journal of biochemistry and molecular biology vol. 40, no

Journal of Biochemistry and Molecular Biology, Vol. 40, No. 5, September 2007, pp. 625-635

Molecular Cloning and Characterization of the Yew Gene EncodingSqualene Synthase from Taxus cuspidata

Zhuoshi Huang1, Keji Jiang1, Yan Pi1, Rong Hou1, Zhihua Liao1,3, Ying Cao1, Xu Han1,

Qian Wang1, Xiaofen Sun1 and Kexuan Tang1,2,*1State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center,

Morgan-Tan International Center for Life Sciences, Fudan University, Shanghai 200433, People’s Republic of China2Plant Biotechnology Research Center, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center,

School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200030, People’s Republic of China3Laboratory of Natural Products and Metabolic Engineering, Institute of Biotechnology, School of Life Sciences,

Southwest China University, Chongqing 400715, People’s Republic of China

Received 18 September 2006, Accepted 28 March 2007

The enzyme squalene synthase (EC 2.5.1.21) catalyzes a

reductive dimerization of two farnesyl diphosphate (FPP)

molecules into squalene, a key precursor for the sterol and

triterpene biosynthesis. A full-length cDNA encoding

squalene synthase (designated as TcSqS) was isolated from

Taxus cuspidata, a kind of important medicinal plants

producing potent anti-cancer drug, taxol. The full-length

cDNA of TcSqS was 1765 bp and contained a 1230 bp open

reading frame (ORF) encoding a polypeptide of 409 amino

acids. Bioinformatic analysis revealed that the deduced

TcSqS protein had high similarity with other plant

squalene synthases and a predicted crystal structure

similar to other class I isoprenoid biosynthetic enzymes.

Southern blot analysis revealed that there was one copy of

TcSqS gene in the genome of T. cuspidata. Semi-

quantitative RT-PCR analysis and northern blotting

analysis showed that TcSqS expressed constitutively in all

tested tissues, with the highest expression in roots. The

promoter region of TcSqS was also isolated by genomic

walking and analysis showed that several cis-acting

elements were present in the promoter region. The results

of treatment experiments by different signaling components

including methyl-jasmonate, salicylic acid and gibberellin

revealed that the TcSqS expression level of treated cells had

a prominent diversity to that of control, which was

consistent with the prediction results of TcSqS promoter

region in the PlantCARE database.

Keywords: RACE, RT-PCR, Squalene synthase, Taxus

cuspidata, TcSqS

Introduction

Isoprenoids, also named as terpenoids, play important roles in

plant metabolism. The plant isoprenoids can be divided into

two classes, primary and secondary metabolites. The best-

known primary metabolites of isoprenoids include sterols,

carotenoids and gibberellins while the secondary metabolites

include various kinds of terpenes, such as mono-, sesqui-, di-

and triterpenes (Chappell, 1995). In plants, the sterols, also

called as phytosterols, not only act as essential molecules for

the primary functioning for eukaryotic organisms (Bloch,

1992), but also exhibit important pharmacological activities

(Jones et al., 1997). The triterpenes in plants also exhibit a

wide range of structural diversity and biological activity, and

their glycosides saponins are considered to be natural

medicines with economical importance (Osbourn, 2003;

Connolly and Hill, 2005). Despite classified into different

classes, the sterols and triterpenes biosynthetic pathways share

Database Accession No: DQ836053 for TcSqS mRNA and

DQ860509 for TcSqS Promoter Region

Abbreviations: CTAB, Cetyl trimethyl ammonium bromide; FPP,

farnesyl diphosphate; GA3, gibberellic acid; GGPP, geranylgeranyl

pyrophosphate; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A

reductase; IPP, isopentenyl diphosphate; MeJA, methyl-jasmonate,

ORF, open reading frame; PCR, Polymerase chain reaction; RACE,

rapid amplification of cDNA ends; RT-PCR, reverse transcriptase-

polymerase chain reaction; SA, salicylic acid, SqS, Squalene syn-

thase.

*To whom correspondence should be addressed.

Tel: 86-21-65642772; Fax: 86-21-65643552

E-mail: [email protected] or [email protected]

626 Zhuoshi Huang et al.

a committed precursor - squalene - which is synthesized from

two molecules of farnesyl diphosphate (FPP) by a two-step

reductive dimerization reaction catalyzed by the enzyme

squalene synthase (SqS) (Abe et al., 1993).

