全 文 ：大丽花花瓣衰老相关蛋白
DpXTH1及其编码基因的分离
张萍萍，阚雪芹，李坚，陈驰，罗成洋，谈建中 *
（苏州大学建筑学院/苏州市建筑与城市环境
重点实验室，江苏苏州 215123）
摘 要 [目的] 为了探讨乙烯非敏感型花卉
衰老相关的分子机理。 [方法] 以大丽花花瓣
为材料，利用双向电泳、质谱分析及分子生物
学技术，分离、鉴定花瓣衰老相关蛋白质及其
编码基因。 [结果] 从大丽花透色期、盛花期和
衰败期花瓣蛋白质的双向电泳图谱中，检测到
44 个表达量差异在 2 倍以上的蛋白质斑点 ，
从中分离、 鉴定了木葡聚糖内糖基转移酶/水
解酶 （xyloglucan endotransglycosylase/hydrolase,
XTH）， 其表达量随花瓣的衰老进程而持续增
加，属于一种衰老相关蛋白质。 以大丽花花瓣
RNA 为材料， 设计一对简并引物， 通过 RT-
PCR 技术克隆了大丽花花瓣 XTH 基因的
cDNA 序列，其编码区序列全长 882 bp，编码
293 个氨基酸残基（基因登录号 HM053613.1，
命名其为 DpXTH1）。 聚类分析表明，DpXTH1
氨基酸序列与其他植物的 XTH 具有较高的同
源性。 [结论] 分离、鉴定的大丽花 DpXTH1 属
于植物 XTH 家族， 其生物学功能与大丽花花
瓣的衰老进程及调控有关。
关键词 大丽花；花瓣；木葡聚糖内糖基转移
酶/水解酶（XTH）；衰老相关蛋白；基因克隆
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（201410285049）。
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 2015-05-22
修回日期 2015-06-21
Identification of Senescence-associated Protein
DpXTH1 and its Gene Cloning in Dahlia Petals
Pingping ZHANG, Xueqin KAN, Jian LI, Chi CHEN, Chengyang LUO, Jianzhong TAN*
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 (201410285049).
*Corresponding author. E-mail: szutjz@hotmail.com
Received: May 22, 2015 Accepted: June 21, 2015A
Agricultural Science & Technology, 2015, 16(7): 1490-1493
Copyright訫 2015, Information Institute of HAAS. All rights reserved Molecular Biology and Tissue Culture
P lant senescence is a develop-mentally regulated and geneti-cally programmed process,
and it is usually observed in many dif-
ferent plant tissues, such as leaves,
petals, roots, seeds and other organs,
ultimately leading to death of a partic-
ular organ or whole plant[1]. Flower ag
ing is accompanied by typical changes
in petal color, shape, fresh weight and
metabolic processes [2]. Flower petals
are good material for studies on plant
senescence and its regulation mecha-
nism, and are also important research
content of ornamental horticulture.
There have been many reports on
molecular mechanism of petal senes-
cence in carnations, petunias, roses
and other ornamental plants[3]. More-
over, a variety of senescence-associ-
ated genes (SAGs) have been cloned,
and they are involved in protein
degradation, ethylene metabolism,
lipid metabolism, cell wall modification
and other physiological functions[3-5].
Previous researches on petal
senescence are mostly concentrated
in ethylene-sensitive flowers[2, 4-6]. Flow-
er senescence is induced and regulat-
ed by the self-generated ethylene.
