全 文 ：武汉植物学研究 2007，25(1)：105—108
Journal D，Wuhan Botanical Research
淫羊藿花蕾 eDNA文库的构建与鉴定
姜天亮 ，黎云祥h，杨子松2，韩素菊 ，陈光登
(1．西华师范大学，四川省环境科学与生物多样性保护重点实验室，四川南充 637002；
2．阿坝高等师范专科学校，四川汶川 623ooo)
摘 要：基于smart技术构建了淫羊藿花蕾eDNA文库并检测了其质量。结果表明，该文库重组率为95％，平均插
入片段大小为 1095 bp，文库滴度为2×10 pfu／mL，是一个高质量的淫羊藿花蕾eDNA文库。此文库的建立将有助
于克隆与次生代谢相关的基因，特别是淫羊藿黄酮特异合成代谢的基因，其次是克隆与花发育相关的基因。
关键词：eDNA文库；花蕾 ；淫羊藿
中图分类号：Q785；Q949．746．8 文献标识码：A 文章编号：1000—470X(2007)O1-0105-04
Construction and Identification of a eDNA Library for the
Flower buds of Epimedium brevicornu M axim
JIANG Tian—Liang ，LI Yun—Xiang ’，YANG Zi—Song2
， HAN Su-Ju ，CHEN Guang—Deng
(1．Slchuan Provincial Key Laboratory ofEnvironmental Scien~and Biological Diversity Conservation，China West Normal
， Nanchong，Sichuan 63700~，China；2．AM Teachers Colege，Wenchuan，Sichuan 623000，China)
Abstract：A cDNA library for flower buds of Epimedium brevicornu Maxim was constructed which based
on SMART technology．Our results showed that the titer of unamplifed library from flower buds was 2 x
10。pfu／mL．The percentage of recombinants was 95％ and the average length of the insert eDNA frag —
ments was 1095 bp．It suggested that one high quahty cDNA library for flower buds of E．brevicornu were
successfuly constructed．Construction of cDNA library wil help to clone the relevan t genes associated
with secondary metabolism ．first，the special genes in the course of flavonoid biosynthesis；second，the
genes related with development of flower buds in brevicornu Maxim．
Key words：eDNA library；Flower buds；Epimedium brevicornu Maxim
As a traditional Chinese medicine and important
genus，Epimedium has been used for a long time，and
is the hotspo t of modem pharmaceutical studies for its
wide an d good curative efects．The recent studies focus
on the chemical constituents an d their correspo nding
chemi cal stmctures，taxonomic system ，ecophysiology，
cultivation．pharmacognosy an d so on[ -4]
． Flavonoids
are main compo nent as a medicine，such as icariin，
anhydroicaritin，epimedoside A ，etc[ 一 。。．
In flowers，seeds，nuts， vegetables and fruits，
flavonoids OCcur naturally． ey belong to a class of
secondary metabolites in plants that are involved in
many impo rtant functions．For examples，protection
against overexpo sure to ultraviolet light，floral pigmen—
ration for attracting po linators and antimierobial aetivi—
ty as phytoalexins【n，’
． Being an integral part of the
human diet，tlavonoids possess health—promoting pro—
perties actiI唱 as an tioxidants and being involved in vaso—
dilator processes．Th e amount an d content of flavonoid
compo unds in plan ts can be mod ifed by altering
expression levels of the enzymes involved in thepath—
wayIn】
． The way of flavonoid biosyn thesis is very clear
in many species such as in bilberry[引 and many enzymes
．
such as chalcone synthase(CHS)。chalcone isomerase
(CHI)，flavanone一3一hydroxylase(F3H)，dihydrofla—
vonol-4一reductase(DFR)，flavonol synthase(FLS)，
rhanmosyl trata~ mse(RT)aIe key enzymes in this
course of biosynthesis[ ‘t 引
．
Constructing cDNA library will lay solid founda—
tion for finding relevant genes an d investigating their
收稿 日期：2006．06-07。修回日期：2006-08．14。
