molecules
Article
Strengthening Triterpene Saponins Biosynthesis by
Over-Expression of Farnesyl Pyrophosphate Synthase
Gene and RNA Interference of Cycloartenol Synthase
Gene in Panax notoginseng Cells
Yan Yang 1,2 , Feng Ge 1, *, Ying Sun 1 , Diqiu Liu 1 and Chaoyin Chen 1
1
2
*
Faculty of Life Science and Technology, Kunming University of Science and Technology,
Kunming 650500, China; [email protected] (Y.Y.); [email protected] (Y.S.);
[email protected] (D.L.); [email protected] (C.C.)
School of Biotechnology and Engineering, Dianxi Science and Technology Normal University,
Lincang 677000, China
Correspondence: [email protected]; Tel.: +86-871-6592-0621
Academic Editor: Derek J. McPhee
Received: 21 February 2017; Accepted: 30 March 2017; Published: 5 April 2017
Abstract: To conform to the multiple regulations of triterpene biosynthesis, the gene encoding
farnesyl pyrophosphate synthase (FPS) was transformed into Panax notoginseng (P. notoginseng) cells
in which RNA interference (RNAi) of the cycloartenol synthase (CAS) gene had been accomplished.
Transgenic cell lines showed both higher expression levels of FPS and lower expression levels of CAS
compared to the wild-type (WT) cells. In the triterpene and phytosterol analysis, transgenic cell lines
provided a higher accumulation of total triterpene saponins, and a lower amount of phytosterols in
comparison with the WT cells. Compared with the cells in which RNAi of the CAS gene was achieved,
the cells with simultaneously over-expressed FPS and silenced CAS showed higher triterpene contents.
These results demonstrate that over-expression of FPS can break the rate-limiting reaction catalyzed
by FPS in the triterpene saponins biosynthetic pathway; and inhibition of CAS expression can
decrease the synthesis metabolic flux of the phytosterol branch. Thus, more precursors flow in the
direction of triterpene synthesis, and ultimately promote the accumulation of P. notoginseng saponins.
Meanwhile, silencing and over-expressing key enzyme genes simultaneously is more effective than
just manipulating one gene in the regulation of saponin biosynthesis.
Keywords: farnesyl pyrophosphate synthase; cycloartenol synthase; Panax; over-expression;
RNA interference; triterpene
1. Introduction
Panax notoginseng is a well-known Chinese medicine herb. Triterpene saponins with a wide
range of structural diversity are the major bioactive components in P. notoginseng. To date, over 70
different kinds of triterpene saponins have been isolated and characterized from different parts of
P. notoginseng [1]. It has been proven that triterpene saponins have applications in anti-cancer [2],
anti-atherosclerotic [3], anti-oxidant [4], anti-diabetic [5], anti-hypolipidemic [6] and some other
pharmacological activities [7,8]. However, difficulties including a narrow habitat (mainly in Wenshan,
China), long maturation period (>3 years) and crop rotation lead to a comparatively low production of
P. notoginseng, and have restrained its pharmacological application. In addition, since the chemical
structures of triterpene saponins are complicated, the chemical synthesis may give rise to high-cost.
Therefore, production of triterpene saponins by metabolic engineering may be an appealing strategy
to manipulate in the future.
Molecules 2017, 22, 581; doi:10.3390/molecules22040581
www.mdpi.com/journal/molecules
Molecules 2017, 22, 581
2 of 12
The
biosynthetic
Molecules
2017, 22, 581 pathway of triterpene saponins in P. notoginseng is shown in Figure
2 of 12 1 [9].
FPS catalyzes the conversion of isoprenyl diophosphate (IPP) and dimethylallyl diphosphate (DMAPP)
The biosynthetic
pathway
of triterpene
saponins
in P.
notoginseng
is shownin
in the
Figure
1 [9]. FPS of
into farnesyl
pyrophosphate
(FPP)
which acts
as the
common
substrate
biosynthesis
catalyzes
the
conversion
of
isoprenyl
diophosphate
(IPP)
and
dimethylallyl
diphosphate
(DMAPP)
sesquiterpenoids, phytosterols and triterpene saponins. The conversion is a rate-determining reaction,
into farnesyl pyrophosphate (FPP) which acts as the common substrate in the biosynthesis of
therefore,
this reaction catalyzed by FPS has been considered as the first pivotal step toward triterpene
sesquiterpenoids, phytosterols and triterpene saponins. The conversion is a rate-determining reaction,
saponins and phytosterols biosynthesis [1]. Recently, the FPS gene has been cloned and characterized
therefore, this reaction catalyzed by FPS has been considered as the first pivotal step toward triterpene
in some
species [10,11], and research has suggested that the expression of FPS exhibits a positive
saponins and phytosterols biosynthesis [1]. Recently, the FPS gene has been cloned and characterized
correlation
triterpene
biosynthesis,
and the
saponins
can be
in somewith
species
[10,11], saponin
and research
has suggested
that accumulation
the expression of
of triterpene
FPS exhibits
a positive
up-regulated
by
the
over-expression
of
FPS
[12,13].
CAS
in
the
pathway
catalyzes
the
conversion
correlation with triterpene saponin biosynthesis, and the accumulation of triterpene saponins can be of
2,3-oxidosqualene
tothe
cycloartenol
which
ultimately
to synthesize
phytosterols.
Although
up-regulated by
over-expression
ofwill
FPS be
[12,13].
CAS inused
the pathway
catalyzes
the conversion
of
2,3-oxidosqualene
to cycloartenol
which will
ultimately
used to synthesize
phytosterols.
Although
CAS does
not participate
in the biosynthesis
of be
triterpene
saponins,
it competes
with dammarenedion-II
CAS does
the biosynthesis
of triterpene saponins,
it competes
with dammarenedion-II
synthase
(DS)not
forparticipate
the sameinprecursor
(2,3-oxidosqualene).
The
2,3-oxidosqualene
is the common
synthase
(DS) for thesaponins
same precursor
(2,3-oxidosqualene).
The 2,3-oxidosqualene
the common
precursor
of triterpene
and phytosterols.
The catalysis
of CAS mayisdirectly
result in
precursor of triterpene saponins and phytosterols. The catalysis of CAS may directly result in the
the decrease of 2,3-oxidosqualene and indirectly reduce the metabolic flux of triterpene saponin
decrease of 2,3-oxidosqualene and indirectly reduce the metabolic flux of triterpene saponin biosynthesis
biosynthesis [14]. Thus, CAS should be considered as a key flux-limiting enzyme in the biosynthesis of
[14]. Thus, CAS should be considered as a key flux-limiting enzyme in the biosynthesis of triterpene
triterpene
saponins. In this study, the over-expression vector of FPS was constructed and integrated into
saponins. In this study, the over-expression vector of FPS was constructed and integrated into the
the genome
cells in
in which
whichRNAi
RNAiofofCAS
CAS
has
been
accomplished;
manipulation
genomeof
of P.
P.notoginseng
notoginseng cells
has
been
accomplished;
suchsuch
manipulation
was used
to
confirm
whether
the
strategy
of
multiple
regulations
was
an
effective
way
to strengthen
was used to confirm whether the strategy of multiple regulations was an effective way to strengthen
the biosynthesis
of triterpene
saponins.
the biosynthesis
of triterpene
saponins.
Figure
1. Biosynthetic
pathwayofoftriterpene
triterpene saponins
AATC:
acetoacetyl-CoA
Figure
1. Biosynthetic
pathway
saponinsininP.P.notoginseng.
notoginseng.
