全 文 ：广 西 植 物 Guihaia 26(6)：608— 616 2006年 11月
Cloning of the segment of flavonoid 3-hydroxylase
gene from the gDNA of Prun us
m um e by degenerate PCR
ZHAO Chang—lingI一，YANG Qing1，CHEN Jun—yu0
(1．College of Li Sciences，Nanjing Agricultural University，Nanjing 210095，China；2．College of
Agricultural Sciences and Biotechnology，Yunnan Agricultural University，Kunming 650201，China；
3．College of Landscape Architecture，Beijing Forestry University，Beijing 100083，China)
Abstract：The gDNA was extracted from the tender leaves of P．DluDle with“Predeimpurity-SDS method”de—
veloped in this study．Based on the highly conserved amino acid regions of the 1 1 deduced amino acid sequences
of the cDNAs of the flavonoid 3-hydroxylase(F3 H )genes which had been openly published and submitted in
the GenBank databases，2 forward degenerate primers and 3 reverse degenerate primers were designed and con—
sisted of 6 sets of primer pair．Only one primer pair could be used in degenerate PCR to amplify three 469 bp
segments from the gDNAs of P．WlUWle‘Nanjing Hongxu’，P．w／uw／e‘Nanjing Hong’and P．mume‘Fenpi Gong—
fen’respectively．Sequence analysis indicated that the 3 segments shared 99．72％ identity among each other
and 65．57 identity with the corresponding regions of the 1 1 cDNAs as a whole．Furthermore，the“GGEK”
motif is pointed out not to be the characteristic sequence of F3 H．It is the first time for the segment of F3 H
gene to be cloned from the gDNA of ligneous plant．This study can underlay the cloning of the ful length of
F3 H gene from P．w／uw／e．
Key words：P．DluDle Sieb．et Zucc．；extraction of gDNA；segment of flavonoid 3-hydroxylase gene；degener—
ate PCR
CLC number：Q949．7 Document code：A Article ID：1000—3142(2006)06—0608—09
Mei(Prunus mume Sieb．et Zucc．)flower is
one of the candidates of the national flower of P．
R．China today． The scientific research of sixty
years on M ei flower in China has obtained superex—
cellent achievements．However。the scientific study
on the flower color of M ei is almost a blank(Chen，
2002；Li，2004)．
The flower color of Mei includes mauve，pink，
pure white，greenish white，light yellow and double
color(Chen，1989)．P．mume‘Nanjing Hongxu’．
belonging to Form Cinnabar，is the typical repre—
sentative of the mauve．P．mume‘Nanjing Hong’
and P．mume‘Fenpi Gongfen’，all belonging to
Form Pink Double，are the typical representatives
of the pink(Chen，1989)．The anthocyanins of the
flower color pigments of these 3 cultivars have
been identified as cyanins(Zhao et a1．，2004a，b，
2006)，indicating that the key enzyme determining
the biosynthesis of the anthocyanins of the flower
color pigment of Mei is flavonoid 3-hydroxylase
(F3 H)．
F3 H is a microsomal cytochrome P450一de—
pendent monooxygenase that requires NADPH as a
co—factor(Forkmann，1991)，which was first dem—
Received date：2005—05—19 Accepted date：2005-10—07
Biography：Zhao Chang-ling(1969一)，male，born in Dujiangyan City，Sichuan Province，Doctor of science，Associate professor
working in plant physiology，biochemistry and molecular biology．
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6期 赵昶灵等：梅花类黄酮 3 一羟化酶基因片段基于基因组 DNA的简并PCR法克隆 609
onstrated in microsomal preparations from cultured
Haplopappus gracilis cells(Fritsch et a1．，1975)．