SqS is an endoplasmic reticulum associated enzyme with

carboxy terminus anchored in the ER membrane and the

amino terminus including active site exposed in the cytosol

(Stamellos et al., 1993). The soluble, bioactive SqS enzymes

have been isolated from rat (Shechter et al., 1992) and

tobacco (Hanley and Chappell, 1992) by methods which

overcome the clag of the hydrophobic ER association for the

purification. cDNA clones for squalene synthase have been

isolated and characterized from various species, such as rice,

maize, soybean (Hata et al., 1997), tobacco (Hanley et al.,

1996; Devarenne et al., 1998), Arabidopsis thaliana

(Nakashima et al., 1995; Kribii et al., 1997) and other plants,

as well as mammals (Robinson et al., 1993; Inoue et al.,

1995) and yeast (Jennings et al., 1991; Robinson et al., 1993;

Merkulov et al., 2000). By the method of the cDNA

expression in E. coli, more soluble squalene synthase proteins

with enzymatically activity have been isolated and characterized,

such as those of human (Thompson et al., 1998), yeast

(Jennings et al.,1991), mouse (Inoue et al., 1995), tobacco

(Hanley et al., 1996; Devarenne et al., 1998), and Arabidopsis

(Nakashima et al., 1995). Since catalyzing the first committed

step in the formation of cholesterols, bile acids and steroid

hormones, human SqS is identified as an attractive target for

therapeutic intervention and draws great attention. Using the

method of x-ray diffraction, the proper crystal structure for

human squalene synthase which is similar to other class I

isoprenoid biosynthetic enzymes has been determined (Pandit

et al., 2000). The interest in the investigation of the squalene

synthase regulation in plants increases steadily in recent years.

The addition of fungal elicitors into tobacco cell cultures

induces the decline in sterol biosynthesis which correlates

with a suppression of SqS activity (Devarenne et al., 1998)

and the overexpression of squalene synthase in Panax ginseng

roots results in a remarkable increase of phytosterols and

triterpene saponins (Lee et al., 2004).

Squalene synthase is commonly described as an incipient

and crucial branch point enzyme away from the main

isoprenoids biosynthetic pathway and a potential regulatory

point that controls carbon flux into sterols and triterpenes

biosynthesis. Lots of evidence support that inhibition of the

squalene synthase enzyme is a potential means of redirecting

FPP away from the sterol biosynthesis, towards the synthesis

of other commercially interesting isoprenoids. The disruption

of sterol biosynthesis at squalene synthase leads a remarkable

accumulation of FPP in an erg9 mutant strain of Saccharomyces

cerevisiae (Song, 2003). Besides FPP, the increase of IPP and

GGPP was also observed when the rat liver cells were treated

with zaragozic acid A, a potent inhibitor of squalene synthase

(Keller, 1996) and similar effects have been observed in plants

(Fulton et al., 1995). A previous study shows that the

inhibition of SqS in tobacco results in up-regulation of mRNA

level and enzyme activity of the HMGR (Wentzinger et al.,

2002), the major limiting-step enzyme in MVA pathway. The

disruption at squalene synthase leads the depletion of squalene

derived products, and the induced compensatory responses

mediated by HMGR contribute to the biosynthesis of other

commercially interesting isoprenoids. The disruption of squalene

synthase as well as the overexpression of downstream

enzymes results in a 7-fold increase in the production of the

carotenoid lycopene in yeast (Shimada et al., 1998).

Taxol, a diterpenoid alkaloid produced by yew (Taxus)

species (Baloglu and Kingston, 1999), is one of the most

efficient anticancer drugs approved by American Food and

Drug Administration (Kohler and Goldspiel, 1994). The

biosynthesis of taxol and squalene derived products share the

same isoprenoids biosynthetic pathway upon the precursor

FPP. As decreasing the flux through competitive pathways is

considered to be one of the trends for the plant secondary

metabolism (Oksman-Caldentey and Inze, 2004), the regulation

of squalene synthase will draw more attentions in the

secondary metabolism of Taxus cuspidata. Here, we report the

molecular cloning and characterization of the squalene

synthase gene from Taxus cuspidata, as the first step for the

regulation of TcSqS in the isoprenoids biosynthetic pathway.

