全 文 ：DNA条形码基因 ITS·ITS2 及
rbcL 在苍耳属可采用相同的
PCR条件
胡伟毅 *，汪连军，盛志超 （连云港出入境检
验检疫局，江苏连云港 222042）
摘 要 [目的]为植物 DNA 条形码标准基因
筛选研究提供参考， 减少植物 DNA 条形码研
究中的工作量。 [方法]对 7 种苍耳属植物 ITS、
ITS2 及 rbcL 基因采用相同的扩增条件（95 ℃
4 min；[35 cycles：94 ℃ 30 s；52 ℃ 45 s；72
℃ 45 s]；72 ℃ 10 min；4 ℃ 保存）。 [结果]3种
DNA 条形码基因同时成功扩增。 [结论]这说明
植物 DNA 条形码基因中 ITS、ITS2 及 rbcL 的
PCR 条件存在合并可能性。
关键词 DNA 条形码；苍耳属；ITS；ITS2；rbcL
基金项目 江苏出入境检验检疫局科技项目
（2012KJ54）。
作者简介 胡伟毅 （1984- ），男 ，河北宣化
人，硕士，主要从事港口外来有害生物截获工
作，E-mail：94087540@qq.com。 * 通讯作者。
收稿日期 2013-08-26
修回日期 2013-09-06
PCR Amplification System of DNA Barcoding
Genes ITS, ITS2 and rbcL from Xanthium
Weiyi HU*, Lianjun WANG, Zhichao SHENG
Lianyungang Entry-exit Inspection and Quarantine Bureau, Lianyungang 222042, China
Supported by Science and Technology Project of Jiangsu Entry-Exit Inspection and
Quarantine Bureau (2012KJ54).
*Corresponding author. E-mail: 94087540@qq.com
Received: August 26, 2013 Accepted: September 6, 2013A
Agricultural Science & Technology, 2013, 14(9): 1212-1214
Copyright訫 2013, Information Institute of HAAS. All rights reserved Molecular Biology and Tissue Culture
Abstract [Objective] This study aimed to provide reference and reduce the workload
for screening standard DNA barcoding genes of plants. [Method] Three DNA bar-
coding genes ITS, ITS2 and rbcL were amplified from seven Xanthium species un-
der the same PCR condition: PCR amplification was started with initial denaturation
at 95 ℃ for 4 min, followed by 35 cycles of denaturation at 94 ℃ for 30 s, anneal-
ing at 52 ℃ for 45 s, and extension at 72 ℃ for 45 s; the amplification was com-
pleted by holding the reaction mixture at 72 ℃ for 10 min to allow complete exten-
sion of PCR, and the PCR products were stored at 4 ℃. [Result] Three DNA bar-
coding genes ITS, ITS2 and rbcL were all amplified successfully. [Conclusion] This
study indicates that PCR amplification conditions for DNA barcoding genes ITS,
ITS2 and rbcL in plants may be consistent.
Key words DNA barcoding; Xanthium; ITS; ITS2; rbcL
P lant DNA barcoding technolo-gy is a technical means foramplification and sequencing
of specific gene fragments in plant
genomes to investigate the nucleotide
variation laws, which was formally
proposed by Canadian scientist Paul
Hebert in 2003[1]. At present, plant DNA
barcoding technology has been ma-
turely applied in researches of animal
CO1 gene. However, due to the con-
servative changes and poor distinction
function of CO1 gene in plant species,
studies of plant DNA barcoding tech-
nology are still concentrated in match-
ing and screening of general gene
fragments [2]. Nevertheless, plant DNA
barcoding technology has played a
groundbreaking role in the protection
of endangered species, food and drug
supervision, herbal resource identifi-
cation, ecology surveys and many
other aspects compared with previous
morphological methods [3]. Genes ITS,
ITS2 and rbcL are most commonly
used in the studies of plant DNA bar-
coding[4]. A large number of PCR reac-
tions should be carried out in different
studies, and the PCR conditions ad-
opted in actual studies are different[5-7].
