant compounds to those of the standards.Authentic farnesol,squalene,squalene epoxide and ergosterol were ob-tained from Sigma -Aldrich Co.
MeJA treatment
To investigate gene expression induced by elicitor treatment,we used in vitro culture of adventitious roots adopted as described by Lee et al.(2004).After 2weeks of culture,adven-titious roots in the vigorous growth phase were elicited by adding various concentrations of MeJA (0for control,5,10,20and 40m M)for 24h.The culture room was maintained at 24±2 C under a 16/8h (light/dark)photoperiod with light supplied by white ?uorescent tubes at an intensity of 24m mol m à2s à2.
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RT–PCR analysis
Total RNAs were isolated from MeJA-treated adventitious roots or from different ginseng plant organs,and reverse-transcribed using an ImProm-II Reverse Transcription System (Promega).The ?rst-strand cDNAs were used as a template for RT–PCR analysis,performed as follows:96 C for 5min,followed by 30cycles of 96 C for 30s,60 C for 30s and 72 C for 1min,with a ?nal 10min extension at 72 C.The primers used were 50-CCA TTT CCA TTT CAC ATT CCA ACT GCA-30and 50-CTG GTA ACC GCT TCC AAG CTC AAG AAA AGC-30for P.ginseng PgSS1;50-GCA ATC TTA TCT AAG AAC AGG T-30and 50-CAT GAT GGA ATT CAT CCA TGA GAA CTT TGT-30for P.ginseng PgSS2;and 50-ATC AAG CAA TCT TAT TTA AGT ATA TAT-30and 50-CGT GAT GGA ACT CAT CCA TGA GAA CTT TGT-30for P.ginseng PgSS3.The cDNA for b -actin was used as a control for RNA integrity and loading accuracy.
RNA in situ hybridization
The 237bp (position 1,104–1,340)PgSS1cDNA was ampli?ed using two oligonucleotide primers:50-GGA GTC TGG AAC CCT GTC CAA A-30and 50-TCA AGA ATA CAA CAT TAA CAG GGT-30.The 235bp (position 1,104–1,338)PgSS3cDNA was ampli?ed using two oligonucleotide primers:50-AAA TTC TGG AGC CCT GAC CAC TA-30and 50-TGA AAG ATA CAT TAA CTG TAC TGT A-30.The PgSS3primers selected were from the C-terminal region,which includes the UTR sequences to avoid binding to PgSS2mRNA.PCR-ampli?ed cDNAs were cloned into pGEM-T (Promega)and used as templates for synthesizing digoxigenin-labeled RNA probes.Sense and anti-sense PgSS1and PgSS3probes were prepared using T7and SP6RNA polymerases after digestion with Bst XI and Sac I,respect-ively.Probes were labeled using the DIG RNA labeling kit (Roche Diagnostics GmbH).Petioles and roots of 2-year-old plants were ?xed and paraf?n embedded.Sectioning,hybrid-ization and detection of hybridization signals were performed as described by Eisel et al.(2000).The sections were analyzed using a light microscope (Olympus BX51).
Isolation of PgSS1and PgSS3promoters from P.ginseng
Genomic DNA was puri?ed from cultured ginseng roots ac-cording to the DNeasy Plant Maxi prep kit (Qiagen)instruc-tions.Genomic DNA was digested with Dra I,Eco RV,Pvu II and Stu I,which are six-cutter enzymes that leave a blunt end at the cutting site,and subsequently used to construct four adaptor-ligated genomic libraries (L1–Dra I,L2–Eco RV,L3–Pvu II and L4–Stu I).
To obtain a 50region ?anking the PgSS1and PgSS3genes,genomic walking was performed according to the manufactur-er’s instructions (Clontech).The primary PCR was carried out using AP1(adaptor primer,50-GTAATACGACTCACTATA GGGC-30)and GSP2(a PgSS1and PgSS3gene-speci?c primer,50-CGAGCTGTTGAATGACGAGGCCGAAAC-30),followed by a second PCR with a nested AP2(50-ACTATAGGGCACGCGTGG
T-3)and a nested GSP1(a PgSS1and PgSS3gene-speci?c primer,50-GATCTGCTTTTCCGCATGCCTAGCCGC-30).The two non-overlapping GSPs were from a conserved region of both PgSS1and PgSS3cDNA sequences and an intronless region of Arabidopsis SQS genes.The PCR was performed in 50m l aliquots containing 1m l of genomic libraries as a template,5m l of 10?PCR buffer,1m l of dNTPs (10mM each),1m l of AP1,1m l of GSP2and 1m l of Ex Taq polymerase (TAKARA).The PCR conditions were:seven cycles of 94 C for 25s and 72 C for 3min;37cycles of 94 C for 25s and 67 C for 3min;and one ?nal cycle of 67 C for 7min.The primary PCR products were diluted 50-fold and used as a template in a second PCR using AP2and GSP1.The PCR conditions were:?ve cycles of 94 C for 25s and 72 C for 3min;20cycles of 94 C for 25s and 67 C for 3min;and one ?nal cycle of 67 C for 7min.The PCR products were cloned into the pGEM-T Easy vector (Promega)and sequenced.
