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Cloning of a PAL Gene of Pennisetum purpureum and Prediction of Its Protein Structure and Function

象草PAL基因克隆及其蛋白质结构与功能预测



全 文 :!24" !1#
 Vol.24  No.1
! " # $
ACTA AGRESTIA SINICA
   2016$  1%
  Jan.  2016
犱狅犻:10.11733/j.issn.10070435.2016.01.019
óŒ犘犃犔NøÌÍfgstÌpÝWÎ5ð²
j¦¦
1,
§  1¨,©ª«1,) ¬1,0$­1,í®x1,2
(1.¡Z()Y+¢+Ðo¸¢+,,,¤ ,Y510642;2.,¤ž-)Ãľ¨/0&!,,¤ ,Y510642)
78

úe…W”…½
(犘犃犔)ÚJKDFu9Kš’ïÂ&T‰½,žDFu、àw26xu?“9oؤ
KTñ’

»/0j
‘N51’R-(犘犲狀狀犻狊犲狋狌犿狆狌狉狆狌狉犲狌犿)n‡ý,<=@”d、3′RACE6hiTailPCRTÒâ,
D犘犃犔®5\‘d,kd犘狆犘犃犔cDNAlj2429bpj|DNAljàŸ3907bp。犘狆犘犃犔³)T󦳻
720M…®W,ÞJEFTGTITASGDLVPLSYIAGúe…W”…½Ð=ɏ®l。ûd9KÔ:+\‘=•,
犘狆犘犃犔®5cDNAlj»z(犣犲犪犿犪狔狊L.)犣犿犘犃犔TˆQÝsn94%,‘Xq»*JKAJìtsTˆ


L…®Wlj]^\ð=•&
,犘狆犘犃犔犣犿犘犃犔 *+ù5Ýä,Ɓ‘Xq»*JK犘犃犔`nßà。
犘狆犘犃犔ó¦Tñ¢epÂF…˜,犘狆犘犃犔ó¦ÞJEFT“ª‰”ep,gh犘狆犘犃犔ÚEFTúe…W”…
½¬

DDFuTš’ªì•«Tcd

9:;

R-

DFu

úe…W”…½

9KÔ:+
<=>?@
:S543.9;Q943.2    ABCDE:A     AFG@:10070435(2016)01013709
犆犾狅狀犻狀犵狅犳犪犘犃犔犌犲狀犲狅犳犘犲狀狀犻狊犲狋狌犿狆狌狉狆狌狉犲狌犿犪狀犱犘狉犲犱犻犮狋犻狅狀狅犳
犐狋狊犘狉狅狋犲犻狀犛狋狉狌犮狋狌狉犲犪狀犱犉狌狀犮狋犻狅狀
CHENWeiwei1,TANGRan1,WUYuyu1,LIFei1,ZHANGXiangqian1,XIEXinming1,2
(1.DepartmentofGrasslandScience,ColegeofForestryandLandscapeArchittecture,SouthChinaAgriculturalUniversity,Guangzhou,Guangdong
Province510642,China;2.GuangdongEngineeringResearchCenterforGrasslandScience,Guangzhou,GuangdongProvince510642,China)
犃犫狊狋狉犪犮狋:Phenylalanineammonia-lyase(犘犃犔)isakeyenzymeinvolvedinligninbiosynthesis.Itcataly
zesthefirststepofphenylpropanoidpathway,whichsuppliesprecursorsforavarietyofsecondarymetab
olitesincludingphenolics,flavonoidsandanthocyanin.Inthisstudyweisolateda犘犃犔genefrom犘犲狀狀犻狊犲
狋狌犿狆狌狉狆狌狉犲狌犿cv.N51usinghomologycloningandRACE(RapidAmplificationofcDNAEnds)methods
aswelashiTailPCR.Thelengthof犘狆犘犃犔cDNAsequencewas2429bp.Thenucleotidesequenceof
犘狆犘犃犔genewas3907bplongandhighsimilarityto犣犿犘犃犔,with94%identity.The犘狆犘犃犔geneen
codeda720aminoacidprotein,whichcontainedaconservedmotifofGTITASGDLVPLSYIAG.Inphylo
genetictree,the犘狆犘犃犔hadaclosegeneticdistancewith犣犿犘犃犔,andwasclusteredinthesamegroup
withothergramineousplantsinvolvedinligninbiosynthesis.Bypredicting,thethree-dimensionalstruc
tureofthe犘狆犘犃犔proteinshowedatypical"seahorse"shape,whichindicatedthat犘狆犘犃犔isatypical
familymemberofphenylalanineammonialyase,shouldplayanimportantroleinligninsynthesis.
犓犲狔狑狅狉犱狊:犘犲狀狀犻狊犲狋狌犿狆狌狉狆狌狉犲狌犿;Lignin;Phenylalanineammonialyase;Bioinformaticanalysis
HIJK
:20141128;LMJK:20150403
NOPQ

