Nghiên cứu ảnh hưởng của chất kích kháng lên sự biểu hiện của một số gen tham gia quá trình sinh tổng hợp curcuminoid ở tế bào nghệ đen (curcuma zedoaria roscoe) tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING
HUE UNIVERSITY
UNIVERSITY OF SCIENCE

TRUONG THI PHUONG LAN

STUDY ON EFFECT OF ELICITORS ON GENES INVOLVED
IN CURCUMIN BIOSYNTHESIS EXPRESSION IN
ZEDOARY CELLS (Curcuma zedoaria Roscoe)

DISSERTATION SUMMARY
Major: Plant Physiology
Code: 9420112

Scientific supervisor:
Prof. Dr. NGUYEN HOANG LOC

HUE – 2019
The study was performed at: Department of Biology
University of Sciences, Hue University


Scientific supervisor: Prof. Dr. Nguyen Hoang Loc
Review 1:Prof. Dr. Duong Tan Nhat
Central Highlands Scientific Research Institute
Reviewer 2: Prof. Dr. Dr. Le Thi Thuy Tien
Ho Chi Minh City University of Technology
Reviewer 3: Prof. Dr. TS Vo Thi Mai Huong
University of Science, Hue University

The dissertation will be defended at scientific Hue University’s

council in Hue University on …. hour …. day …. month … year
2019

For more information about dissertation, please visit:
- Library of University of Sciences, Hue University
- Vietnam national library


INTRODUCTION
1. The necessity of the study
Curcuma

zedoaria,

belonging

to

the

ginger

family

(Zingiberaceae), has been using in traditional medicine in various
countries to treat inflammation, aches, skin diseases such as wounds
and spots sores, as well as abnormalities of the menstrual cycle.
Curcuminoids, a mixture of curcumin and its delivery including
demethoxycurcumin,

and

bisdemethoxycurcumin,

is

major

compound having bioactivity of Curcuma. Previous studies
suggested that curcuminoids, especially curcumin, have valuable
biological

activities

such

as

antioxidant,

antitumor,

anti-

inflammatory, anti-acidogenic, radioprotective and neuroprotective
(anti β-amyloid) properties.
Genes that involve the pathway of curcuminoid metabolism in
C. longa have been identified and qualified the expression level,
including two type III polyketide synthase genes, DCS gene and
CURS1, CURS2 and CURS3 genes. However to our knowledge,

genes which metabolize curcuminoids in zedoary (C. zedoaria) were
not reported yet.
Elicitors are the chemical compounds which have been used to
modify the pathway of secondary metabolism in order to enhance of
the biosynthesis of pharmaceutically significant metabolites or
phytopharmaceuticals in plant cell cultures.
Thus, we carried out the research entitles “Study the effect of
elicitors on expression of genes involved curcuminoid biosynthesis
pathway in Curcuma zedoaria cells” to find out the elicitor and
optimal concentration to enhance the expression level of type III
polyketide synthase gene in the phenylpropanoid pathway of


Curcuma zedoaria cells. Our results firstly provide scientific
evidence on the role of SA, YE and MeJA as the positive regulation
compounds for gene expression in this medicinal plant.
2. Objectives of study
Theoretical objectives
Improvement the expression level of CzDCS, CzCURS1,
CzCURS2 and CzCURS3 genes involved in phenylpropanoid
metabolism pathway for curcuminoid biosynthesis in Curcuma
zedoaria cells cultured in vitro by elicitors.
Practical objectives
Enhancement the curcumin biosynthesis, major compound of
curcuminoid and being widely applied in pharmaceutical from
Curcuma zedoaria cells cultured in vitro.
3. Research scope and content
Research content
- Establishing in vitro Curcuma zedoaria cells culture in
supplement with AgNO3 into medium culture.