AATC:
acetoacetyl-CoA
acyltransferase;
DXPS:
1-deoxyD-xyulose-5-phosphate synthase); HMGS: 3-Hydroxy-3-methylglutarylacyltransferase; DXPS: 1-deoxy-D-xyulose-5-phosphate synthase); HMGS: 3-Hydroxy-3-methylglutarylCoA synthase; DXR: 1-deoxy-D-xyulose-5-phosphate reductoisomerase; HMGR: 3-hydroxy-3CoA synthase; DXR: 1-deoxy-D-xyulose-5-phosphate reductoisomerase; HMGR: 3-hydroxy-3methylglutaryl-CoA reductase; CMS: 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase; GPPS:
methylglutaryl-CoA reductase; CMS: 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase; GPPS:
geranyl pyrophosphate synthase; FPS: farnesyl pyrophosphate synthase; SS: squalene synthase; SE:
geranyl pyrophosphate synthase; FPS: farnesyl pyrophosphate synthase; SS: squalene synthase;
squalene epoxidase; DS: dammarenediol-II synthase; P450: P450-monooxygenase; CAS: cycloartenol
SE: squalene
dammarenediol-II
synthase;
P450: P450-monooxygenase; CAS: cycloartenol
synthase.epoxidase;
The dottedDS:
lines
indicate several enzyme
reactions.
synthase. The dotted lines indicate several enzyme reactions.
2. Results and Discussion
2. Results and Discussion
2.1. RNAi and Over-Expression Vector Construction
2.1. RNAiThere
and Over-Expression
Vector Construction
are several methods
to induce gene silencing in plants, such as anti-sense and
co-suppression,
but these
constructs
a modest proportion
of silenced
There
are several
methods
to usually
induceresult
genein silencing
in plants,
such asindividuals.
anti-senseIt and
has
been
reported
that
efficient
gene
silencing
can
be
achieved
by
using
hpRNA
construct
which
co-suppression, but these constructs usually result in a modest proportion of silenced individuals.
contains a sense arm, anti-sense arm and an intron [15,16]. Based on these rules, the pHellsgate2
It has been reported that efficient gene silencing can be achieved by using hpRNA construct which
vector was designed to accept the CAS RNAi fragment (with attB1 and attB2 sites) via homologous
contains
a sense arm, anti-sense arm and an intron [15,16]. Based on these rules, the pHellsgate2
recombination to form two arms of the hairpin and generate an ihpRNA construct.
vector was designed to accept the CAS RNAi fragment (with attB1 and attB2 sites) via homologous
recombination to form two arms of the hairpin and generate an ihpRNA construct.
Molecules 2017, 22, 581
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Several members of a gene family can be simultaneously silenced by targeting the highly
conserved sequence
domainofofinaagene
the
In order to silenced
guarantee
the
specificity
of CAS
Several
family
can
the
Severalmembers
members
geneRNAi
familyprocess.
canbe
besimultaneously
simultaneously
silencedby
bytargeting
targeting
thehighly
highly
silencing,
the RNAi
fragment
was
selected
from
the
non-conservative
of of
the
CAS
sequence,
conserved
sequence
domain
ininthe
InInorder
specificity
silencing,
conserved
sequence
domain
theRNAi
RNAiprocess.
process.
ordertotoguarantee
guaranteethe
theregion
specificity
ofCAS
CAS
silencing,
the
fragment
was
from
non-conservative
region
ofof the
CAS
and
the RNAi
RNAi
fragment
was selected
selected
from
the
non-conservative
region
thevector
CAS sequence,
sequence,
and
and recombined
into
pHellsgate2
(Figure
2) tothe
construct
the RNAi
expression
pHellsgate-CAS.
recombined
pHellsgate2
(Figure
construct
the
vector
The
recombinedinto
into
pHellsgate2
(Figure
construct
theRNAi
RNAi
expression
vectorpHellsgate-CAS.
pHellsgate-CAS.
The
The pHellsgate-CAS
was
transferred
into2)2)
E.toto
coli
(Escherichia
coli)expression
DH5α. Positive
clones were screened.
pHellsgate-CAS
was
into
(Escherichia
coli)
clones
screened.
pHellsgate-CAS
wastransferred
transferred
intoE.E.coli
coli
(Escherichia
coli)DH5α.
DH5α.Positive
Positive
cloneswere
were
screened.
The plasmid
(pHellsgate-CAS)
was isolated
from
the positive
clone,
and
digested
by
XbaI
and XhoI
The
Theplasmid
plasmid(pHellsgate-CAS)
(pHellsgate-CAS)was
wasisolated
isolatedfrom
fromthe
thepositive
positiveclone,
clone,and
anddigested
digestedby
byXbaI
XbaIand
andXhoI
XhoI
separately. Two fragments, 1.8 kb and 0.66 kb (the fragment of 0.66 kb contained 438 bp CAS RNAi
separately.
separately.Two
Twofragments,
fragments,1.8
1.8kb
kband
and0.66
0.66kb
kb(the
(thefragment
fragmentofof0.66
0.66kb
kbcontained
contained438
438bp
bpCAS
CASRNAi
RNAi
fragments
and there
222
bp
fragments
between
the
enzyme
digestion
sites
and
recombination
fragments
and
there
222
bp
between
the
enzyme
and
recombination
fragments
andwere
therewere
were
222
bpfragments
fragments
between
the
enzymedigestion
digestionsites
sites
and
recombination
sites), were
detected
by
agarose
gel,
and
the
size
of
the
fragments
digested
by
XbaI
and
XhoI
sites),
was
the
sites),were
weredetected
detectedby
byagarose
agarosegel,
gel,and
andthe
thesize
sizeofofthe
thefragments
fragmentsdigested
digestedby
byXbaI
XbaIand
andXhoI
XhoI
was
thewas
the same.
TheThe
RNAi
expression
vector
was
constructed
correctly
(Figure
same.
expression
vector
(Figure
3).
same.
TheRNAi
RNAi
expression
vectorwas
wasconstructed
constructedcorrectly
correctly
(Figure
3). 3).
The
sequence
ofofFPS
(1033
bp)
was
amplified
cDNA
ofofP.
Theencoding
encoding
region
sequence
FPS
(1033
bp)
wasamplified
amplifiedfrom
fromthe
the
cDNA
P.notoginseng
notoginseng
The encoding
regionregion
sequence
of FPS
(1033
bp)
was
from
the
cDNA
of
P. notoginseng
and
successfully
inserted
into
the
pCAMBIA1300S
vector.
and
successfully
inserted
into
the
pCAMBIA1300S
vector.
and successfully inserted into the pCAMBIA1300S vector.
Figure 2. Schematic diagram of the pHellsgate-CAS RNAi vector. LB: left border; P nos: NOS promoter;
Figure
2.2.Schematic
diagram
the
RNAi
border;
PPnos:
NOS
promoter;
Figure
Schematic
diagram
thepHellsgate-CAS
pHellsgate-CAS
RNAivector.
vector.LB:
LB:left
left
border;
nos:into
NOSthe
promoter;
T nos: NOS
terminator;
RB:
rightofof
border.
The CAS RNAi
fragment
was
inserted
XbaI sites
TTnos:
nos:NOS
NOSterminator;
terminator;RB:
RB:right
rightborder.
border.The
TheCAS
CASRNAi
RNAifragment
fragmentwas
wasinserted
insertedinto
intothe
theXbaI
XbaIsites
sites
and the XhoI sites of the pHellsgate2 by means of inverted repeats. After this step of the homologous
and
andthe
theXhoI
XhoIsites
sitesofofthe
thepHellsgate2
pHellsgate2by
bymeans
meansofofinverted
invertedrepeats.
repeats.After
Afterthis
thisstep
stepofofthe
thehomologous
homologous
recombination
reaction,
the hairpin
structure
(contains
ananantisense
target
sequence,
the
PDK
intron
recombination
antisense
recombinationreaction,
reaction,the
thehairpin
hairpinstructure
structure(contains
(containsan
antisensetarget
targetsequence,
sequence,the
thePDK
PDKintron
intron
and a sense
target
sequence)
was
inserted
between
the
35S
promoter
(P
35S)
and
OCS
terminator
(T ocs)
and
andaasense
sensetarget
targetsequence)
sequence)was
wasinserted
insertedbetween
betweenthe
the35S
35Spromoter
promoter(P(P35S)
35S)and
andOCS
OCSterminator
terminator(T(Tocs)
ocs)
of the pHellsgate2
vector,
and
fulfilled
the
function
of
gene
silencing
[15,16].
ofofthe
thepHellsgate2
pHellsgate2vector,
vector,and
andfulfilled
fulfilledthe
thefunction
functionofofgene
genesilencing
silencing[15,16].