Materials and Methods

Plant materials. T. cuspidata plants were grown in greenhouse at

Fudan University, China. T. cuspidata cultured callus lines, initiated

from hypocotyl of yew seeds, were maintained in improved B5

solid medium (Sigma, USA) supplemented with 1 mg/L naphthalene-

acetic acid (NAA), 0.5 mg/L thidiazuron (TDZ), 1 mg/L 2,4-

dichlorophenoxy-acetic acid (2, 4-D) and 5 basic amino acids:

0.8 g/L tyrosine, 0.4 g/L alanine, 0.4 g/L praline, 0.4 glutamic acid

and 0.4 g/L lysine.

RNA and DNA isolation. Total RNAs were extracted from

different tissues including roots, stems and leaves of 3-year-old T.

cuspidata plants and from calluses using the special method for

Taxus species (Liao et al., 2004) and then stored at −80oC prior to

cDNA cloning and semi-quantitative RT-PCR analysis. Genomic

DNA was isolated using a CTAB-based method (Rechards et al.,

1995) for the walking library construction and Southern blot

analysis. The quality and concentration of RNA and DNA samples

were examined by EB-stained agarose gel electrophoresis and

spectrophotometer analysis.

Isolation of the TcSqS full-length cDNA. According to the

manufacturer’s protocol (PowerScriptTM Reverse Transcriptase),

single-strand cDNA library was constructed by reverse

transcription from total RNA isolated from T. cuspidata cultured

cell lines. After RNaseH treatment, the cDNA library was used as

templates for PCR amplification of the conserved region of SqS

from T. Cuspidata. A pair of degenerate oligonucleotide primers,

TcSQSF and TcSQSR, designed according to the conserved

sequences of other plant and fungal SqS genes, was used for the

Squalene synthase Gene from Taxus cuspidata 627

amplification of the core cDNA fragment of TcSqS by standard

gradient PCR amplification. All primers used in the study were

listed in Table 1. The PCR products were analyzed in 0.8% agarose/

EB gel, along with DL2000 DNA size markers (TaKaRa) and the

result showed a single major product sized about 0.4 kb was

amplified via PCR. The amplified PCR product was purified and

cloned into pMD18-T vector (TaKaRa, Japan) followed by

sequencing. BLAST analysis showed that the 450 bp fragment

sequenced above had high homology to other plant SqS genes. This

fragment was subsequently used for designing gene specific

primers for the cloning of 5' end of the cDNA of TcSqS by RACE.

The 5'-RACE-Ready first-strand cDNA library was synthesized

following the protocol provided by the manufacturer (SMARTTM

RACE). The 5'-end of TcSqS cDNA was amplified using two 5'-end

gene specific primers (GSP) and the universal primers provided by

the kit. For the first PCR amplification, TcSQS5-1 and UPM

(Universal Primer A Mix, Table 1) were used as the primers and 5'-

RACE-Ready cDNA library as the template. For the nested PCR

amplification, TcSQS5-2 and NUP (Nested Universal Primer A,

Table 1) were used as the nested PCR primers, while the products

of the first PCR amplification were used as templates. Both the first

and the nested PCR amplification procedures were carried out at

the following condition: 3 min at 94oC, 28 cycles (35 s at 94oC, 35 s

at 60oC, 2 min at 72oC) and 8 min at 72oC. The nested amplification

products were purified and subcloned into pMD18-T vector

followed by sequencing. The sequences of the 5'-end and the core

fragment were aligned and assembled perfectly on Contig Express

(Vector NTI 9.0), and an 800 bp-length cDNA sequence including

the whole 5'-UTR and part of the CDs of the TcSqS gene was

deduced. This fragment was subsequently used for designing gene

specific primers for the cloning of the full-length cDNA of TcSqS

by RT-PCR.

To amplify the full-length cDNA of TcSqS, two gene specific

primers, TcSQSFull-1 and TcSQSFull-2, were synthesized following

the 5'-UTR sequence. The RT-PCR amplification was carried out

following the protocol of One Step RNA PCR Kit (TaKaRa) in

which the AMV RTase XL was used to reversely transcribe mRNA

to cDNA. TcSQSFull-1 and adapter primer (AP) with a poly (T) tail

were used as RT-PCR primers and 1 µg total RNA as template.

PCR amplification procedure was carried out at the following

condition: 30 min at 50oC for reverse transcription, 2 min at 94oC

for RTase denaturation followed by amplification of 30 cycles

(94oC for 30 s, 65oC for 30 s, and 72oC for 3 min). The products of

the RT-PCR were diluted and used as the template for the nested

amplification PCR. The nested PCR used TcSQSF-2 and AP as

primers, and the PCR amplification procedure was carried out as

following: 3 min at 94oC followed by 30 cycles of amplification

(94oC for 30 s, 65oC for 30 s, 72oC for 3 min) and by 72oC for

8 min. The 1.8 kb-length nested amplification products were

purified and subcloned into pMD18-T vector followed by

sequencing.