¬¶·*+®¸
(31272491,30972138);öJús?+˜ÊË+*­©´*/®¸(20124404110011)“Å
5RST

t˜˜
(1990),È,šË/09,Ì«ÍÎ?-®5¦§/0,Email:1039553248@qq.com;@?|}c™,Ó·(1988),
—

ÊË/09

Ì«ÍÎ?-®5¦§/0
,Email:tr880213@163.com;<Ôc™ Authorforcorrespondence,Email:
xiexmbs@scau.edu.cn
    R - (犘犲狀狀犻狊犲狋狌犿 狆狌狉狆狌狉犲狌犿)n q » *
(Gramineae)DE-¬$9C4 -»JK。.¤
épÜȒYÜ6 BiokoÎg,Pþ,-GJé
pÜ6*p܎
[1]。
5ÞJ¤`s

EDë
ì

K,Ò`

KŠ

øt6U9§¥–

9KF¤`6ps?8­

‘’npÜ

*
pÜ,-GJTHҋd?-

•«T+í.ý
j|ÝÞN¥T§”cK
[23]。
·n

DFu»
`6ä’zLûYÄAS×ìR-TÏd

~
UV?-,0

íî+풻

9KìéfÝ
j|Hl´µ
[47]。
5.

<=®5Ãľ¨D
R-DFuš’‰½®5\‘ab

Ín;<
! " # $ !24"
DFu»`$ä’

D;ÒR-Â1GÞJ•«
T÷ø

DFuÚßà䒁epæ=*CT
s=m`û`šK

LJKT9Ÿ°J=Ä&
ªì•«Tcd
[8]。
WMDFu9Kš’=Ä
&JŽ½®5Tž

XÛt@ab¢øJK
&DFuT»`|‘ä’’=
[9]。
úe … W ” … ½
(phenylalanineammonia
lyase,犘犃犔)ÚDFuš’ª½hT‰½,
Ÿéúe(ïÂTƒ,
。犘犃犔å#ì89oØ
6úe(àoØ

Ú%ðúe(àoØ!ßu
ŠT ½

A Ú ú e ( à o Ø T A Ø ½
[1011]。
Koukol?[12]Œj͕pJK&°Pƛf=5
¡犘犃犔,._‰é犘犃犔 ®5d?ÒÓT/0
6i½gر¼

ä$ç

J‰ 犘犃犔 ®5T/
0¯&L®5d|gW8ÒÓ

~LQ§
(犗狉狔狕犪.狊犪狋犻狏犪L.)[13]、»z(犣犲犪犿犪狔狊L.)[14]、
•äi
(犔犲犾犲犫犪狅犾犱犺犪犿犻)[1516]、6¾(犛犪犮犮犺犪狉狌犿
狅犳犳犻犮犻狀犪狉狌犿)[17]、 E é (犘犻狀狌狊 犿犪狊狊狅狀犻犪狀犪
犔犪犿犫.)[18]、wR(犕狅狀犵犺狅犾犻犮狌狊)[19]、wj(犆狌犮狌
犿犻狊狊犪狋犻狏狌狊犔犻狀狀)[20]、k Ô (犚犻犮犻狀狌狊犮狅犿犿狌狀犻狊
犔.)[21]?J K & € J 犘犃犔 ® 5 T ˆ ‰ / 0。
犘犃犔DFu、¬fu、xu6àw2?“9
oؤKTš’Ɔˆ‰
[22],
nòJKTø
J앫T ]