[15,16].
Figure
Figure3.3.The
Theenzymatic
enzymaticdetection
detectionofofpHellsgate-CAS
pHellsgate-CASby
byXbaI
XbaIand
andXhoI.
XhoI.1:1:XbaI
XbaIdigestion
digestionproducts;
products;2:2:
Figure 3. The enzymatic detection of pHellsgate-CAS by XbaI and XhoI. 1: XbaI digestion products;
XhoI
XhoIdigestion
digestionproducts.
products.
2: XhoI digestion products.
2.2.
2.2.Genetic
GeneticTransformation
TransformationofofP.P.notoginseng
notoginseng
2.2. Genetic Transformation
of P. notoginseng
The
Thecells
cellscultured
culturedininvitro
vitrocan
canbe
beaauseful
usefulsystem
systemfor
forgenetic
geneticstudy
studyand
andhave
havebeen
beenproven
proventoto
possess
many
advantages,
including
high
growth
rate,
genetic
and
biochemical
stabilities
and
possess
many
advantages,
including
high
growth
rate,
genetic
and
biochemical
stabilities
and
the
The cells cultured in vitro can be a useful system for genetic study and have been proven
to the
possess
totipotency
totipotencyofofsecondary
secondarymetabolism
metabolism[17].
[17].Previous
Previousresearch
researchhas
hasconfirmed
confirmedthat
thatcell
cellcultures
culturesofof
many advantages, including high growth rate, genetic and biochemical stabilities and the totipotency
P.P.notoginseng
notoginsengcould
couldincrease
increasethe
theproduction
productionofoftriterpene
triterpenesaponins
saponins[18,19].
[18,19].InInour
ourstudy,
study,on
onthe
thebase
baseofof
of secondary metabolism [17]. Previous research has confirmed that cell cultures of P. notoginseng
callus
calluscells,
cells,pHellsgate-CAS
pHellsgate-CASwas
wastransformed
transformedinto
intothe
theWT
WTcells
cellsand
andpCAMBIA1300S-FPS
pCAMBIA1300S-FPSwas
wasthen
then
could increase
theinto
production
of triterpene
saponins
[18,19].
In rounds
our
study,
on the (each
base
of
callus cells,
introduced
transformed
cells.
After
ofofscreening
introduced
intothe
thepHellsgate-CAS
pHellsgate-CAS
transformed
cells.
After5~7
5~7
rounds
screening
(eachsubculture
subculture
pHellsgate-CAS was transformed into the WT cells and pCAMBIA1300S-FPS was then introduced
into the pHellsgate-CAS transformed cells. After 5~7 rounds of screening (each subculture period
lasted about 4 weeks), the transgenic cell lines possessing kanamycin and hygromycin-resistance were
obtained, and no morphological difference between transgenic and WT cells was observed.
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2017, 22,
58122, 581
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4 of 12
period lasted about 4 weeks), the transgenic cell lines possessing kanamycin and hygromycin-resistance
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2.3. PCR
Analysis
Phosphotransferase
(nptII) transgenic
Gene and Hygromycin
Phosphotransferase
were
obtained,
and
no morphological
differenceIIbetween
and WT cells was
observed.4 of 12 II
(hptII) Gene
period lasted about 4 weeks), the transgenic cell lines possessing kanamycin and hygromycin-resistance
2.3. PCR Analysis of Neomycin Phosphotransferase II (nptII) Gene and Hygromycin Phosphotransferase II
were
obtained,
no morphological
difference
between
transgenic
WTT-DNA,
cells wasthe
observed.
Since
the
438 bpand
fragment
of CAS and
the nptII
possessed
theand
same
integration of
(hptII) Gene
the exogenous 438 bp fragment of CAS in the genome of P. notoginseng cells could be confirmed by
the 438 bp
fragmentPhosphotransferase
of CAS and the nptII
possessed
the Hygromycin
same T-DNA,
the integration IIof
2.3.Since
PCR Analysis
of Neomycin
II (nptII)
Gene and
Phosphotransferase
PCR analysis
of nptII. Similarly,
the FPS and the hptII
possessed
the same T-DNA,
so the integration
the
exogenous
(hptII)
Gene 438 bp fragment of CAS in the genome of P. notoginseng cells could be confirmed by
of exogenous
FPS in the genome could be confirmed by PCR analysis of hptII. The genomic DNA of
PCR analysis of nptII. Similarly, the FPS and the hptII possessed the same T-DNA, so the integration
Since
the 438
bpisolated
fragmentand
of CAS
and
possessed
same
T-DNA, hptII
the integration
of
transgenic
cell
lines
was
used
asthe
thenptII
template
for the
both
PCR.
Both
of exogenous
FPS
in the
genome could
be confirmed
by PCR analysis
ofnptII
hptII. and
The genomic
DNA
of nptII
the exogenous 438 bp fragment of CAS in the genome of P. notoginseng cells could be confirmed by
and hptII
fragments
werewas
detected
all used
transgenic
cell lines,
no signal
wasPCR.
observed
in the WT
transgenic
cell lines
isolatedinand
as the template
forwhile
both nptII
and hptII
Both nptII
PCR analysis of nptII. Similarly, the FPS and the hptII possessed the same T-DNA, so the integration
and hptII
fragments
detected
in all that
transgenic
cell lines,
no signal
was
observed
in the WT
cells (Figure
4A,B).
Thiswere
result
indicated
the CAS
RNAiwhile
fragment
and
FPS
were simultaneously
of exogenous FPS in the genome could be confirmed by PCR analysis of hptII. The genomic DNA of
cells (Figure
4A,B). This result
indicated
that
CAS RNAi
FPS
were
simultaneously
transformed
into cell
transgenic
lines.
With
thethe
offragment
the
two and
lines
with
poor
growth,
the other
transgenic
lines wascell
isolated
and
used
asexception
the template
for both
nptII and
hptII
PCR.
Both nptII
transformed into transgenic cell lines. With the exception of the two lines with poor growth, the
five PCR-positive
cell lines
(named
T-1,
T-2,
T-3, T-4 cell
andlines,
T-5)while
wereno
used
forwas
further
study.
and hptII fragments
were
detected
in all
transgenic
signal
observed
in the WT
other five PCR-positive cell lines (named T-1, T-2, T-3, T-4 and T-5) were used for further study.
cells (Figure 4A,B). This result indicated that the CAS RNAi fragment and FPS were simultaneously
transformed into transgenic cell lines. With the exception of the two lines with poor growth, the
other five PCR-positive cell lines (named T-1, T-2, T-3, T-4 and T-5) were used for further study.
Figure 4. PCR detections of nptII and hptII in transgenic cell lines. (A) PCR detection of nptII; (B) PCR
Figure 4. PCR detections of nptII®and hptII in transgenic cell lines. (A) PCR detection of nptII; (B) PCR
detection of hptII; M: Trans2K
DNA Marker; 1~7: Seven transgenic cell lines; 8: Positive control; 9:
detection
of hptII; M: Trans2K® DNA Marker; 1~7: Seven transgenic cell lines; 8: Positive control;
wild-type (WT).
Figure(WT).
4. PCR detections of nptII and hptII in transgenic cell lines. (A) PCR detection of nptII; (B) PCR
9: wild-type
detection of hptII; M: Trans2K® DNAAnalysis
Marker; 1~7: Seven transgenic cell lines; 8: Positive control; 9:
2.4. Reverse-Transcriptase-PCR(RT-PCR)
wild-type (WT).