Construction of plasmids
PgSS1and PgSS3promoter constructs used to drive GUS gene expression were prepared as follows.Sal I/Bam HI fragments of PgSS1and PgSS3promoters were synthesized by PCR with for-ward (50-ACTATAGGGCACGCGTGGT-30)and reverse (50-CGG GATCCTGCCCCCAAACTTCCCAT-30)primers,respectively.The ampli?ed fragments contained the 30end of PgSS1and PgSS3promoter fragments ending at the ATG start codon of the gene fused with the ATG start codon of the GUS reporter gene.PCR ampli?cation consisted of 30cycles at 94 C for 30s,56 C for 1min and 72 C for 1min 30s.The PCR products were cloned into the Sal I/Bam HI sites of pBI101bearing the GUS reporter gene.The two resulting constructs were utilized to transform ginseng by Agrobacterium tumefaciens infection.
Construction of transgenic ginseng and GUS assay
Genetic transformation of P.ginseng was carried out as described in our previous report (Choi et al.2001).Because the growth of P.ginseng is extremely slow in the ?eld,an in vitro culture system for adventitious roots was adopted to analyze the characteristics of transgenic plants,as described by Lee et al.(2004).Culture conditions and line selection were per-formed as in our previous report (Lee et al.2004).The in vitro cultured adventitious roots derived from independent trans-genic ginseng plants were regarded as an independent line.There were >20transgenic lines,from which PgSS22,-3and -4were selected for further analysis.
Expression of GUS was detected by histochemical staining according to the method used by Jefferson et al.(1987).In vitro cultured plantlets and adventitious roots were stained for GUS in media containing 0.5mg ml à1X-glucuronide (Clontech),0.5mM K +-ferrocyanide,0.5mM K +-ferricyanide,10mM Na 2EDTA,50mM phosphate buffer (pH 7.0)and 0.1%(w/v)Triton X-100.Endogenous glucuronidase activity was not de-tected in plants or adventitious roots from ginseng.
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Histological observation
Samples were ?xed at 4 C for 24h in 1.5%glutaraldehyde and 1.6%paraformaldehyde,buffered with 0.05M phosphate,pH 6.8.They were dehydrated in an ethanol series (30,50,60,70,80,90,95and 100%)and embedded in epoxy resin.The samples were semi-thin (3mm)and sectioned using an autocut micro-tome (Leica RM 2165).The sections were observed with a light microscope (Olympus BX51).
Supplementary data
Supplementary data are available at PCP online.
Funding
This work was supported by grants from Biogreen 21(20100301-061-001-04),Rural Development Administration,and from the WCU project (R33-10157)of the Ministry of Education,Science &Technology (MEST),Republic of Korea.
Acknowledgments
We thank Francis Karst (INRA,Colmar,France)for the yeast strain 2C1.
References
Abe,I.,Rohmer,M.and Prestwich,G.D.(1993)Enzymatic cyclization of squalene and oxidosqualene to sterols and triterpenes.Chem.Rev.93:2189–2206.
Baxter,A.,Fitzgerald,B.J.,Hutson,J.L.,McCarthy,A.D.,Motteram,J.M.,Ross,B.C.et al.(1992)Squalestatin 1,a potent inhibitor of squalene synthase,which lowers serum cholesterol in vivo.J.Biol.Chem.267:11705–11708.
Bergstrom,J.D.,Kurtz,M.M.,Rew,D.J.,Amend,A.M.,Karkas,J.D.,Bostedor,R.G.et al.(1993)Zaragozic acids:a family of fungal me-tabolites that are picomolar competitive inhibitors of squalene syn-thase.Proc.Natl https://www.doczj.com/doc/8a4223842.html,A 90:80–84.
Bloch,K.(1992)Sterol molecule:structure,biosynthesis and function.Steroids 57:378–383.
Briskin,D.P.(2000)Medicinal plants and phytomedicines.Linking plant biochemistry and physiology to human health.Plant Physiol.124:507–14.
Busquets,A.,Keim,V.,Closa,M.,del Arco,A.,Boronat,A.,Arro
′,M.et al.(2009)Arabidopsis thaliana contains a single gene encoding squalene synthase.Plant Mol.Biol.67:25–36.
Chang,H.K.(1998)Changes of saponin contents in Panax ginseng leaves by different harvesting months.Korean J.Food Nutr.11:82–86.Choi,Y.E.,Yang,D.C.,Kusano,T.and Sano,H.(2001)Rapid and ef?cient Agrobacterium-mediated genetic transformation by plas-molyzing pretreatment of cotyledons in Panax Ginseng .Plant Cell Rep.20:616–621.