Isolation of the TcSqS promoter. GenomeWalker DNA libraries

were constructed following the user manual provided by the

manufacturer (Universal GenomeWalkerTM Kit). The T. cuspidata

genomic DNA isolated from fresh leaves was completely digested

separately with six different blunt-end restriction enzymes (EcoRV,

HpaI, NaeI, NruI, SnaBI and StuI). After purification, each of the

blunt-end digestions was ligated to the GenomeWalker adaptor by

T4 DNA ligase. The adaptor-ligated genomic DNA fragments

referred as GenomeWalker libraries were used as the template in

the following PCR-based DNA walking.

The amplification of TcSqS promoter region consisted of two

walking amplifications. The primary PCR used the outer adaptor

primer AP1 provided in the kit and the gene specific primer

TcSqSW-1, and the amplification was performed in the process of 7

cycles (94oC for 30 s; 72oC for 3 min), followed by 32 cycles (94oC

for 25 s; 67oC for 3 min) and by 67oC for 8 min. The primary PCR

mixture was then diluted 50-fold and used as the template for

nested PCR with the nested adaptor primer AP2 provided in the kit

and a nested gene-specific primer TcSqSW-2. The amplification

was performed in the process of 5 cycles (94oC for 30 s, 72oC for

3 min), followed by 32 cycles (94oC for 30 s, 67oC for 3 min) and

by 67oC for 8 min. The PCR products were analyzed by

Table 1. The PCR primers used in the study

Primer Direction Sequence

TcSQSF + 5'-GT(C/T)GA GGA(C/T)G A(C/T)AC(A/T/C/G) AG(C/T)AT (A/T)(C/G)C-3'

TcSQSR - 5'-(T/G)(G/A)T(T/C)T TCTG(A/C/G) AGAAA (C/T)A(A/G)(G/A)C CCAT-3'

TcSQS5-1 - 5'-CAGTT GACAC GTGAT GAAAT TGGTC-3'

TcSQS5-2 - 5'-AAGAG AAGTG CCACG AAGGA TCATA-3'

TcSQSFull-F1 + 5'-GCCCT AACTT CTGAA TCACA CAGTT C-3'

TcSQSFull-F2 + 5'-GGCTG GGACG GGCTC AATTT TGATC AAT-3'

TcSQSW-1 - 5'-CTGTG TGATT CAGAA GTTAG GGCGC TAGAA-3'

TcSQSW-2 - 5'-GCCGT GCGCT TATAA ATTGT TTGAC ATCA-3'

UPM* +Long: 5'-CTAAT ACGAC TCACT ATAGG GCAAG CAGTG GTATC AACGC AGAGT-3'Short: 5'-CTAAT ACGAC TCACT ATAGG GC-3'

NUP* + 5'-AAGCA GTGGT ATCAA CGCAG AGT-3'

AP* - 5'-GGCCA CGCGT CGACT AGTAC TTTTT TTTTT TTTTT TT-3'

Walker-AP1* + 5'-GTAAT ACGAC TCACT ATAGG GC-3'

Walker-AP2* + 5'-ACTAT AGGGC ACGCG TGGT-3'

The primers provided by commercial kits were marked with *.


electrophoresis, purified from agarose gel, and then cloned into

pMD18-T vector followed by sequencing.

Semi-quantitative RT-PCR analysis. Semi-quantitative RT-PCR

was carried out to investigate the mRNA expression profiles of

TcSqS in different tissues of T. Cuspidata and under different

treatments. All RNA templates were digested with DNaseI and then

purified. RT-PCR was performed under the following condition:

50oC for 30 min for reverse transcription, 94oC for 2 min for RTase

denaturation followed by the amplifications (94oC for 30 s; 58oC for

30 s and 72oC for 50 s) with the cycles ranged from 20-30,

according to the protocol of One Step RNA PCR Kit (TaKaRa).

