全 文 ：Cloning and Analysis of a Chalone Synthase
Gene from Fagopyrum tataricum
Kai LIU1, 2, Yaohui HU1, Guan WANG3, Hansong YU1*
1. College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China;
2. Jilin Agricultural Comprehensive Development Office, Changchun 130118, China;
3. Academy of Life Science, Jilin Agricultural University, Changchun 130118, China
Supported by 948 Program, Ministry of Agriculture of China (2008-z27).
*Corresponding author. E-mail: yuhansong@jluhp.edu.cn
Received: January 31, 2012 Accepted: February 11, 2012A
Abstract [Objective] This study aimed to clone a full-length CHS gene from buck-
wheat. [Method] With total RNA extracted from buckwheat as the template, CHS cD-
NA sequence was cloned from buckwheat by using RACE technology and CODEHOP
primer design method, the full length gene was obtained by primers which were de-
signed for amplification of full-length gene sequence with buckwheat DNA template.
Clustalxl.81 and MEGA4 software were used for sequence analysis and construction
of phylogenetic tree; NCBI Blastn and Blastp programs were applied for homology
analysis of nucleic acid and protein. [Result] Bioinformatics analysis showed that the
full length of this gene is 1 906 bp, containing a 463 bp intron sequence and a 1 188
bp coding region, encoding 395 amino acids. Blastn sequence alignment revealed that
the CHS gene sequence obtained in this study shared 86% homology with the CHS
gene of closely related species. [Conclusion] This study laid the foundation to clarify
molecular basis of the synthesis of buckwheat bioflavanoids and explore an effective
way to improve the content of buckwheat bioflavanoids.
Key words Fagopyrum tataricum; CHS; RACE; Intron; Cladogram
Agricultural Science & Technology, 2012, 13(4): 708-710，726
Copyright訫 2012, Information Institute of HAAS. All rights reserved Agricultural Biotechnology
T atar buckwheat (Fagopyrum tat-aricum), commonly known asbuckwheat, belongs to family of
Polygonaceae, genus of Fagopyrum
esculentum [1-2]. Previous studies show
that buckwheat has a variety of physio-
logical functions including prevention
of diabetes and coronary heart disease,
anti-cancer, prevention of internal blee-
ding, blood lipids-lowering, blood-glu-
cose-lowering and anti-aging[3-7], these
physiological functions and medicinal
value were found closely related to the
abundant flavonoid in buckwheat[8].
CHS (chalcone synthase) is the
first enzyme in the flavonoid biosyn-
thetic pathway and also a key enzyme
in the secondary metabolic pathway of
plants, which catalyzes condensation
between three acetoxy of malonyl-
coenzyme A and a acetoxy of A4-cou-
maroyl-CoA[9] and synthesizes ch-al-
cone with 4-coumaric acid-CoA as the
best substrate [10]. Chalcone is the sy-
nthetic precursor for plant flavonoid [11].
Improving the expression level of this
key enzyme can increase the chalcone
content in plants or plant cells, thus in-
creasing the content of flavonoid.
Therefore, cloning CHS gene of buck-
wheat has important significance for
regulating the synthesis of flavonoid at
the molecular level.
So far, many researches have
been conducted on the determination,
synthesis pathway and regulation of
flavonoid in buckwheat. However, no
research has been reported on regu-
lating the synthesis of flavonoid at the
molecular level and cloning of full-
length CHS gene sequence from
buckwheat. Therefore, in this study,
buckwheat was used as experimental
material for cloning the full-length
cDNA sequence of CHS gene by using
RACE (rapid-amplification of cDNA
ends) technology and CODEHOP
(consensus-degenerate hybrid oligon-
ucleotide primers) primer design
method and bioinformatics analysis, to
provide an initial basis for regulation of
the flavonoid content in buckwheat at
the molecular level.
Materials and Methods
Materials
Experimental material Buckwheat
from Ukraine Irina was used as the
experiment material in this study.
Strains and reagents Escherichia
coli DH5α, which was stored in our
laboratory, was used as the recipient
strain for transformation. M-MLV re-
verse transcriptase was the product of
Promega Corporation; Ex Taq DNA
polymerase, dNTPs, DNA Marker and
pMD18-T vector kit were purchased
from TaKaRa Biotechnology Co., Ltd.
Methods
Extraction of total RNA from buck-
wheat leaves The total RNA from
buckwheat leaves were extracted by
using modified CTAB-LiCl method and
the specific steps were in accordance
with the previous literature[12].
