全 文 ：广 西 植 物 Guihaia 27(3)：518— 521 2007年 5月
Construction of cDNA library in Epimedium
brevicornum leaves
QIAN Bao-Ying1,2，LI Yun-Xiang ，YANG Zi—Song ，FENG Tu
(1．Sichuan Provincial Key Laboratory of Environmental Science and Biodiversity Conservation，
China West Normal University，Nanchong 637002，China；2．College of Life
Sciences，Taizhou University，Taizhou 317000，China)
Abstract：The total RNA was extracted from young leaves of Epimedium brevicornum ，and mRNA was puri—
fled by oligo(dT)一celulose column chromatography，which was reverse transeripted into eDNA using SM ART
(the Switch M echanism At the 5’end of RNA Templates)technique． The resulting eDNA was digested by
Sfi，the digested fragments were fraetionated by CHROMA SPIN 400，and then ligated to XTriplEx2 vector，
the mixture of ligation was finally packed into Lambda virons with packaging extracts．6 X 10 reeombinants
had been obtained．8O percent of products were over 500bp．2o clones were chosen randomly from the library
and were screened eDNA inserts using PCR．The results showed that the eDNA library of young leaves of E．
brevicornum had been constructed．and it could be used to screening to isolate particular clones．
Key words：Epimedium brevicornum ；young leaf；eDNA library
CLC number：Q943 Document code：A Article ID：1OOO一3142(2OO7)03-0518-04
Epirnedium brevicornum 。Yin Yang-Huo in Chi—
nese，is traditional Chinese herbal medidne，the aerial
parts of E．brevicornum is listed as source plants in Chi—
nese pharmacopoeia(the state Pharmacopoeia commis—
sion of PRC，2000)． “Yin Yang-Huo”was frequently
used for treating senile functional disease and enhancing
kidney function，it can also strengthen physique，cure
rheumatism．In recent years，domestic researchers have
done further work on E brevicornum and have ma de
great progress in physiological study，especially the study
of the flavonoids the important class of secondary prod—
ucts which are widely distributed inspermatophytic
plants and have a variety of roles in plants，anima ls and
micro-organisms(Andrea et a1．，2002)．Although the
study of the flavonoids of the E brevicornum is abun—
dant，it is mainly focus on the study on the effect of
physiology，and no study touch upon to the molecular of
the flavonoids．
Numerous flavonoids accumulate abundantly in E．
brevicornum，it play great role in protecting the plant
from UV，provide pigmentation to attract pollina tors，and
it also act as antibiotics in plant defense responses(Ku—
basek eta1．，1992；Koes et a1．，1994；Shirley，1996)．In—
dividual plant species can synthesize a variety of fla—
vonoid compounds．“Yin Yang-Huo”species are of much
interest because they accumulate a large quantity of fla—
vonoids．especialy in Yin Yang-Huo’S green tissues and
flowers． The biosynthetic pathway for the flavonoids
were well established(Schram et a1．，1984；Wiering et
a1．，1984)，and these flavonoids must be catalyzed by
ma ny key enzymes such as chalcone synthase(chs)，fla—
vanone~3一hydroxylase(f3h)and flavonol synthase(fls)
etc．before they play the function．
A cDNA library is a collection of different DNA se—
Received date：2005—09—22 Accepted date：2006—05—28
基金项目：四川省杰出青年学科带头人培养计划(04ZQ026—047)；四川省科技厅应用基础项目(03JY029—021—22)[Supported bythe SichuanOut-
standing Youth Academic Leader Cultivation hem(04ZQ026—047)；Science and Technology Department of Sichuan Provlnce(03JY029—021—22)]
作者简介：钱宝英(1979-)，女，浙江绍兴人，硕士研究生，从事植物分子生态学研究。
。通讯作者(Authorfor c0rresp0ndence，E—mail：yx_li@263．net，wutongye1979@tom．corn)
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3期 钱宝英等：淫羊藿嫩叶cDNA文库的构建 519
quences from an organism each of which has been cloned
into a vector for ease of purification，storage and analysis