The consistency of each example was measured by Ultraviolet-

Visible Spectrophotometer to make sure that fixed quantificational

RNA of each example (500 ng per reaction) was used for RT-PCR

analysis. Two gene-specific primers, TcSQS5-1 and TcSQSFull-F1,

were used to amplify a 500 bp length fragment in the semi-

quantitative RT-PCR as the consults of TcSqS expression levels. At

the same time, the RT-PCR reaction for the house-keeping gene

(18S gene) using specific primers 18SF and 18SR designed

according to the conserved regions of plant 18S genes was

performed to estimate if equal amounts of RNA among samples

were used in RT-PCR as an internal control. Amplifications were

performed under the following conditions: 50oC for 30 min, 94oC

for 2 min followed by 20 cycles of amplification (94oC for 30 s,

58oC for 30 s and 72oC for 50 s). The amplified products were

separated on 1% agarose gel and analyzed with Gene analysis

software package (Gene Company).

Southern blot analysis. Total genomic DNA was isolated from

fresh T. cuspidata leaves following CTAB based method. Aliquots

of DNA (30 µg/sample) were digested respectively at 37oC with

EcoRI, HindIII and XbaI, which did not cut within the probe

region. Fully digested samples were fractionated by 0.85% agarose

gel electrophoresis, and transferred to a positively charged Hybond-

N+ nylon membrane (Amersham Pharmacia). The TcSqS 5'-end

cDNA fragment amplified with the primers TcSqSFullF-1 and

TcSqS5-1 was used as probe labeling. Probe labeling (biotin),

hybridization and signal detection were carried out using Gene

images random prime labeling module and CDP-Star detection

module following the manufacturer’s instructions (Amersham

Pharmacia). The filter was washed under high stringency condition

at the temperature of 60oC and the hybridized signals were

visualized by exposure to Fuji X-ray film at room temperature for

0.5 h.

Northern blotting analysis. To investigate the TcSqS expression

pattern in different tissues of T. cuspidata, high quality total RNAs

were extracted from root, stem, and leaf of three-year-old T.

cuspidata plants. Equivalent of each RNA sample (20 µg/sample)

was denatured and separated on 1% agarose gel containing 0.66M

formaldehyde, and blotted onto a positively charged Hybond-N+

nylon membrane (Amersham Pharmacia). The Probe labeling

(biotin), hybridization and signal detection were carried out with

the same protocol and same probe in Southern analysis. The

membrane was washed under high stringency condition at the

temperature of 65oC and the hybridized signals were visualized by

exposure to Fuji X-ray film at room temperature for 1 h.

Results and Discussion

Amplification of the TcSqS conserved region. Agarose gel

analysis revealed that gradient PCR amplification with

primers TcSqS-F and TcSqS-R generated a single major

product of 390 bp in length with the annealing temperature of

54oC. BLAST-n analysis indicated that it had wide similarity

to known squalene synthases from A. thaliana, Glycine max

and Oryza sativa, implying that it was probably a part of the

squalene synthase gene.

Rapid amplification of TcSqS 5'-cDNA end. Electrophoresis

analysis of primary PCR product of 5' RACE showed a

specific band of about 600 bp in length. Nested amplification

was carried out using primary PCR product as template and a

specific DNA band of about 550 bp was amplified. After

cutting away the adapter sequences, the final 5' cDNA end of

TcSqS turned out to be 540 bp. An overlap of 94 bp was found

between the 5' and the conserved fragments. BLAST-n analysis

of this sequence resulted in a similar result as that of the

conserved region. Based on the sequence of the 5' UTR region

of TcSqS, two pairs of primers were designed: TcSqSFull-F1

and TcSqSFull-F2 for the full-length cDNA amplification,

TcSqSW-1 and TcSqSW-2 for the genomic walking.

Isolation and analysis of TcSqS full-length cDNA. Agarose

gel analysis revealed that amplification with primers TcSqSFull-

F1 and poly(T) primer AP resulted in a DNA band of about

1.9 kb accompanied by some faint bands and smearing.

Nested amplification resulted in a bright band of about 1.8 kb

as anticipated. Sequencing result of this sequence coincided

with the 5'-end of TcSqS cDNA sequence amplified before.

After cutting away the adapter sequences, the 1765 bp cDNA

of TcSqS possessed a 1530-bp open reading frame (ORF)

from 60 bp to 1289 bp of the sequence besides a 59-bp 5'

UTR and a 476-bp 3' UTR including a PolyA tail. Nucleotide-

nucleotide BLAST of the TcSqS full-length cDNA sequence

on NCBI website indicated that its conserved segments had

similarity to the known SqS of other plant species such as G.

max, Lotus japonicus, and Panax ginseng.

Characterization of the TcSqS protein. The open reading

frame of TcSqS was obtained by the ORF Finder on the NCBI

Web (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The predicted

TcSqS protein precursor was 409 aa in length with a theoretical

molecular weight of 46.46 kDa and pI value of 6.45.

