全 文 ：大丽花 DpXTH1 和 DpXTH2
基因的组织特异性表达分析
张萍萍，王蕾，陈驰，王利芬，郑必平，钱力鑫，
谈建中 * （苏州大学建筑学院园艺系/苏州市
建筑与城市环境重点实验室 ， 江苏苏州
215123）
摘 要 [目的] 探讨木葡聚糖内转糖苷酶/水
解酶在大丽花生长发育过程中的功能及作用
机制 。 [方法 ] 以 β-actin 为内参基因 ， 应用
Real-time RT-PCR 技术， 分析了 DpXTH1 和
DpXTH2 基因在大丽花根、茎、叶、花中的相对
转录水平。 [结果] DpXTH1 和 DpXTH2 在根中
均不表达，在茎和叶片中表达丰度较低，在花
瓣中高丰度表达。 [结论] 大丽花 DpXTH1 和
DpXTH2 为花瓣组织特异性基因，与花瓣细胞
的生长发育密切相关。
关键词 大丽花； 木葡聚糖内转糖苷酶/水解
酶；XTH 基因； 表达特异性； 实时荧光定量
PCR
基金项目 苏 州 市 应 用 基 础 研 究 计 划
（SYN201405）；国家级大学生创新创业训练计
划项目（201410285049Z）。
作者简介 张萍萍（1990-），女，山东即墨人，
硕士研究生，研究方向：园林植物资源与应用，
E-mail：zhangpingping151@163.com。 * 通讯作
者，教授，从事园林植物资源与生物技术方面
的研究，E-mail：szutjz@hotmail.com。
收稿日期 2015-06-18
修回日期 2015-07-27
Analysis on Tissue-specific Expression of
DpXTH1 and DpXTH2 Genes in Dahlia
Pingping ZHANG, Lei WANG, Chi CHEN, Lifen WANG, Biping ZHENG, Lixin QIAN, Jianzhong TAN*
Department of Horticulture, College of Architecture, Soochow University/Key Laboratory of Architecture and Urban Environment of
Suzhou City, Suzhou 215123, China
Supported by Applied Basic Research Project of Suzhou City (SYN201405); National
College Students’ Innovative Entrepreneurial Training Program (201410285049Z).
*Corresponding author. E-mail: szutjz@hotmail.com
Received: June 18, 2015 Accepted: July 27, 2015A
Agricultural Science & Technology, 2015, 16(8): 1596-1599
Copyright訫 2015, Information Institute of HAAS. All rights reserved Molecular Biology and Tissue Culture
X yloglucan endotransglycosy-lase/hydrolase (XTH) is a kindof cell wall remodeling en-
zyme, and it can change the structure
of cellulose in cell wall. XTHs are en-
coded by a variety of XTH genes.
They are involved in the expansion
and degradation of cell walls[1], hydrol-
ysis of xyloglucans for energy supply[2]
and changes in fluidity of cell plates [3].
In addition, they are closely related to
the elongation of plant tissues, soften-
ing process of fruits, programmed
death of cells, etc. At present, XTH
family has been found in a variety of
plants, and in various tissues of the
plants, the activities of XTHs have
been detected[4]. Total 25 XTHs have
been found in tomato. Among them,
SlXTH5 is highly expressed in mature
fruits[5]. It is predicted that there are 29
open reading frames in rice [6]. Among
them, OsXTH8 is found to be ex-
pressed in sheaths [7], and OsXTH5,
OsXTH19, OsXTH20, OsXTH24 and
OsXTH28 are involved in the exten-
sion of rice pedicels [ 8 ] . Among the
cloned 33 XTH genes from Arabidop-
sis thaliana, most are concentrated to
be expressed in root elongation zone;
but AtXTH2 is only expressed in
pollen, and AtXTH3 is expressed
abundantly in inflorescences, flowers
and stamens [9]. Different XTH genes
show significant differences in expres-
sion and regulation model among dif-
ferent organs and tissues of plants,
and they constitute the entire XTH
gene family.