- Isolation of CzDCS, CzCURS1, CzCURS2, and CzCURS
genes involved in curcuminoid biosytheis from Curcuma zedoaria.
- Investigation the effect of elicitors including YE, SA and
MeJA on expression level of gene for curcuminoid synthesis and
curcumin accumulation in Curcuma zedoaria cells cultured in vitro.
Research limit
Studies were conducted with the laboratory scale.
4. Novelty of dissertation
- Perivious studies on in vitro propagation and callus cells
culture did not use AgNO3 to improve the effect of culture. In this
study, AgNO3 supplementation at 1.5 mg/L into in vitro propagation


and callus cells culture enhanced the results in compare to previous
studies.
- Succesfully isolated four genes involved in curcuminoid
biosynthesis, showing 99% similarity to corresponding genes in C.
longa and had deposited on GenBank with accession number
MF663785, MF402846, MF402847, and MF987835. These genes
expressed in Zedoary turmeric and callus.
- DCS gene played most importance role in the genes involved
in curcumin biosynthesis in Zedoary, expression of this gene directly
regulated curcumin accumulation.
- Studied the effect of elicitors (yeast extract and salicilic
acid) on curcumin accumulation and related genes expression. The
optimal values reached under treatment by 1 g/L YE after the day 5
of culture, gene expression level was 2.78 fold higher than that of
control.
5. Dissertation structure
Dissertation

is

presented

in

127 A4

pages

without

suplementary. In which, the introduction section consists of 4 pages,
litterateur review section contains 22 pages, materials and methods
section involves 11 pages, result section accounts for 40 pages,
discussion section is 16 pages, conclusion and suggestion section is 1
page, list of publication section is 1 pages, reference section is 21
pages and supplementary section is 13 pages. Dissertation referred
169 references including 161 English references. The results section
consists of 9 tables and 26 figures.


CHAPER 1. LITTERATEUR REVIEW
1. Zedoary
Zedoary (Curcuma zedoaria Roscoe) is a valuable medicinal
plant; the essential oil obtained from its rhizomes is reported to have
antimicrobial activity. It is also used clinically in the treatment of
cervical cancer, as the aqueous extract of zedoary has antimutagenic
activity. In traditional Asian medicine, zedoary (Zedoariae rhizoma)

is also used for treatment of stomach diseases, hepato-protection, the
treatment of blood stagnation. Furthermore, zedoary has antiinflammatory potency related to its antioxidant effect.
Zedoary is medicinal plant which its root mainly contains
emission compounds (sesquiterpene and monosesquiterpene) and
curcuminoid (curcumin, demethoxy-curcumin and bisdemethoxycurcumin). In addition, Zedoary roots is consisted of starch, stick
compounds and phenolic compunds such as tannin, flavonoid.
2. Plant cells culture for secondary metabolite production
Plant cells culture has potential appling for secondary
metabolite production, especially medicinal compounds. This
approach could lead the stable in quality of products, resulting in less
dependence to the nature production. Moreover, it provides materials
for biophysical, biochemistry experiements and application on
extraction metabolite compounds.
3. Elicitor and application
Elicitor is defined as a molecule that initials or enhances
secondary metabolite biosynthesis in the plant cells. Elicitation is an
induction process that increases the synthesis bioactive compounds
under trigger of elicitor, leading plant in against the stress
environment such as pathogen infection or adverse ecological


conditions.
Up to day, numerous studies have been reported on application
elicitor to induce secondary metabolite compounds production from
in vitro plant. Most studies demonstrate the increasing accumulation
of secondary metabolite compounds in compared to control after
being treated by elicitor under optimal condition. Numerous plants
have been cultured under elicitor treament including Dioscorea
zingiberensis, Digitalis lanata, Hypericum triquetrifolium, Portulaca
oleracea, Psoralea corylifolia, Silene vulgaris, Alstonia scholaris,

