全 文 ：西北农业学报 2008 , 17(2):218-224
Acta A griculturae Boreali-occidentalis Sinica
Cloning and Sequence Analysis of a Phenylalanine
Ammonia-lyase cDNA from Pittosporum tobira
XU Feng1 , CHEN Liu-ji1 , CA I Rong1 , DU He-wei2 and CH ENG Shui-yuan1 , 3＊
(1.C ollege of Ho rticulture and Gardening , Yangtze Universi ty , Jingzhou Hubei 434025 , C hina;
2.College of Life S cien ce , Yang tze University , Jingzhou H ubei 434025 , China;3.College of Life
Science and Engineering , H uanggan g Normal University , Huangzh ou H ubei 438000 , China)
Abstract:A pheny lalanine ammonia-lyase(PAL)gene(designated as P itPAL)was cloned from P it-
tosporum tobira .The P itPAL cDNA fragment w as 866bp and it encoded a 289 amino acid pro tein.
PitPAL was found to have extensive homology w ith those of other plant PAL genes via multiple a-
lig nments.T he dominat ion si tes and ca talyt ic active si tes in PAL pro tein of Oryza sativa and Zea
mays were also found in PitPAL.Phylo genetic t ree analy sis revealed that P itPAL had closer relat ion-
ship w ith PA Ls from arbor plants than from o ther plants.The arbor plant is N erium oleander and
Camell ia sinensis .P itPAL i s a useful to ol to study the regulat ion o f anthocyanin metabolism in P.to-
bira.
Key words:P itPAL ;PAL ;Pit tosporum tobira ;Molecular cloning;Sequence analy sis
CLC number:Q785 Document code:A Article ID:1004-1389(2008)02-0218-07
＊
海桐花苯丙氨酸解氨酶的基因克隆与序列分析
许 锋1 ,陈柳吉1 ,蔡 荣1 ,杜何为2 ,程水源1 , 3＊
(1.长江大学园艺园林学院 ,荆州 434025;2.长江大学生命科学学院 ,荆州 434025;
3.黄冈师范学院生命科学与工程学院 ,黄冈 438000)
摘 要:从海桐中克隆得到苯丙氨酸解氨酶(P AL)基因 cDNA 片段 , 并命名为 PitP AL , GenBank 登录号为
AY747678。 PitP AL 长 866 个 bp , 编码 289 个氨基酸。通过核苷酸和蛋白质序列多重比较发现 PitPA L 与
其他植物的P AL 基因高度同源。 PitP AL 编码的蛋白质序列包含与水稻 、玉米 P AL 蛋白质相同的脱氨基位
点和催化活性位点。 P AL 系统进化树表明P itPA L 与乔木类植物(如夹竹桃 、山茶)的 PA L基因聚类关系最
近。 PitP AL 基因的克隆为利用基因工程技术来调控海桐花青苷的合成代谢 、以及海桐花的颜色调控提供了
基因资源。
关键词:P itPA L;苯丙氨酸解氨酶;海桐;基因克隆;序列分析
Anthocyanis are the dominant pigments in
plants that show red , orange , blue and purple
colo rs.T he accumulat ion o f anthocyanin is af-
fected by various environmental factors [ 1] .The
pathw ay of anthocyanin bio synthe sis is w ell
studied and most of the enzymes and the gene s
＊Received date:2007-10-22 Accepted date:2007-11-23
Foundation i tem:The w ork w as supported by Prog ram fo r New Centu ry E xcellen t T alents in Universi ty (NCET-04-0746), the Re-
gion Techn ology Developmen t Program of Chines e Minis t ry of Educat ion (02095), the Natural S cience Foundat ion
of H ubei Province(2002AB094), the Youth Talent Foundation of Hub ei Province(2003AB014)and the Education-
al Of fice Key Research Prog ram of H ubei Province(Z200627002).
