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元宝枫MYB转录因子基因的克隆(英文)



全 文 :Cloning of AtrMYB Transcription Factor Gene
from Acer truncatum
Yanling WANG, Xueying HAO, Zhen FENG*
College of Forestry, Shandong Agricultural University, Taian 271018, China
Supported by Agricultural Elite Cultivar Project of Shandong Province (lkz2014[96]).
*Corresponding author. E-mail: fengzh408@qq.com
Received: December 12, 2015 Accepted: March 11, 2016A
Agricultural Science & Technology, 2016, 17(4): 792-796, 821
Copyright訫 2016, Information Institute of HAAS. All rights reserved Molecular Biology and Tissue Culture
C olored-leaf tree species attractmuch attention in landscapeplanning and greening due to
their colorful leaves, and their applica-
tion is becoming more and more ex-
tensive. Acer truncatum Bunge is an
important arbor species in Acer of Ac-
eraceae having both ornamental and
economic value, but most Acer trun-
catum plants perform ordinary in leaf
color. Therefore, our previous study
established its hybrid segregation
population, and found a number of
lines with good ornamental traits, am-
ong which ‘Luhong No.1’ showed
bright red leaves in autumn for a long
time period with high ornamental val-
ue, providing a precious experimental
material for the research of the
molecular mechanism of leaf color.
There have been many reports about
the synthesis and regulation of antho-
cyanin in plants. It was reported that
the development of the red color of
plant organs was related to antho-
cyanin metabolism, the synthesis of
anthocyanin was a coordinated ex-
pression process of its structural gene
under the regulation of corresponding
transcription factor [1-4], the expression
of the structural gene was mainly reg-
ulated by the MYB transcription factor,
and furthermore, MYB, BHLH and
WD40 also could form a protein com-
plex for the co-regulation of the ex-
pression of the anthocyanin structural
gene [4-6]. The MYB transcription factor
is one of the largest transcription factor
families, and 130 kinds have been
found only in Arabidopsis thaliana, ac-
counting for 0.2%-0.6% of the coding
genes of the genome of Arabidopsis
thaliana. The MYB transcription factor
widely participates in the regulation of
plant secondary metabolism and re-
sponses to hormones and environ-
mental factors, and plays an important
role in regulation of cell differentiation,
cell cycle, organ formation and mor-
phogenesis of plant leaves. According
to the number of contained MYB
structural domains, the MYB transcrip-
Abstract [Objective] Cloning of the AtrMYB transcription factor gene from Acer
truncatum was conducted to further explore the red leaf development mechanism
and breed cultivars of colored-leaf maple. [Method] The Acer truncatum ‘Luhong
No.1’ cultivar was used as the material for cloning the MYB gene by mean of RT-
PCR and RACE-PCR. [Results] Sequence analysis showed that the fragment con-
tained a full coding region of 831 bp encoding 276 amino acid residues with a
molecular weight of 32.17kD and a molecular formula C1430H14052N2247O406S14. The gene
was named as AtrMYB with a GenBank accession number of 1825712. This coded
protein had a pI of 9.44. The results showed that the AtrMYB exhibited typical fea-
tures of the R2R3-MYB domain. The AtrMYB was highly homologous with the MYB
of other species at nucleotide and amino acid levels. The AtrMYB had no signal
peptide, but a nuclear localization signal. The phylogenetic tree showed that the
AtrMYB was at the same clade as the MYB from Citrus sinensis. [Conclusion] The
AtrMYB was cloned from Acer truncatum ‘Luhong No.1’ cultivar. These results
have provided a foundation for further purification and identification of target protein
and function study of the AtrMYB.