According to NCBI conserved domain search, a conserved

domain “Trans_IPPS_HH” (cd00683) was found to penetrate

through the core of predicted TcSqS protein.

The NCBI protein-protein BLAST showed that the deduced

TcSqS amino acid sequence had 72% (282/387) identities and

84% (327/387) positives in local alignments to G. max SqS. It

also shared very broad and high local identities and positives

to other plant species of broad classes such as Panax

notoginseng, P. ginseng, Solanum tuberosum, L. japonicus,

Nicotiana benthamiana and A. thaliana. The deduced TcSqS


protein shared a less identity to the squalene synthases of

mammalian (human and rat) and fungi (yeast). Multi-

alignment of TcSqS with other SqS was conducted (Fig. 2).

Six highly conserved peptide domains of 1423 amino acids

which might be important for catalytic/functional activity

(Robinson et al., 1993) were discernible in the TcSqS amino

acid sequence (Fig. 2). Domains III, IV, and V showed a

highly conserved consensus sequence with other SqS enzymes,

while domains I and II were much less conserved. Domain II

was highly conserved with an aspartate-rich-motif among the

other SqS proteins (Devarenne et al., 1998). Similar motifs

had been noted in other isoprenoid biosynthetic enzymes,

such as FPP synthase (Song and Poulter, 1994) and

trichodiene synthase (Cane et al., 1995). These aspartate-rich

motifs were proposed to coordinate/facilitate FPP binding

through a magnesium ion requirement. Despite of a low level

of sequence identity in the aligned SqS enzymes, the domain

IV in the carboxyl terminus of proteins exhibited very

hydrophobic characteristics and might be able to perform a

putatively similar function (Gu et al., 1998). The TMpred

analysis (Hofmann and Stoffel, 1993) for the deduced TcSqS

protein predicted a transmembrane region in the carboxyl

terminus. The carboxyl-terminal deletion of residues resulted

in functionally soluble SqS enzyme activity and SqS protein

accumulation in tobacco (Devarenne et al., 1998), capsicum

(Lee et al., 2002) and yeast (LoGrasso et al., 1993). Therefore,

the domain VI was implicated as a membrane targeting and

anchoring signal for the SqS enzyme.

The 3D model of the predicted TcSqS protein was built

with ESyPred3D Web Server 1.0 (http://www.fundp.ac.be/

sciences/biologie/urbm/bioinfo/esypred) (Lambert et al., 2002),

using the 3D structure of human SqS (PDB accession code

1EZF) as template. The template shared 40.8% identities with

the TcSqS sequence (using the ALIGN program). The

predicted structure of TcSqS is entirely á-helices, with the

axes of all the helices somewhat aligned and arranged in three

layers (Fig. 3). The protein is folded as a single domain, with

a large channel running through the center, surrounded by

helices C, F, G, H, and K. On the basis of reaction

mechanisms, the enzymes involved in isoprenoid biosynthesis

were subdivided into two classes. Class I enzymes generate a

comparatively stable allylic carbocation species by the release

of a pyrophosphate group. Class II enzymes generate the

carbocation species by protonating a C-C double bond or the

corresponding epoxide (Wendt and Schulz, 1998). The TcSqS

core structure is similar to that of the several class I isoprenoid

biosynthetic enzymes whose crystal structures are known and

the conserved feature in all the structures is a α-helical core

surrounding a central active site cavity (Pandit et al., 2000).

The structural homology of TcSqS with other class I enzymes

is consistent with conclusions that most class I isoprenoid

biosynthetic enzymes have developed similar structures through

the divergence of evolution regardless of the homologous

degree at amino acid level (Akamine et al., 2003).

Expression profile of TcSqS in different tissues. To

investigate the TcSqS expression pattern in different tissues of

T. cuspidata, total RNAs extracted from roots, stems, and

leaves were used as the template in semi-quantitative RT-PCR

analysis. With the amplification of twenty-five cycles, the

difference of expression levels among the tissues was too

ambiguous to be defined through agarose/EtBr gel

electrophoresis. After five additional cycles, the electrophoresis

Fig. 1. Nucleotide and predicted amino acid sequences of TcSqS

gene from T. cuspidata. Nucleotides are numbered starting at the

first nucleotide of the PCR Primer TcSqsFull5-2 (shown by

arrows). The ATG start codon and TAA terminate codon are

underlined. An open reading frame of 1227 bp from 60 to 1289

encoding 409 amino acids is shown below the nucleotide

sequence. Genbank Accession DQ836053.


result indicated that TcSqS expressed in a constitutive manner

in the tissues of roots, stems and leaves, with the highest

expression in roots (Fig. 4a).