Biography:XU Feng(1979-), Assis tant Lectu rer , th e major research f ield w as the genet ic engineering of plan t secondly m etab o-
lism.Email:xufeng198@126.com .
＊Corresponding au th or:CHENG Shui-yuan (1965-), Professor , Doctor Supervi sor.Emai l:s y cheng@sina.com
invo lved have been characterized [ 2] .The fi rst
step in the bio synthesis is a reaction by which
pheny lalanine is conve rted to t rans-cinnamic
acid.This step is catalyzed by phenylalanine am-
monia-lyase(PAL , EC 4.3 .1.5).Because of i ts
impo rtant ro le in the bio synthesis o f f lav onoids ,
lignins and phy toalexins , PAL and its g ene are
widely studied [ 3-6] .T he expression of PAL is
regulated by va rious environmental facto rs such
as light , low temperature , fungal infection and
w ounding [ 6-8] .
PAL genes have been isolated and se-
quenced from a number o f plant species , inclu-
ding a gymnosperm , monoco ts , and herbaceous
and w oody dicots.In most plant species , PAL
genes occur in a smal l gene fami ly
[ 9-16] .Excep-
tions to this include potato , which appears to
contain over fo rty PAL genes [ 17] , and loblolly
pine , which has been repo rted to contain a single
PAL gene
[ 18] .Each member of the PAL family
show s a dist inctive expression pat te rn .In bean ,
PAL 7 is expressed in roo ts , shoots and leaves ,
PAL2 in roots , shoo ts and petals , and PAL3
only in roots.P AL1 and PAL3 we re induced by
fungal infect ion and PAL7 and PAL2 w ere in-
duced by light.All three genes are induced by
mechanical w ounding
[ 19] .In parsley , PAL is
encoded by a small family of at least four genes
and one o f the PAL gene s is expressed in re-
sponse to UV irradiation and elicitor t reatment.
The kinetics of mRNA expression induced by
light is completely different from that induced by
elici to r t reatment[ 10] .Besides the expression
pat tern , the promo ter region of the PAL gene is
also characterized in several plants.Sequences
such as box1 (box L)and box2 (box P)a re re-
po rted to be conserved among promoter s o f the
gene fo r the pheny lpropanoid pathw ay
[ 10-12] .
Pittosporum tobira L ., which is a common
ornamental plant in China due to i ts w hite flow-
er , has been used for experiments on f low ering
[ 20] .Howeve r , li tt le is known about the role of
PAL gene in f lavonoid and anthocyanin accumu-
lation in P .tobira , and there is no repo rt on the
cloning of PAL gene f rom P.tobira.In this pa-
per , we repo rt fo r the fi rst t ime the cloning of
the PAL gene f rom P.tobira .The sequences a-
nalysis including the alignment of amino acid
predicted f rom PAL cDNA and the evolutional
relationship are also presented , which wi ll be
useful for unveiling the overall dow nst ream met-
abolic pathw ay s o f f lavonoid and anthocyanin in
P.tobira.
1 Materials and methods
1.1 Plant materials and reagents
Petals o f P .tobira were co llected f rom the
garden o f the School o f Ho rticul ture and Gar-
dening , Yang tze University .
Primers used in the experiment we re syn-
thesized by Shanghai Sangon Biotechno logy
Company , China and DNA sequencing w as per-
fo rmed by Dalian TaKaRa , China.The high-pu-
ri ty DNA gel ext raction kit w as obtained from
AxyPrep , Union city , USA .The pMD18-T vec-
to r w as purchased from Invit rogen , Hong
Kong , China.The avian myeloblasto sis vi rus
(AMV)reverse t ranscriptase , dN TPs , RNasin
and Taq DNA polymerase w ere obtained from
Promega (Madison , WI , USA).
1.2 Extraction of total RNA
To tal RNA was ex tracted f rom petals of P.
tobira using CTAB method
[ 21] .The quality and
concentration of the RNA was detected by agar-
o se gel elect ropho resis and Eppendorf Biopho-
tometer before using .The RNA samples w ere
sto red at -80 ℃prior to RT-PCR analy sis.