Key words Acer truncatum; MYB transcription factor; Gene cloning
元宝枫 MYB 转录因子基因的
克隆
王延玲,郝雪英,丰震* (山东农业大学林学
院,山东泰安 271018)
摘 要 [目的 ]克隆元宝枫转录因子 MYB 基
因, 为进一步研究元宝枫 MYB 基因功能和对
其花青苷结构靶基因的调控研究奠定基础。 [方
法]以元宝枫‘鲁红 1 号’为材料,采用 RT-PCR
和 RACE-PCR 方法,克隆元宝枫‘鲁红 1 号’中
MYB 基因。 [结果] 测序结果显示该基因全长
831 bp, 编码 276 个氨基酸, 该蛋白分子量为
32.17 kDa,分子式为 C1430H14052N2247O406S14,原子
总数为 4 510 个,等电点为 9.44,GenBank 登录
号为 1825712, 命名为 AtrMYB。 该蛋白具有
R2R3MYB 结构域,该蛋白偏疏水性,没有信号
肽,具有核定位信号。 氨基酸序列比对发现与
其它物种的 MYB 有较高的同源性, 进化树分
析表明,AtrMYB 与调控橙子花青苷合成的转
录因子 MYB 亲缘关系最近,处在同一进化枝。
[结论]从元宝枫‘鲁红 1 号’中成功获得了元宝
枫转录因子 MYB 基因。
关键词 元宝枫;MYB 转录因子;基因克隆
基金项目 山东省农业良种工程项目,鲁科字
(2014[96 号])。
作者简介 王延玲(1968-),女,山东莱芜人 ,
副教授 , 主要从事园林植物研究 , E-mail:
wangyl219@126.com。 *通讯作者 ,E-mail:feng-
zh408@qq.com。
收稿日期 2015-12-12
修回日期 2016-03-11
DOI:10.16175/j.cnki.1009-4229.2016.04.006
Agricultural Science & Technology2016
tion factor is divided into proteins with
single MYB structural domain, proteins
with 2 repeated MYB structural do-
mains and proteins with 3 repeated
MYB structural domains, and in plants,
the MYB transcription factor is mainly
the proteins with 2 repeated MYB
structural domains (R2R3MYB), which
are divided into 24 subgroups accord-
ing to their functions. Among them, the
tenth subgroup MYB10 mainly partici-
pates in the regulation of anthocyanin
synthesis metabolism. The MYB
genes for the regulation of antho-
cyanin synthesis in many plants have
been cloned and identified, and the
metabolism and regulation of antho-
cyanin have become a research
hotspot[7-11].
Currently, the studies on coloring
of Acer truncatum have focused on the
metabolism of anthocyanin mainly[11-15],
and the genes MYB, BHLH and WD40
in Acer truncatum for the regulation of
anthocyanin synthesis have not been
reported so far. The study was con-
ducted to obtain the MYB transcription
factor gene for the regulation of anoth-
ocyanin synthesis by cloning and se-
quence analysis of the MYB gene from
Acer truncatum ‘Luhong No.1’, so as
to lay a foundation for further studies
on the function of Acer truncatum MYB
gene and the regulation of target gene
for anthocyanin synthesis .
Materials and Methods
Plant materials
This experiment was carried out
in 2014 in Biotechnology Research
Center of Shandong Agricultural Uni-
versity. The tested line was five-year-
old Acer truncatum ‘Luhong No.1’,
and the stock was Acer truncatum
seedlings. These experimental mate-
rials were all collected from the Land-
scape Plant Experiment Station of
Shandong Agricultural University. Col-
lected fresh plant materials were
placed in a refrigerator at -70 ℃ for
later use.
Main reagent including strain and
vector
The Escherichia coli strain DH5
and pMD18-T Vector for cloning were
both purchased from Takara Biotech-
nology (Dalian) Co. Ltd. TRIZOL Kit
was purchased from Beijing Dingguo
Changsheng Biotechnology Co., Ltd.;
EASY spin Plus Plant RNA Rapid Ex-
traction Kit was purchased from Aidlab
Biotechnologies Co., Ltd.; Agarose
Gel Extraction Kit (DP209-02) was
purchased from Tiangen Biotech
(Beijing) Co., Ltd.; MightyAmp DNA
Polymerase Ver.2 Kit and EasyTaqTM
First-Strand cDNA Synthesis Super-
Mix Kit were both purchased from Bei-
jing TransGen Biotech Co., Ltd.; 5’
RACE System for Rapid Amplification
of cDNA Ends,Version 2.0 (18374 -
058) Kit was purchased from Invitro-
gen corporation; and SMARTerTM
RACE cDNA Amplification Kit
(634923) was purchased from Clon-
tech corporation.
Primer design
The degenerate primers (F and R)
designed by Feng et al. [2] were used,
and the anticipated fragment was 250
bp in size. According to the intermedi-
ate fragment sequence obtained by
sequencing, 5’ RACE primers (A157-
1 (GSP1), A157-2 (GSP2) and A157-3
(GSP3) of the gene were designed ac-
cording to the design principle of the
specific primers of the 5’ RACE Kit of
Invitrogen corporation; and 3’ RACE
primers (3’ 744-1 and 3’ 744-2) of the
gene were designed according to the
design principle of the specific primers
of the 3’ RACE Kit of Clontech corpo-
ration, and they were synthesized by
Sangon Biotech (Shanghai) Co., Ltd.
The primers were shown in Table 1.
RNA extraction and cloning of Atr-
MYB
The total DNA from the bright red
leaves of Acer truncatum ‘Luhong No.