Primer design
Design of degenerate primers for
3’-RACE Design of upstream
primer: six CHS amino acid sequences
published on GenBank were selected,
including Fagopyrum esculentum (ac-
cession number: ABV72596.1), Aquil-
aria sinensis (accession number: AB-
M73434.1), Rheum palmatum (acces-
sion number: ABB13607.1), Dictam-
nus albus (accession number: CAH-
61575.1), Daucus carota (accession
number: BAA03784.1) and Populus
alba (accession number: ABD24222.1).
These sequences were submitted to
http:// bioinformatics. weizmann. ac. Il /
blocks / blockmkr / www / make_blocks.
html in FASTA format, to obtain the
conserved sequences of these six
DOI:10.16175/j.cnki.1009-4229.2012.04.030
Agricultural Science & Technology
Vol.13, No.4, 2012 Agricultural Science & Technology
2012
CHS amino acids, the most
conservative sequence was submitted
to the online software CODEHOP
(http:// bioi-nformatics. weizmann. ac.
il/blocks/codehop. html) for primer
design, and the obtained primer was
named J3CHS, with sequence of 5’-
CATGATGTACCAG-CARGGTTGC-
3’. Design of downstr-eam primer:
according to the sequence of reverse
transcription primer P1 (5’-GAGGAC-
TCGAGCTCAAGCTTTTTTTTTTTTT-
3’), downstream primer P2 was desi-
gned, with sequence of 5’-GAGGAC-
TCGAGCTCAAGC-3’.
Design of degenerate primers for
5’-RACE Design of upstream primer:
bioinformatics analysis showed that 5’
sequence of buckwheat CHS was rel-
atively conservative, so the primer was
designed according to the conserva-
tive sequence and named J5CHSup,
with sequence of 5’-ATGGCACCGA-
CGGTCCAGGA-3’. Design of down-
stream primer: downstream J5CHS-
down was designed according to the
cloned 3’ cDNA, with sequence of 5’-
CAGACGACGAGGACACGAGC-3’.
Design of specific primers for am-
plification of full-length RaCHS
gene According to the open reading
frame (ORF) of the cDNA sequence,
specific primers for amplification of full-
length gene were designed. Upstream
primer CHSup: 5’-ATGGCACCGAC-
GGTCCAGGA-3’; downstream primer
CHSdown: 5’-AGTTGGCTAGGGTG-
GTGG-3’.
PCR amplification conditions First
strand cDNA synthesis by reverse
transcription was used as the template
for 3’-RACE experiment. The 3’-RACE
PCR amplification was started with
predenaturation at 94℃ for 3 min, fol-
lowed by 30 cycles of denaturation at
94 ℃ for 30 s, annealing at 56 ℃ for
45 s, and extension at 72 ℃ for 1 min;
the amplification was completed by
holding the reaction mixture at 72 ℃
for 10 min to allow complete extension
of PCR products. The 5’-RACE PCR
amplification was started with prede-
naturation at 94 ℃ for 3 min, followed
by 30 cycles of denaturation at 94 ℃
for 30 s, annealing at 60 ℃ for 45 s,
and extension at 72 ℃ for 1 min; the
amplification was completed by hold-
ing the reaction mixture at 72 ℃ for 10
min to allow complete extension of
PCR products. The PCR amplification
for full’-length RaCHS gene was
started with predenaturation at 94 ℃
for 3 min, followed by 35 cycles of de-
naturation at 94 ℃ for 30 s, 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 to allow complete
extension of PCR products. Recovery,
ligation, transformation and identifica-
tion of the target gene were conducted
in accordance with routine method [13],
the sequencing was completed by
Shanghai Sangon Biological Engi-
neering Technology Services Co., Ltd.
Bioinformatics analysis of RaCHS
Clustalxl.81 and MEGA4 software
were used for sequence analysis and
construction of phylogenetic tree;
NCBI Blastn and Blastp programs
were applied for homology analysis of
nucleic acid and protein.
Results and Analysis
RNA extraction
Total RNA of buckwheat leaves
was extracted with modified CTAB-
LiCl method, electrophoresis results
were shown in Fig.1. Lithium ion could
selectively precipitate nucleic acids
under certain pH conditions, which
easily causes loss of small RNA, so
only the 28S and 18S rRNA bands
were shown in Fig.1. However, smear
bands were observed around the 18S
bands, indicating that mRNA had rela-
tively high abundance and could be
used as a template for RT-PCR ex-
periment. Purity was determined by
UV spectrophotometer, which showed
that the OD260/OD280 value was 1.91, in-
dicating the high purity of total RNA.