基金项目：I~1)11省杰出青年学科带头人培养计划项 目(04ZQ026-047)资助；四川省科技厅应用基础项目(03JY029-021-22)资助；四川省
重点学科建设项目(SZD0420)资助。
作者简介：姜天亮(1979一)，男，吉林四平人，硕士研究生，从事植物分子生态学研究。
· 通讯作者(E-mail：yx—li@263．net)。
维普资讯 http://www.cqvip.com
106 武 汉 植 物 学 研 究 第 25卷
functions．The eDNA library cal be used not only to
screen target genes required+but also to express them．
A eDNA library for E． br~icornu flower buds can be
used to find the relevant genes of the biosynthesis of
flavonoids and interesting proteins associated with the
development of E brevicomu[16 3
． Then．altering expres-
sion levels of the enzymes involved in the pathway of
the biosynthesis of flavonoids，such as icarlin，can be
used to increase the production of icariin to make more
people be healed of their sickness．
1 M aterial and m ethods
The flower buds of E brevicornu were sampled
and then were placed into hquid nitrogen immediately，
at last stored at一80 ．
Th e total RNAs for the flower buds ofE ~reu／comu
were extracted using Trizol Reagent(Life Technologies．
USA)．Then，the folowing procedure．was performed
according to the manufacturer’B recommendation of the
SMARr cDNA Librau"Construetion Kit User Manual
(PT3000—1，PRI5738)．At first，Using SMART tech—
nique，CDS I／1／3’primer WRg used to s)mthesize the
first—strand eDNA． Long distance polymerase chain
reaction f LD PCR)was used to synthesize the double—
strand eDNA that was then digested by Sfi I and frae—
fionated by CHROMA SPIN-400 column．The longer
than 0，5 kb cDNAs ware collected and ligated to
XTriplEx2 vector Then phage packaging reaction
and library amplification were performed．The quality
of the unamplifed eDNA library was strictly checked
by conventional titer determination． Twen~"plaques
were randomly picked and tested using PCR with uni—
versal primers derived from the ~quence flanking of
the rector．
2 Results
2·1 Extraction and purification of total RNAs
The total RNAs for flower buds of E 6r鲫 0mH
were ex~acted using Trizol method The ratio of
OD2∞／OD of total RNA wB．s 1．82．Th en，the integ-
rip of the total RNA was analyzed by agarose gel elec—
txophoresis(Figure 1)．According to figure 1，the
bands of 28S and 18S were obvious and the brightness
0fthe band of山c 28S wastwice ofthe 18S atleast．
Fig．1 TBtal RNA ofE brevicornu flower buds
2．2 eDNA synthesis
Using 2 Ixg high qu alily total RNAs，First—Strand
eDNAs were synthesized according to the pretoeol of
SMART eDNA Library Construction Kit(Clontech．
USA)．Then．1／5 ofthe ss cDNA were used to s}mthe—
size the ds eDNAs．After 22 cycles．5 of】00 L was
analyzed by agarose gel electrophoresis(Figure 2)．The
bands of ds cDNAs were dispersed and the length of ds
cDNA was mainly bounded o12 500—4500 bp． The
bands were natural and theirs．brightness of山e bands
0fthe ds eDNA were s1】ffi【-jeT】t．
Fig．2 Ds eDNA of五 brevicornuflower buds
2．3 Fraetionation of ds eDNA by gel filtration
through CHROM A SP ．4O0
As shown in the Figure 3．fifteen tube s of ds
cDNA were obtained by fractionation of ds cDNA of the
flower buds of E，brevicornu．The cDNA 0f the tube of
No．4 was not obvious．but it was also colleeted in
order to avoid the losing of big fragment of eDNA．At
the same time．the cDNA of山e tube s of No．9一lO
