全 文 ：Cloning and Sequence Analysis of CHS Gene
Fragment from Acer truncatum
Limin MA, Zhen FENG*, Xinling QI
College of Forestry, Shandong Agricultural University, Tai’an 271018, China
Supported by Agricultural Improved Variety Project of Shandong Province (LKZ[2014]
No.96).
*Corresponding author. E-mail: fengzh@sdau.edu.cn
Received: April 30, 2016 Accepted: June 26, 2016A
Agricultural Science & Technology, 2016, 17(7): 1658-1662, 1678
Copyright訫 2016, Information Institute of HAAS. All rights reserved Molecular Biology and Tissue Culture
A cer truncatum Bunge is aperennial woody plant belong-ing to the genus Acer of family
Aceraceae, which has been widely
used and concerned in landscaping as
a famous ornamental tree with beauti-
ful shape. The tender leaves are red
and turn yellow or red in autumn. At
present, little information is available
about the molecular mechanism of leaf
color formation of A. truncatum, and
no studies have been reported on
cloning and sequence analysis of leaf
color formation-related genes in A.
truncatum, which greatly limits molec-
ular breeding of A. truncatum and uti-
lization of leaf color formation-related
genes. In this study, using leaves of A.
truncatum cultivars No.1-6 as experi-
mental materials, CHS, a key gene in-
volved in anthocyanin synthesis, was
isolated by RT-PCR for sequence
analysis, aiming at revealing the
mechanism of anthocyanin synthesis
in A. truncatum leaves at the nucleic
acid and protein levels, which not only
laid the foundation for subsequent
analysis of the transcriptional expres-
sion of key genes involved in different
developmental stages and breeding of
new transgenic varieties, but also pro-
vided important molecular biological
basis for the research of colored-leaf
plants in China[1-8].
Materials and Methods
Materials
Leaves of A. truncatum cultivars
No.1 -6 were collected from the test
base of Shandong Agricultural Univer-
sity in summer and autumn.
Primer design
(1) Upstream fragment
Forward primer CHS5-8: ACYC-
CNCCNAAYTGTGTBGATC
Reverse primer CHS5-2: TTCG-
GAACACACGACGAGGAC
(2) Intermediate fragment
Forward primer CHSZ-1: YGC-
NAGRCARGAYATGGTGGT
Reverse primer CHSZ-2: CCR-
AANCCRAANAGVACNCC
(3) 3’Race
GSP-1: AGCCTTGTTCGGT-
Abstract [Objective] This study aimed to clone and analyze the sequence of CHS
gene from Acer truncatum leaves. [Method] Using A. truncatum cultivars No.1-6 as
experimental materials, total RNA was extracted from A. truncatum leaves with the
modified CTAB method. CHS gene sequences were downloaded from the NCBI and
aligned by BLAST. Degenerate primers were designed by DNAMAN and Primer-
premier5 to amplify the target band. CHS gene fragment was amplified by RT-PCR
and ligated to pMD18-T vector. The identified positive colonies were sequenced.
[Result] A 1 365 bp fragment was amplified. Sequence analysis suggested that the
obtained fragment encoded 365 amino acids and shared above 90% homology to
nucleotide sequence of CHS gene from A. palmatum and A. maximowiczianum.
[Conclusion] In this study, CHS gene was successfully cloned from A. truncatum for
the first time, which laid the foundation for efficient utilization of CHS gene.
Key words Acer truncatum; Chalcone synthase; Cloning; Sequence analysis; RT-
PCR
元宝枫 CHS 基因片段的克隆
及序列分析
马立敏，丰震*，齐新玲 （山东农业大学林学
院，山东泰安 271018）
摘 要 [目的] 对元宝枫叶片查尔酮合成酶
（CHS）基因片段进行克隆及序列分析。[方法]以
元宝枫的红叶新品系 1~6 号为试材 ， 采用
CTAB 法提取其夏季与秋季叶片总 RNA， 搜索
GenBank 数据库中已报道的 CHS 基因序列 ，
BLAST 比对 ，然后用 DNAMAN、Primerpremier5
软件设计兼并引物来扩增其目的片段 ， 采用
RT-PCR 方法扩增 CHS 基因片段并连接到
pMD18-T 载体上，阳性克隆经 PCR 检测后进行
测序。 [结果]得到一段 1 365 bp 的序列，序列
分析表明，该片段编码 365 个氨基酸，与鸡爪
槭、毛果槭的查尔酮合成酶（CHS）基因核苷酸
序列同源性在 90%以上。 [结论]该研究首次从
元宝枫中克隆出了 CHS 基因，为有效利用该基
因奠定了基础。
关键词 元宝枫查尔酮；合成酶；基因克隆序
列分析；RT-PCR
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 马立敏(1990-)，女，山东德州人，硕
士研究生， 研究方向为园林植物资源及利用。
E-mail:283501950@qq.com。*通讯作者。E-mail:
fengzh@sdau.edu.cn。
收稿日期 2016-04-30
修回日期 2016-06-26
DOI:10.16175/j.cnki.1009-4229.2016.07.029
Agricultural Science & Technology2016
GATGGTG
GSP-2: GGGCTCACATTCCAC-
CTCCT
B26: GACTCTAGACGACATC-
GATTTTTTTTTTTTTTT
Methods
Primer design
The amino acid and nucleic acid
sequences of CHS gene from several
plant species were downloaded from
the NCBI. The conserved regions
were compared by DNAMAN. Degen-
erate primers were designed with
Primer premier5.0 based on con-
served region sequences.