1.3 cDNA cloning
O ne microg ram of to tal RN A iso lated f rom
P.tobira petals w as used to synthesize fi rst
st rand cDNA acco rding to the manual o f the
AMV Reverse T ranscriptase ki t.To iso late
PAL cDNA fragments , a pai r of degenerated o li-
gonucleotide primers , PALsP :5′-GC(A/ T/C)
TC(T/C/G)GGT GAT (C/T)T(A/G)GT(T/
C)-3′and PA LaP:5′-ACA TCT TGG TT(A/
G)TG (T/C)TGC TC-3′, co rresponding to
conserved regions of PAL were synthesized.A
part ial PAL cDNA fragment of P.tobira was
·219·2 期 XU Feng , et a l:Cloning and Sequence Analy sis of a Phenylalanine Ammonia-ly ase ……
obtained by using the cDNA as the template and
PA LsP , PALaP as the tw o primers.The param-
eters for PCR were 35 cycles o f 1 min at 94 ℃, 1
min at 48 ℃, 1 min at 72 ℃.After the last cy-
cle , the amplif ication w as extended fo r 10 min at
72 ℃.The amplified products w ere analy zed by
1% gel electrophoresis and purified by a
AxyPrep DNA Gel Ex traction Ki t.The purif ied
product w as cloned into the pMD18-T vector by
method described in the instruction manual and
sequenced in both directions.
1.4 Sequence and bioinformation analysis
Sequence assembly w as performed w ith pro-
g rams from DNAstar (ht tp ://www .dnastar.
com).Protein and DNA homology searches
were perfo rmed using TBLAS TN , TBLASTX ,
BLASTP and BLAS TN prog rams (ht tp://
www .ncbi.nlm.nih .gov/BLAS T/).Multiple
sequence alignment w as perfo rmed w ith the sof t-
ware Vector NTI suit 6 prog ram .Phy logenetic
analysis of PAL from P.tobira and o ther PA Ls
from o ther plant w ere aligned w ith CLUS TAL
W 1 .83 and subsequent ly a phy log enetic t ree
w as const ructed by the neighbo r-joining (NJ)
method using the sof tw are o f M EGA 3.1 [ 22] .
The reliability of the tree w as measured by boot-
st rap analysis w ith 100 replicates .
2 Results and discussion
2.1 Cloning of the cDNA fragment of PitPAL
Using an RT-PCR approach , a cDN A frag-
ment encoding a PAL , designated P itPAL
(GenBank acce ssion no.AY747678), was isola-
ted and cha racterized.It w as 866 bp long (Fig.
1), contained G/C content of 51.2% and enco-
ded a 289 amino acid pro tein (Fig.2), and had
high similarity wi th o ther PAL genes o f repo rted
plants (Table 1).The nucleo tide sequence of
PitPAL1 w as 82% identical to PAL gene f rom
Nerium o leander , 81% ident ical to PAL gene
f rom Petunia hybrida , 80% identical to PAL
gene f rom Tri folium pratense , Prunus av ium ,
Cicer arietinum , andUlmus pum ila , and 77%i-
dent ical to PAL gene from Vitis vini f era , and
Juglans regia , implying PitPAL was the PAL
gene f ragment of oleande r.Fur thermore , the
homolo gous sequence of PAL gene among differ-
ent species showed the PAL gene might keep a
st rong conservation during the mo lecular evolu-
tion
[ 16] .
M , DNA marker(100bp Ladder);CK , negative con trol;1 ,
2 and 3:amplif ication by PALsP and PALaP
Fig.1 Electrophoresis of RT-PCR of PitPAL gene
2.2 Characterization of the deduced PitPAL
protein
The deduced amino acid sequence fo r the
PitPAL polypept ide w as also show n in Fig .3.