were not collected because the cDNA of them were too
short and the numbers of the cDNA 0f No．5—8 were
enough to use．Th e eDNA of tubes of No 4—8 were
colected and mixed togetheF．
1● 衄 =：I—
维普资讯 http://www.cqvip.com
第l期 姜天亮等：淫羊落花蕾 eDNA文库的构建与鉴定(英) l07
Fig．3 los eDNA afterfractionafion ofds eDNA
碧d filtrationt 0ugh CIIROMA SPIN-400
2．4 Calculate the total quantity of cDNA
After sample was collected，it was deposited by
adding Droper vdlime of 100％ ethoa3o1．at last dis—
solved in 7 L dd H2O．TheO．5 L 0f7 山 was used
to measure the concentration of山e cDNA by absor-
bance at 260 nm by comparing it with standard cam—
pare eDNA(Figure 4)．The eoneen~afion of ds eDNA
of the E brevicornu flower buds was about 100 ng／gL．
So．the total quantitv 0f cDNA w且s eDoElgh to constrnct
the corresponding eDNA library．
Fig．4 Detec廿0n of the t~ncentration
nfthe purifi~l eDNA
2．5 Measurement of the titers and capacity of the
the eDNA library
After the ds cDNAs were hgated to．kTrlplEx2 VeC—
tor，the titer of unamplifod 1 brat was measured．The
titer of unamplifed library was 2 ×10。pfu／mL． e
capacity of the eDNA library was 1×10 pfu．for the
volume of each eDNA librar~,i5 5o0 LLIJ_
2．6 Identification of the percentages of recombi·
nants of the library
Mter titer the amplified libra~r．the percentage of
recombinant clones of amplified librarv need to be de—
termined．In order to analyze the size of the construe—
ted 1ibrarv and the diversity of cDNA inserts．twenty-
one pJaques"~qeFe randomly picked with sterilized tooth
sticks from plate of the librm~／ into PCR tube with
20 LLL defonized H O and incubated at 100屯 for
10 min．And then Pyrobest DNA polymerase(TaKaRa，
Japan)，Pymbest bufer，dNTP mixture，s~nse primer
(synthesised by Sangon Company，sequence：5’一CTC—
CCAGATCTGGACGAGC-3’) and antisense primer
f 5’ rAATACGACTCACTATAGGG．3’) were added
into the tube．PCR was then carried out according to
thefoHowing pDOgTam：95℃ 3 inin；94℃ 30 s，55
30 s．72oc 2 min for 30 cycles；72℃ 5 rain． PCR
products were checked by running agarose gel along-
side DNA marker(Figure 5)． percentage of re—
comblnants from the library,was 95％ in amplified flow—
er buds library． e
．
average 1
．
ength of the inserts was
1095 bp in flower buds libra,
Fig．5 Detection of im~erts fragment of the library
and recombinallt rate
3 Discussion
Epimedium brevicoraum’§ flo~&-er buds cDNA
librarv was constructed successfully in this experiment．
In the progress of construeting eDNA libraD．the quan—
tity and the quality ofthe total RNAs were vet),impor—
taut．and the presence of file molecular weight material
in the size．fractionated ds eDNA must he over 0．4 kb．
these would decide the quality of library． kTriplEx2
vector WflS chosen because of its many advantages：
high titer libraries，blue／white screening for reeombi—
nants，regulated expression of cloned inserts，and ease
of converting clones from phage to a phsmid vector via
Cre lox—mediated subcloning．
The exploitation and utilization of genetic re—
SOLU~es plays an important role in research on structure
and hinction of genes．In the seeonda~metabohsm of
flavornoids．$ome genes were largely expressed in the