2.4. Reverse-Transcriptase-PCR(RT-PCR)
Analysis
As the expressions of genes related to the biosynthesis of triterpene saponins in different growth
periods
were different,
expressions
FPS
CAS in different
growth periods
werein
examined
bygrowth
2.4.
As
theReverse-Transcriptase-PCR(RT-PCR)
expressions
of the
genes
related of
toAnalysis
the and
biosynthesis
of triterpene
saponins
different
RT-PCR. The results showed that the expression levels of FPS and CAS on the 35th day were all higher
periods were
the expressions
of to
FPS
CAS in of
different
growth
periods
were
examined
Asdifferent,
the expressions
of genes related
theand
biosynthesis
triterpene
saponins
in different
growth
than those in other growth periods (Figure 5). According to the result, the sampling time of the
periods
were
different,
the expressions
of
FPS and CAS
in different
growth
periods
were35th
examined
by
by RT-PCR.
The
results
showed
that
the
expression
levels
of
FPS
and
CAS
on
the
day
were
all
follow-up experiments was unified.
RT-PCR.
The
results
showed
that
the
expression
levels
of
FPS
and
CAS
on
the
35th
day
were
all
higher
higher than those in other growth periods (Figure 5). According to the result, the sampling time of the
than those in other growth periods (Figure 5). According to the result, the sampling time of the
follow-up
experiments was unified.
follow-up experiments was unified.
Figure 5. The expressions of FPS and CAS at different growth periods in P. notoginseng cells.
Among the five nptII and hptII PCR-positive transgenic cell lines, T-3, T-4, and T-5 showed both
5.expressions
The expressions
of FPS
and
CASatatdifferent
different growth
P. notoginseng
Figure
5.and
The
of FPS
and
CAS
growth
periods
in
P.(Figure
notoginseng
cells.
higher
FPSFigure
lower
CAS transcription
levels
in comparison
withperiods
the
WTincells
6).cells.
The
result
demonstrated that the over-expression of FPS and CAS silencing were simultaneously accomplished
Among the five nptII and hptII PCR-positive transgenic cell lines, T-3, T-4, and T-5 showed both
in T-3, the
T-4, five
and nptII
T-5. Although
expression levels
of FPScell
in T-1
andT-3,
T-2T-4,
were
increased,
the both
Among
and hptIIthe
PCR-positive
transgenic
lines,
and
T-5 showed
higher FPS and lower CAS transcription levels in comparison with the WT cells (Figure 6). The result
expression
levels
of
CAS
did
not
show
significant
reduction.
Possible
reasons
for
this
result
may
be
(1) result
higher FPS
and lower
CAS
comparison
with
thesimultaneously
WT cells (Figure
6). The
demonstrated
that
the transcription
over-expressionlevels
of FPSinand
CAS silencing
were
accomplished
the insertion site of exogenous FPS into the genome happens to be a certain point in the CAS RNAi
demonstrated
FPS and CAS
were
accomplished
in
in T-3, that
T-4, the
and over-expression
T-5. Although theofexpression
levelssilencing
of FPS in
T-1 simultaneously
and T-2 were increased,
the
fragment, and the insertion of exogenous FPS blocks the expression of the CAS RNAi fragment,
of CAS
didexpression
not show significant
Possible
reasons
this result may
be (1)
T-3, T-4,expression
and T-5. levels
Although
the
levels ofreduction.
FPS in T-1
and T-2
wereforincreased,
the expression
resulting in the ineffective inhibition of CAS expression; (2) during the screening process of transgenic
the insertion
site
of exogenous
FPSreduction.
into the genome
happens
to be
certain
point
in the
CASthe
RNAi
levels of
did not
show
significant
Possible
reasons
forathis
result
may
be (1)
insertion
P. CAS
notoginseng
cells,
genetic
mutation
of the RNAi
fragment
occurs
in some
cells,
leading
to the
fragment,
and
the
insertion
of
exogenous
FPS
blocks
the
expression
of
the
CAS
RNAi
fragment,
site of invalid
exogenous
FPS
into
the
genome
happens
to
be
a
certain
point
in
the
CAS
RNAi
fragment,
expression of the RNAi fragment; (3) after several generations of continuous screening, theand the
resulting in the ineffective inhibition of CAS expression; (2) during the screening process of transgenic
insertion
exogenous
FPS
expression
of thecell
CAS
RNAi fragment, resulting in the ineffective
CASofRNAi
fragment
fellblocks
off the the
genome
of transgenic
lines.
P. notoginseng cells, genetic mutation of the RNAi fragment occurs in some cells, leading to the
inhibition
of
CAS
expression;
(2)
during
the
screening
process
of transgenic
P. notoginseng
cells,the
genetic
invalid expression of the RNAi fragment; (3) after several generations
of continuous
screening,
mutation
of
the
RNAi
fragment
occurs
in
some
cells,
leading
to
the
invalid
expression
of
the
RNAi
CAS RNAi fragment fell off the genome of transgenic cell lines.
fragment; (3) after several generations of continuous screening, the CAS RNAi fragment fell off the
genome of transgenic cell lines.
Besides, compared with the cell line (T-Ri) which was only transformed by the CAS RNAi
fragment, the transcription levels of FPS in T-3, T-4 and T-5 were all dramatically increased, and the
Molecules 2017, 22, 581
5 of 12
Molecules
2017,
Molecules
2017, 22,
58122, 581
Besides,
compared
5 of 512of 12
with the cell line (T-Ri) which was only transformed by the CAS
RNAi
fragment, the
transcription
of FPS
T-3,
T-4 and
T-5
were
dramatically
and the
Besides,
comparedlevels
with the
cell in
line
(T-Ri)
which
was
onlyalltransformed
by increased,
the CAS RNAi
expression
of
CAS
showed
a
downward
trend.
This
indicated
that
the
expression
of
these
two
genes
fragment,
the showed
transcription
levels of FPS
in T-3,
T-4indicated
and T-5 were
dramatically
and the
expression
of CAS
a downward
trend.
This
thatall
the
expressionincreased,
of these two
genes
was
not
mutually
exclusive
in
T-3,
T-4
and
T-5,
and
the
three
transgenic
cell
lines
were
then
selected
expression
CAS showed
a downward
trend.
This
indicated
that the expression
thesethen
two genes
was not
mutuallyofexclusive
in T-3,
T-4 and T-5,
and
the
three transgenic
cell linesofwere
selected
for
further
qPCR
analysis.
was
not
mutually
exclusive
in
T-3,
T-4
and
T-5,
and
the
three
transgenic
cell
lines
were
then
selected
for further qPCR analysis.
for further qPCR analysis.
FigureFigure
6. The
The6.RT-PCR
RT-PCR
results
of FPS
FPS
andand
CAS
inin
P.P.
notoginseng
cells.
T-Ri:cells
cellstransformed
transformed
by
the
The RT-PCR
results
of FPS
CAS
notoginsengcells.
cells. T-Ri:
by by
the the
Figure
6.
results
of
and
CAS
in
P.
notoginseng
T-Ri:
cells
transformed
CAS
RNAi
fragment
alone;
T-1,
T-2,
T-3,
T-4
and
T-5:
cells
transformed
by
the
CAS
RNAi
and
FPS
CAS
RNAi
fragment
alone;
T-1,
T-2,
T-3,
T-4
and
T-5:
cells
transformed
by
the
CAS
RNAi
and
FPS
CAS RNAi fragment alone; T-1, T-2, T-3, T-4 and T-5: cells transformed by the CAS RNAi and FPS
over-expression
fragment
simultaneously.
over-expression
fragment
simultaneously.
over-expression
fragment
simultaneously.
2.5. qPCR Analysis
2.5. qPCR
qPCR Analysis
Analysis
2.5.