When the amount of ethylene in plants
reaches or exceeds a certain thresh-
old, a large amount of ethylene will be
induced to be generated, initiating res-
piratory climacterics and the whole
ripening and senescence pro cesses
in flowers [ 6 ] . However , in ethy lene-
insensitive flowers, petal senescence
is initiated by a small amount, instead
of a large amount of ethylene [6 ] . So
Abstract [Objective] This study aimed to explore the molecular mechanism of
senescence in ethylene-insensitive flowers. [Method] The dahlia petals were used as
material, and the senescence-associated proteins were isolated and identified using
two-dimensional electrophoresis, mass spectrometry and an encoding gene was
cloned using molecular biology techniques. [Result] In the two-dimensional elec-
trophorogram of proteins from dahlia petals at building color, full flowering and flow-
er senescence periods, a total of 44 protein spots with differences in expression
level more than two times were detected. From the 44 protein spots, xyloglucan
endotransglycosylase/hydrolases (XTHs), a senescence-associated protein, was iso-
lated and identified and its expression level was increased continuously with the
senescence process of dahlia petals. By using the total RNA of dahlia petals as
material and a pair of degenerate primers, the cDNA sequence of XTH gene was
cloned by RT-PCR. The encoding region of XTH gene has a full length of 882 bp,
encoding 293 amino acid residues, and is named as DpXTH1 (Accession number:
HM053613.1). The cluster analysis showed that the amino acid sequence of
DpXTH1 has high homology with those of XTHs in other plants. [Conclusion] The
isolated and identified DpXTH1 from dahlia petals belonged to the XTH family in
plants, and its biological function was associated with the senescence process and
regulation of dahlia petals.
Key words Dahlia; Petals; Xyloglucan endotransglycosylase/hydrolase; Senescence-
associated protein; Gene cloning
DOI:10.16175/j.cnki.1009-4229.2015.07.039
Agricultural Science & Technology2015
there may be different regulatory path-
ways for petal senescence in ethylene-
insensitive flowers [1]. In this study, the
ethylene-insensitive dahlia petals were
used as material, and a senescence-
associated protein xyloglucan endo-
transglycosylase/hydrolase (XTH) was
isolated from the total petal proteins by
two-dimensional electrophoresis and
identified by mass spectrometry. On
this basis, the encoding sequence of
XTH gene in dahlia was cloned using
experimental techniques in bioinfor-
matics and molecular biology, and
then, the structure characteristics of
XTH gene in dahlia and homology with
different plant species were analyzed,
providing a new experimental basis for
elucidating the molecular mechanism
of petal senescence in dahlia.
Materials and Methods
Plant materials
The tested material was
monopetalous Dahlia (Dahlia pinnata
Cav.) variety ‘Danbanhuang’. Accord-
ing to the degrees of development and
senescence of dahlia petals, the flow-
ering process was divided into three
stages: building color stage (B), full
flowering stage (F) and flower senes-
cence stage (S). The collected petals
were rinsed with double distilled water,
dried, rapidly frozen by liquid nitrogen
and preserved at -70℃.
Protein extraction and two-dimen-
sional electrophoresis
The Dahlia petals were sampled
at B, F and S stages, respectively. The
proteins in Dahlia petals were extract-
ed by phenol-methanol/ammonium
acetate precipitation method[7], and the
proteins concentrations were deter-
mined by Bradford method. The two-
dimensional electrophoresis was per-
formed according to the operational
guidelines of two-dimensional elec-
trophoresis apparatus Ettan IPGphor
(GE Healthcare).
Mass spectrometric analysis of
proteins
The two-dimensional elec-
trophoresis gel was scanned and ana-
lyzed using Image Master 5.0 (GE
Healthcare). The differences in petal
protein expression level among differ-
ent flowering stages were compared.
The in-gel digestion and mass spec-
trometry analysis were completed by
Shanghai Applied Protein Technology
Co., Ltd.
Total RNA extraction from dahlia
petals
Taking the dahlia petals at the full
flowering stage as material, and the
petal total RNA was extracted. The
concentration of total RNA was deter-
mined by spectrophotometry, and the
quality was examined by gel elec-
trophoresis. The extracted total RNA
was reverse transcribed into cDNA us-
ing reverse transcription kit produced
by the Sangon Biotech (Shanghai)
Co., Ltd. After the subpackage, the
cDNA was preserved at -70 ℃ for
use.