1’ was extracted by the improved
CTAB method, and subjected to re-
verse transcription according to the in-
struction of the EasyScriptTM First-
Strand cDNA Synthesis SuperMix Kit,
obtaining cDNA which was stored at -
20℃ for later use. With the cDNA as a
template, the degenerate primers
(Table 1) were designed according to
the conserved sequence of the MYB-
gene, and PCR amplification was per-
formed. The PCR conditions were as
follows: 94℃ for 2 min; 35 cycles of 94
℃ for 30 s, 45 ℃ for 30 s and 72℃ for
1 min; and extension at 72 ℃ for 5
min. The intermediate fragment highly
homologous with the MYB was select-
ed, primers were designed to perform
5’ and 3’Race, and PCR was per-
formed according to the instruction of
Smart Race. The 5’ and 3’end frag-
ments were ligated, and specific
primers were designed to perform
PCR for the full-length gene. The PCR
conditions were as follows: 94 ℃ for 2
min; and 35 cycles of 94 ℃ for 30 s,
56.2 ℃ for 30 s and 72 ℃ for 1 min.
The PCR product was recovered ac-
cording to Agarose Gel Extraction Kit
(DP209-02) from Tiangen Biotech
Table 1 Specific primers used in gene cloning
Classification Primer name Primer sequence
Intermediate fragment F TGYATHRAYAARTAYGGIGARGGIAARTGG
R GTRTTCCARTARTTYTTIACRTCRTTNGC
5 RACE primer A157-1(GSP1) TGCAGCCTCAAAATCA
A157-2(GSP2) CTCCTCTCTTGATATTTGGC
AAP GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG
A157-3(GSP3) CAATCTACAACTTTTCCGGC
AUAP GGCCACGCGTCGACTAGTAC
3 RACE primer 3 CDS primer A AAGCAGTGGTATCAACGCAGAGTAC(T)30VN
UPM CTAATACGACTCACTATAGGGC
3744-1 ATGAAGTCGATATGATTTTGAGGCTGCA
3744-2 GTTGGGAAACAGATGGTCATTGATTGC
793
Agricultural Science & Technology 2016
A: Intermediate fragment; B: 5 RACE-PCR; C: 3 RACE-PCR; M: DL2000 Marker.
Fig. 1 Gel electrophoresis of PCR amplification product
Fig. 2 Signal peptide of the protein coded by the MYB transcription factor gene
Table 2 Secondary structure ofthe protein coded by theAtrMYB transcription factor gene
Secondary structure Number of amino acids Contents
Alpha helix (Hh) 93 33.70%
Extended strand (Ee) 51 18.48%
Beta turn (Tt) 19 6.88%
Random coil (Cc) 113 40.94%
(Beijing) Co., Ltd. The recovered
product was ligated with the pMD18-T
vector and transformed into E. coli
DH5α competent cells, and positive
clones were screened, confirmed by
PCR, and sequenced by Beijing Ge-
nomics Institute.
Bioinformatics analysis of AtrMYB
Homologous sequence alignment
was performed with the BLASTn pro-
gram on NCBI, DNAMAN was used
for analyzing AtrMYB ORF, the amino
acid sequence was then deduced, and
the online software SOPMA was used
for predicting the secondary structure
of the protein coded by the MYB tran-
scription factor gene. A phylogenetic
tree was established with MEGA5.0.
Results
Cloning of full-length cDNA of Atr-
MYB gene from Acer truncatum
‘Luhong No.1’
The degenerate primers F and R
(Table 1) were designed according to
the conserved sequence of the MYB
gene, and RT-PCR was performed
with the cDNA of Acer truncatum
‘Luhong No.1’ as a template, obtain-
ing a band of about 250 bp (lane 1 in
Fig. 8-A); and the first chain cDNA of
the target gene was synthesized with
primer GSP-1 and SUPERSCCRIPT II
RT enzyme. Amplification was per-
formed with GSP-2/APP as primers
and the first chain cDNA as a tem-
plate, and the obtained product was
used as the template for nested PCR
with GSP-3/AUAP as primers, by
which a band of about 260 bp (lane 1
in Fig. 8-B) was obtained; and
SMARTScribeTM Reverse Transcrip-
tase and the 3’ CDS primer were used
for the reverse transcription of RNA,
obtaining desired cDNA. Then, ampli-
fication was performed with 3’744-1/
UPM as primers and the cDNA as a
template, and the product was used as
the template for nested PCR with 3’
744-2/UPM as primers, obtaining a
band of about 730 bp (lane 1 of Fig.8-
C). The intermediate fragment, 5’ter-
minal sequence and 3’terminal se-
quence were spliced, giving the gene
sequence with a total length of 831 bp,
which was designated as AtrMYB with
a GenBank accession number of
1825712.