Total RNA extraction
A. truncatum leaves are rich in
polysaccharides and polyphenols,
which may restrict the extraction of to-
tal RNA. In this study, total RNA was
extracted from A. truncatum leaves
with the modified CTAB method[9].
RT-PCR assay
cDNA was obtained by reverse
transcription according to kit introduc-
tions. RNA for 3’Race was added with
B26 before reverse transcription.
PCR reaction system for amplifi-
cation of upstream fragment:
Double distilled water was added
to a final volume of 25 μl.
The PCR amplification was start-
ed with initial denaturation at 94 ℃ for
5 min, followed by 35 cycles of denat-
uration at 94 ℃ for 1 min, annealing at
58 ℃ for 45 s, and extension at 72 ℃
for 1 min; the amplification was com-
pleted by holding the reaction mixture
at 72℃ for 10 min.
PCR reaction system for amplifi-
cation of intermediate fragment:
Double distilled water was added
to a final volume of 25 μl.
The PCR amplification was start-
ed with initial denaturation at 94 ℃ for
5 min, followed by 35 cycles of denat-
uration at 94 ℃ for 1 min, annealing at
57 ℃ for 45 s, and extension at 72 ℃
for 1 min; the amplification was com-
pleted by holding the reaction mixture
at 72℃ for 10 min.
3’Race PCR reaction system:
Double distilled water was added
to a final volume of 25 μl.
The PCR amplification was start-
ed with initial denaturation at 94 ℃ for
5 min, followed by 35 cycles of denat-
uration at 94 ℃ for 1 min, annealing at
57 ℃ for 45 s, and extension at 72 ℃
for 1 min; the amplification was com-
pleted by holding the reaction mixture
at 72℃ for 10 min.
The above products were diluted
500 times as the template for PCR.
Double distilled water was added
to a final volume of 25 μl.
The PCR amplification was start-
ed with initial denaturation at 94 ℃ for
5 min, followed by 35 cycles of denat-
uration at 94 ℃ for 1 min, annealing at
57 ℃ for 45 s, and extension at 72 ℃
for 1 min; the amplification was com-
a: Upstream fragment; b: intermediate fragment; c: 3Race fragment; M: Marker DL2000
Fig. 2 Agarose gel electrophoresis of PCR product
Fig. 1 Agarose gel electrophoresis of total
RNA extracted from A. truncatum leaves
Component Volume
Template cDNA 1.0 μl
PCR SuperMix 12.5 μl
CHS3-2 1.0 μl
B26 1.0 μl
Component Volume
Template cDNA 1.0 μl
PCR SuperMix 12.5 μl
CHSZ-1 1.0 μl
CHSZ-2 1.0 μl
Component Volume
Template cDNA 1.0 μl
PCR SuperMix 12.5 μl
CHS5-8 1.0 μl
CHS5-2 1.0 μl
Component Volume
Template cDNA 1.0 μl
PCR SuperMix 12.5 μl
CHS3-1 1.0 μl
B26 1.0 μl
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Agricultural Science & Technology 2016
Fig. 3 Nucleotide and deduced amino acid sequences of CHS gene fragment from A. truncatum
pleted by holding the reaction mixture
at 72℃ for 10 min.
Ligation and transformation
RT-PCR products were detected
by agarose gel electrophoresis and
purified. The ligation volume was 10 μl,
containing 5 μl of Solution I, 4.5 μl of
purified product, and 0.5 μl of pMD18-
T vector. The mixture was incubated
for 4-5 h.
The 10 μl aliquot of ligation prod-
uct was mixed evenly with 50 μl of E.
coli competent cells, incubated at 42
℃ for 90 s, allowed to stand in an ice
bath for 3 min, added with 750 μl of LB
liquid medium (without AMP), and cul-
tured with shaking for 1 h; the bacterial
liquid was coated on LB solid medium
(containing AMP) and cultured upside
down at 37 ℃ for 12 -16 h. Single
colonies were selected, inoculated to
LB liquid medium (containing AMP),
and cultured in a shaker at 37℃ for 10
h. The plasmid was extracted and de-
tected by PCR. Positive plasmid was
sequenced by BGI Tech.
Results and Analysis
Total RNA extraction
The electrophorogram of total
RNA extracted from A. truncatum
leaves was shown in Fig. 1. The con-
centration and OD value were in ac-
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Agricultural Science & Technology2016
Fig. 4 Multiple comparisons between amino acid sequences of CHS genes from A. truncatum and other plants
cordance with the requirements for
subsequent analysis.