It controls the hydroxylation of dihydrokaempferol
to dihydroquercetin and of naringenin to eriodictyol
(Brugliera et a1．，1999)． As a result，F3 H de—
termines the shift from pelargonidin to cyanidin(Tana—
ka P￡口Z．。1998)and results in the accumulation of cya-
nidin derivatives，which lcads to red flower color．
As membrane-bound and P450_-dependent pro·
tein，F3 H is difficult to isolate．Furthermore，clo—
ning and identification of flavonoid 3-hydroxylase
gene(F3 H )by homology to the highly conserved
regions shared by the P450 family in plants is corn—
plicated by the large number of P450_-like se·
quences(Schuler，1996；Chapplet 1998)． Till 1999，
isolation of a F3 H cDNA clone from Petunia hy—
brida was first reported(Brugliera et a1．，1999)．
Degenerate polymerase chain reaction(PCR)，
namely degenerate oligonucleotide—primed PCR
(DOP—PCR)(Telenius et a1．，1992)，uses degener-
ate primers to accomplish PCR．Degenerate prim-
ers are a set of primers which have a number of op—
tions at several positions in the template sequence
so as to allow annealing to and amplification of a
variety of related sequences(Innis et a1．，1 990)．
They are radically resulted from the degeneracy of
the genetic code because primers targeted to parti-
cular amino acid sequences must be degenerate to
encode the possible permutations in that sequence．
The obvious advantage of degenerate PCR is that
the nucleotide sequences of the target genes are ob—
tained by amplifying according to the conserved a—
mino acid sequences of the target genes’homolo-
gous protein． As a result，it has been widely ap-
plied in the cloning of new gene，the analysis of
gene expression and so on(Shi et a1．，2004)．
W e report in this paper the amplification of
the segment of F3 H from the genomic DNA(gD—
NA)of P．mume by degenerate PCR with the objec—
tive to underlay the starting point for cloning the
complete sequence of the F3 H ．It is the first time
for the segment of F3 H to be cloned from the gD—
NA of ligneous plant．
1 M aterials and methods
1．1 M aterials
1．I．1 Plant materials All leaves of P．mume
‘Nanjing Hongxu’。P．mume‘Nanjing Hong’and
P．mume‘Fenpi Gongfen’were obtained in the Re—
search Centre of Mei flower of Sun Yat—sen M auso—
laura Administrative Clffiee of Nanjing． Tender
leaves were collected randomly，encased in white
gauze pocket，immediately frozen in liquid nitro—
gen． Then the leaves were quickly ground into
powder in a white porcelain pestle after adding ap—
proximate 1 5 mL liquid nitrogen and frozen at一80C
until the extraction of gDNA．
1．1．2 Plasmid and strain DNA cloning pMD18-T
vector kit was the product of TaKaRa Biotechnolo—
gy(Dalian)Co．，Ltd．Escherichia coli(E．coli)
DH5 0【was conserved in our laboratory．
1．1．3 Enzyme and reagents Thermus aquaticus
(Taq)DNA polymerase，namely T K口R口 Taq ，
and TaKaRa Agarose Gel DNA Purification Kit
Ver．2．0 were the products of TaKaRa Biotechnol—
ogy(Dalian)Co．，Ltd．EcoR I，Sodium Dodeeyl Sul—
phate(SDS)，I3一mercaptoethanol(BME)and tetram-
ethyl ammonium chloride(TMAC)were the prod—
ucts of Amresco． RNase A and agarose were the
products of Sigma． Distilled deionized water
(ddH2 O)was used for all buffers and reagents．
Ampieillin (Amp)，Isopropyl—J3一D-thiogalactoside