RT-PCR
Based on the identified XTH
amino acid sequence and according to
Blast analysis, one pair of degenerate
primers was designed. The sequence
of forward primer was as follows: 5’-
AAGATT (A/G) C (A/G) TTAGT (A/G)
CATGG-3’; while the sequence of re-
verse primer was as follows: 5’-TCAA
(C/T)TCAAATTCCGGC-3’. With the
synthesized cDNA as template, PCR
was carried out. The reaction system
(25 μl) was as follows: 10 × reaction
buffer 2.5 μl, 2.5 mmol/L dNTP 2.5 μl,
Ex Taq DNA polymerase 0.125 μl, for-
ward primer 0.5 μl, reverse primer 0.5
μl, cDNA 1.5 μl, distilled water 17.375
μl. The reaction conditions were as fol-
lows: pre-denaturation at 94 ℃ for 3
min; denaturation at 94 ℃ for 1 min,
annealing at 50℃ for 2 min, extension
at 72℃ for 2 min, 35 cycles; extension
at 72 ℃ for 10 min. The PCR products
were preserved at 4℃.
Cloning and sequencing of PCR
products
The PCR products were exam-
ined by 1.6% agarose gel elec-
trophoresis. The target fragment was
recovered by Agarose Gel DNA Ex-
traction Kit (Sangon Biotech (Shang-
hai) Co., Ltd.), ligated with pUCm-T
and transformed into E. coli TOP10
competent cells. The positive colons
were screened on LB plates with ampi-
cillin (60 μg/ml). The sequencing was
completed by the Sangon Biotech
(Shanghai) Co., Ltd.
Sequence analysis
The sequencing results were
compared with the corresponding nu-
cleic acid and protein sequences in
GenBank using NCBI BLAST. The
multiple comparisons and phylogenet-
ic analysis were performed using
DNAMAN Version 6.
Results
Isolation and mass spectrometric
identification of senescence-asso-
ciated protein XTH in dahlia petals
As shown in Fig.1, the protein
components from dahlia petals were
mainly distributed in the PI range of 5-
8 and molecular weight range of 14-
90 kDa. A total of 432, 421 and 387
protein spots were detected at B, F
and S stages, respectively. And there
were 44 protein spots among which
the differences in expression level
were more than two times. From the
44 protein spots, total 15 spots with
different expression levels and clear
separation were selected for in-gel di-
gestion and MALDI-TOF/TOF mass
spectrometry. The results showed that
the No.D11 protein spot was identified
as xyloglucan glycosyltransferase/hy-
drolase (XTH); the expression level of
XTH was increased with the develop-
ment process of dahlia petals, and the
expression levels at full flower and
flower senescence stages were in-
B, building color stage; F, full flowering stage; S, flower senescence stage.
Fig.1 Morphological characteristics and protein two-dimensional electrophorogram of
dahlia petals
1491
Agricultural Science & Technology 2015
creased by 1.17 and 2.83 times re-
spectively compared with that at build-
ing color stage. According expression
characteristics of senescence-associ-
ated genes in plants [1], XTH in dahlia
could be considered as a kind of petal
senescence-associated protein.
Cloning of cDNA sequence of XTH
gene in dahlia petals
The extracted total RNA from
dahlia petals was reverse transcribed
into cDNA, which was amplified by
PCR using a pair of degenerate
primers. An amplified band in full
length of 900 bp was obtained (Fig.2).
The recovered objective band was lig-
ated with pUCm-T vector and then
transformed. The screening of blue-
white spots was carried out on LB
plates containing ampicillin. The posi-
tive clones were cultured and then
verified by PCR. The results showed
that a clear band, in the same molec-
ular weight with target band, was final-
ly amplified (not shown).
Analysis of cDNA sequence of XTH
gene in dahlia petals
The sequencing results showed
that the size of cloned fragment
was 882 bp; the target fragment
encoded 293 amino acid residues
and 1 stop codon (Fig .3) and was
named as DpXTH1 (accession num-
ber: HM053613.1). The Blast analysis
of encoding sequence in DpXTH1
showed that the homologies between
DpXTH1 and other 100 XTH gene
sequences ranged from 82% to 91%.
Most of the plants belonged to
Compositae. The XTH gene derived
from Helianthus petiolaris showed the
highest homology (91%) with DpXTH1,
indicating that the genetic relationship
between the two XTH genes was
closest.
Homology analysis of XTH amino
acid sequences among different
plants
XTH is a kind of protein that has a
highly conserved structure and is en-
coded by a very large family of genes.