To accurately verify the effects of FPS over-expression and CAS suppression, qPCR was carried
To accurately
accurately verify the
the effects of
of FPS
FPS over-expression
over-expression and
and CAS
CAS suppression,
suppression, qPCR
qPCR was
was carried
carried
To
out within theverify
WT cells, effects
T-Ri, T-3, T-4
and T-5 after cells had
been cultured
for 35 days. The
results
out within
within
thedecreased
WT cells,
cells,accumulation
T-Ri, T-3,
T-3, T-4
T-4
and T-5
T-5after
aftercells
cells
had
been
cultured
for 35
35days.
days.
The
results
showed
of and
CAS
transcript
in
T-Ri,
T-3,
T-4cultured
and T-5 compared
withThe
the results
WT
out
the
WT
T-Ri,
had
been
for
showed
decreased
accumulation
of
CAS
transcript
in
T-Ri,
T-3,
T-4
and
T-5
compared
with
the
WT
cells
(0.58, 0.65,accumulation
0.33 and 0.39 folds
of the
WT cells, in
respectively).
confirmed
that thewith
designed
showed
decreased
of CAS
transcript
T-Ri, T-3, It
T-4
and T-5again
compared
the WT
cells (0.58,
(0.58,
0.65,
0.33
and 0.39
0.39
folds
of the
the
WT
cells,
respectively).
Itthe
confirmed
again
that(Figure
the designed
designed
CAS 0.65,
RNAi0.33
fragment
wasfolds
efficient
andWT
could
successfully
inhibitIt
expression
of CAS
7A).
cells
and
of
cells,
respectively).
confirmed
again
that
the
Meanwhile,
the
transcription
levels
of
FPS
in
T-3,
T-4
and
T-5
all
manifested
an
increasing
trend
CAS
RNAi
fragment
was
efficient
and
could
successfully
inhibit
the
expression
of
CAS
(Figure
7A).
CAS RNAi fragment was efficient and could successfully inhibit the expression of CAS (Figure 7A).
compared
with
the WT cellslevels
(4.22, 3.34
and 3.51
foldsT-4
of the
WTT-5
cells,all
respectively),
which
demonstrated
Meanwhile,
the
transcription
of
FPS
in
T-3,
and
manifested
an
increasing
trend
Meanwhile, the transcription levels of FPS in T-3, T-4 and T-5 all manifested an increasing trend
that with
over-expression
of FPS
T-3, and
T-4 and
had
realized
7B). Besides,
in demonstrated
contrast to
compared
the WT
WT cells
cells
(4.22,in3.34
3.34
3.51T-5
folds
ofbeen
theWT
WT
cells,(Figure
respectively),
which
compared
with the
(4.22,
and 3.51
folds
of
the
cells,
respectively),
which
demonstrated
T-Ri
in
which
only
a
single
transgene
was
transformed,
the
decrease
of
CAS
expression
level
in contrast
T-4 and to
that over-expression
over-expression of
of FPS
FPS in
in T-3,
T-3, T-4
T-4 and
and T-5
T-5 had
had been
been realized
realized (Figure
(Figure 7B).
7B). Besides,
Besides, in
in
that
contrast
T-5 was more significant.
This suggested
a possible
interaction
occurring between
two transgenes,
and to
T-Ri in
in which
only
aasingle
transgene
was
transformed,
thethe
decrease
of CAS
expression
levellevel
in T-4
T-Ri
which
only
single
transgene
was
transformed,
decrease
of
CAS
expression
in and
T-4
the same phenomenon had been observed in the co-overexpression of SS and DS in P. notoginseng
T-5
was
more
significant.
This
suggested
a
possible
interaction
occurring
between
two
transgenes,
and
and T-5cells
was[20].
more
This suggested
possible interaction
occurring between
two transgenes,
Thesignificant.
detailed mechanism
of such ainteraction
should be investigated
in the future.
the same
phenomenon
hadhad
been
observed
in in
thethe
co-overexpression
notoginseng
and
the same
phenomenon
been
observed
co-overexpressionofofSS
SSand
andDS
DSin
in P.
P. notoginseng
cells [20].
[20]. The
The detailed
detailed mechanism
mechanism of
of such
such interaction
interaction should
should be
be investigated
investigated in
in the
the future.
future.
cells
Figure 7. The relative transcription levels of FPS and CAS in P. notoginseng cells. (A) The relative
transcription levels of FPS; (B) The relative transcription levels of CAS; The internal reference is
GAPDH. Vertical bars indicate mean values ± standard deviations (SDs) from three independent
experiments (n = 3). The symbol * represents that there is a significant difference between WT
Figure
7.
transcription
levels of
FPS and CAS in P. notoginseng cells. (A) The relative
Figure(control)
7. The
The relative
relative
transcription
and
transgenic
cells at p <levels
0.05. of FPS and CAS in P. notoginseng cells. (A) The relative
transcription
transcription levels
levels of
of FPS;
FPS; (B)
(B) The
The relative
relative transcription
transcription levels
levels of
of CAS;
CAS; The
The internal
internal reference
reference is
is
GAPDH.
GAPDH. Vertical
Vertical bars
bars indicate
indicate mean
mean values
values ±± standard
standard deviations
deviations (SDs)
(SDs) from
from three
three independent
independent
experiments
symbol
* represents
that there
is a significant
difference
betweenbetween
WT (control)
The
symbol
* represents
that there
is a significant
difference
WT
experiments (n(n==3).3).The
and
transgenic
cells
at
p
<
0.05.
(control) and transgenic cells at p < 0.05.
Molecules 2017, 22, 581
Molecules 2017, 22, 581
6 of 12
6 of 12
Metabolic
Analysis
2.6.2.6.
Metabolic
Analysis
investigate
whether
over-expression
FPS
silencing
of CAS
could
influence
triterpene
To To
investigate
whether
over-expression
of of
FPS
andand
silencing
of CAS
could
influence
triterpene
phytosterol
biosynthesis,
metabolites
from
WT,
T-3,T-5
T-4
and
T-5analyzed.
cells were
andand
phytosterol
biosynthesis,
thesethese
metabolites
from the
WT,the
T-Ri,
T-3,T-Ri,
T-4 and
cells
were
The data
that allcell
transgenic
cell lines
(T-Ri,
T-4
and T-5) significantly
produced significantly
Theanalyzed.
data showed
thatshowed
all transgenic
lines (T-Ri,
T-3, T-4
andT-3,
T-5)
produced
higher
higher of
amounts
of total triterpene
in comparison
withcells
the WT
(1.46,
2.46,
2, 2.08
folds
amounts
total triterpene
saponins saponins
in comparison
with the WT
(1.46,cells
2.46,
2, 2.08
folds
of the
the WT
cells, respectively)
(Figure
8A). It the
confirmed
the that
hypothesis
FPS in P.was
notoginseng
WTofcells,
respectively)
(Figure 8A).
It confirmed
hypothesis
the FPSthat
in P.the
notoginseng
a key
was a key
regulatory
in the
triterpene
saponinspathway
biosynthetic
pathway
and its over-expression
regulatory
enzyme
in theenzyme
triterpene
saponins
biosynthetic
and its
over-expression
could raise
the fluxand
of the
pathwayupregulated
and accordingly
upregulated
accumulation
triterpene
thecould
flux ofraise
the pathway
accordingly
the accumulation
of the
triterpene
saponins.ofWith
the
saponins.
With
the exception
increased
contents of
saponins,
contents
were
exception
of the
increased
contentsofofthe
saponins,
phytosterols
contents
werephytosterols
reduced (0.77,
0.88, 0.58
(0.77,
0.88,WT
0.58cells,
and respectively)
0.61 folds of the
WT cells,
respectively)
(Figure 8B).
confirmed
andreduced
0.61 folds
of the
(Figure
8B). The
result confirmed
that The
CASresult
played
a key
that
a key role notofonly
in the biosynthesis
phytosterols
but of
also
in the accumulation
role
notCAS
onlyplayed
in the biosynthesis
phytosterols
but also inofthe
accumulation
triterpene
saponins.
triterpene saponins.