(IPTG)，tris—acetic acid—EDTA buffer(TAE)and
tris—boracic acid—EDTA buffer(TE)(PH8．0))were
filtered though 0．2／xm membrane．5一bromo-4-chlo—
ro-3一indolyl—J3一D-galaetoside(X—Ga1)was dissolved
in N，N-dimethylformamide at 20 mg／mL，and
stored at一20℃ in a foil wrapped tube．
Other reagents were of analytical grade made
in China．
1．2 M ethods
1．2．1 General A1l plastic supplies，including mi—
crofuge tubes and pipet tips，were sterilized by au-
toclaving． Centrifugation was performed in 3K30
centrifuge(Sigma)．The absorption values of DNA
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were determined at room temperature(RT)in a l
cm pathlength quartz cell using a Ultrospec R 3000
UV—Vis spectrophotometer(Amersham Pharmacia
Biotech)．
Agarose gel electrophoresis was carried out in
horizontal electrophoretic trough using l× TAE
(pH8．0)as electrophoresis buffer．Ethidium bro—
mide(EB)was directly mixed into the ge1．6× load—
ing buffer was composed of 30 mmol／L EDTA，
36 glycerol，0．05 xylene cyanol FF and 0．05
bromophenol blue。The voltage was 5 V／cm。
Thermal cycling of degenerate PCR was per—
formed in the PTC-200 Pehier Themal Cycler(M J
Research，W atertown，M A，USA)．
1．2．2 Extraction of gDNA from leaf The gDNAs
of the three cultivars were isolated from the tender
leaf with“Predeimpurity—SDS method”developed in
this study．
5 g leaf powder was ground to be mushy after
mixing with 5 mL“deimpurity buffer(0．4 mol／L
glucose，3 polyvinylpyrrolidone(PVP)(Couch et
nZ．，1990；Kim et a1．，l997；Fu et a1．，1998)，0．3
B—mercaptoethanol(BME))’，and centrifuged at
10 000 g for 10 min at 4~C． The supernatant was
discarded．
After being treated again with the same buffer
and same method，the precipitate was added 10 mL
preheated at 65℃“extraction buffer(100 mmol／L
Tris—HCl pH8．0，500 mmol／L NaCl，50 mmol／L
EDTA pH8．0，1．5 SDS，0．3 BME)(Dellaporta
et a1．，1983)”，mixed reversedly and gently and
heated in 65℃ water bath for l h while being shak—
an gently now and then．The mixture was cooled
to RT，added 5 mL of 5 mol／L KCl，mixed re—
versedly and gently again，then ice—bathed for 20
min．The icy mixture was centrifuged at 7 000 g
for 20 min at 4℃ and the corresponding superna—
tant was taken out and added equivalent volume of
chloroform—iso—pentanol(24：l，v／v)and reversed
slowly for several times to make it adequate． The
solution was centrifuged at 12 000 g for 10 min at
4℃ ，the supernatant(water phase)was added 0．6
volume of iso—propanol which was frozen in 一20℃
in advance，reversed slowly for several times and
placed quietly at RT for l h．The white floc was
drawn out with a glassy hook and blotted up on
germfree filter paper． After being washed with
70 ethanol for three times，each for 10 min，the
floc was dried at RT and dissolved in 500 L TE，
then added 10／*L RNase A and placed in 37℃ wa-
ter bath for l h．The solution was added equivalent
volume of chloroform—iso—pentanol(24：1，v／v)，re—
versed slowly for several times and centrifuged at
12 000 g for 15 min at 4C．The supernatant(water
phase)was added 0．1 volume of 3 mol／L NaAc
and 2 volume of absolute ethanol which was fro-
zen in 一20℃ in advance，
and centrifuged at 7 000
placed at RT for 30 min
g for 15 min at 4E ．The
corresponding precipitate was washed with 70 eth—