One possible reason to explain this was that the TcSqS usually

existed as a low expression gene in common cells. Through

additional PCR cycles solved this problem, but the plateau

effect in PCR amplification make this result unconvincing. To

affirm the TcSqS expression pattern in different tissues,

Northern blotting analysis was performed and similar result

was gained (Fig. 4b). The Result indicated that the expression

level of TcSqS was much higher in root than in stem and leaf.

It was close to those of other plants such as P. ginseng (Lee et

al., 2004) and L. japonicus (Akamine et al., 2003).

Southern blot analysis. Previous work reported that one

single form of squalene synthase was present in no-plant

Fig. 2. Multi-alignment by Vector NTI 9.0 of the deduced TcSqS amino acid sequence with those of 8 other plant SqSs as well as the

mammals and fungi. The aligned sequences are from Arabidopsis thaliana SqS1 (BAA06103, Nakashima et al., 1995), Glycine max

(BAA22559, Hata et al., 1997), Lotus japonicus (BAC56854, Akamine et al., 2003), Nicotiana tabacum (AAB08578, Devarenne et al.,

1998), Oryza sativa (BAA22557, Hata et al., 1997), Panax ginseng (BAD08242, Lee et al., 2004), Solanum tuberosum (BAA82093,

Yoshioka, et al., 1999), Zea mays (BAA22558, Hata et al., 1997), Taxus cuspidata, Homo sapiens (AAA60582, Robinson et al., 1993),

Mus musculus (BAA06102, Inoue et al., 1995), Saccharomyces cerevisiae (AAA34597, Jennings et al., 1991). The six highly conserved

peptide domains discernible in the aligned amino acid sequence are underlined.


species, such as human and yeast (Robinson et al., 1993).

Rice (Hata et al., 1997) and green microalga (Okada et al.,

2000) genomes also seemed to contain a single form of SqS

gene. However, cDNA for a second form of squalene synthase

was isolated from several plants, such as from G. glabra

(Hayashi et al., 1999) and A. thaliana (Kribii et al., 1997). To

investigate how many copies of TcSqS presented in T. cuspidata

genome, southern blot analysis was performed by digesting T.

cuspidata genomic DNA with restriction endonucleases

EcoRI, HindIII and XbaI respectively. A 488-bp 5’-end cDNA

Fig. 2. Continued.


fragment amplified with primers TcSqSFull-F1 and TcSqS5-1

was used as probe. The result showed that only one hybridization

band was present in each lane (Fig. 5), indicating that TcSqS is

a single copy gene in the genome of T. cuspidata.

Isolation and characterization of TcSqS promoter region.

Agarose gel electrophoresis analysis showed the nested PCR

using the product of the primary PCR of HpaI library as

template generated a band of DNA sized about 1.2 kb. After

cutting away the genomic walker adapter sequence, the 1132

bp genomic sequence upstream TcSqS (Fig. 6) was assembled

with the 5'-end sequence of TcSqS cDNA acquired in the 5'-

RACE by the software Vector NTI 9.0. A 63 bp overlap

fragment was found and the transcriptional start site (TSS) of

the TcSqS mRNA was confirmed at the site “A” of the

putative initiator sequence “TTCATTCG”.

The upstream region of TcSqS possessed a typically high

A + T content of 65.9%, which was commonly found in other

plant promoters. The sequence was analyzed in the PlantCARE

database (http://bioinformatics.psb.ugent.be/webtools/plantcare/

html/) (Lescot et al., 2002) for cis-acting regulatory elements.

A short fragment “TAATATATAT” which was consisted

completely of A and T was found around the −30 bp position

upstream from the TSS and was predicted to be the most

probable sequence including the TATA Box. Eighteen putative

CAAT sequences were predicted upstream from the start site

of transcription. These sequences might correspond to the

CAAT box, which was sometimes important for the efficiency

of eukaryotic transcription (Kazok, 1987).

