全 文 ：Received： 2008－12－24
Biography： JIANG Wen（1978－），male，born in Luchuan，Guangxi，Postergraduate，major in Plant physiology and molecular biolo－
gy． * for corresponding author，E－mail：liyr＠gxaas．net．
荸荠基因组DNA的提取及RAPD反应体系的建立
江 文1，2，3，陈丽娟2，李杨瑞1，3 *，杨丽涛1
（1 广西大学农学院，南宁 530005；2 广西农业科学院生物技术研究所，南宁 530007；
3 广西农业科学院广西作物遗传改良生物技术重点开放实验室，南宁 530007）
摘要：以荸荠叶状茎为试验材料，采用改良的SDS法提取其基因组DNA，并对其RAPD反应体系进行优化，建立了
荸荠的RAPD－PCR优化反应体系和程序。结果表明，提取的基因组DNA纯度和完整性较好，OD260/OD280 值在1．8～2．0之
间，DNA无降解现象，完全可以满足RAPD－PCR扩增要求。建立了荸荠RAPD反应体系：总体积为25μl，各有关成分的
最佳浓度分别为25mmol/L Mg2＋，1．0U Taq DNA聚合酶，0．2mmol/L dNTPs，1μmol/μl 引物，1．5ng/μl DNA模板。PCR反应
程序为：94℃预变性3min；94℃变性1min，37℃退火30s，72℃延伸60s，40个循环；最后72℃延伸10min。
关键词：荸荠；基因组DNA；提取；RAPD；反应体系
Extraction of genomic DNA and establishment of RAPD reaction
system for water chestnut（Eleocharis tuberose
Roem et Schult）
JIANG Wen1，2，3，CHEN Li－juan2，LI Yang－rui1，3 *，YANG Li－tao1
（1 Agricultural College， Guangxi University， Nanning 530005，China； 2 Biotechnology Research Institute， Guangxi
Academy of Agricultural Sciences， Nanning 530007，China； 3 Guangxi Crop Genetic Improvement and Biotechnology
Laboratory， Guangxi Academy of Agricultural Sciences， Nanning 530007，China）
Abstract： The young phylloclade of water chestnut was used as experimental material， and the improved SDS method
was used to extract the genomic DNA of young phylloclade successfully． The results showed that the extracted genomic
DNA was pure and integral， and the ratio of OD260/OD280 was from 1．8 to 2．0， and the degradation of extracted genomic
DNA was not found． Therefore， the extracted genomic DNA was suitable for RAPD－PCR reaction． The conditions for
RAPD－PCR were optimized， and the best RAPD reaction system was established as follows： total volume being 25μl，
containing 25 mmol/L Mg2＋， 1．0 U Taq DNA polymerase， 0．2 mmol/L dNTPs， 1 μmol/μl primer and 1．5 ng/μl template
DNA． The PCR program was 3 min at 94℃ for predenaturalization， then 40 cycles of 1min at 94℃ for denaturation， 30s at
37℃ for annealing and 60s at 72℃ for extension， finally extension at 72℃ for 10min．
Key words： water chestnut（Eleocharis tuberose Roem et Schult）； genomic DNA； extraction； RAPD； reaction system
CLC number：S645.3 Document code：A Article：1002－8161（2009）03－0229－04
Guangxi Agricultural Sciences Vol.40 No.3 2009
Water chestnut （Eleocharis tuberose Roem et
Schult） belongs to the Cyperaceae， and is a kind of
shallow perennial herb in China Yangtze River and
southern provinces． The bulbs underground are fitted
to be eaten， that has high nutritional value in the
processing of canned and starch extraction． In
medicine， it is helpful to health［1］．
However， water chestnuts are not well classified
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广西农业科学 2009年第 40卷第 3期
and few research on cultivars and resources with
molecular biology technique． RAPD （random ampli-
fied polymorphic DNA） markers have been proved
useful in many genetic studies． It was already widely
applied to gene location， genome DNA fingerprint，
and polymorphism analysis on genetic resource［2－4］． In
this technique， it is important to extract genomic
DNA and access high－quality DNA． The phylloclade
of water chestnut contains high level of polysaccha-
ride， polyphenol and secondary metabolites． The in-
tegrity and high purity of DNA is not easy to be ob-
tained in the experiment． In the present study，
RAPD markers was used to investigate the distribu-
tion of genetic variability in water chestnuts from
Guangxi，Guangdong，Hubei，Hunan，Jiangsu，Yunnan，
and other places． The methods of DNA extraction