anol for three times，each for 10 min，then dissolved
in 500“L Tris—EDTA buffer and stored at一20℃ ．
The quality and concentration of gDNA ex—
tracted were determined by spectrophotometry，di—
gestion of restriction endonuclease(EcoR I)and
1 agarose gel electrophoresis analyses．
1．2．3 Design of degenerate primers Degenerate
primers were designed on the basis of the multiple
alignment of the deduced amino acid sequences of
the cDNAs of the F3 H s which had been openly
published and submitted into the GenBank of Na—
tional Centre for Biotechnology Information(NC—
BI，America)．M ultiple alignment was constructed
with DNassist 2．2．
1．2．4 Amplification of the segment of the F3 H by
degenerate PCR The degenerate PCR reactions
were conducted in 200 L thin—walled micro—centri—
fuge tubes and performed in 50 ／*L reaction vol—
umes(Table 1)． In degenerate PCR，TMAC has
been used to increase the yield and specificity of
PCR products because it is thought to alter the ki—
netics reactions of primers and templates so as to
increase the frequency of the specific annealing be—
tween primers and templates(Chevet et a1．，1995)．
Hot start PCR was as follows：Apart from
TaKaRa Taq M．all reaction components were con—
fected on ice and mixed equably． Soon after being
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6期 赵昶灵等：梅花类黄酮 3-羟化酶基因片段基于基因组 DNA的简并 PCR法克隆 611
heated at 96℃ for 5 min，the system was immedi—
ately cooled to 0*C in icy water bath，then added
TaKaRa TaqTM．The subsequent programs of PCR
were as follows：35 cycles of 95℃ for 1 min，47～
5 1℃ (The concrete annealing temperature was set
according to the special melting temperatures
(Tms)of the primer pairs，and was about 5C be—
low the lowest Tm of the primer pair to be used
(Innis et a1．，1990))for 2 min and 72℃ for 2 min，
followed by a 7 min extension at 72℃ ．
Table 1 Reaction system of degenerate PCR
Reacfion
com ponents
S
．
toc
．k w? ki Fina1
soIution solution
concen_
concen— prepara- ．
trati0n t—ion一(tlL) t at 0n
Note： PCR bufer(Mg + free)(10×)：100 mmol／L Tris-HC1
pH8．3(25℃)，500 mmol／LKC1．bPCR buffer(Mgz+free)(1×)：
10 mmol／L Tris-HCl pH8．3(25℃)，5O mmol／L KC1．
1．2．5 Inspection，purification and cloning of the de-
generate PCR product The PCR products were an—
alyzed by 2 agarose gel electrophoresis． The
band was excised from the gel and purified with the
TaKaRa Agarose Gel DNA Purification Kit．then
ligated into pMD18一T Vector according to the
manufacturer’s recommendations．
E．coli DH5a competent cells were prepared
by CaC12 method in advance(Sambrook et a1．，
2002a)．
1O “L recombinant plasmid was transformed
into 100,uL the competent cells using heat—shock
method，added 900 L liquid luria—bertani(LB)me—
dium(Amp free)and cultured at 150 rpm for 1 h at
37℃ ．100 “L bateria solution was mixed with 4O
L X—Gal and 4 L of 200 mg／mL IPTG，inocula—
ted on LB-Amp plate(O9．0 cm)and stored re—
versedly at 37℃ for 12～ 16 h．
1．2．6 Selection and identification of positive clones
The white colony was randomly selected and in—
oculated in liquid LB medium which contained 100
／~g／mL Amp，and cultured at 200 rpm for 6 h at
37℃ ．5 L of the subsequent bacteria solution was
used as PCR template solution to identify the posi—
tive clone(Sambrook et a1．，2002b)，and the corre—