Hypericum perforatum, Scrophularia kakudensis, Taxus baccata,
Artemisia annua… Cultivation the curcuma genus under elidictor
supplementation have been reported on C. aeruginosa, C. longa, and
C. mangga.
4. Curcuminoid biosynthesis genes
The curcuminoid biosynthesis in C. longa has been involved
different enzymes require for several reactions in which most of
genes/enzymes already has been characterized on properties and
fuctions as well as recombinant expression. Among them, polyketide
synthase type III plays most importance role. Diketide-CoA synthase
gene (DCS) encodes for diketide-CoA synthase, a enzyme belonging
to polyketide synthase type III catalyzes for reaction to form
feruloyldiketide-CoA by feruloyl-CoA and malonyl-CoA. Curcumin
synthase gene (CURS) encodes for enzyme catalyzes the formation
of curcuminoid by cinnamoyldiketide-N-acetylcysteamine and
feruloyl-CoA. The co-reaction of DCS and CURS in the presence of
feruloyl-CoA and malonyl-CoA is resulted in plenty curcumin
accumulation, whereas CURS alone exhibits low curcumin synthesis
in the presence of feruloyl-CoA and malonyl-CoA.


CHAPTER 2. MATERIALS AND METHODS
2.1. Materials
Study qualified the expression level of genes involved in
curcuminoid biosynthesis pathway in Zedoary culture cells
(Curcuma zedoaria Roscoe).
2.2. Methods
2.2.1. In vitro zedoary plants culture
Leaf-base and root in vitro cultureo
Sterilized leaf-base explants (approx. 1 × 1.5 cm) was cultured

on MS medium containing 2% (w/v) sucrose, 0.8% (w/v) agar,
supplemented with 3 mg/L BAP, 0.5 mg/L IBA and 20% (v/v)
coconut water. In vitro leaf-base was transferred on the same medium
with addition of 0.5-2.5 mg/L AgNO3 to investigate the generation of
multi-shoot. Các chồi in vitro (3-4 cm) was cultivated on MS
medium supplemented with 2 mg/L NAA and AgNO 3 range from 0.5
to 2.5 mg/L AgNO3 to examine the root generation.
Callus culture
Leaf-base explants (approx. 0.2 × 1 cm) of 4 weeks old in
vitro zedoary plants were tranferred on MS medium supplemented
with 3 mg/L BAP and 3 mg/L 2,4-D to induce callus formation. The
primary calli were sliced into pcieces (approx. 2 mm diameter) and
subcultured on fresh medium containing 1 mg/L 2,4-D and KIN, or
NAA (0.5-2 mg/L), or AgNO 3 (0.5-2.5 mg/L) for biomass
production.
Supension cell culture
3 g callus after precultivation for 2 weeks was transferred into
250ml shaking flask containing 50 mL MS medium with 20 sucrose
g/L, 3 mg/L 2,4-D and 3 mg/L BAP, shaking of 150 rpm for 18 day.
2.2.2. Curcuminoid biosynthesis genes isolation


DNA isolation
Total DNA of zedoary was extracted from young leaves of in vitro
plants by the CTAB method according to Babu et al. (2014) with
slight modification.
PCR amplification
Total DNA of zedoary was used as template to amplify the
coding gene based on specific primers designed for corresponding
genes in Curcuma longa (Table 2.1).

Table 2.1. Primers used for PCR amplification of the coding DNA
sequences of curcuminoid genes in C. zedoaria.
Genes

Primers

Nucleotide sequences (5’- 3’)

DCS-F

GTCGTTTCTGTGACCTTCTC

DCS-R

CTTTTGGATGCAGACTGGAACA

CURS1-F

CTGCGACTGCGAGAAGAAGC

CURS1-R

CAGATAGACAGCCATACAAACC

CURS2-F

GCACGCGTTTTCTTGCTAATC

CURS2-R

GATCGTGTTCATAATTCACTGG

CURS3-F

CTAGCTAGCTGCAATTCGTT

CURS3-R

GTGCTAGCTTAGCTTGACGTA

Putative length
(nu)

CzDCS
CzCURS1
CzCURS2
CzCURS3

~ 1400
~ 1250
~ 1300
~ 1250

Gene cloning and Phylogenetic analysis
PCR products were purified, ligated to pGEM®T-Easy vector
using T4 DNA ligase (Promega, USA) and transferred into E. coli
TOP10 by heat-sock method. Nucleotide sequences were sequenced
by dideoxy terminator method.
Phylogenetic analysis
Coding region of DCS, CURS1, CURS2, CURS3 genes of C.