)犘犃犔ɏŒŒ5ÀìJ
KTȇP9Ÿ

$™OP“9oØKFJ‰
THlKŠ6øtœ?PR
[2324]。
µv
þJ/0gh

Lf*JKj|u^~&DFu
TÖ¹犘犃犔Tɏ„‡ˆ‰[2526],Æòþk
d)“-犘犃犔TÚ®5Jr[27]。
Ýä

‰éR-DFuepä’8·
[28]、
DF
l¢»`![29]、
DFuš’‰½Tɏ![30]
j|6@1GR-DFu»`T!\<ð
[31]
þÀ
kd/0

R-TDFu»`|ep8·þ®»
€

R-DFuš’‰½ 4犆犔 ®5ú=l
j
[32]、犆犆犚®5lj[33]6犆犃犇 ®5lj[34]©þ
kdd

IR-DFuš’‰½犘狆犘犃犔®5
Td|‘¦§=•¢öri½

5.»/0j
犘狆犘犃犔®5n/0DR,Œ“d¡犘狆犘犃犔 ®
5TcDNA|DNAlj,\‘9KÔ:+T=•,
àÓ¡”¡犘狆犘犃犔®51³)Tó¦,³´®»
ùðF

å9ep

ñ9ep

*H.©Ÿ|‘]
^\ð°9

¡”¡犘犃犔®5|‘gWó¦Tß
}®»8

n\ß;ÒR-DFu»`j|Â
1G0©®¯

1 UVWXY
1.1 jkUV
»/01d‡ýn
‘N51’R-(犘犲狀狀犻狊犲狋狌犿
狆狌狉狆狌狉犲狌犿cv.‘N51’),f¶¡Z()Y+-)*
+]ÊG¸

fR-TêXn‡ý

1.2 cDNA28ywNøxDNA2Žô
ÏdQÚRNA›fBÜß(:;³nð"9
K*¾ºp

\‘RNAT›f;cDNATš’Ïd
TaKaRa(‰9KÃÄYåJAºp)TuÚNBÜ
ßUhm\‘
(RNAPCRKit(AMV)Ver.3.0)。
ÏdÂFJK®5äDNA›fBÜß(:;³n
ð"9K*¾ºp

\‘DNA›f。
1.3 ESTÑY2Z[
d@”dTÒâ\‘R-犘狆犘犃犔 ®5
cDNAljTd,<=þãTq»*JKT犘犃犔
lj

\‘Blast,kd@”tsTlj,dClust
alW2D.}¨Wlj\‘D¸,QdÐ=lj,²
²ŽÆSœÝÊKPF16PR1(g1)。ÏdYD
ÊK

jR-cDNAnÂP\‘¡PCRií,uŠ
N]n20μL:(10μL2×TaqMasterMix,0.5μL
PF1,0.5μLPR1,2.8μLddH2O,1μLcDNA )。
uŠÄl

!195℃ 5min,1MGH;!2
94℃30s,58℃30s,72℃1min,30MGH;!3
72℃10min,1MGH。
1.4 犘狆犘犃犔Nø3′õö2Z[
犘狆犘犃犔®53'y?TiížÒTaKaRa(‰9
KÃÄYåJAºp

T3'Ful RACECoreSet
Ver.2.0BÜßTÒâ\‘,1dÊKn GSPout
er,GSPinner(g1)。
1.5 犘狆犘犃犔Nø5′ö2ÌÍ
Lþ—kTESTljT®¯S,²²8ºœ
ÝÊKPALR2,}Ï@”dTÒâ,L!1Me
…mTÐ=ŽS²²ŽÆÊKPALF2(g1),
ÏdYDÊK

jR-cDNA nÂP\‘¡PCR


@2dhiTailâ[35]\‘ª½†)mTi
í

1dÊKnSP1,SP2,SP3(g1)。PCR¤KÀ
1%Taô¿ij`b×Ã_,dDNAijÊ_B
Üß

:;³nð"9K*¾ºp

Ê_

å#d
pMD18T/N,ÚðÍY0®Þ,gfd÷ÞP
831
!1# t˜˜?:R-犘犃犔®5d|‘ó¦Fep¦§SÃ
ÊSª°nu®9K¾¨ºpÃl

1.6 cDNA÷‡2ÌÍ
}ÏþÀdOT3'65'?lj,Lª½†)
mATGÓ²²SÝ8ºÊKPALQCF,L.[
†)mTGAÓ²²œÝ8ºÊKPALQCR,j
R-cDNAnÂP,PALQCF6PALQCR(g1)n
SœÝÊK\‘PCRií,uŠN]6uŠÄlž
ÒSŠ
。PCR¤KT/NpžÒSŠ。
1.7 ޞ2ÌÍ
²²1D8ºÊK PALS16 PALA1(g
1),=¾Ÿé!1Me…m6!2Me…mS,Ïd
1²²TÊKjR-TDNAnÂP\‘2»mT
d