(Turner et a1．，2003)． In order to investigate the key
enzymes in the flavonoids biosynthesis pathway of the
E brevicornurn，the cDNA library of E brevicornum’s
young leaf was constructed in this experiment．
1 Materials and Methods
1．1 Plant materials
Young leaves of E brevicoFnuTn were collected
and dehydrated in liquid nitrogen。then stored at一80℃
for use．
1．2 Isolation of total RNA and mRNA
Total RNAs of young leaves were extracted by
the method of Trizol，and then stored in一20℃ after
purified，and then analyzed by gel eletrophoresis and
UV spectrophotometer．Poly(A)RNA was purified by
oligo(dT)一cellulose column chromatography according
to the rnanual of Pharmacia mRNA purification kit．
After washing with 0．5 mmol／L NaC1。0．1 mmol／L
NaCl，the mRNA was colected with TE buffer，and the
concentration of the mRNA was measured through an
UV spectrophotometer．
1．3 cDNA synthesis and library construction
The sscDNA and dscDNA synthesis and library
construction were carried out according to the manual of
SMART~ cDNA Library Construction Kit(Clontech)．
About 2 g n1I A was reverse transcribed to single
stranded cDNA by SuperscriptI](Gibco BRL)at 42℃
for 1}L One fifth of the first-strand cDNA was used to
carry out dscDNA synthesis by a 22 cycles of PCR(95℃
for 15 s．68℃ for 6 rnin)．A1l of the dscDNA were di—
gested with sfi．The reaction product was fractionated
by CHRC帆 SPIN-400 and the fractions containng
dscDNA larger than 500bp were collected，and re-dis—
solved in dd H2 O after deposited in alcoho1．And at last，
these dscDNAs were ligated into lamb& TriplEx 2 vec—
tor．
1．4 Tittering the unanplified library an d determining the
percentage of recombinant clones
A single，isolated colony from the streak plate was
picked out randomly，which was used to tittering the li—
brary according to the manual of SMARTr cDNA Li—
brary Co nstruction Kit，and in order to test for ligation
efficiency，20 negative single clones were picked out ran—
domly used to PCR，and then checked by gel eletro—
phoresis．
2 Results and Discussing
The E．brevicornum’s young leaves were colected
in early January．It was not only because flavonoids
were expressed ma ximally，but also ma ny valuable
genes were expressed in this period．From 30 g young
leaves，about 1 50／lg total RNA was obtained．The e—
lectrophoresis analysis showed that there were appar—
ent integrate bands of 28S and 18S rRNA，and the 28S
band exhibited nearly about twice of the 18S(Fig．1)．
From UV photometric measurement．it could be seen
that A26o nm／A28o nm 1．82．These characters demon—
strated a high quality of the total RNA．2．0／lg mRNA
could be obtained after Oligo(dT)一cellulose column
chromatography from 150／lg total RNA．
28S
18S
5S／5．8S
Fig．1 Total tLNA of the Epimedium
brevicornum’s young leaves
A modified oligo(dT)primer(CDS$／3’PCR
primer)primed the first-strand syn thesis reaction by use
of 2／lg mRNA．One fifth of the resulting sscDNA was
amplified by PCR using CDS$／3’PCR primer and 5’
PCR primer derived from SMART Ⅲoligonucleotide and
obtained the dscDNA． The products were checked by
gel electrophoresis，the result showed that it produced a
dispersive molecules bands from about 0．5 kb to 4．5 kb
(Fig．2)，it showed that the quantity of the&eDNA was
enough．Co mparing with other ones，a higher percentage
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520 广 西 植 物 27卷
of full-length eDNA could be obtained with this method
(I_j a1．，2004；Bai et a1．，2002)．
Ma rke r dSDNA
p
p
Fig．2 dscDNA synthesis by a 22 cycles of PCR
The dscDNA was digested by sfi。and the resulting
of eDNAsize fractionation by CHROMA APIN一4OO。l7
tubes of diferent sizes of fragments were separated．