Sequence analysis of AtrMYB gene
of Acer truncatum ‘Luhong No.1’
The sequence of the cloned gene
and the sequence of the coded protein
were analyzed with the EditSeq pro-
gram of DNAStar software package,
Prot-Param tool in ExPasy server,
CDS program on NCBI, SignalP 4.1
Server andsoftware DNAMAN; ORF
Finder was used for finding the largest
open reading frame (ORF); and
BLASTp and B1astX analysis were
performed on the nucleotide sequence
of the MYB transcription factor by
NCBI, to verify the correctness of the
ORF. It was found by the analysis that
AtrMYB had a total length of 831 bp
coding 276 amino acid residues, the
coded protein had a molecular weight
of 32 169.9 Da and a molecular for-
mula C1430H14052N2247O406S14, and con-
tained 4 510 atoms in total, and its pI
was 9.44.The protein showed an insta-
bility index of 48.46 (>40), and it was
thus speculated as an instable protein.
The fat index was 72.07, and the aver-
age hydrophilic index was -0.738, indi-
cating that the protein was weakly hy-
drophobic. From the point of signal
peptide (as shown in Fig. 2), the Y val-
ue and C value were tend to be 0; and
as to the S value, the condition of the
S value before the splice site higher
than that after the splice site was not
satisfied, the score of the amino acid
at the N terminal was also lower, and it
was thus speculated that the protein
coded by the MYB transcription factor
gene contained no signal peptide. It
was found by the alignment between
the AtrMYB amino acids of ‘Luhong
No.1’ and other MYB transcription fac-
tors that the AtrMYB contained obvi-
ously 2 repeated MYB domains
(R2R3MYB), which consisted of 46
and 44 amino acids, respectively; and
the AtrMYB transcription factor was
highly homologous with other MYB
transcription factors (Fig. 3). For in-
stance, it had the homology of 54%
with the MYB transcription factor of
Citrus sinensis, the homology of 49%
794
Agricultural Science & Technology2016
Fig. 3 Alignment of Amino acid sequences of MYB domains between the AtrMYB transcription factor and other MYB proteins
Fig.4 Phylogenetic tree of the AtrMYB of Acer truncatum ‘Luhong No. 1’ and some other
MYB proteins
Fig. 5 Secondary structure of the protein coded by the AtrMYB transcription factor gene
with the R2R3MYB transcription factor
of Fragaria vesca, the homology of
47% with the MYB10 of Ribes nigrum,
the homology of 50 % with the MYB1
of Morella rubra, the homology of 48%
with the MYB10 V2 of Prunus avium,
the homology of 47% with the MYB10
of Garcinia mangostana L., the ho-
mology of 47% with the MYB10a of
Malus domestica, and the homology of
50% with the Petunia hybrid ‘PURPLE
HAZE’. The conservative tryptophan
residue W existed in both the R2 and
R3 domains. The coding region in-
cluded a transactivation domain rich in
acidic amino acids at the C-terminal
besides the typical R2R3MYB con-
served domain, and it was thus specu-
lated that the gene was a typical
R2R3MYB transcription factor gene.
Phylogenetic tree analysis of Atr-
MYB from Acer truncatum Luhong-
No. 1
In order to determine the evolu-
tionary relationship between the Atr-
MYB protein coded by the cloned gene
and other MYB proteins, a phylogenet-
ic tree of the AtrMYB and other MYB
transcription factors was established
and analyzed with the software
MEGA5.0. The result showed that
‘Luhong No.1’ and Citrus sinensis
were located on the same clade, indi-
cating that the genetic relationship be-
tween them was closer, i.e., the ho-
mology between them were higher;
and ‘Luhong No.1’ exhibited closer
genetic relationships with such species
as Morella rubra, Ribes nigrum, Vitis
vinifera and Theobroma cacao, but
farther genetic relationships with such
species as Capsicum annuum, Ipo-
moea batata and Fragaria vesca (Fig.
4), which accorded with conventional
classification, and the MYB genes of
these plants were all proved to regu-
late the biosynthesis of anthocyanin.
These results interpreted the molecu-
lar evolution process of the protein
coded by the AtrMYB transcription
factor gene of ‘Luhong No.1’, and al-
so indicated that the AtrMYB transcrip-
tion factor gene of ‘Luhong No.1’ had
the function of regulating the biosyn-
thesis of anthocyanin.