In this study, three DNA barcoding
genes ITS, ITS2 and rbcL were suc-
cessfully amplified from seven Xanthi-
um species under the same PCR re-
action system, indicating that PCR
amplification conditions for DNA bar-
coding genes ITS, ITS2 and rbcL in
plants may be consistent, which will re-
duce the workload and working hours
in application of plant DNA barcoding
technology and provide reference for
matching and screening of standard
DNA barcoding genes from plants if it
can be successfully used in other plant
species.
Materials and Methods
Materials
Experimental materials Seven Xan-
thium species were used as experi-
mental samples, including Xanthium
chinese, Xanthium sibiricum, Xanthi-
um spinosum, Xanthium occidentale,
Xanthium brasilicum, Xanthium speci-
osum and Xanthium pensylvanicum,
which were intercepted in imported
DOI:10.16175/j.cnki.1009-4229.2013.09.001
Agricultural Science & Technology2013
1, Xanthium pensylvanicum; 2, Xanthium
occidentale; 3, Xanthium brasilicum; 4,
Xanthium speciosum; 5, Xanthium sibir-
icum; 6, Xanthium spinosum; 7, Xanthium
chinese; 8, control.
Fig.3 Amplification results of rbcL gene
soybeans in 2012 and identified by
Chinese Academy of Inspection and
Quarantine.
Reagents and primers DNeasy誖
Plant Mini Kit, 2 × Taq master mix,
agarose, Goldenview I and other
reagents were used in this study.
ITS primers：
ITSf：5′ -CCTTATCATTTAGAGG-
AAGGAG-3′
ITSr：5′ -TCCTCCGCTTATTGA-
TATGC-3′
ITS2 primers：
ITS2f：5′-ATGCGATACTTGGTG-
TGAAT-3′
ITS2r：5′-GACGCTTCTCCAGAC-
TACAAT-3′
rbcL primers：
rbcLf：5′ -ATGTCACCACAAACA-
GAAAC-3′
rbcLr：5′ -TCGCATGTACCTGCA-
GTAG-3′
Above primers were all synthe-
sized by Sino-US Taihe Biotechnology
(Beijing) Co., Ltd.
Methods
DNA extraction DNA was extract-
ed using the modified DNeasy誖 Plant
Mini Kit extraction method: ① Xanthi-
um samples were cut off using plant
scissors, and the seeds were collected
and ground in a mortar; ② ground
samples were separately placed in 2
ml centrifugal tubes, added with 400 μl
of Buffer AP1 and 4 μl of RNase A,
mixed evenly, and lysed in a 65 ℃
water bath for 30 min; ③ lysates were
added with 130 μl of Buffer AP2 and
lysed in an ice bath for 20 min;④ lysa-
tes were centrifuged at 14 000 r/min
for 5 min; ⑤ after centrifugation, the
supernatant was collected, added into
QIAshredder spin columns and cen-
trifuged at 14 000 r/min for 2 min; ⑥
liquid phases were transferred into
new centrifuge tubes, added with 1.5
times the volume of Buffer AP3/E, and
mixed evenly;⑦ 650 μl of mixture was
added into DNeasy mini spin columns
and centrifuged at 8 000 r/min for 1
min; ⑧ the previous step was repeat-
ed until all the mixture was processed;
⑨ DNeasy mini spin columns were
placed into new 2 ml collection tubes,
added with 500 μl of Buffer AW and
centrifuged at 8 000 r/min for 1 min,
liquid phases were removed; ⑩ 500 μl
of Buffer AW was added into the
columns and centrifuged at 14 000
r/min for 2 min, liquid phases were re-
moved; DNeasy mini spin columns
were placed into new 2 ml collection
tubes, added into 100 μl of Buffer
AE, placed at the room temperature
for 5 min, and centrifuged at 8 000
r/min for 1min; the previous step was
repeated once, 200 μl of DNA sam-
ples were obtained and preserved in
-20 ℃ refrigerator.