Several important cis-acting elements for gene regulation

were predicted within TcSqS promoter region, which include:

1) 1 site of CGTCA-motif and 1 site of TGACG-motif, both

of which were cis-acting regulatory element involved in the

MeJA-responsiveness; 2) 2 sites of TCA-element, a cis-acting

Fig. 3. The 3D model of the predicted TcSqS protein built with

ESyPred3D Web Server, using the human SqS 3D structure

(PDB accession code 1EZF) as template. The main α-helices are

numbered with A-M from the N-terminal to the C-terminal.

Fig. 4. Expression profile of TcSqS in different tissues of T.

cuspidata including root, stem and leaf. (A) Semi-quantitative

RT-PCR analysis (30 cycles). The 18S rRNA is used as internal

control (20 cycles). (B) Northern blotting analysis. Aliquots of

20 µg/sample total RNA isolated from root, stem and leaf tissues

of Taxus cuspidata was hybridized with the biotin-labeled TcSqS

5' end cDNA fragment (upper panel). Electrophoresis bands of

18S and 28S rRNAs were used as internal control (lower panel).

Fig. 5. Southern blot analysis. Total genomic DNA isolated from

fresh leaves of Taxus cuspidata was digested EcoRI, HindIII and

XbaI respectively and hybridized with the biotin-labeled TcSqS

5’-end cDNA fragment.


element involved in salicylic acid responsiveness, with conform

sequence “CCATCTTTTT”; 3) 2 sites of P-box, gibberellin-

responsive element, with conform sequence “CCTTTTG”; 4)

1 site of HSE with conform sequence “AAAAAATTTC”, a

kind of cis-acting element involved in heat stress responsiveness.

Expression of TcSqS under MeJA, SA and GA3 treatments.

To validate the predicted cis-acting elements, the expression

of TcSqS under MeJA, SA and GA3 treatments was analyzed

by semi-quantitative RT-PCR. Total RNA isolated from

calluses of T. cuspidata treated respectively with SA, MeJA

and GA3 for different durations, including 30 min, 1 h, 2 h,

4 h, 8 h and 16 h, was used in analysis. MeJA was identified

earlier as a signal of altered gene expression in various plant

responses to biotic and abiotic stresses as well as of distinct

stages of plant development (Creelman and Mullet, 1997;

Wasternack and Parthier, 1997). Previous work indicated that

the transcript levels of P. ginseng SqS increased remarkably

under the treatment of MeJA (Lee et al., 2004). Subjected to

treatment by 0.1 mM MeJA, the expression of TcSqS in T.

cuspidata was up-regulated markedly compared to the untreated

control (Fig. 7a). However, the up-regulated expression level

was not influenced by the treating duration. The potential

reason to explain this was that the TcSqS gene induced a rapid,

steady and long-lasting respondence to the treatment of

MeJA. Similar results were also observed when T. cuspidata

callus was treated with 0.1 mM SA, an important component

of signal transduction cascades activating plants’ defense

response against pathogen attack (Durner et al., 1997), though

the increasing ranges of transcript levels were not so

remarkable compared to MeJA under the same concentration

(Fig. 7b). Like MeJA, gibberellic acid (GA3) was known as a

signaling molecule inducing defense- or stress-related genes

in plants. Like the results from MeJA and SA treatments,

under GA3 (250 mg/L) treatment, TcSqS transcript levels were

up-regulated compared to the untreated control (Fig. 7c). All

Fig. 6. Genomic nucleotide sequence of the promoter region for the TcSqS gene amplified from the HpaI genomic walking library. The

digested HpaI site is found at the 5’ end of the sequence. Nucleotides are numbered starting at the transcriptional start site “A”. The

putative initiator, TATA-box, and other important cis-acting elements for gene regulation predicted in the PlantCARE are marked out.

Genbank Accession DQ860509.


these results reflect well with the TcSqS promoter analysis results

and indicate that TcSqS is signaling molecules-responsive gene,

which may be involved in the defense responses and regulation of

secondary metabolites in T. cuspidata.

Squalene synthase catalyzes the first enzymatic step

committing carbon away from the backbone pathway of

isoprenoid toward sterols and triterpenes biosynthesis.

Regulation of squalene synthase gene in plants presents as a

key of the regulation of isoprenoid biosynthetic pathways.

The cloning and characterization of TcSqS as well as its

promoter region from T. cuspidata not only constitutes an

important step unraveling the key committed step of the

sterols and triterpenes biosynthetic pathway, but also reveals

an advance in understanding of the regulation of the

isoprenoid pathway genes in this medicinal plant.

Acknowledgments This research was funded by China

National “863” High-Tech Program, China Ministry of

Education and Shanghai Science and Technology Committee.

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