RT-PCR amplification
By using primers CHS5-8 and
CHS5-2, a 500 bp upstream fragment
was amplified (Fig. 2-a); by using
primers CHSZ-1 and CHSZ-2, a 850
bp intermediate fragment was ampli-
fied (Fig. 2-b). After 3’ nested PCR, a
600 bp specific fragment was amplified
(Fig. 2-c).
Sequence analysis
According to the sequencing re-
sults, a 1 365 bp nucleotide sequence
was obtained. The open reading frame
was searched in the NCBI, and the
coding region of the sequence was
found at 1-1 098 bp. It was speculated
that the cDNA sequence encoded 365
amino acids (Fig. 3). By using DNA-
man software, the coding region of the
sequence was translated into amino
acid sequence for protein Blast in the
NCBI. The results indicated that amino
acid sequence encoded by the ob-
tained gene fragment shared above
92% similarity to proteins encoded by
CHS genes from Acer maximow-
iczianum, Acer palmatum, Gossypium
hirsutum, Theobroma cacao, Actinidia
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Agricultural Science & Technology 2016
Fig. 5 Phylogenetic tree of CHS gene from A. truncatum
chinensis, Populus euphratica, Abel-
moschus manihot, Citrus sinensis,
Rhus chinensis, Mangifera indica and
Camellia japonica, and the nucleotide
similarity was above 78% (Fig. 4).
The conserved domains in the ob-
tained fragment were searched in the
NCBI. The results showed that the
fragment contained CHS-Like domain
(cd00831), suggesting that the ob-
tained fragment belonged to the chal-
cone synthase gene family.
Phylogenetic analysis of amino
acid sequence of CHS gene from A.
truncatum
By using Alignment and Phyloge-
ny procedures of MEGA5.10 software,
a phylogenetic tree was constructed
based on amino acid sequences of
CHS genes from Acer maximow-
iczianum, Acer palmatum, Gossypium
hirsutum, Theobroma cacao, Actinidia
chinensis, Populus euphratica, Abel-
moschus manihot, Citrus sinensis,
Rhus chinensis, Mangifera indica and
Camellia japonica and the obtained
fragment with neighbor-joining method
(Fig. 5).
As shown in Fig. 5, as three
species from the family Aceraceae, A.
maximowiczianum, A. palmatum and
A. truncatum shared the closest genet-
ic relationship. Moreover, A. truncatum
had a closer relationship with A. pal-
matum compared to A. maximow-
iczianum. These three species were
clustered into the same category as
Citrus sinensis. Therefore, A. maxi-
mowiczianum, A. palmatum and A.
truncatum had a closer relationship
with Citrus sinensis than other
species.
Discussion
In this study, by RT-PCR and 3’
Race, CHS gene fragment was suc-
cessfully cloned from A. truncatum
leaves for the first time. Through se-
quence alignment in the NCBI, it could
be inferred that the obtained cDNA se-
quence could encode a CHS protein,
which provided reference for molecular
biological research of colored-leaf tree
species.
As a class of natural secondary
metabolites, flavonoids are widely dis-
tributed in various terrestrial plants,
which can not only play an important
role in anthocyanin accumulation,
stress resistance, antibiosis and cell
development in plants, but also im-
prove human health significantly, such
as treating and preventing cardiovas-
cular diseases and tumors [10]. Chal-
cone synthase is a key enzyme in-
volved in biosynthetic pathways of
flavonoids such as anthocyanin in
plants. The mutation, silencing or over-
expression of chalcone synthase gene
may directly or indirectly affect biosyn-
thesis of various flavonoids, thereby
exerting certain effects on flower color,
leaf color, stress resistance and
flavonoid content of plants. So far, a
large number of studies have been
carried out about characteristics of
chalcone synthase and regulation of
the expression of chalcone synthase
genes, most of which are focused on
model plants such as Antirrhinum ma-
jus and Petunia hybrida, but little is
known about chalcone synthase gene
from A. truncatum. A. truncatum is not
only a beautiful colored-leaf tree
species, but also a novel oil crop.
Therefore, the research of chalcone
synthase could not only lay the foun-
dation for regulation and control of leaf
color and improvement of anthocyanin
content of A. truncatum, but also pro-
mote cloning of other key enzyme
genes and transcription factor genes
involved in anthocyanin biosynthesis.
Conclusion
In this study, total RNA was suc-
cessfully extracted from A. truncatum
leaves with the modified CTABmethod.
By RT-PCR and 3’Race, a 1 365 bp
fragment was amplified, containing an
open reading frame at 1 -1 098 bp.
The obtained fragment encoded 365
amino acids and shared above 90%
homology to nucleotide sequence of
CHS gene from A. palmatum and A.
maximowiczianum. The obtained frag-
ment contained CHS-Like domain
(cd00831), indicating that it belonged
to the chalcone synthase gene family.
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