The biosynthesis
of triterpene
could
be raised
when the formation
Theofbiosynthesis
of triterpene
saponins could
be raisedsaponins
when the
formation
of phytosterols
was
of phytosterols
was blocked.
This
attractive
conclusion
was ginseng
also confirmed
in Panax
ginseng hairy
blocked.
This attractive
conclusion
was
also confirmed
in Panax
hairy roots
[14]. Meanwhile,
rootsanalysis
[14]. Meanwhile,
HPLC
that the
contents of(Rb1,
five major
ginsenosides
(Rb1,
HPLC
revealed that
theanalysis
contentsrevealed
of five major
ginsenosides
Rg1, Rh1,
Re and Rd)
Rh1, Re
and
Rd)were
in transgenic
cell lines
were allwith
increased
in thethe
WTfive
cells.
in Rg1,
transgenic
cell
lines
all increased
compared
those compared
in the WTwith
cells.those
Among
Among theRe
five
ginsenosides,
Re showed
the highest
of increase
(the
contents
ofand
Re in
ginsenosides,
showed
the highest
rate of increase
(therate
contents
of Re in
T-Ri,
T-3, T-4
T-5T-Ri,
wereT-3,
T-44.06,
and2.66
T-5and
were
2.03,
4.06,
2.66inand
of that in(Figure
WT cells,
respectively)
(Figure 9). The
2.03,
3.62
folds
of that
WT3.62
cells,folds
respectively)
9). The
monomer ginsenosides
monomerabove
ginsenosides
mentioned
are thewhich
main belong
bioactive
components
which belong
mentioned
are the main
bioactiveabove
components
to tetracyclic
triterpenoids
and to
tetracyclic
triterpenoids
and have
been suggested
to possess
important
pharmacological
effects,
such
have
been suggested
to possess
important
pharmacological
effects,
such as
neuroprotection
activity
as
neuroprotection
activity
(Rb1)
[21,22],
hypoglycemic
activity
(Rg1)
[23],
anti-tumor
activity
(Rh1)
(Rb1) [21,22], hypoglycemic activity (Rg1) [23], anti-tumor activity (Rh1) [24], antioxidant activity
[24],
activity
(Re)
[25],nervous
and protective
effects on
the nervous
system,
immune system,
(Re)
[25],antioxidant
and protective
effects
on the
system, immune
system,
and against
cardiovascular
and
and against cardiovascular
and cerebrovascular
(Rd)
[26].
this study,
the metabolic
flux
cerebrovascular
diseases (Rd) [26].
In this study, thediseases
metabolic
flux
for In
triterpene
skeleton
formation
forenhanced
triterpeneby
skeleton
formation wasof
enhanced
theRNAi
over-expression
of FPS
and the
RNAi ofthe
CAS,
was
the over-expression
FPS andbythe
of CAS, which
finally
promoted
which finally
promoted the
biosynthesisHowever,
of tetracyclic
triterpenoids.
However,
after the formation
biosynthesis
of tetracyclic
triterpenoids.
after
the formation
of the triterpene
skeleton, of
the triterpene
different
sorts of
tetracyclic
triterpenoids
(Rb1,
Rg1,
Rh1, Re andundergo
Rd) would
different
sorts of skeleton,
tetracyclic
triterpenoids
(Rb1,
Rg1, Rh1,
Re and Rd)
would
subsequently
subsequently
undergo different
post-modification
was related
to a series
complex
different
post-modification
processes.
It was related toprocesses.
a series ofItcomplex
enzymatic
actionsofduring
enzymatic
actions
these processes,
and consequently
to differentin
increases
of ginsenosides
these
processes,
and during
consequently
led to different
increases ofled
ginsenosides
transgenic
cell lines.
in transgenic
cell lines.
addition,
cell lineswith
which
transformed
with both
CAS
In addition,
transgenic
cellIn
lines
which transgenic
were transformed
bothwere
the CAS
RNAi fragment
andthe
FPS
RNAi fragment
and FPSsaponins
producedcompared
more triterpene
saponins
compared
with transgenic
the CAS RNAi
produced
more triterpene
with the
CAS RNAi
fragment
line fragment
(T-Ri).
transgenic
line (T-Ri).
There
was two
a synergistic
among two
transgenes
in triterpene
saponin
There
was a synergistic
effect
among
transgeneseffect
in triterpene
saponin
biosynthesis.
The multiple
biosynthesis.
The
multiple
regulations
including
over-expressing
key
enzyme
genes
and
blocking
regulations including over-expressing key enzyme genes and blocking the by-product pathway werethe
by-product
pathway
were effective
strategies
to strengthen
saponin biosynthesis in P. notoginseng.
effective
strategies
to strengthen
saponin
biosynthesis
in P. notoginseng.
Figure
8. The
contents
of total
triterpenes
phytosterols
innotoginseng
P. notoginseng
cells.
contents
Figure
8. The
contents
of total
triterpenes
andand
phytosterols
in P.
cells.
(A)(A)
TheThe
contents
of of
total
triterpene;
contents
of phytosterol;
Vertical
indicate
mean
values
± SD
from
three
total
triterpene;
(B)(B)
TheThe
contents
of phytosterol;
Vertical
barsbars
indicate
mean
values
± SD
from
three
independent
experiments
= 3).
symbol
* represents
there
a significant
difference
between
independent
experiments
(n =(n3).
TheThe
symbol
* represents
thatthat
there
is aissignificant
difference
between
(control)
transgenic
< 0.05.
WTWT
(control)
andand
transgenic
cellscells
at pat< p0.05.
Molecules 2017, 22, 581
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7 of 12
7 of 12
Figure 9. The contents of five major ginsenosides (Rb1, Rg1, Rh1, Re and Rd) in P. notoginseng cell lines.
Figure 9. The contents of five major ginsenosides (Rb1, Rg1, Rh1, Re and Rd) in P. notoginseng cell lines.
Vertical bars indicate mean values ± SD from three independent experiments (n = 3). The symbol *
Vertical bars indicate mean values ± SD from three independent experiments (n = 3). The symbol *
represents that there is a significant difference between WT (control) and transgenic cells at p < 0.05.
represents that there is a significant difference between WT (control) and transgenic cells at p < 0.05.
Previous
studies
mainly
focused
onon
manipulating
Previous
studies
mainly
focused
manipulatinga asingle
singlegene
geneby
bymeans
meansof
of over-expression
over-expression or
or silencing, such as
RNAi
of
CAS,
thus
enhancing
the
accumulation
of
triterpene
saponins
[14].
as RNAi of CAS, thus enhancing the accumulation of triterpene
saponins
[14].
However,
thethe
amount
of triterpene
saponins
produced
by by
engineering
a single
gene
is limited,
so we
However,
amount
of triterpene
saponins
produced
engineering
a single
gene
is limited,
so we
suggest
thatthat
manipulating
twotwo
genes,
governing
thethe
expression
of rate-limiting
enzymes,
may
result
suggest
manipulating
genes,
governing
expression
of rate-limiting
enzymes,
may
result
in ainpositive
cooperative
or
synergistic
effect
to
promote
the
accumulation
of
target
compounds.
a positive cooperative or synergistic effect to promote the accumulation of target compounds.
Multigene
transformation
may
be be
an an
appealing
strategy
to further
enhance
thethe
triterpene
saponin
Multigene
transformation
may
appealing
strategy
to further
enhance
triterpene
saponin
biosynthesis
in
some
medicinal
plants.
biosynthesis in some medicinal plants.