from water chestnuts were discussed and RAPD reac-
tion system was established and optimized in our
study．
1 Materials and methods
1． 1 Experimental materials
Water chestnut varieties sampled from different
areas including Guangxi， Guangdong， Hubei， Hunan，
Jiangsu and Yunnan， etc． were employed as the experi-
mental materials， which were offered by Biotechnology
Research Institute， Guangxi Academy of Agricultural
Sciences． The corms were planted in greenhouse in
Guangxi Academy of Agricultural Sciences． The phyl-
loclade was sampled for the extraction of genomic
DNA when the plants grew to 20－30 cm high．
1． 2 Extraction of genomic DNA
Approximately 0．1g of young phylloclade materi-
al of each variety was used to extract and purify ge-
nomic DNA through the SDS method developed by
Wang and Fang［5］． The materials were cut into pieces
and grinded into fine powder in liquid N2， and then
the powder was transferred into a 10 mL centrifuge
tube immediately， and 2．8mL DNA extraction buffer
（500mmol/L NaCl， 100mmol/L Tris－HCl pH7．5， and
50mmol/L EDTA pH7．5） was added and well mixed
in tube． Then， 480μl of 10％ SDS were added and
rotated for 30s． The samples were incubated at 65℃
for 20min for cell lysis and vortex gently upside down
for 2－3min． After the tube cooled to room tempera-
ture， 440μl 5mol/L KAC were added and mixed，
then was incubated in ice for 30 min and centrifuged
at 12，000 r/min for 12 min at 4℃． 0．1 vol of 3mol/L
sodium acetate （pH5．2） and 2．5 vol of ice－cold iso-
propanol （－20℃） were added to the supernatant and
mixed gently by inversion． The tube was placed at －20℃
for 30min to precipitate the extracted DNA， and then
centrifuged at 12，000r/min for 10min at 4℃ again．
The DNA pellets were collected and washed with 70％
ethanol and air －dried for several minutes， and the
DNA was suspended in 500μl pH8．0 TE buffer again
and added with 5μl RNase A， then incubated at 37℃
for 30min or more． The same volume phenol/chloro-
form（25∶24）was added and centrifuged at 12000r/min
for 10min， then the DNA was purified with chloro-
form， 0．1 vol of 3mol/L sodium acetate （pH5．2）and
2．5 vol of ice－cold isopropanol （－20℃）were added to
the supernatant and mixed gently by inversion to pre-
cipitate the DNA． The pellet DNA was washed twice
with 70％ ethanol， then dried by air and suspended
in 200μl TE buffer （pH8．0）again and stored at －20℃
for further use． The concentration and purity of DNA
were determined with the ratios of A260/A230 and A260/A280
by a UV－visible spectrophotometer and also quanti-
fied in 1％ （w/v）agarose gels by comparison with
known quantities of the lane maker DNA and stored at
－20℃ in aliquots．
1． 3 RAPD－PCR protocol
Random decamer oligonucleotide primers were
screened by polymerase chain reaction（PCR） for use-
fulness in an initial survey． Of these， clear repro-
ducible polymorphic profiles after checking for re-
peatability of band patterns were obtained in our
study． And the PCR method was used as described by
Williams ［6］ and Welsh ［7］ with some modification， that
is 40 cycles of amplification in a 25 μl reaction mixture
containing 25 mmol/L Mg2 ＋ ，1．0 U Taq DNA poly-
merase，0．2 mmol /L dNTPs ，1 μmol/μl primer an d
1．5 ng/μl template DNA．
Guangxi Agricultural Sciences Vol.40 No.3 2009230· ·
JIANGWen et al：Extraction of genomicDNAand establishment of RAPD reaction system for water chestnut（Eleocharis tuberoseRoem et Schult）
3 Discussion
Williams et al.[6] and Welsh et al.［7］ first applied
RAPD －PCR technique to study the DNA polymor-
phism on the whole genome in 1990． This technique