PCR reaction system and amplifi-
cation conditions Three DNA Bar-
coding genes ITS, ITS2 and rbcL were
amplified using 2 × Taq master mix
(Sino-US Taihe). The total PCR reac-
tion volume was 25 μl, containing 3 μl
of DNA template, 1 μl of primer 1, 1 μl
of primer 2, 12.5 μl of 2 × Taq master
mix and 7.5 μl of ddH2O. The PCR
amplification was started with initial
denaturation at 95 ℃ for 4 min, fol-
lowed by 35 cycles of denaturation at
94 ℃ for 30 s, annealing at 52 ℃ for
45 s, and extension at 72 ℃ for 45 s;
the amplification was completed by
holding the reaction mixture at 72 ℃
for 10 min to allow complete extension
of PCR. The PCR products were
stored at 4℃.
Agarose gel preparation and elec-
trophoresis Firstly, 1.5 g of regular
agarose G-10 was weighed and trans-
ferred into the flask, added with 100 ml
of ddH2O and 2 ml of 50 × TAE buffer,
heated for 4 min in a microwave oven,
placed into a 75 ℃ water bath for 5
min, added with 10 μl of Goldenview I,
mixed evenly, stood for 10 min, and
poured into the gel mold. The agarose
gel was placed at the room tempera-
ture for 30 min before use.
After amplification, 4 μl of PCR
products were applied for elec-
trophoresis under 100 V for 45 min.
Subsequently, the electrophoresis gels
were observed and photographed in
the gel imaging system.
Results and Analysis
As can be seen from the elec-
trophoresis patterns of DNA barcoding
genes ITS, ITS2 and rbcL from seven
Xanthium species (Fig.1-Fig.3), under
the above PCR condition, three DNA
barcoding genes ITS, ITS2 and rbcL
could be successfully amplified from
seven Xanthium species; especially,
electrophoretic bands of genes ITS
and ITS2 were relatively bright, indicat-
ing that the PCR condition adopted is
appropriate for amplification of ITS and
ITS2 fragments.
Discussions
The selection of amplified frag-
ments has become the bottleneck of
plant DNA barcoding technology. In
this study, PCR amplification condi-
1, Xanthium pensylvanicum; 2, Xanthium
occidentale; 3, Xanthium brasilicum; 4,
Xanthium speciosum; 5, Xanthium sibir-
icum; 6, Xanthium spinosum; 7, Xanthium
chinese; 8, control.
Fig.1 Amplification results of ITS gene
1, Xanthium pensylvanicum; 2, Xanthium
occidentale; 3, Xanthium brasilicum; 4,
Xanthium speciosum; 5, Xanthium sibir-
icum; 6, Xanthium spinosum; 7, Xanthium
chinese; 8, control.
Fig.2 Amplification results of ITS2 gene
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Agricultural Science & Technology 2013
Responsible editor: Qingqing YIN Responsible proofreader: Xiaoyan WU
QGCFAGGTVLR, and three other
motif sites. The results were consistent
with other plants.
Mulberry fruit is one of the most
popular fruits in the world because of
its high nutrient level and pleasant fla-
vor, which includes many cultivated
species according to the fruit color,
such as puce, milk white and atropur-
pureus. The color changes from green,
dark green, red, dark red to atropur-
pureus during the course of fruit ripen-
ing, and chalcone synthetase plays a
main role. In young fruit period, total
phenolics and total flavonoids in mul-
berry are more, with the maturity of the
fruit, the synthesis rate become slowly.
At full red period the phenolics and
flavonoids begin to accumulate. Syn-
thesis of anthocyanins was controlled
by chalcone synthase, and from red
period to the purple black peak phase
the rate of synthesis is accelerated.
So, through the study of CHS can re-
veal the physiological and biochemical
changes during the fruit ripening. Fur-
ther study is needed to genetically and
physiologically characterize the CHS
in Morus and to gain a better under-
standing of its function and relationship
with change in fruit color.
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Responsible editor: Xiaohui FAN Responsible proofreader: Xiaoyan WU
tions for DNA barcoding genes ITS,
ITS2 and rbcL in plants are the same,
which can not only bring convenience
to experimental operations, but also
provide guidance for subsequent
screening of standard amplified frag-
ments of plant DNA barcoding. How-
ever, due to the limited scope of the
study, the PCR amplification system
has only been used in Xanthium
species, while its applicability in other
families and genus requires further
verification.
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