uŠN]6uŠÄlžÒSŠ
。PCR¤K
T/NpžÒSŠ

`1 犘狆犘犃犔ÑYZ[\,
Table1 Primersusedforamplificationof犘狆犘犃犔gene
ÊKïS lj
(5'→3')
PALF1 AGCTGAAGCACCAYCCN
PALR1 GGCRCCCTTGAARCCGT
PALF2 GABGAGGTGAAGMGGATGGT
PALR2 CAACTTCCAGGGCACTCC
GSPouter AGTCATCCGATTCGCCACCAAGTCC
GSPinner ATGGACAACACCCGTCTCGCCCTCG
PALS1 GTGAAGCGGATGGTGGCGGAGTT
PALA1 AAGGGGTGACGTTGGCGTTGAGC
PALQCF ATGGAGTGCGAGACCGGCCTCGTCG
PALQCR TCAGCAGAGCGGCAGGGGCTCGCCG
SP1 TAGCTGTCGGTGCCGTTCATCAT
SP2 ACGATGGACTCCAGTCACCCAGTCGCTGCTGGCCTTGAC
SP3 TCCGCCACCATCCGCTTCACCTC
1.8 ÑY²³f,éê÷>r
¨WljTÃlèSª°nu®9K¾¨ºp
œ’

ÏdNCBITBlastN6BlastPD¨Wlj
6…®Wlj\‘×î$¸D=•

ÆSÃó¦Ð
=ep

ÏdDNAMAN6ClustalXD¨W$
…®Wlj\‘¸D=•

Ïd MEGA6.0\‘
…®Wlj]^\ð{Tp

=¾dÝYQ·â
6ú#â\‘\ð{p

Ædbootstrap×C。
<=œjÃÞ
:Translate(http://web.expasy.
org/cgi-bin/translate/dna_aa)ШWljmX’
…®Wlj
;ProtParam(http://web.expasy.org/
protparam/)Dó¦T®»ùð8·\‘=•;
Protscale(http://web.expasy.org/protscale/)D
ó¦T<ÏQ\‘=•
;PredictProtein(ht
tps://www.predictprotein.org/)Dó¦Tå9e
p\‘SÃ
;SwissModel(http://swissmodel.ex
pasy.org/)Dó¦ñ9ep\‘SÃ[36]。dPy
MolÕÖDó¦\‘ñ¢ep’l。
2 pqW>r
2.1 犘狆犘犃犔Nø2ÌÍ
<=@”d

kd EST lj 455bp,À
BlastN ¸D_,°Puz (犛犲狋犪狉犻犪犻狋犪犾犻犮犪 )
犘犃犔犾犻犽犲(ÙN{ XM_004953097)TˆQn
96%。
ûd3′RACE@âPCRTÒâ\‘犘犃犔 ®
53'?d,ÀÃl—k1009bpTÍÜ(ë1A)。
<=BlastN¸D=•,uz犘犃犔犾犻犽犲TˆQ
n96%。
5'?dkd´,Yun1100bp(ë1B)。Ã
l_<=BlastN¸D,°Puz犘犃犔犾犻犽犲Tˆ
Qn96%。\ß]^1/0ljnR-犘犃犔
®5

BC<=hi-TailPCRTÒâ,D5'?öã
lj\‘d

kd465bpTlj,‘&³»!ß
Me…mTöãlj6ú=Ú!mlj

<=P#1—kTlj

²²1DiàŸTÊ
K

SÝÊKͪ½†)mATG¼½,œÝÊKd
.[†)mTGAe5,jcDNAnÂPd犘狆
犘犃犔®5cDNA ljàŸ,ÃlcDNA Yun
2163bp(ë1C)。
D2»m\‘ií

kd×n2000bpTµT
ÍÜ

ë1D),ÀÃl_P#,]^n犘狆犘犃犔®5
2»m

Yun1744bp。
2.2 犘狆犘犃犔Nø2,éê÷>r
2.2.1 犘狆犘犃犔ڎE¯ôŒ »/0—kd
lj4293bp,dLfÃÞGENSCAN=•,k
ãY®5J2M ORF,=¾Ÿé305~720bp6
931
! " # $ !24"
2465~4211bp,ORFàŸ2163bp,³)1M»720
M…®WTó¦F
,GC»`n66.44%。7e,2
»mn1744bp,5'?ÈmXŽ184bp,3'ÈmXŽ
82bp(ë2)j|Ú!mlj120bp。<=NCBIT
BlastND犘狆犘犃犔®5 ORF\‘¸D,°P犘狆
犘犃犔®5 ORFljuz犘犃犔犾犻犽犲、»z (犣犲犪
犿犪狔狊)犘犃犔(ÙN{NM_001158010)、Q§(犗狉狔
狕犪狊犪狋犻狏犪)犘犃犔(ÙN{NM_001054017)TCDsÞ
JtsTˆQ