zedoaria and C. longa were used to generate the phylogenetic tree by
MEGA7 software.
2.2.3. Expression qualification of curcuminoid biosynthesis genes


Elicitor treatment
Elicitors were added into medicum culture at difference
conccentration (25-150 µM MeJA, 50-150 µM SA and 0.1-1.5 g/L
YE) and difference inoculation time (initiation or at the day of 5).
RT-PCR
Expression level of curcuminoid genes in various samples
was analysed by RT-PCR with the primers that designed based on
their specific regions (Table 2.2).
Table 2.2. Primers used for RT-PCR amplification of the specific regions of
curcuminoid genes in C. zedoaria.
Genes

CzDCS

Primer Nucleotide sequences (5’- 3’)

Length of

Annealing

s

indicators

temperature

(nu)

(oC)

272

55

286

55

211

55

202

50

ID-F

TGCTCCGAGGTCACCGTGC

ID-R

GGTCAGCCCAATTTCGCGG

IC1-F CCGCTGGAAGGAATTGAAA

CzCURS
1

AA
IC1-R GAGCTTGTCCGGGCTCAGCT
G
IC2-F CCACCTCCGCGAGGTGGGG

CzCURS
2

CT
IC2-R GCGGTGGCCAGCTTGCTCTG
T
IC3-F CACCTGAGGGAAATCGGCTG

CzCURS
3

G
IC3-R GCGAGCTTCCCCTGTTCCAG
C

2.2.3.3. HPLC
Curcumin was isoalted as described by Paramapojn và
Gritsanapan (2009). Curcumin concentration was determined


according to curcumin standard curve.
2.2.4. Statistical analysis

The experiments were randomly designed and done in
triplicate. The data were analyzed as means followed by one-way
ANOVA (Duncan’s test, p<0.05) using SPSS software.
CHAPTER 3. RESULTS
3.1. Cells culture
3.1.1. Zedoary in vitro propagation
3.1.1.1. In vitro propagation


Whole in vitro bud. In the medium control, the apical bud initial
elongated at the day 3 and the axillary buds appeared at the day 5.
The apical bug elongated on medium supplemented with AgNO 3 a
day late. Zedoary cultured on AgNO 3 medium resulted in higher or
similar number buds generation in compared with control.
Table 3.1. Effect of AgNO3 on C. zedoary in vitro propagation
AgNO3 (mg/L)

Propagation
generation (%)

Bud/sample

Elongation (cm)

Control

100

5.4ab

6.3ab

0.5

100

5.6ab

7.1a

1.0

100

6.1ab

6.9ab

1.5

100

6.4a

6.6ab

2.0

100

5.1b

6.2b

Control: without AgNO3 supplementation. The note is same for table 3.2
and 3.3. Different letters in a column indicate significantly different means
(Duncan’s test, p < 0.05). The note is same for all table except table 3.7.




Spited in vitro bud. The results showing in table 3.2 indicates all
examined media effectively mediated bud propagation as the same as
using whole in vitro bud. In particularly, the spited in vitro bud
displayed higher bud propagation than whole in vitro bud (7,5/6,4
buds).


Table 3.2. Effect of AgNO3 on C. zedoary bud propagation from spited in vitro
bud.

AgNO3 (mg/L)

Propagation

Bud/sample

Elongation (cm)

generation (%)

Control

100

5.6c

3.4c

0.5

100

5.9bc

3.6bc

1.0

100

6.6b

3.9a

1.5

100

7.5a

3.7b

2.0

100

6.1bc

3.6bc

3.1.1.2. In vitro root propagation
All examined media containing NAA and AgNO 3 showed in
vitro root propagation. In the medium containing AgNO 3, root
appeared early than control with only 3 day after inoculation and the
root number also was higher than control.
Table 3.3. Effect of AgNO3 on C. zedoary root propagation from in vitro
bud.
AgNO3 (mg/L) Root propagation (%) Root/sample Elongation (cm)
Control