3. Materials
andand
Methods
3. Materials
Methods
3.1. Plant Materials
3.1. Plant Materials
P. notoginseng seeds were collected from Wenshan, Yunnan Province, China. The callus cells were
P. notoginseng seeds were collected from Wenshan, Yunnan Province, China. The callus cells
induced from the seeds and cultured on Murashige and Skoog (MS) medium supplemented with
were induced from the seeds and cultured on Murashige and Skoog (MS) medium supplemented
2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mg/L kinetin (KT), 3% sucrose and 0.75% agar at
with 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mg/L kinetin (KT), 3% sucrose and 0.75% agar
24 ± 1 ◦ C in the dark. After continuous subculture, the callus cells in the vigorous growth period were
at 24 ± 1 °C in the dark. After continuous subculture, the callus cells in the vigorous growth period
employed as experimental materials during the whole research.
were employed as experimental materials during the whole research.
3.2. RNA Isolation and cDNA Synthesis
3.2. RNA Isolation and cDNA Synthesis
Total RNA was isolated from the fresh P. notoginseng cells by Tri-Reagent (MRC, Cincinnati,
Total RNA was isolated from the fresh P. notoginseng cells by Tri-Reagent (MRC, Cincinnati, OH,
OH, USA) and the RNeasy plant Mini Kit (Qiagen, Duesseldorf, Germany) according to the
USA) and the RNeasy plant Mini Kit (Qiagen, Duesseldorf, Germany) according to the manufacturer’s
manufacturer’s protocol. The integrality and concentration of total RNA samples were tested by
protocol. The integrality and concentration of total RNA samples were tested by using agarose gel
using agarose gel electrophoresis and a UV/Visible spectrophotometer (GE, Fairfield, CT, USA),
electrophoresis and a UV/Visible spectrophotometer (GE, Fairfield, CT, USA), respectively. Then, 2
respectively. Then, 2 µg of total RNA was reverse transcribed with M-MLV reverse transcriptase
μg of total RNA was reverse transcribed with M-MLV reverse transcriptase (Promega, Madison, WI,
(Promega, Madison, WI, USA). The obtained cDNA was used as a RT-PCR template to achieve the
USA). The obtained cDNA was used as a RT-PCR template to achieve the encoding region of FPS
encoding region of FPS and the RNAi fragment of CAS.
and the RNAi fragment of CAS.
3.3. RNAi Vector of CAS Construction
3.3. RNAi Vector of CAS Construction
Suppression of CAS (GenBank accession number: EU342419) was achieved by the expression
of CAS (GenBank
accessionwas
number:
EU342419)
wasnon-conservative
achieved by the expression
of a 438Suppression
bp RNAi fragment.
The fragment
selected
from the
region of of
a 438
bp RNAi
fragment. The
was selected
non-conservative
region
of CAS inwas
order
CAS
in order
to guarantee
the fragment
specific inhibition
of from
CAS the
transcription.
The RNAi
fragment
to guarantee the specific inhibition of CAS transcription. The RNAi fragment was amplified with a
pair of specific primers, and the forward and reverse sequences of the primers were 5′-GGGGA
Molecules 2017, 22, 581
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amplified with a pair of specific primers, and the forward and reverse sequences of the primers
were 50 -GGGGACAAGTTTGTACAAAAAAGCAGGCTGCGAGATGGTGGGTGGGGTTTG-30 and
50 -GGGGACCACTTTGTACAAGAAAGCTGGGTCCACATAGGTTGCGTGCTTGATTC-30 , respectively.
The PCR mixture was incubated in a 20 µL volume using a DNA thermal cycler (2720 thermal cycler,
ABI, Foster City, CA, USA). The PCR conditions were as follows: 94 ◦ C for 3 min; 32 cycles of 94 ◦ C for
30 s, 57 ◦ C for 30 s, 72 ◦ C for 30 s; a final 5 min extension at 72 ◦ C. After sequencing appraisal, the PCR
products with the correct DNA sequences were inserted into the RNAi vector (pHellsgate2 which
contained a nptII gene and conferred kanamycin resistance to plant cells) by a step of homologous
recombination reaction mediated by Gateway® BP Clonase™ II Enzyme Mix (Invitrogen, Carlsbad,
CA, USA). The homologous recombination reaction was performed in a 10 µL volume containing
0.15 µg of PCR products, 0.15 µg of the pHellsgate2 vector, and 2 µL of BP Clonase™ II Enzyme Mix.
After 4 h of incubation at 25 ◦ C, the reaction was terminated by adding proteinase K and then incubated
at 37 ◦ C for 10 min. The recombinant vector pHellsgate-CAS was identified by enzyme digestion and
subsequently transformed into Agrobacterium tumefaciens (A. tumefaciens) EHA105 competent cells by
the freeze-thaw method [27].
3.4. Over-Expression Vector of FPS Construction
The complete encoding region of FPS (GenBank accession number: DQ059550.1) was amplified
with a pair of specific primers carrying KpnI and XbaI sites, respectively. The forward and
reverse sequences of the primers were 50 -GGTACCAGAATGAGCGATCTGAAGACGAG-30 and
50 -TCTAGAACAGACAACAACTTCCCCTCCAT-30 , respectively. The PCR mixture was incubated in
a 20 µL volume, and the PCR conditions were as follows: 94 ◦ C for 5 min; 32 cycles of 94 ◦ C for 30 s,
58 ◦ C for 30 s and 72 ◦ C for 1 min 20 s; a final 15 min extension at 72 ◦ C. After DNA sequence analysis,
the PCR products with no mutations were inserted into pCAMBIA1300S which contained an hptII
gene and conferred hygromycin resistance. The recombinant vector pCAMBIA1300S-FPS was then
transformed into A. tumefaciens EHA105 competent cells via the above method [27].
3.5. Genetic Transformation of P. notoginseng Cells
The A. tumefaciens EHA105 harboring pHellsgate-CAS was firstly cultured at 28 ◦ C for 3 days
on solid LB medium with 50 mg/L kanamycin and 25 mg/L rifampicin. Then, the bacteria were
collected with a vaccination needle and further cultured in liquid MGL medium containing 40 mg/L
acetosyringone at 28 ◦ C, 220 rpm/min until a final A600 between 0.6 and 0.8 was achieved. Meanwhile,
the fresh wild-type (WT) cells were preincubated on MS solid medium with 40 mg/L acetosyringone,
2 mg/L 2,4-D, 1 mg/L KT at 25 ◦ C for 3 days in darkness. Next, cells were collected and immersed in
the A. tumefaciens suspension at 28 ◦ C, 90 rpm/min for 25 min. After removing bacteria liquid, the cells
were subsequently placed on MS solid medium with 40 mg/L acetosyringone, 2 mg/L 2,4-D, 1 mg/L
KT and co-cultured at 25 ◦ C for 3 days in darkness. Then, the cells were washed by sterile distilled
water (repeated 3~4 times) and then incubated on MS solid medium with 400 mg/L cefotaxime,
2 mg/L 2,4-D, 1 mg/L KT for 15 days to inhibit the growth of EHA105. The final transformants with
pHellsgate-CAS were selected on MS solid medium with 2 mg/L 2,4-D, 1 mg/L KT and 50 mg/L
kanamycin. A total of 5~7 rounds of screening were conducted and each round lasted 35~50 days.
To obtain the cell lines harboring both pHellsgate-CAS and pCAMBIA1300S-FPS,
the pCAMBIA1300S-FPS were secondly transformed into the kanamycin-resistant transformants
according to the method described above except the selective MS solid medium containing 75 mg/L
kanamycin, 30 mg/L hygromycin, 2 mg/L 2,4-D and 1 mg/L KT.