Devarenne,T.P.,Ghosh,A.and Chappell,J.(2002)Regulation of squa-lene synthase,a key enzyme of sterol biosynthesis,in tobacco.Plant Physiol.129:1096–1106.
Devarenne,T.P.,Shin,D.H.,Back,K.,Yin,S.H.and Chappell,J.(1998)Molecular characterization of tobacco squalene synthase and regulation in response to fungal elicitor.Arch.Biochem.Biophys.349:205–215.
Eisel,D.,Grunewald-Janho,S.and Kruchen,B.(2000)Transcriptional labeling of RNA probes:procedures for non radioactive labeling and detection.In:DIG Application Manual.Roche Diagnostics Corporation,Germany.
Favre,B.and Ryder,N.S.(1996)Characterization of squalene epoxidase activity from the dermatophyte Trichophyton rubrum and its in-hibition by terbina E ne and other antimycotic agents.Antimicrob.Agents 40:443–447.
Gu,P.,Ishii,Y.,Spencer,T.A.and Shechter,I.(1998)Function–structure studies and identi?cation of three enzyme domains involved in the catalytic activity in rat hepatic squalene synthase.J.Biol.Chem.273:12515–12525.
Gundlach,H.,Mueller,M.J.,Kutchan,T.M.and Zenk,M.H.(1992)Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures.Proc.Natl https://www.doczj.com/doc/8a4223842.html,A 89:2389–2393.
Han,J.Y.,In,J.G.,Kwon,Y.S.and Choi,Y.E.(2010)Regulation of ginsenoside and phytosterol biosynthesis by RNA interferences of squalene epoxidase gene in Panax ginseng .Phytochemistry 71:36–46.
Han,J.Y.,Jung,S.J.,Kim,S.W.,Kwon,Y.S.,Yi,M.J.,Yi,J.S.et al.(2006a)Induction of adventitious roots,analysis of ginsenoside and genes involved in triterpene biosynthesis in Panax ginseg .J.Plant Biol.49:26–33.
Han,J.Y.,Kwon,Y.S.,Yang,D.C.,Jung,Y.R.and Choi,Y.E.(2006b)Expression and RNA interference-induced silencing of the dam-marenediol synthase gene in Panax ginseng .Plant Cell Physiol.47:1653–1662.
Haralampidis,K.,Trojanowska,M.and Osbourn, A.E.(2002)Biosynthesis of triterpenoid saponins in plants.Adv.Biochem.Eng.Biotechnol.75:31–49.
Hartmann,M.A.and Benveniste,P.(1987)Plant membrane sterols:isolation,identi?cation,and biosynthesis.Methods Enzymol.148:632–650.
Hata,S.,Sanmiya,K.,Kouchi,H.,Matsuoka,M.,Yamamoto,N.and Izui,K.(1997)cDNA cloning of squalene synthase genes from mono-and dicotyledonous plants,and expression of the gene in rice.Plant Cell Physiol.38:1409–1413.
Hayashi,H.,Hirota, A.,Hiraoka,N.and Ikeshiro,Y.(1999)Molecular cloning and characterization of two cDNAs for Glycyrrhiza glabra squalene synthase.Biol.Pharm.Bull.22:947–950.
Higo,K.,Ugawa,Y.,Iwamoto,M.and Korenaga,T.(1999)Plant cis-acting regulatory DNA elements (PLACE)database.Nucleic Acids Res.27:297–300.
Hostettmann,K.and Marston, A.(1995)Saponins.Cambridge University Press,Cambridge.
Huang,Z.,Jiang,K.,Pi,Y.,Hou,R.,Liao,Z.,Cao,Y.et al.(2007)Molecular cloning and characterization of the yew gene encoding squalene synthase from Taxus cuspidate .J.Biochem.Mol.Biol.40:625–635.
Jefferson,R.A.,Kavanagh,T.A.and Bevan,M.W.(1987)GUS fusions:b -glucoronidase as a sensitive and versatile gene fusion marker in higher plants.EMBO J.6:3901–3907.
Jennings,S.M.,Tsay,Y.H.,Fisch,T.M.and Robinson,G.W.(1991)Molecular cloning and characterization of the yeast gene for squa-lene synthetase.Proc.Natl https://www.doczj.com/doc/8a4223842.html,A 88:6038–6042.
T.-D.Kim et al .
by guest on February 15, 2012
https://www.doczj.com/doc/8a4223842.html,/Downloaded from
Jones,P.J.H.,MacDougall,D.E.,Ntanios,F.and Vanstone,C.A.(1997)Dietary phytosterols as cholesterol-lowering agents in humans.Can.J.Physiol.Pharmacol.75:217–227.