In this study, the Dahlia pinnata
Cav. ‘Danbanhuang’ was used as
tested material, and the expression
levels of DpXTH1 and DpXTH2 in dif-
ferent tissues and organs were de-
tected by real-time PCR. The expres-
sion characters of XTH gene in dahlia
were analyzed so as to provide certain
experimental basis for revealing the bi-
ological functions of DpXTH1 and
DpXTH2 genes.
Materials and Methods
Plant material
The tested material was Dahlia
pinnata Cav. ‘Danbanhuang’. At the
full blooming stage, the roots, stems,
Abstract [Objective] This study aimed to investigate the functions and related
mechanisms of xyloglucan Endotransglycosylase/hydrolases (XTHs) during the growth
and development of dahlia. [Method] Using β-actin as the reference gene, the rela-
tive transcription levels of DpXTH1 and DpXTH2 genes in roots, stems, leaves and
petals of dahlia were analyzed by real-time RT-PCR. [Result] The DpXTH1 and
DpXTH2 were not expressed in the roots, but expressed abundantly in the petals of
dahlia. There were little expressions in the stems and leaves of dahlia. [Conclusion]
The DpXTH1 and DpXTH2 were petal-specific genes and closely related to the
growth and development of petals in dahlia.
Key words Dahlia; Xyloglucan endotransglycosylase/hydrolases; XTH gene; Expres-
sion specificity; Real-time fluorescence quantitative PCR
DOI:10.16175/j.cnki.1009-4229.2015.08.008
Agricultural Science & Technology2015
leaves and petals of dahlia were col-
lected. They were cleaned with dis-
tilled water, and dried for the extraction
of total RNA.
Extraction of total RNA and synthe-
sis of first-strand cDNA
The total RNA in the four tissues
of dahlia was extracted with RNAprep
pure Plant Kit (Tiangen). The integrity
of extracted RNA was examined by
1% agarose gel electrophoresis. The
OD values of RNA at the wavelengths
of 260 and 280 nm were measured,
and according to the OD260/OD280, the
concentrations and purities of total
RNA were assessed. The first-strand
cDNA synthesis was performed by
transcription with First Strand cDNA
Synthesis Kit (Thermo Scientific).
Primer design and synthesis
According to the design principles
for quantitative PCR primers, the
primers were designed using Primer
5.0 based on the registered gene se-
quence of dahlia. The primer se-
quences of β-actin (ID: AB621922.1),
with target fragment size of 117 bp,
were as follows: F, 5’-CCCGACT-
GTCCCTGTT-3’; R, 5’-CGGCGTTG-
TTACTTT G-3’. The primer sequ-
ences of DpXTH1 (ID: HM053613.1),
with target fragment size of 239 bp,
were as follows: F, 5’-GGTCG-
GATTCTCACAT-3’; R, 5’-TTGCC-
CAC TTCGGTTT-3’. The primer seq-
uences of DpXTH2 (ID: JQ948092.1),
with target fragment size of 132 bp,
were as follows: F, 5’-AGACAACTT-
GAGGGTGGTAG -3’; R, 5’-CGCA-
GAGTCTCCAGGAATAA-3’. The pri-
mers were all synthesized by the
Sangon Biological Engineering Tech-
nology & Services Co., Ltd (Shanghai,
China).
Standard preparation and real-time
PCR
The cDNA, obtained by reverse
transcription, was used as template for
verifying the specificities of synthe-
sized primers. The PCR system (25 μl)
was as follows: 10× Taq Buffer 2.5 μl,
dNTP Mix (2 mmol/L for each) 2 μl,
forward primer (10 μmol/μl) 0.4 μl, re-
verse primer (10 μmol/μl) 0.4 μl, MgCl2
(25 mmol/L) 2 μl, Taq DNA Poly-
merase 0.125 μl, template cDNA 1.5
μl, sterile distilled water 16.075 μl. The
reaction program was as follows: pre-
denaturation at 94℃ for 3 min; denat-
uration at 94 ℃ for 30 s, annealing at
50℃ for 30 s, extension at 72℃ for 45
s. 35 cycles; extension at 72 ℃ for 7
min. The specificities of amplification
products were examined by 1.6% a-
garose gel electrophoresis.