3.6. PCR Analysis of nptII and hptII Genes
Positive transgenic cell lines with both pHellsgate-CAS and pCAMBIA1300S-FPS were confirmed
by genomic PCR analysis. Genomic DNA from transgenic cells and WT cells was isolated as described
by Luo et al. [28]. The primers were designed to detect the 400 bp and 450 bp fragments of nptII and
Molecules 2017, 22, 581
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hptII, respectively, thus determining the presence of T-DNA in transgenic cells (Table 1). The PCR
conditions of nptII and hptII were all performed as follows: 94 ◦ C for 5 min; then 32 cycles of 94 ◦ C for
30 s, 58 ◦ C for 30 s, and 72 ◦ C for 1 min; with a final 5 min extension at 72 ◦ C. The PCR products were
analyzed by agarose gel electrophoresis.
Table 1. Primer sequences used for genomic PCR.
Gene
Forward Primer Sequence (50 –30 )
Reverse Primer Sequence (50 –30 )
nptII
hptII
CTCTGATGCCGCCGTGTT
GAAGTGCTTGACATTGGGGAAT
CCCTGATGCTCTTCGTCCA
AGATGTTGGCGACCTCGTATT
3.7. Semiquantitative RT-PCR Analysis
To unify the sampling time of the follow-up experiments, the expressions of FPS and CAS were
analyzed in different growth periods. When the cells grew to 20 days, total RNA was extracted
every 5 days for 45 days via the method described above. An amount of 2.0 µg of total RNA was
reverse-transcribed by M-MLV Reverse Transcriptase (Promega, Madison, WI, USA). The primers
used for RT-PCR are listed in Table 2. The GAPDH was used as a reference standard to normalize the
expression level of each gene [29]. The PCR conditions were performed as follows: 94 ◦ C for 5 min;
then 32 cycles of 94 ◦ C for 30 s, 58 ◦ C for 30 s, and 72 ◦ C for 30 s; with a final 5 min extension at 72 ◦ C.
The PCR products were analyzed by agarose gel electrophoresis. Thus, the best period of FPS and CAS
expression was ascertained.
Next, the total RNAs from transgenic cell lines were isolated and used for further semi-quantitative
RT-PCR analysis. According to the results, the transgenic cell lines with simultaneously enhanced FPS
expression level and decreased CAS expression level were preliminarily screened.
Table 2. Primer sequences of CAS, FPS and GAPDH.
Gene
Forward Primer Sequence (50 –30 )
Reverse Primer Sequence (50 –30 )
FPS
CAS
GAPDH
CGGATGCTGGACTATAATGTG
CGGATGGTTTATCTGCCTATGTC
CTACCAACTGTCTTGCTCCCCT
ATTTACGGCAATCATACCAACC
GGTTGCGTGCTTGATTCCA
TGATGCAGCTCTTCCACCTCTC
3.8. Expression Analysis of FPS and CAS by Quantitative Real-Time PCR (qPCR)
Total RNAs from transgenic cells and WT cells were isolated at the best time of FPS and CAS
expression. qPCR and RT-PCR shared the same gene-specific primers (Table 2). The qPCR was
performed in a 20 µL volume containing 20 ng of cDNA, 10 µL of 2× GoTaq® qPCR Master Mix
(Promega, Madison, WI, USA), and 0.4 µL of 10 µM qPCR primers. The qPCR mixture was then
programmed at 95 ◦ C for 2 min, followed by 40 cycles of 95 ◦ C 15 s, 60 ◦ C for 30 s on a real-time
system (ABI 7500, Foster City, CA, USA). The target quantity in each sample was normalized to the
reference control (GAPDH) using the comparative method (2−∆∆Ct ). Three independent experiments
were performed.
3.9. Quantitative Analysis of Total Triterpene Saponins
The preparation of total triterpene saponins solution: WT cells, cells transformed by the CAS
RNAi fragment (T-Ri) and cells with both FPS and the CAS RNAi fragment were collected and dried
to the constant weight at 50 ◦ C. Next, 0.5 g of these three kinds of dried cells were crumbled and
transformed into a tube. After soaking in 50 mL 70%~75% methanol overnight, the cells were then
treated with ultrasonic waves for 90 min (60 W, 4 s/5 s). Subsequently, the filtrate of methanol extract
was collected and then evaporated. The dried residue was dissolved in 10 mL distilled water and then
extracted with the same volume of water-saturated butanol 2~3 times. The residue was dissolved in
Molecules 2017, 22, 581
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methanol to constant volume of 25 mL after the solvent was evaporated. Finally, the evenly mixed
liquid was filtered by 0.45 µm filter membrane.
The mixture of vanillin-perchloric acid as a coloring reagent was added to the total triterpene
saponins solution, and the coloring reaction lasted 15 min at 60 ◦ C. The absorbance at 550 nm of the
reaction product was detected. According to the absorbance at 550 nm and standard curve, it was easy
to obtain the content of the total triterpene saponins.
3.10. High-Performance Liquid Chromatography (HPLC) Analysis of Four Major Ginsenosides
The separation method of ginsennosides was described as follows: 0.1 g milled powder of dried
cells was soaked in 70% methanol at 50 ◦ C. After the liquid evaporated, the residue was dissolved
in water and extracted with water-saturated butanol. The butanol layer was evaporated to produce
a saponin fraction and dissolved in methanol. The determination of monomer ginsennoside content
was carried out on an ULTIMATE 3000 LPG-3400A system (Dionex, Sunnyvale, CA, USA). The HPLC
analysis was performed on a Waters Symmetry C18 column (5 µm, 4.6 mm × 250 mm) with water and
acetonitrile as the mobile phase. Identification and quantification of ginsenosides were carried out by
comparing their retention times and peak areas with those of standard ginsenosides purchased from
National Institutes for Food and Drug Control (Beijing, China).
3.11. Quantitative Analysis of Totalphytosterols
The preparation of total phytosterols solution: WT cells, cells transformed by the CAS RNAi
fragment (T-Ri) and cells with both FPS and the CAS RNAi fragment were collected and dried to the
constant weight at 50 ◦ C. Total phytosterols were extracted from 1.0 g of these three kinds of dried
cells according to the alkali saponification method, and the details were elaborated by Liang [14].
The obtained purified total phytosterols were dissolved with 2 mL acetic anhydride at 60 ◦ C.
A drop of concentrated sulfuric acid was then added into the above solution, and the color of the
mixture gradually changed from colorless to light green, and changed further from light green to
dark green. The absorbance of the mixture with stabilized color at 650 nm was detected. According to
the absorbance at 650 nm and standard curve, it was easy to obtain the content of the total phytosterols.
3.12. Statistical Analysis
The data were the averages of three independent sample measurements. The error bars indicated
the standard deviations from the means of triplicates. The data were analyzed with the Student’s t test.
The difference between WT (control) and transgenic cells was considered significant when p was <0.05
in a two-tailed analysis.
4. Conclusions
In this research, simultaneously over-expressing FPS and silencing CAS in P. notoginseng cells could
regulate the expression levels of corresponding genes and thus enhance the accumulation of triterpenes.
It was suggested that the over-expression of FPS could help to break the rate-limiting reaction
catalyzed by FPS, thus providing sufficient precursors for triterpene biosynthesis. The inhibition
of CAS expression decreased the synthesis metabolic flux of the phytosterol branch with allowing
more precursors to flow in the direction of triterpene synthesis, and ultimately promoting saponin
production. Furthermore, compared with the interference of a single gene, the simultaneous
interference and over-expression of double genes led to further improvement of triterpene biosynthesis.
This study lays good foundations for the future commercial large-scale production of triterpene
saponins via a multi-gene transformation system in P. notoginseng cells.
Acknowledgments: This work was supported by the National Natural Science Foundation of China (No. 31260070
and 31060044).
Molecules 2017, 22, 581
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Author Contributions: Feng Ge and Yan Yang conceived of and designed the experiments. Yan Yang and
Ying Sun performed the experiments. Yan Yang analyzed the data. Feng Ge, Diqiu Liu and Chaoyin Chen
provided technical guidance and offered constructive advice to the experiments and data analysis. Yan Yang
wrote the paper. Feng Ge revised the manuscript and approved the final version.
Conflicts of Interest: The authors declare no conflict of interest.
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