By using the sof tw are o f Computer pI/Mw Too l
at www.expasy .org , the calculated isoelect ric
point (pI)and mo lecular w eight of the deduced
PitPAL polypept ide w ere predicted to be about
5.71 and 31.0 kD , respectively.A database
search w i th BlastP2 .2.14 (National Center fo r
Bio technology Info rmation databases)and multi-
alignment by Vector NT I show ed that the de-
duced PitPAL polypept ide had high similarity
wi th o ther PALs from o ther plant species (Fig.
3).The amino acid sequence of PitPAL was
94% identical to PAL from Vitis vini f era , 93%
identical to PA L from Populus balsami f era and
Rubus idaeus , 92% identical to PA L from
Prunus avium , Lactuca sativa , Jatropha cur-
cas , Pyrus communis and Camell ia sinensis ,
91% identical to PAL from Daucus carota , Petu-
nia hybrida , Petrosel inum crispum and Manihot
esculenta , show ing PitPAL had very close rela-
tionship w ith PA Ls from other plants in this as-
pect .In addi tion , many sites essential fo r PAL
activi ties conserved in di ffe rent plant specie s
we re also found in PitPAL.Fo r example , re si-
dues of the deaminat ion site , including the site s
·220· 西 北 农 业 学 报 17 卷
of L5 , V6 , L56 , A57 w ere found in P itPAL
(Fig .3).The catalyt ic active sites w ere found
at N60 , G61 , N180 , D181 , N182 , H196 ,
HNQDV (285-289).All the active sites men-
tioned above w ere the same w ith tho se o f PAL
f rom Oryza sativa and Zea mays
[ 11 , 23] , indica-
ting NoPA L1 w as a member of PAL fam ily.
The primer sequences are underlined , and the f irst amino acid i s begin ning w ith the thi rd nucleot ide.
Fig.2 Nucleotide sequence and deduced amino acid sequence of PitPAL
Table 1 Nucleotide sequence of PitPAL similarity to the PAL gene of other plant species
Species GenBank Acces sion N o. Ident ity No.bp overlap E-value
Ner ium oleander A Y747677 82% 719 0
Petunia h ybr ida A Y705976 81% 610 6e-163
Tri fol ium pra tense DQ073809 80% 595 5e-154
P runu savium AF036948 80% 590 2e-152
Cicer ar iet in um AJ250836 80% 596 7e-148
Ulmus pumi la DQ078280 80% 595 2e-147
Vi t is vini fera EF192469 77% 675 9e-142
J ug lans r eg ia A Y747676 77% 683 1e-140
2.3 Molecular evolution analysis
To investiga te the evolut ionary relat ion-
ships among PitPAL and other PAL proteins , a
phylo genetic t ree w as const ructed based on the
deduced amino acid sequense of PitPAL and
other PAL pro teins f rom o ther plant species , u-
sing histidine ammonia-ly ase f rom Pseudomonas
sy ringae as an outg roup .The analy sis result
highlighted three impo rtant features in all in-
ferred species (Fig .4).All the PAL pro teins ,
which shared the same family signature sequence
and the conserved motif s , were derived f rom a
common ancesto r in the evolution using HAL as
outg roup , no mat ter w he ther they belonged to
the gymnospe rm or angio sperm plants.Second-
ly , PAL sequences fo rm several distinct species-
specific clusters.For example , Pittosporum to-
bira , together w i th othe r arbo r species(N .ole-
·221·2 期 XU Feng , et a l:Cloning and Sequence Analy sis of a Phenylalanine Ammonia-ly ase ……
ander and Camel lia sinensis), formed a cluster ,
indicating that PA L of P it tosporum tobira had
clo ser relat ionship wi th other arbor species than
wi th non-arbor species (Fig.4).