And then checked by gel electrophoresis(Fig．3)，the
cDAN in 7～ 1O tube were colected and combinated to—
gether，here，the cDNA in 7 tube was also be colected
although it was faint in order to avoid losing the big
fragm ent because of the lower resolving power of the ge—
lose,The 11 and 12 tube were not be colected be—
cause the fragm ents of them were smal1．
The collected cDNA was deposited in alcohol。and
then re-dissolve in 7 L dd H2Q O．5 L dissolved cD-
1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7
Fig．3 The dscDNA after size fractionation by CHROMAAPIN-400
7 5 n g／la 1 50n g／la g 25 n g／la g
Fig．4 Detection of the concentration of the purified cDNA
The lower one is the sample cDNA，and the upper are the control eDNA
NA was compared wi th the control cDNA。the concen—
tration of the dissolved cDNA was about 68 ng／／~L after
the UV photometric measurement(Fig．4)，SO the total
cDNA will be about 900 ng．The quantity of the sample
cDNA could be used to construct the cDNA library．
The eligible dscDNA was ligated to XTriplEx 2 vec—
tor，here，the ratio of cDNA to vector in the ligation reae—
tion was a critical factor in determining transformation
efficiency，and ultimately the number of independent
clones in the library．An d the optima l ratio of cDNA to
vector in ligation reactions must be determined empiri—
DL2000 1 2 3 4 5 6 7 8 9 10 1 1 1 2 13 14 15 1 6 1 7 1 8 1 9 20
2000bp
250bp
1OObp
Fig．5 De tection of inserts of the library and the combining rate
cally for each vector／cDNA combination．According to
t}-e ma nual of SMART~”cDNA Library Construction
Kit，the dscDAN was successfuly ligated to the
XTriplEx2 vector，and then packed it into Lambda virons
with packaging extracts finally．The titer of the cDNA
library was 1．2×10 Pfu／mL In order to test the effi—
ciency of the ligation，randomly 20 negative single clones
were picked out used to PCR after the cDNA was cut in
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3期 钱宝英等：淫羊藿嫩叶eDNA文库的构建 521
intro。the sizes of the products of PCR were mainly 0．5
～ 2 kb(Hg．5)，the average size of the fragments is about
975 bp，efficient ligation of the cDNA to the kTriplEx 2
vector resuh in 80 9，6 recombinants．
E brevicornum ’s young leaf cDNA library was
constructed successfully in this experiment．The quan—
tity and the quality of the total RNA and the mRNA
were very important in constructing the cDNA library。
and the presence of the molecular weight material in
the size-fractionated dseDNA must be over 0．4 kb．
And when chose the vector，the),TriplEx 2 vector was
wel be chosen to use，this vector allow digestion with
multiple enzymes which make the stuffer fragment un—
clonable，and it also provides other advantages，such as
high titer libraries，blue／white screening for recombi—
nants，regulated expression of cloned inserts，and every
cDNA inserted into the MCS of),TriplEx 2 is expressed
in all three reading frames．
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Andrea G Prescott。Nicholas PJ Stamford，Guy wheeler。et a1．
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378— 382
Kubasek WL，Shirley BW ，Mckilop A，et a1．1992．Regulation of
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65
淫羊藿嫩叶 cDNA文库的构建
钱宝英 ，2，黎云祥1*，杨子松1，冯 图1
(1．西华师范大学 环境科学与生物多样性保护省级重点实验室，四川 南充
637002；2．台州学院 生命科学学院，浙江 台州 317000)
摘 要：以淫羊藿嫩叶为实验材料，用 Trizol方法提取植物总 RNA，纯化出 mRNA，用 SMART(the Switch
Mechanism At the 5 end of RNA Templates)技术反转录成cDNA，同时使用 CHROMA SPIN-400凝胶柱层析纯
化 cDNA，最后将片断连人 kTriplEx2 vector，经包装得到 500 L原始文库，文库的滴度为 1．2×106 Pfu／mL。经
体内切割后，随机挑选文库的2O个阳性克隆进行PCR鉴定，算出文库的重组率为8O ，扩增出的片断主要集中
在 0．5～2 kb之间。结果说明文库质量较好 ，可以用于基因筛选 。
关键词：淫羊藿；嫩叶；cDNA文库
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