sponding primer pair was used as the PCR prim—
ers．The PCR program was as follows：95C for 5
min。followed by 35 cycles of 95℃ for 1 min，47～
51℃ for 2 min and 72℃ for 2 min，followed by a fi—
nal extension of 72℃ for 7 min．During the denat—
uration of 5 min，the E．coli cells rived and released
the recombinant plasmid(Sambrook et a1．，2002b)．
1．2．7 DNA sequencing and analysis The positive
clone solution was added 50 glycerol till the final
concentration of glycerol was 1 5 ，and subj ected
to sequencing by Invitrogen Biotechnology Co．
Ltd．M ultiple alignments of the 3 segments of the
F3 H s amplified from the 3 cultivars of P．mume
and of the 3 segments with the corresponding re—
gions of the 1 1 cDNAs were constructed with
DNAM AN 4．O．
2 Results and analyses
2．1 Extraction of the gDNA from the tender leaves
The gDNAs extracted from the tender leaves
of the 3 cultivars of P．mume are all purely white
solid or transparent gelatinoid．Their A26o／A28o s
range from 1．7 to 1．9(Table 2)，indicating that the
biomacromolecule impurities such as proteins and
polysaccharids have been eliminated to a great ex—
tent．The A26o／A23o s of the gDNAs are all bigger
than 2．0(Table 2)，indicating that the small mole—
cules such as phenols and amino acids have also
been wiped off(Clark，1998)．So the gDNAs have
basically met the demands of downstream manipu—
lations．
1 6 agarose gel electrophoresis analysis re—
vealed the high quality of the gDNAs extracted
(Fig．1：A)． The clear，none—dispersive and none—
tailing bands showed the gDNAs are comparatively
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6l2 广 西 植 物 26卷
unabridged．Comparing with the DNA marker．the
gDNAs are found to he longer than 50 kb．There is
not any fluorescence before the band of bromophe—
nol blue，denoting that no RNA 1eftover exist s in
the gDNAs． Moreover．no fluorescence was ob—
served in the holes of sample—adding．implying I10
viscous impurities t e．g．p0lysaccharjde，remains in
the gDNAs．It；vas also showed by electr0ph0resls
that the gDNAs cal be completely digested by
EcoR I(Fig．1：B)．
Table 2 Appearance and ultraviolet absorption
characteristics of the gDNAs extracted from
the tender leaves of 3 cujtivars of P． “m
P ⋯ ‘Na [ng Hongxu’ transparent gelatinoid 1 735 S．088
P．⋯ ‘Nanjing Hong’ transparent gelatinold 1 738 2．tC4
尸 Fenpi Gongfen’ purely w hile solld 1．S0 9 2 0E6
Note： A06c／A?s。and A26c／A03c Bre the average va[u
four experiments respectively
2．2 Design of the degenerate prim ers based on multi—
pie alignm ent
1 1 F3 H s，including those of Arabidopsis
thaHana ecotype= ‘Columbia”．Arabidopsis thali
dⅡ ecotype一 “I andsberg erecta”．Glycine 71ax
‘Wiltiams 一Glycine dz ‘1、o7B’。Ipvmoea P“r—
pur Ⅱ (common morning—glory)，I．nil Magen．
(Japanese morning glorY)．J． icolor‘Heavenly
Blue’，J．quamoctit，Petunia h ida}Torenia h 一
brida；Oryza sativa(Japonica eultivar group)，had
been openly published and submitted into t}le Gen
Bank of NCB1．It was a great pity that all of these
F3 H s’cDNAs were obtained only from herba
ceous plants，Do DNA sequence of F3 H was ob—
tained and I1o F3 H was cloned from Rosaceae
By aligning the 1 1 complete deduced amino
acid sequences of the cDNAs of the F3‘H s．tour
most highly conserved amino acid regions were ob