process of flowering，for examples，CHS-A，CHS—J，
and so on【”-
．
S0．fhe eDNA libra~ constructed from
flower huds 0f E． brevicormt will not only provide a
usefol tool for studying the molecular mechanisms of
the seconda~"metabolism of flavonoids of E brevicor-
．
but also offer help for finding related genes and in—
teresting proteins that might play important roles in the
development of E．brevieornu，and increasing the pro—
duction of ieariin by altering expression levels of the
eaz)q-nes invelred in the pathway of the flavonoid blo—
维普资讯 http://www.cqvip.com
1O8 武 汉 植 物 学 研 究 第 25卷
synthesis．
References：
[1] Li z z，Xu Y Q，Wang Y，Husag H W．Status sad p t of
H目eaIch Oilmedicinal plants ofEpimedium L[J]．CMnese Tva~-
a．onal and Herbal Drug，2905，36(2)：289—295．
[2] Hou JR，Sheng JM，WangXQ，TianYX，WuG．Advsacesin
studies on heatm epimedium[J]．J脚 Agriadmml Univerd,cy，
2O04，26(1)：59—65．
[3] Shi J X，Li H M，Liu Y D，Chen J．Advance in research of
medium L Ecophysiolngy[J]．JJishou Un&,erJ ，20O0，21(1)：
84—85．
[4] Zhang JX，SuzX，Liu S J．Advancein咖dy onKpbned／u[J]．
JSichuanTeachers CoO*We(Natund Science)，2003，24(2)：
160—166．
[5] IjW S，ChertX．Study on extraction procesa~oficariin[J]．L／
shi-ahea Medicine and Materia Medica R~ reh，2OOO，11(11)：
971—972．
[6] u W K，Xisa P G，Lisa M c．Csahuoside-Efromthe aerial parts
ofEpimedium kooanl127g Nakal[J]．Chemical J Chinese Uniz~rsi-
ties，1995，16(1O)：1575—1576．
[7] Iju c M，Iju z Q，Dou J P，Li L，Liu S Y．Isolation sad 8Uuc*
tuIe identification ofthe novel alkaloid constituent from
m J}c矾砸，ll，INakai[J]．Chem／ca／JCh／nese Un／verdaes，2OO3。24
(12)：2215—2217．
[8] Fukai T，NomuraT．Seven prenylatedflavonol glycoside~sfromtwo
m specie~s[J]．Phytochemlstry，1988，27：259—266．
[9] Guo B L，Xisa PG．Comment∞main species ofhed)a印 m
[J]．Ch／aa J Chinese Materia Medica，2003，28(4)：303—307．
[10] Sun P Y，Xu Y，Wen Y，Pei Y P，Chen Y J，Nofiko Shimim，Ta-
dahiro Takeda．The chemical constituents of 幽 m korea-
，ll，I NakaiⅡ[J]．Chinese J Medicinal Chemistry，1998，8(4)：
281—284．
[11] Dooner H K，Robbins T P，Genetic sad developmental control of
sathocyanin biosynthesis[J]．Ann Rev Gena，1991，25：173—
199．
[12] Druka A，Kudma D，Rostoks N，Bmeggeman R，yon Wetstein
D，KleinhofsA．Chalconeisomerase genefrom rice(D sat／-
t )sad barley(Hor&~a帆 )：physical，genetic sad muta-
tion mapping[J]．Cene，2003，302：171—178．
[13] Jaakola L，Pirtilt A M，Halonen M，Hohtola A．Isolation ofhi．gh
quality RNA from the bilbery(Vacc／n／um m删 L．)fruit
[J]．MolBiotechnol，2001，19：201—203．
[14] Jaakola L，M丑越t苴K，Pirtila A M，TOrOnen R，Karenlampi S，
Hohtola A．Expression of genes involved in sathocysa in biosyn-
thesis in relation to sathocyanin，proanthocyanidin，sad flavonol
levels during bilberyfruit development[J]．PlantPhysiol，2002，
130：729—739．
[15] Winkel—Shirley B．Biosynthesis offlavonoids sad efects of 8t1"~88
[J]．Curr PlantBiol，2002，s(3)：218—223．
[16] Ying J S．Petal evolution sad distribution paterns of ，l础 m
L．(Berbefidacesa)[J]．Acta Phytotaxonom／ca S／n／ca，2002，40
(6)：481—489．
[17] Ronald E K，Rikvsa B，Francesca Q，Arijen J V。Joseph N M M．
Chalcone synthase promoters in petunia are active in pigmented
sad unpigmented cdl type[J]．P／ant Ce／，1990，2：379—392．
维普资讯 http://www.cqvip.com