Karst, F.and Lacroute, F.(1977)Ergosterol biosynthesis in Saccharomyces cerevisiae :mutants de?cient in the early steps of the pathway.Mol.Gen.Genet.154:269–277.
Kribii,R.,Arro
′,M.,Del Arco,A.,Gonza ′lez,V.,Balcells,L.,Delourme,D.et al.(1997)Cloning and characterization of the Arabidopsis thali-ana SQS1gene encoding squalene synthase,involvement of the C-terminal region of the enzyme in the channeling of squalene through the sterol pathway.Eur.J.Biochem.249:61–69.
Krogh,A.,Larsson,B.,von Heijne,G.and Sonnhammer,E.L.(2001)Predicting transmembrane protein topology with a hidden Markov model:application to complete genomes.J.Mol.Biol.305:567–580.
Kushiro,T.,Shibuya,M.and Ebizuka,Y.(1998)b -Amyrin synthase:cloning of oxidosqualene cyclase that catalyzes the formation of the most popular triterpene among higher plants.Eur.J.Biochem.256:238–244.
Lee,M.H.,Jeong,J.H.,Seo,J.W.,Shin,C.G.,Kim,Y.S.,In,J.G.et al.(2004)Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squalene synthase gene.Plant Cell Physiol.45:976–984.
Madey,E.,Nowack,L.M.and Thompson,J.E.(2002)Isolation and char-acterization of lipid in phloem sap of canola.Planta 214:625–634.Robinson,G.W.,Tsay,Y.H.,Kienzle,B.K.,Smith-Montroy,C.A.and Bishop,R.W.(1993)Conservation between human and fungal squalene synthatases:similarities in structure,function,and regu-lation.Mol.Cell.Biol.13:2706–2717.
Seo,J.W.,Jeong,J.H.,Shin,C.G.,Lo,S.C.,Han,S.S.,Yu,K.W.et al.(2005)Overexpression of squalene synthase in Eleutherococcus senticosus increases phytosterol and triterpene accumulation.Phytochemistry 66:869–877.
Shibata,S.(2001)Chemistry and cancer preventing activities of ginseng saponins and some related triterpenoid compounds.J.Korean Med.Sci.16:S28–S37.Suzuki,H.,Achnine,L.,Xu,R.,Matsuda,S.P.T.and Dixon,R.A.(2002)A genomics approach to the early stages of triterpene saponin biosynthesis in Medicago truncatula .Plant J.32:1033–1048.
Threlfall,D.R.and Whitehead,I.M.(1988)Coordinated inhibition of squalene synthetase and induction of enzymes of sesquiterpenoids phytoalexin biosynthesis in cultures of Nicotiana tabacum .Phytochemistry 27:2567–2580.
Tozawa,R.,Ishibashi,S.,Osuga,J.,Yagyu,H.,Oka,T.,Chen,Z.et al.(1999)Embryonic lethality and defective neural tube closure in mice lacking squalene synthase.J.Biol.Chem.274:30843–30848.
Uchida,H.,Yamashita,H.,Kajikawa,M.,Ohyama,K.,Nakayachi,O.,Sugiyama,R.et al.(2009)Cloning and characterization of a squalene synthase gene from a petroleum plant,Euphorbia tirucalli L.Planta 229:1243–1252.Vo ¨geli,U.and Chappell,J.(1988)Induction of sesquiterpene cyclase and suppression of squalene synthetase activities in plant cell cultures treated with fungal elicitor.Plant Physiol.88:1291–1296.
Vogler,B.K.,Pittler,M.H.and Ernst,E.(1999)The ef?cacy of ginseng.A systematic review of randomized clinical trials.Eur.J.Clin.Pharmacol.55:567–575.
Wentzinger,L.F.,Bach,T.J.and Hartmann,M.A.(2002)Inhibition of squalene synthase and squalene epoxidase in tobacco cells triggers an up-regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.Plant Physiol.130:334–346.
Wen,J.and Zimmer,E.A.(1996)Phylogeny and biogeography of Panax L.(the ginseng genus,Araliaceae):inferences from ITS sequences of nuclear ribosomal DNA.Mol.Phylogenet.Evol.6:167–177.
Yue,P.Y.,Wong,D.Y.,Wu,P.K.,Leung,P.Y.,Mak,N.K.,Yeung,H.W.et al.(2006)The angiosuppressive effects of 20(R)-ginsenoside Rg3.Biochem.Pharmacol.72:437–445.
Zhao,M.W.,Liang,W.Q.,Zhang,D.B.,Wang,N.,Wang,C.G.and Pan,Y.J.(2007)Cloning and characterization of squalene synthase (SQS)gene from Ganoderma lucidum .J.Microbiol.Biotechnol.17:1106–1112.
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