100

18.3c

0.6c

0.5

100

20.0bc

0.7ab

1.0

100

22.5b

0.7ab

1.5

100

27.0a

0.8a

2.0

100

20.8bc

0.8a

3.1.2. Callus cells culture

3.1.2.1. Effect of 2,4-D and KIN
The results showing in table 3.4 indicated 2,4-D and KIN
significantly affected on callus cells growth. In particularly, callus
grew faster than control on medium supplemented with 2,4-D (1


mg/L) and KIN (1-1,5 mg/L), resulting in average diameter of 1,531,56 cm). Fresh weight and dry weight of callus were 0.84-0.9 g and
83.15-84.11 mg, respectively.
3.1.2.2. Effect of 2,4-D and NAA
Table 3.5 shows the effect of 2,4-D and NAA on callus growth
and the effect is slightly lower than the formula 2,4-D and KIN.
Table 3.4. Effect of KIN (0.5-2 mg/L ) and 2,4-D (1 mg/L) on the effect of
callus cells growth.
KIN (mg/L)

Diameter (cm)
c

Weight
Fresh (g)

Dry (mg)

c

61.24c

Control

1.35

0.68

0.5

1.48abc

0.79b

79.03b

1.0

1.53ab

0.84ab

83.15a

1.5

1.56a

0.90a

84.11a

2.0

1.38bc

0.85ab

80.37b

Control: medium containing 2,4-D (3 mg/L) and (BA 3 mg/L). This note is
same for table 3.5 and 3.6.
Table 3.5. Effect of NAA (0.5-2 mg/L) and 2,4-D (1 mg/L) on the growth of
callus cells.
NAA (mg/L)

Diameter (cm)
c

Weight
Fresh (g)
c

Dry (mg)

Control

1.35

0.65

60.89c

0.5

1.45abc

0.70abc

64.39c

1.0

1.56a

0.79a

80.16a

1.5

1.50ab

0.76ab

73.91b

2.0

1.44bc

0.69bc

63.23c

3.1.2.3. Effect of 2,4-D and AgNO3
The results showed that examined media enhanced the callus


growth in compared with control. Among that, medium containing
AgNO3 (1.5 mg/L) callus reached highest dry weight of 62,43 mg
(Table 3.6).
Table 3.6. Effect of AgNO3 (0.5-2 mg/L) and 2,4-D (1 mg/L) on the growth
of callus cells.
AgNO3 (mg/L)

Diameter (cm)

Control
0.5
1.0
1.5
2.0

b

1.28
1.38ab
1.41a
1.45a
1.35ab

Weight
Fresh (g)
c

0.53
0.59bc
0.65ab
0.71a
0.67ab

Dry (mg)
48.21c
49.78c
56.87b
62.43a
58.18b

3.1.3. Callus cells culture
The results showing in Fig. 3.3 indicated the lag phase is short. The
cells changed to growth phase and continuously exponential from
day 2 to day 14 of culture, then dramatically decreased. Fresh cells
biomass reached approximately 170g/L, equally to 15 g/L of dry
biomass weight.

Figure 3.3. The growth curve of zedoary callus cells.

3.2. Identification of curcuminoid biosynthesis genes


3.2.1. Gene isolation
The full length of DCS, CURS1, CURS2 và CURS3 genes of
zedoary are 1382 bp, 1240 bp, 1288 bp and 1265 bp, respectively
(Fig. 3.6). Hene, the genes were named as CzDCS, CzCURS1,

CzCURS2 and CzCURS3, and deposited on GenBank with accession
number of MF663785, MF402846, MF402847 and MF987835,
respectively.
To identify intron/exon in our genomic DNA sequences of
CzDCS, CzCURS1, CzCURS2 and CzCURS3, we used several
strategies to ensure high accuracy of identity assignment. The results
showed that CzCURS1, CzCURS2 and CzCURS3 have one intron
whereas CzDCS has two introns. The putative intron/exon regions
are shown in figure 3.7.

Figure 3.6. PCR products of genes involved in curcuminoid biosynthesis
amplified from total genomic DNA. Lane M1 and M2: DNA size marker
(100 bp (BioRad) and 1 kb DNA Ladder (Geneaid)), 1: CzDCS, 2:
CzCURS1, 3: CzCURS2, 4: CzCURS3.