3’-RACE results
With the extracted total RNA of
buckwheat leaves as the template for
reverse transcription by using primers
P1 and PCR amplification by using
primers P2 and J3CHS. Subsequently,
the products were detected by using
agarose gel electrophoresis, the re-
sults(Fig.2) showed that an 800 bp band
was obtained by the 3’-RACE experi-
ment. The obtained fragment was in-
serted into pMD18-T simple vector and
transformed into E. coli, and the posi-
tive clones were selected and sent for
sequencing. The sequencing results
showed that the length of this se-
quence was 891 bp, which shared
88% homology with Rheum palmatum
(accession number: DQ205352.1) ac-
cording to the sequence alignment by
Blastn program, indicating a new
gene, which was named RaCHS.
5’-RACE results
With the extracted total RNA of
buckwheat leaves as the template for
reverse transcription by using primers
P1 and PCR amplification by using
primers J5CHSup and J5CHSdown,
the products were detected by using
agarose gel electrophoresis (Fig.3),
which showed that a 550 bp band was
obtained by the 5’-RACE experiment.
The obtained fragment was inserted
into pMD18-T simple vector and trans-
formed into E. coli, and the positive
clones were selected and sent for se-
quencing. The sequencing results
Fig.1 Electrophoresis of the total RNA of
buckwheat leaves
M, DL 2000 marker; 1, PCR amplification
product.
Fig.2 3’-RACE results of RaCHS
M, DL 2000 marker; 1, PCR amplification
product.
Fig.3 5’-RACE results of RaCHS
709
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Agricultural Science & Technology Vol.13, No.4, 2012
2012
showed that the length of this se-
quence was 572 bp, which shared
87% homology with Rheum palmatum
(accession number: DQ205352.1) ac-
cording to the sequence alignment by
Blastn.
RaCHS gene merger and full-length
cDNA cloning
DNAman software was used for
splicing the 3’ cDNA sequence and 5’
cDNA sequence of the isolated
RaCHS gene, a 1 443 bp sequence
was obtained. By using primers
CHSup and CHSdown, a gene frag-
ment with length of 1 906 bp was am-
plified (Fig.4). Similarly, the fragment
was recovered, inserted into pMD18-T
simple vector, transformed into E. coli,
identified and sequenced. The se-
quencing result was consistent with
the splicing result except the base se-
quence between + 182 and + 644 bp,
which indicated that there was a 463
bp intron sequence between + 182 and
+ 644 bp.
Bioinformatic analysis of RaCHS
gene
Domain analysis of RaCHS gene
By using Bankit tool on the NCBI web-
site, the obtained DNA sequence of
RaCHS gene was submitted to Gen-
Bank (accession number: HQ434624).
The cDNA sequence was translated
into amino acid sequence for analysis
by using the NCBI Blastp program,
and the results (Fig.5) indicated this
gene belonging to the CHS superfam-
ily. Translated RaCHS protein had
BcsA-type domain, indicating that
RaCHS might belong to chalcone syn-
thase-like protein.
Evolutionary analysis of RaCHS
gene Clustal X software was used
for multiple sequence alignment be-
tween RaCHS gene-deduced amino
acid sequence and CHS amino acid
sequences from other plant species
with identified function[14-16]. Neighbor-
Joining algorithm in Bootstrap test of
MEGA4 software was used for con-
struction of phylogenetic tree (Fig.6),
number of branches indicated the
Bootstrap supporting rate of the
branch. As can be seen from Fig.6,
RaCHS gene showed the highest ho-
mology to Rheum palmatum and rela-
tively high homology to Gypsophila
paniculata, while it was distantly ho-
mologous with other plant species.
Conclusion and Discussion
Chalcone synthase (CHS) is a
key enzyme in flavonoid biosynthetic
pathway, which catalyzes the synthe-
sis of chalcone with 4-Coumaroyl-CoA
as the best substrate, thereby synthe-
sizing flavonoid through a series of
pathways. Improving the expression
level of CHS can increase the chal-
cone content in plants or plant cells,
thus increasing the content of
flavonoid. Therefore, cloning CHS
gene of buckwheat has important sig-
nificance for regulating the synthesis
of flavonoid at the molecular level.