served and converted into DNA sequence，from
which degenerate primers，namely two forward
primers(FPs)and three reverse primers(RPs)．
vcere designed(Fig．2，Tabte 3)．W hat needs to be
explained is that two of the four conserved amino
acid regions are thought to be important to the ac—
tivitv of F3’H．The“P”of“LPPGP’is the conserved
proline—rich residues that are important for P450
topology and“LPPGP is thought to act 8s a hinge
that【s important for the optimal orientation and
targeting of the enzyme to the mierosomal mere
brahe (Murakami “ a1．， 1994)． The “IPF—
GAGRR1C”is the signalure sequence for the heme
binding domain(FxxGxxxCxG)of P450 enzymes
and the C’。of the‘IPFGAGRRIC”s the conserved
heine—hinding cysteine residue serving as the rifth
1igand to heme iron(Chapplet 1 998；Lewis eg a1．，
1 998)．In【he designed primers，the use of inosines
for substit uting 4 base wobbles instead of using al[
4 base substitutions Call lower the degeneracy of
primers，resulting in the increase of the coneentra—
tion of a specific primer and the decrease of the
background noise(Innis et“ ．，1 990)．
1 2 3 M 1 2 3 M
A
52856b0
1 6日41 b口
7233bp
6770bp
6527bD
5626 bP
5505bO
B
52B56bp
1 6B41 bp
7233 bP
6770bp
6527bp
5626 b0
5505 bD
Fig 1 l agarose get e【ec【r0ph。re gra[】1 0f
gDNAs extracted from tl／e~ender leaves o
the 3 cultiva rs ol P．171“
A Imacl gDN,＼sl B ＆1．R[dlgc~ted gDN,＼s Lane I P． ⋯
Na ing Hongx,t。；Lane 2： 1⋯ Nanjins Hong’}Lane 3 P．
：㈣ ‘FcnpiGongfen’i Lane 31：DNA n1arker Th⋯ lc sizes of
thc bands pr~queed by DNA marker w sh0wrt 0n the right
It WaS reported by Brugliera“ a1．(1 999)and
Zabala et nr．(2003)that“GGEK”motif was the
characteristic sequelce of F3 I{by which F3 H and
flavonoid 3 ，5 hydroxylaseiF3 +5 H )were distin
guished．But in this study，muhipIe alignment of
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6期 赵昶灵等 ：梅花类黄酮 3 一羟化酶基因片段基于基因组 DNA的简并 PCR法克隆 613
the 1 1 deduced amino acid sequences of the cDNAs
of the F3 H s showed，for the first time，the
“GGEK” was not the characteristic sequence of
F3 H． This was because．in the amino acid se-
(32)
(30)
(30)
(30)
(31)
(31)
(31)
(31)
(32)
(33)
(40)
· · ·LPPGP···
· · ·LPPGP···
· · ·LPPGP···
· · ·LPPGP···
· · ·LPPGP···
· · ·LPPGP···
· · ·LPPGP···
· · ·LPPGP···
· · ·LPPGP···
· - ·LPPGP···
· - ·LPPGP···
Consensus：LPPGP
(255)
(255)
(256)
(256)
(263)
(263)
266)
263)
257)
259)
271)
· · ·IKALLLN···
⋯ IKALLLN⋯
⋯ IKALLLN⋯
⋯ IKALLLN⋯
· · ·IKALLLN···
· · ·IKALLLN···
⋯ IKALLLN⋯
⋯ IKALLLN⋯
⋯ IKALLLN⋯
⋯ IKALLLN⋯
· · ·IKALLLN···
IKALLLN
quence of the F3 H of Oryza sativa(japonica culti—
var-group)，“GGEK’ was replaced by“GRM H”．
All primers were synthesized by Invitrogen Bi-
otechnology Co．，Ltd．．
(137)⋯ IPFGAGRRIC⋯
(137)⋯ IPFGAGRRIC⋯
(137)⋯ IPFGAGRRIC⋯
(137)⋯ IPFGAGRRIC⋯
(137)⋯ IPFGAGRRIC⋯
(137)⋯ IPFGAGRRIC⋯
(137)⋯ IPFGAGRRIC⋯
(137)·一IPFGAGRRIC⋯
(137)⋯ IPFGAGRRIC⋯
(137)⋯ IPFGAGRRIC⋯
(137)⋯ IPFGAGRRIC⋯
I PFGAGRRIC
^IIJCC＼rl YG(IN(；C＼(：(： M(：＼M(：＼^ lI¨ ( Y
匝 巫 囹
IcJ’：；
(39)⋯YGL FLQRAV⋯5l3
(39)⋯YGLTLQRAV⋯5l3
(39)⋯YGLq、LQRAV⋯5 13
(39)⋯YGL FLQRAV⋯5 l3
(39)⋯YGLTLQRAV⋯519
(39)⋯YGLTLQRAV⋯519
(39)⋯YGL1、LQRAV⋯522
(39)⋯YGL FLQRAV⋯519
(39)⋯YGLTLQRAD⋯5 12
(39)⋯YGLTLQRAE⋯5l2
(39)⋯YGLTLQRAV⋯526
YGLTLQRA
Fig．2 Design of the degenerate primers used to amplify the segments of the F3 Hs from the 3 cultivars of P