We employed phylogenetic analysis as a tool to correctly
assign identity for C. zedoaria curcuminoid biosynthesis genes. The
results are shown in figure 3.16 indicated that corresponding genes
cluster with very high bootstrap values, demonstrating the orthologs


and paralogs of these genes in C. longa and C. zedoaria. In the
phylogenetic tree, CURS genes classify as group with high identity,
the difference are only range 0.1 to 0.2. The DCS genes also have
high similarity and form as group.

Figure 3.7. Schematic diagram of intron/exon arrangement. The diagram
shows intron (line), exon (box) and their boundaries in four curcuminoid
biosynthesis genes in C. zedoaria. Number 1 indicates the first nucleotide of
the start codon. The general structure of the encoding proteins is also shown

below. Domain assignment was obtained using Interproscan.

Figure 3.16. Molecular phylogenetic analysis of curcuminoid genes

3.2.2. Expression of curcuminoid genes
To assess the expression of curcuminoid biosynthesis genes in


C. zedoaria (rhizome and callus), we opted RT-PCR method and
analyzed the intensity of DNA bands from gel electrophoresis.
The data of RT-PCR of four curcuminoid genes (CzDCS,
CzCURS1, 2, and 3) indicated all the genes expressed in both the
rhizome and callus (Fig. 3.17). The results suggest that the
curcuminoid metabolism of C. zedoaria also occurs in in vitro
culture, and callus is a suitable material source for establishing plant
cell suspension culture to produce curcumin.

Figure 3.17. Transcription expression of CzDCS, CzCURS1,
CzCURS2 and CzCURS3 genes in various tissues of C. zedoaria. M: DNA
size marker (1 kb), 1-3-5-7: rhizome, 2-4-6-8: callus, A: CzDCS, B:
CzCURS1, C: CzCURS2, and D: CzCURS3.

To ensure the above hypothesis, we analyzed the curcumin
concentration, the major compound of curcuminoid in rhizome and
callus. HPLC analysis indicatea both the rhizome and callus extracts
contain the peak of curcumin with the retention time is similarity to
that of standard curcumin.
3.3. Effect of elicitor on curcuminoid biosynthesis
3.3.1. Effect of type of elicitors on curcuminoid
biosynthesis genes expression

RT-PCR results showed treatment with methyl jasmonate (25150 mM) did not change the CURS1 expression in compared to
control (Fig. 3.22A) but increased CURS3 expression level (Fig.


3.22B). Under treatment by salicylic acid (50-150 mM), the
expression level of these genes were higher than control. CURS1
expression level strongest enhanced when the cells are being treated
with 100 mM SA while CURS3 showed highest expression at the 150
mM SA treatment. Yeast extract treatment increased genes
expression level at the concentration of 0.7-1.0 g/L, whereas other
treatments reduced gene expression level.
Based on these results, we found MeJA shows less effect on
CURS expression, while SA and YE had higher effect on expression
level of these genes. The slected elicitor treament concentration are
salicylic acid 100 mM and yeast extract 1 g/L to examine gene
expression level at different period of treament.
3.3.2. Expression of curcuminoid genes
RT-PCR amplification in Figure 3.23 exhibited that expression
of curcuminoid genes in treated cells of C. zedoaria is higher than
that of untreated cells. Expression level of genes was evaluated
through the intensity of DNA bands showed that they are 1.14-3.64fold higher than the control (Table 3.8). In general, YE showed a
higher elicitation effect compare to SA, the total intensity of DNA
bands reached a maximum value of 71528 versus 31625 (approx.
2.3-fold).
Table 3.8. Intensities of DNA bands from RT-PCR amplification of
the specific regions of curcuminoid genes.
Gene

Control

SA (100 µM)

YE (1 g/L)

0

5

0

5

CzDCS
CzCURS1
CzCURS2
CzCURS3

4706
6881
4646
4414

5312
7810
6186
4146

6146
10766
8510

6203

5113
32576
12139
21700

13425
13524
17783
12664

Total

20647

23455

31625

71528

57397