To analyze the homology in XTH be-
tween dahlia and other plants, the
cluster analysis of XTH amino acid
sequences was carried out among
dahlia and other 28 plants. As shown
in Fig.4, the 29 XTH gene sequences
were divided into 4 groups. The
DpXTH1 (HM053613) from dahlia,
XET (DQ235255) from gerbera, XET4
(AF093507) from alfalfa, EXGT
Fig.4 Phylogenetic relationship analysis of amino acid sequences between DpXTH1 from dahlia and XTHs from other plants
Fig.2 Gel electrophorograms of extracted
total RNA (left) and RT-PCR prod-
ucts (right)
Shaded area indicates conserved region;* indicates stop codon.
Fig.3 Nucleotide acid sequence and deduced amino acid sequence of DpXTH1 in Dahlia
petals
1492
Agricultural Science & Technology2015
(Continued from page 1489)
Responsible editor: Xiaoxue WANG Responsible proofreader: Xiaoyan WU
(Senna obtusifolia) [J]. Biocontrol Sci-
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1057-1066.
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WEAVER M A. Biocontrol efficacy of
Colletotrichum truncatum for hemp
sesbania (Sesbania exaltata) is en-
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!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
Responsible editor: Tingting XU Responsible proofreader: Xiaoyan WU
(D88413) from upland cotton and
AtXTH (NM119942) from Arabidopsis
belonged to one group. Among them,
DpXTH1 showed the highest homolo-
gy with TpXTH (JN164663) from
tagetes, followed by CmXET
(HM752243) from chrysanthemum,
GeXET (DQ235255) from gerbera,
GeXET (D884135) from gossypium
and the like. However, the homologies
in XTH amino acid sequence between
dahlia and monocots, such as corn
(U15781), and barely (X93174,
X93175), were relatively low, and the
homologies between DpXTH and
three AtXETs from arabidopsis and
TmXG1 (X68254) from nasturtium
were lowest.
Discussion
Xyloglucan endotransglycosy-
lases/hydrolases (XTHs) are a class of
important cell wall modifying enzymes
associated with plant cell growth and
development[7-8]. Their function is to cut
glycosidic bond in glucan constituting
the cell wall microfibers and transfer
and link the cut oligosaccharide
molecules to ends of similar
molecules. XTHs also have the func-
tion of hydrolytic enzymes, leading to
relaxation and reconstruction of plant
cell walls, thereby participating in a
variety of metabolic processes of plant
growth and development [8-9]. Corollas
are a vital organ for ornamental
plants, and they are also involved in
cell wall modification during petal
senescence[10]. Toikkanen et al. cloned
a XTH gene from gerbera, and they
determined the activity of XTH en-
zyme[11]. Sane et al. found that the ex-
pression level of RbEXPA1 gene was
all up-regulated during the opening
and shedding of rose petals, so they
speculated that XTH was involved in
petal cell wall modification, resulting in
petal falling[12]. In this study, it was de-
tected that the expression level of XTH
was increased with the senescence
process of petals on the two-dimen-
sional electrophorogram of proteins
from dahlia petals. According to ex-
pression characteristics of senes-
cence-associated genes and their defi-
nitions in plants, DpXTH1 can be con-
sidered as a senescence-associated
protein in dahlia petals.
DpXTH1 shows high homologies
among different species. Most of the
plant-derived XTH proteins contain a
highly conserved amino acid residue
region DEIDFEFLG and two double
and half cystine sites[8-9]. In current re-
ports about plant XTH, the encoding
sequences of XTH genes are ulti-
mately obtained with their conserved
sequences combined with RACE tech-
nique. In this study, from the perspec-
tive of differential proteomics, the dif-
ferences in XTH expression level dur-
ing flower senescence were detected.
Based on plant-derived XTH amino
acid sequences, XTH gene sequences
were obtained by electronic cloning. In
addition, one pair of degenerate
primers locating between the start
codon and stop codon was designed,
and the cDNA sequence of XTH gene
coding region in Dahlia petals was di-
rectly cloned by RT-PCR technology.
All the above will lay a foundation for
further study on expression character-
istics of XTH gene at the protein and
mRNA levels.
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