In this experiment, a full-length
CHS gene was first cloned from buck-
wheat, and the full-length cDNA of
buckwheat CHS gene was success-
fully cloned by using RACE technology
and CODEHOP primer design method.
The full length of cDNA sequence was
1 906 bp, containing a 463 bp intron
sequence and a 1 188 bp coding re-
gion, encoding 395 amino acids.
Blastn sequence alignment revealed
that RaCHS gene sequence showed
86% homology to the CHS genes of
closely related species Rheum palma-
tum (accession number: DQ205352.1).
This study laid the foundation to clarify
molecular basis of the synthesis of
buckwheat bioflavanoids and explore
an effective way improve the content
of buckwheat bioflavanoids.
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Agricultural Science & Technology Vol.13, No.4, 2012
2012
苦荞中查尔酮合成酶全长基因的克隆及序列分析
刘 凯 1，2，胡耀辉 1，王 冠 3，于寒松 1* （1. 吉林农业大学食品科学与工程学院，吉林长春 130118；2. 吉林省农业综合开发办公室，吉林长春
130118; 3.吉林农业大学生命科学学院，吉林长春 130118）
摘 要 [目的]该研究旨在克隆苦荞中查尔酮合成酶全长基因。[方法]选用乌克兰伊琳娜苦荞为试验材料，以从叶片中提取的 RNA 为模板，应用
RACE 技术结合 CODEHOP 引物设计方法克隆苦荞中查尔酮合成酶 cDNA 序列，通过电子合并获得其全长。设计基因全长特异性引物，以 DNA
为模板进行 PCR 扩增出基因序列。应用 Clustalxl. 81 和 MEGA4 软件进行序列分析和进化树的建立; 核酸和蛋白质序列同源性分析应用
NCBI的 Blastn 和 Blastp 完成。[结果]生物信息学分析表明，该基因全长 1 906 bp，具有一个 463 bp 的内含子序列，编码区长度为 1 188 bp，
编码 395个氨基酸。Blastn 序列比对发现该试验所获得的 CHS基因序列与相近物种 Rheum palmatum(登录号:DQ205352.1)的 CHS基因同源性达
86%。[结论]该研究为阐明苦荞生物类黄酮合成的分子基础，探索提高苦荞生物类黄酮含量的有效途径奠定基础。
关键词 苦荞；查尔酮合成酶；RACE；内含子；进化树
基金项目 国家农业部“948”项目(2008-z27)。
作者简介 刘凯（1970-），男，河北沧县人，在读博士，研究方向：植物生物反应器与功能性食品。*通讯作者，博士，副教授，研究方向：食品生物技
术；E-mail: yuhansong@jluhp．edu．cn。
收稿日期 2012-01-31 修回日期 2012-02-11
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Responsible editor: Xiaohui FAN Responsible proofreader: Xiaoyan WU
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大肠杆菌 NrfA蛋白表达、纯化及多克隆抗体的制备
何婷婷 1，2，龚钢明 1*，高 然 3 （1．上海应用技术学院生物工程系，上海 200235；2.上海师范大学生命与环境科学学院，上海 200234；3．浙江理工大
学生命科学学院生物化学研究所，浙江杭州 310018）
摘 要 [目的]为克隆大肠杆菌 NrfA基因，构建 pET-28a（+）-NrfA表达载体，制备相应的多克隆抗体并鉴定。[方法]以以大肠杆菌基因组 DNA为
模板, PCR扩增得到 NrfA基因编码区, 构建 pET-28a（+）-NrfA表达载体；经 IPTG 诱导表达并纯化重组蛋白；再免疫新西兰雄兔，制备多克隆抗
体；用 ELISA方法检测抗体的效价，Western Βlotting检测抗体的特异性。[结果]适构建的表达载体 pET-28a（+）-NrfA在大肠杆菌中诱导后可以
高效表达 NrfA蛋白，免疫获得的多克隆抗体用 ELISA检测，其效价为 1：204 900，经Western Blotting分析，抗体的特异性较好。[结论]成功克隆大
肠杆菌的 NrfA基因，构建了表达载体，制备的 NrfA 多克隆抗体具有较高的效价和良好的特异性。为研究细菌有关 NrfA奠定了基础。
关键词 NrfA基因；原核表达；多克隆抗体
作者简介 何婷婷（1987-），女，安徽安庆人，硕士研究生，研究方向：微生物分子生物学。*通讯作者，E-mail: Kming512@yahoo.cn。
收稿日期 2012-01-19 修回日期 2012-02-22
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