． 仇 Ⅲ 7le
Top’the deduced amino acid sequences of 11 F3 H were aligned by DNassist 2
．
2．The four most highly conserved regions were shown，sepa．
ted by the number of intervening amino acids(shown in parentheses)
． Middle，the consensus amino acids were shown below the aligned se-
quences·lIottomthe degenerate nucleotide sequences were determined according to part of the consensus amino acid sequences
． FPs and RPs
were designed and shown in the panes．The affixation of the restriction endonuclease sites of EcoR I or BamH I in the primers was used to
dj“ t th。Ims of the primers and administered to recognizing the PCR products in the pMD18一T vectors
． Abbreviations：ate：ArabidD由
thaliana ecotype=“Columbia”；atl；Arabidopsisthaliana ecotype= “Landsberg erecta”；grow：Glyci mnz‘Williams’；gmt
： vc ，，I(【z
‘To7B’；ip；Ipomoea purpurea；into：f
． nil Magen；ith：f．tricolor‘Heavenly Blue’；iq：f
． quamDclit；ph：P t in hybrida；th：n ^v-
brida；os：Oryza sativa．
Table 3 Primer design in degenerate PCR
2．3 Screening of the effective primers
In order to screen suitable primers to realize
the efficient amplification of the segment of the F3
H from P．mume，2 FPs and 3 RPs were combined
to constitute total 6 sets of primer pairs in the de—
generate PCR reactions．Pairs of FP and RP were
selected or discarded according to the length corn—
parison of the fragments obtained by PCR and those
predicted by the deuced amSno acid sequences which
were spanned by the special pair of FP and RP．
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6I4 广 西 植 物 26卷
It was found that，for atI of the 3 cultivars of P
m ” ．only the primer set of FP2 and RP3 was suit
able for arnplifying the segment o1the F3 H because
only the length of the product amplified by FP2 and
RP3 was longer than the theoretical length of the seg—
ment spanned by FP2 and RP3(Table 4)．
2 agarose gel electroph0resis analysis indica—
ted that．when the primer set of FP2 and RP3 was
used in degenerate PCR，one about 470bp—length band
was observed for at1 of the 3 euhivars(Fig．3)．
Table 4 Amplification results of degenerate PCR w】小
djfferent combinations of FP and RP
Note． The approxlmale lengths 0f the products amplified by de
generate PCR wefe determ ined by the comparison of the 1)r(x!ucts
with the bands produced by DNA murker． The theoretical length of
the segment spanned by the specific pair of FP and RP was~ lcutated
ac 口rding to the amlno acid number[oc~ting between the[wo sites
99．72 identityamong each other(Fig．4)．More—
over．they also shared 65 57 idenlily with t Lte corre
sponding regions of the 11 cDNAs of ttie F3 H s
which have been openly published and submitted in
the GenBank databases(data hot shown)．Howev—
er，the above alignments could not be used to eom—
plelely verify the characteristics of the segments of
the F3 H s。heeause so far．rio isolation of a F3 H
DNA clone had been reported．
2 匹
T000bP
800b口
6O0bP
5O0bp
400bp
300bP
2O0 bP
Fig．3 2 agarose gel ele r()ph0re LDgra[】l of the three
segmen!s of t1c F3 H s amplified fronl：he
gDNAs of 3 cu1 vars ol P．，¨“¨
Lane 1：P． ：“ ‘Nanjing Hongxu’}Lane 2：P ，⋯ l ‘Na ing
Hong’：Lane 3：P t Ⅲ ‘FenpiGonsfen ：LaneM ：DNA mark—