Using the standards of the 3
genes and cDNA from 4 tissues as the
templates, real-time quantitative PCR
was performed with QuantiNova
SYBR Green PCR Kit in a PCR instru-
ment (Prism 7300, ABI). The reaction
system (20 μl) was as follows: SYBR
Premix Ex TaqTM (2× , TaKaRa) 10μl,
forward primer (10 μmol/μl) 0.4 μl, re-
verse primer (10 μmol/μl) 0.4 μl, ROX
Reference Dye (50 ×) 0.4 μl, cDNA 2.0
μl, dH2O 6.8 μl. The PCR program was
as follows: 95 ℃ 1 min; 95 ℃ 15 s, 60
℃ 31 s, 45 cycles. After the amplifica-
tion, the melting curves were ana-
lyzed. There were 3 replicates for each
sample.
Preparation of standard curve and
data analysis
The amplified cDNA product was
diluted by 10, 102, 103, 104 and 105
times, respectively for the preparation
of standard curve. Taking the Ct val-
ues of β-actin, DpXTH1 and DpXTH2
as the abscissa and the logarithms of
their concentrations as the ordinate,
the standard curves were drawn. The
copies of β-actin, DpXTH1 and
DpXTH2 in different tissues of dahlia
were calculated using Microsoft Excel
2007, and the expression differences
in DpXTH1 and DpXTH2 among differ-
ent tissues of dahlia were analyzed.
Results and Analysis
Quality analysis of total RNA from
different tissues of dahlia
A certain amount (3 μl) of extract-
ed RNA from each tissue was exam-
ined by 1% agarose gel electrophore-
sis. Apparent 18 S and 28 S rRNA
bands were obtained. The OD260/OD280
values of total RNA from roots, stems,
leaves and petals were 1.734, 1.800,
1.813 and 1.900, respectively. It indi-
cated that the extracted RNA had mild
degradation, high purity and high
quality, and it had not been contami-
nated by genomic DNA. Thus the ex-
tracted RNA could be used for reverse
transcription.
RT-PCR results
Using the cDNA from roots,
stems, leaves and petals of dahlia as
the templates, the real-time PCR was
performed with the corresponding
primers for β-actin, DpXTH1 and
DpXTH2. The results showed that the
bands of DpXTH1 and DpXTH2 of
roots were relatively dim, and the ex-
pected target fragments were detected
in all the stems, leaves and petals of
dahlia (Fig.1). It indicated that the de-
signed primers were suitable for SYBR
Green real-time quantitative PCR.
Amplification results of the sam-
ples and standard curves
The different-concentration stan-
dards of β-actin, DpXTH1 and
DpXTH2 were amplified by PCR. As
shown in Fig.2, the amplification
curves were all smooth, and only sin-
gle peaks were shown in the melting
curves. So it was further illustrated that
the designed 3 pairs of primers all had
good specificities.
M, D2000 Marker; Lane 1, root; Lane 2, stem; Lane 3, leaf; Lane 4, petal.
Fig.1 Amplification products of total RNA from different tissues and organs of dahlia
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Agricultural Science & Technology 2015
Based on the obtained Ct values
and calculated copies of β-actin,
DpXTH1 and DpXTH2, the regression
equations were deduced: β-actin, y =
-2.807x+31.24 (R2=0.998); DpXTH1,
y=-3.185x+33.14(R2=0.996); DpXTH2,
y=-2.889x+29.94 (R2=0.995). Accord-
ing to the following formula:
E = 10(-1/slope) -1
The amplification efficiencies
were calculated as 1.27, 1.06 and
1.22, respectively. There were no sig-
nificant differences in amplification ef-
ficiency between the reference gene
and target genes, indicating that the
prepared standard curves met the re-
quirements by real-time quantitative
PCR and they could be used for rela-
tively quantitative analysis.