has many advantages， such as speediness， simple-
ness， safety， efficiency， sensitivity and little amount
of DNA sample． RAPD has already been applied to
gene location widely， the genome DNA fingerprint，
2 Results and analysis
2． 1 Extraction and purification of DNA from
water chestnut
In the present experiment， the OD260/OD230 ratio
of the extracted DNA of water chestnut varieties ob-
tained with UV －visible spectrophotometer was over
2．0， and the OD260/OD280 ratio was 1．8－2．0， and the
genomic DNA fragment separated in 1．0％ TBE a-
garose gels were clear and intact， which showed that
the DNA fragment were good enough for the RAPD
analysis （Fig．1）．
2． 2 Genetic polymorphism and establishment of
RAPD reaction system for water chestnut
The RAPD－PCR results for different water chest-
nut varieties showed that the amplified DNA fragment
sizes were from 250 to 2000bp． The conditions for
RAPD－PCR were optimized， and the best RAPD reac-
tion system was established as follows： total volume be-
ing 25 μl， containing 25 mmol/L Mg2＋，1．0 U Taq DNA
polymerase，0．2 mmol/L dNTPs， 1 μmol/μl primer and
1．5 ng/μl template DNA． Moreover， the PCR program
was 3 min at 94℃ for predenaturalization， then 40 cy-
cles of 1 min at 94℃ for denaturation， 30s at 37℃ for
annealing and 60s at 72℃ for extension， finally exten-
sion at 72℃ for 10min． The best results for 16 water
chustnut varieties amplied with the primers S382 and
S374 were showed in Fig． 2．
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广西农业科学 2009年第 40卷第 3期Guangxi Agricultural Sciences Vol.40 No.3 2009
polymorphism analysis on genetic resource had be-
come an important method for genetic study． And the
RAPD－PCR was influenced by the amount of DNA
template，dNTPs，MgCl2，and Taq DNA polymerase．
In the present study， the improved SDS method
was used to successfully extract the genomic DNA
from young phylloclade material of different water
chestnut varieties． The results showed that the ex-
tracted genomic DNA was pure and integral， the
ratio of OD26 0/OD280 was from 1．8 to 2．0， and the
degradation of extracted genomic DNA was not found．
It showed that the extracted genomic DNA was suit-
able for RAPD －PCR reaction． The conditions for
RAPD－PCR were optimized， and the best RAPD re-
action system was well established． It would provide a
rapid， accurate identification technique for water
chestnut resources．
Reference：
［1］ Li F，Ke W D，Liu Y M． Advances in research of com－
mon spikesege［J］． Journal of Changjiang Vegetables，2006
（8）：39－43．
［2］ Zheng G S，Chen X M，Meng L． RAPD－PCR analysis on
genetic relationships between cultivars of tree peony［J］．
Agricultural Sciences in China，2002，1（7）：792－797．
［3］ Deng J Y，Wang X，Fang F X，Zhang M，Sun D L． Ef－
fective extraction of genomic DNA and RAPD analysis on
different sex types of Momordica charantia L［J］． Guangxi
Agricultural Sciences，2007，38（3）：223－226．
［4］ Yue Q N，Ge J，Wang L，Zhang X，Wang S M，Wang J
M． Genetic diversity in main cultivars of Safflower in Xin－
jiang Uighur autonomous region based on RAPD analysis［J］．
Agricultural Science＆Technology，2008，9（4）：34－38．
［5］ Wang G L，Fang H J． Plant genetic engineering［M］． Bei－
jing：Science Press，2002：742－744．
［6］ Williams J G K，Kubelik A R，Livak K J，Rafalshi J A，
Tingey S V． DNA polymorphisms amplified by arbitrary
primers are useful as genetic markers［J］． Nucleic Acids
Res，1990，18：6531－6535．
［7］ Welsh J，McClelland M． Fingerprinting genomes using
PCR with arbitrary primers［J］． Nucleic Acids Res，1990，
18：7213－7218．
（责任编辑 韦莉萍；审 校 李杨瑞）
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