Pi tPA L , P.tobira , (accession no.AAX18626);LsPAL , Lactuca sativa , (accession no.AAL55242);VvPAL , Vit i s vini fera ,
(acces sion no.CAO18169);JcPAL , J atropha curcas , (acces sion no.ABI33979);PcPAL , P yruscommunis , (acces sion no.ABB70117);
RiPAL , Rubus idaeus , (accession no.AAF40224);PbPA L , Populus ba lsami fera , (accession n o.AAQ74878).T he deamination sites
are in dicated in the boxes.And the catalyt ic active sites are m ark ed w i th asteris k signs.The alignment w as performed wi th the C LUST-
A L W program.
Fig.3 Comparison of predicted amino acid sequences of PAL protein
It i s obvious that PAL of gymnosperm spe-
cie s including pine and ginkgo w ere g rouped into
a cluster.As could be seen from the tree , PA Ls
from Pteridophyta species such as Pel lia
epiphy lla , Isoetes lacustris , Huperz ia lucid ula
and Lycopodium tristachyum also naturally fo rm
ano ther cluster.Monocoty ledon species such as
Oryz a sat iva and Trit icum aestivum also form a
cluster.Meanwhile , PA Ls from Beta vulgaris ,
Petunia hybrida , Solanum tuberosum , Nicoti-
ana tabacum , I pomoea ni l , Daucus carota and
Lactuca sativa , the Dico ty ledon species , were
grouped into a cluster in the t ree.Thirdly , sev-
eral other species such as Bromheadia f inlayso-
niana and Arabidopsis thal iana f rom different
families had a shorter genet ic distance among
·222· 西 北 农 业 学 报 17 卷
each other (Fig.4).P itPAL had the close st re-
lationship w ith tho se of othe r arbo r specie s and
i t w as g rouped in the cluster of arbo r plants.It
i s suggested that P itPAL shared a common evo-
lut ionary o riginals w ith the arbo r species as w ell
as the conserved sequences mo tif s.
In this w o rk , a novel PitPAL cDNA frag-
ment w as cloned and characterized for the fi rst
time.Mult iple alignments show ed that the de-
duced PitP AL was homo logous w ith o ther
known PAL proteins , and it contained conse rved
active sites possessed by PAL pro tein family.
The anthocyanis and f lavonoids we re main com-
ponents of pigments in P.tobira and they played
very impo rtant ro le s by show ing dif ferent colors
of P.tobira f low er.The PAL is a key enzyme
in anthocyanis biosynthetic pathw ay.Although
much research about PAL has been fo cused on
plant-microo rg anism , lig ht-inducing and w oun-
ding-inducing , there has been an explosion o f in-
terests in f loral pigments of plant [ 24-26] .With
the increasing know ledge of anthocyanin biosyn-
thetic pathw ay , it wi ll be possible to have a
complete understanding of the w hole pathway ,
no t only in the model plants but also in some
no rmal f lo ral plant fo r e xample Pittosporum to-
bira .Fo r the specific ornamental propert ies of
P.tobira f low er , the know ledge on the seconda-
ry metabolic pathw ay , including the related gene
cloning and characterization , will be a g reat mo-
tivat ion for bioengineering the secondary meta-
bolics in P.tobira.The iso lation o f full-leng th
cDNA of PAL gene in Pittosporum tobira and
the functional characterization o f PitPAL gene
w as also been recent ly pro ceeding .
The numbers at each node repres ented the boots t rap values.There are 5 b ranches incu lding Gymnosperm , Pteridophyta , M onocoty-
l edon , Dicotyledon and Arbor.Th e P it tosporum tobira belon gs to the Arbor wi th Camel lia sinensi s and Ner ium o leander .
Fig.4 Neighbor-joining phylogenetic tree of the sequences of PitPAL and other PAL
proteins using PsHAL as outgroup
·223·2 期 XU Feng , et a l:Cloning and Sequence Analy sis of a Phenylalanine Ammonia-ly ase ……
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·224· 西 北 农 业 学 报 17 卷