er The c0ncr c sizes of 1he bnnds prcduced by DNA mark r
eFe shown Ol the right
where the FP and RP were designed． Two obvious bands we⋯ oh
e the de pheretog⋯ 。 the CR pr。 s- 3 Discussion
2．4 Efficiency of recombination and transform ation
The recombination efficiency of the PCR prod—
ucts with 1he pM D18-T Vector was very high．be—
ing demonstrated by the facl that．in one LB-Amp
plate，about 600 wbite colonies were found every—
where and on Ly 3～ 7 bI Lie colonies were observed．
The transformation of the above recombinant
vector to E ct， i DHj口 competent cells was also
very efficient．PCR inspection indicated thai，in Ehe
2l randomly selected white colonies，about 1 9．4
colonies were positive ones averagely．
2．5 DNA sequence analysis
DNA sequencing revealed that the segments of
the F3 H s of the 3 cuhivars of P．， ‘ are at1 469
bplong．
A multiple alignment showed t}tat they shared
～一～一～一～一一一～ ～
一一一～一一
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6期 赵昶灵等：梅花类黄酮 3-羟化酶基因片段基于基因组 DNA的简并 PCR法克隆 615
the 3 cultivars of P．mume respectively．Sequence
analysis indicated that the 3 segments shared
99．72 identity among each other，and 65．57％ i—
dentity with the corresponding regions of the 1 1
cDNAs．It is the first time for the segment of F3 H
to be cloned from the gDNA of ligneous plant．
Meanwhile，it was found that the“GGEK’was not
the characteristic sequence of F3 H ，which was not
consistent with the previous viewpoint．
In degenerate PCR，it is impossible to foresee
F 90
HX 9o
H 90
F 179
H)【 179
F 268
HX 268
H 268
F 357
m【 357
H 357
F 446
}D( 446
H 446 簿罅鹳搿艨瓣瀑辨搿避蹬鞘《 瓣 窖辫糍 鱼歉 潍 §l墟鬈 池遣轰：
I从 RccIcGIccIKcIKcIT^ DAC
469
469
469
Fig．4 M ultiple alignment of the 3 segments of the F3 H s amplified from the gDNAs of 3 cultivars of P．mume
FP is shown by arrow above its corresponding sequence~RP is shown by arrow below its complementary sequence．Abbreviations=F：The
segment from P．inuln~‘Fenpi Gongfen’；H：The segment from P．raum~‘Nanjing Hong’；HX：The segment from P·ml~me‘Naning
Hongxu’．
the efficiency of primers．It was found in this stud—
y that the targeted fragment might not be amplified
by using theoretically reasonable degenerate prim—
ers．For example，the degenerate PCR in which the
primer pair of FP1 and RP1 was used produced a
strange short fragment which was consumedly
shorter than the theoretically predicted length(data
not shown)．It may be because the conserved se—
quences by which the degenerate primers were de—
signed are，by the square，be possessed by another
genes．So it is always necessary to design more
than one pair of degenerate primer to accomplish
the demanded amplification．
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梅花类黄酮 3-羟化酶基因片段基于
基因组 DNA的简并 PCR法克隆
赵昶灵1，2，杨 清1，陈俊愉3
(1．南京农业大学 生命科学学院，江苏 南京 210095；2．云南农业大学农学与生物技术
学院，云南 昆明 650201；3．北京林业大学 园林学院，北京 100083)
摘 要：用该研究设计的“预先去杂-SDS法”从梅花嫩叶提取到高质量的基因组DNA。根据 ll条已公开发
表的并提交到GenBank的类黄酮3，_羟化酶基因cDNA的假定氨基酸序列的保守区设计 2个正向简并引物和
3个反向简并引物组成 6对引物，仅有 1对引物能以PCR法同时从梅花‘南京红须’、‘南京红’和‘粉皮宫粉，
的基因组 DNA扩增到一个 469 bp的核苷酸片段，这 3个片段在总体上有 99．72 的一致性，与 11条类黄酮
3 一羟化酶基因 cDNA的相应区域有 65．57 的一致性。同时，“GGEK”并非类黄酮 3 一羟化酶的特征性模体。
这是首次从木本植物的基因组 DNA克隆到类黄酮 3 一羟化酶基因片段 该研究结果可为梅花类黄酮 3 一羟化
酶基 因全长的克隆奠定基础。
关键词：梅花 ；基因组 DNA提取；类黄酮 3 一羟化酶基 因片段 ；简并 PCR
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