Expression differences of DpXTH1
and DpXTH2 genes among different
tissues
In order to determine the expres-
sion differences between DpXTH1 and
DpXTH2 among different tissues and
organs of dahlia, the total RNA from
roots, stems, leaves and petals of
dahlia was extracted, and the real-time
quantitative PCR was performed using
β-actin as the reference gene. The Ct
values of DpXTH1 and DpXTH2 from
roots were all higher than 35, so it
could be concluded that the DpXTH1
and DpXTH2 genes were not ex-
pressed in dahlia roots. The relative
expression levels of DpXTH1 and
DpXTH2 in stems, leaves and petals
of dahlia were analyzed. The results
showed that there were significant dif-
ferences in relative expression levels
of DpXTH1 and DpXTH2 among differ-
ent tissues of dahlia (Fig.3). Both
DpXTH1 and DpXTH2 genes were
highly expressed in petals of dahlia,
and their expression levels were sig-
nificantly higher than those in the
same-growth period stems and leaves.
It suggested that at the full blooming
stage, the transcription of DpXTH1
and DpXTH2 was mainly concentrated
in petal cells; at that time, the expres-
sion level of DpXTH1 was moderate in
leaves of dahlia, but the expression
level of DpXTH2 was relatively low in
stems and leaves of dahlia. In addition,
the relative expression level of
DpXTH1 was higher than that of
DpXTH2, indicating that the roles of
DpXTH1 and DpXTH2 might be differ-
ent in the physiological metabolism of
dahlia at the full blooming stage.
Therefore, it could be speculated that
the expression and regulation of
DpXTH2 were dominant during the
growth and development of dahlia at
the full blooming stage.
Discussion
Xyloglucan endotransglycosy-
lase/hydrolases (XTHs) are a typical
family of proteins encoded by multiple
genes. They are closely associated
with the growth and development of
plants, and participate in the elonga-
tion of tissues[10-12], growth and senes-
cence [13], fruit ripening [14] and other
physiological processes. The expres-
sion of XTH genes is tissue- and or-
gan-specific [15]. Moreover, the expres-
sion peaks of different XTH genes may
be different in the same tissue among
different growth periods. For example,
in Arabidopsis thaliana, AtXTH1 gene
is expressed in siliques; AtXTH9 is ex-
pressed in shoot apical meristems,
flower buds and flowering branches,
and it is involved in the elongation of
A, B: β-actin; C, D: DpXTH1; E, F: DpXTH2.
Fig.2 Amplification curves (left) and melting curves (right) of β-actin, DpXTH1 and DpXTH2
Fig.3 Expression levels of DpXTH1 (left) and DpXTH2 (right) in different tissues of dahlia
1598
Agricultural Science & Technology2015
(Continued from page 1587)
Responsible editor: Tingting XU Responsible proofreader: Xiaoyan WU
(食品中单核增生李斯特氏菌检测研究
进展) [J]. Journal of Microbiology (微生
物学杂志), 2001, 21(2): 36-38.
[8] GIAMMARINI C, ANDREONI F, AMA-
GLIANI G, et al. Purification and char-
acterization of a recombinant Listeri-
olysin O expressed in Escherichia coil
and possible diagnostic applications [J].
Journal of Biotechnology, 2004, 109(1-
2): 13-20.
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
Responsible editor: Tingting XU Responsible proofreader: Xiaoyan WU
stems and flowering branches;
AtXTH27 is expressed in vein chan-
nels and expanded leaves, and it takes
part in the development of vessels[12, 16].
In the ripening persimmon fruits, the
expression peak of DkXTH1 appears
before the softening of fruits, while the
expression peak of DkXTH2 appears
in the mid of ripening period[14]. In this
study, the DpXTH1 and DpXTH2
genes are not expressed in roots of
dahlia, but they are highly expressed
in petals. So it can be concluded that
the DpXTH1 and DpXTH2 genes are
petal-specific genes, and they are
closely associated with the growth and
development of petal cells.
On the other hand, in Arabidopsis
thaliana, most XTH genes are abun-
dantly expressed in floral organs, as
well as in other tissues and organs.
However, AtXTH3 is only highly ex-
pressed in floral organs, and its tran-
scription level is relatively low in oth-
er tissues and organs. Therefore, it
can be speculated that AtXTH3 has
a flower-specific promoter [17]. In this
study, compared with roots, stems and
leaves of dahlia, DpXTH1 and
DpXTH2 show higher transcription lev-
els in petals. So it is speculated that
there may be also a flower-specific
promoter in the gene structure, and it
regulates the expression levels of
DpXTH1 and DpXTH2 genes during
the development of dahlia petals.
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