Prediction of secondary structure
of protein coded by AtrMYB tran-
scription factor gene of Acer trun-
catum ‘Luhong No.1’
The secondary structure of the
protein coded by the MYB transcription
factor gene was predicted with the on-
795
Agricultural Science & Technology 2016
line software SOPMA, and the result
was shown in Table 4 and Fig. 5. The
secondary structure of the protein
contained alpha helix, extended
strand, beta turn and random coil,
which accounted for 33.70%, 18.48%,
6.88% and 40.94% , respectively.
Therefore, the protein coded by the At-
rMYB transcription factor gene con-
tained random coil mainly, and α-helix
secondarily.
Conclusions
This study successfully acquired
from ‘Luhong No.1’, the Acer trunca-
tum transcription factor MYB gene with
a total length of 831 bp, which was
designated as AtrhMYB with a Gen-
Bank accession number of 1825712.
The gene coded 276 amino acids, and
the coded protein had a molecular
weight of 32 169.9 Da and a molecular
formula C1430H14052N2247O406S14, and con-
tained 4 510 atoms in total, and its pI
was 9.44. The protein included a
R2R3MYB domain, and was weakly
hydrophobic. It had no signal peptide,
but a nuclear localization signal. It was
found by the amino acid sequence
alignment that the AtrhMYB was highly
homologous with the MYB transcrip-
tion factors of other species. The phy-
logenetic tree analysis showed that the
AtrhMYB had the closest genetic rela-
tionship with the MYB transcription
factor regulating the anthocyanin syn-
thesis of Citrus sinensis as they were
located on the same clade.
Discussion
Since the isolation of the MYB
transcription factor C1 for the regula-
tion of plant anthocyanin metabolism[16],
hundreds of MYB genes have been
isolated and cloned from a variety of
plants such as Arabidopsis thaliana,
maize, Petunia xhybrida, Antirrhinum
majus, Vitis vinifera and Malus do-
mestica. Theses MYB genes are con-
servative in sequence, but similar in
structure to a certain degree. However,
the function of the MYB protein varies
greatly in different varieties, different
individuals and even between different
organs of the same individual [2,10,17 -23].
Currently, the functions of many MYB
transcription factors are unclear yet,
and therefore, it is very important to
understand their functions in the as-
pects of regulation of plant secondary
metabolism and responses to hor-
mones and environment stress. This
study isolated an MYB transcription
factor gene from the autumn leaves of
Acer truncatum ‘Luhong No.1’ by RT-
PCR and RACE-PCR. The structural
analysis showed that the gene had a to-
tal length of 831 bp coding 276 amino
acid, the coded protein had a molecular
weight of 32 169.9 Da and a chemical
formula of C1430H14052N2247O406S14, and
contained 4510 atoms in total, and
its pI was 9.44. The protein showed
an instability index of 48.46 (>40),
and it was thus speculated as an in-
stable protein. The fat index was
72.07, and the average hydrophilic
index was -0.738, indicating that the
protein was weakly hydrophobic. From
the point of signal peptide, the Y value
and C value were tend to be 0; and as
to the S value, the condition of the S
value before the splice site higher than
that after the splice site was not satis-
fied, the score of the amino acid at the
N terminal was also lower, and it was
thus speculated that the protein coded
by the MYB transcription factor gene
contained no signal peptide. The cod-
ing region of the AtrMYB transcription
factor gene contained 2 MYB do-
mains, which consisted of 46 and 44
amino acids, respectively; and the con-
servative tryptophan residue W exist-
ed in both the R2 and R3 domains.
The coding region included a transac-
tivation domain rich in acidic amino
acids at the C-terminal besides the
typical R2R3MYB conserved domain,
and it was thus speculated that the
gene was a typical R2R3MYB tran-
scription factor gene.The phylogenetic
tree analysis showed that ‘Luhong No.
1’ and Citrus sinensis were located on
the same clade, indicating that the ge-
netic relationship between them were
closer, i.e., the homology between
them were higher; and ‘Luhong No.1’
exhibited closer genetic relationships
with such species as Morella rubra,
Ribesnigrum, Vitisvinifera and Theo-
broma cacao, but farther genetic rela-
tionships with such species as Cap-
sicum annuum, Ipomoea batatas and
Fragaria vesca, and the MYB genes of
these plants were all proved to regu-
late the biosynthesis of anthocyanin. It
was thus speculated that the AtrMYB
transcription factor gene of ‘Luhong
No.1’ might participated in the regula-
tion of the transcription of the antho-
cyanin structural gene. In order to un-
derstand the specific biochemical and
molecular biological functions of the
AtrMYB protein in the red color devel-
opment in the leaves of ‘Luhong No.
1’ in autumn, further studies about the
gene expression, gene function verifi-
cation and protein expression are
needed.
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