=¾n96%,94%,90%,\ß
"©1kdT犘狆犘犃犔®5ORFnœWT³)Ž
lj
。犘狆犘犃犔®5Tàljþ›æÍ NCBIëÏ
˜
,GenBankÙN{nKF447872。
=1 犘狆犘犃犔Nø犮犇犖犃ÑY§fޞ2ÌÍ
Fig.1 cDNAandintroncloningof犘.狆狌狉狆狌狉犲狌犿
犘犃犔gene
ü
:M:DL2000;A:3'RACEií¤K;B:5'?ií¤K;
C:cDNAàŸií¤K;D:2»mií¤K
Note:M:DL2000;A:PCRresultof3'RACE;
B:PCRresultof5'RACE;C:PCRresultofcDNA;
D:PCRresultofDNAintron
=2 犘狆犘犃犔NøpÝøù=
Fig.2 Schematicstructureof犘狆犘犃犔gene
ü
:E1e…m1;E2e…m2;I12»m
Note:E1exon1;E2exon2;I1intron
2.2.2 犘狆犘犃犔çèEÚ°½¾a¿Œ <=
ProtParam=•,犘狆犘犃犔®5t³)¡720M…®
W

=m`n77.7kD,ùy?`­n5.97,1ñ’T
犘狆犘犃犔ó¦=mânC3413H5484N962O1037S34。d
ÅÝtsT…®WJAla,Leu,Gly6Glu,=¾Øc
…®WT11.9%,10.3%,8.6%68.1%;ÅÝt€
T…®WJ Trp,Cys6 Tyr,=¾Øc…®WT
0.6%,1.7%61.9%。‘&W…®W84M,*
…®W74M。犘狆犘犃犔ó¦T6¨©]ën32.19,
Ñà騩ó¦

ÏdProtScaleD犘狆犘犃犔ó¦‘
ÏQ=•

‘cTÏQR©]ën
-0.102,SÃn—¨9K&犘狆犘犃犔ó¦+LéH.F&。
2.2.3 犘狆犘犃犔çèE±—² ÏdPredictPro
teinLf=•ÕÖD犘狆犘犃犔ó¦\‘=•SÃ,S
Ãefgh
,犘狆犘犃犔 ó¦å9ep&α;Ø
53.2%,βC\Ø3.3%,ï£("éØ43.5%,SÃ
efA…˜O¡犘狆犘犃犔ó¦TešŸ­(ë3)。
ó¦TOoŸ­LH.2bÁ&ª•«c
d

~jWðUVÔ{+"

¿®ð;<ó¦FTˆ
>#6µFžK

nò]§[\H.H.$H.
H.e®FTˆöcd

<=SÃefLã

犘狆犘犃犔ó¦A»J.:TŸ­,=¾Ú3M N-
¿®ðŸ­
、6Mó¦M½CjWðŸ­、5Mpó
¦M½ⅡjWðŸ­、1Mp…WM½jWðŸ­、
18MN-f7gðŸ­、1MghðŸ­,@2L
202~218aaÓ°P¡úe…W”…½6ä…W”
…½FqljGTITASGDLVPLSYIAG,/³eL
½É&!

ٚ
[GS][STG][LIVM][STG]
[SAC]SG[DH]L.[PN]L[SA].{2,3}[SAGVTL]
Túe…W”…½FqljÂâ

ÏdSwissModelD犘狆犘犃犔ó¦Tñ9ep
\‘SÃ
,犘狆犘犃犔ó¦ñ9epÂ&¶!r–j
¬s
(犘犲狋狉狅狊犲犾犻狀狌犿犮狉犻狊狆狌犿 )犘犮犘犃犔 ó¦(PDB
No.1W27.1)nžÒ\‘[37],¶!9’T犘狆犘犃犔
󦁞^…@”n76.21%。犘狆犘犃犔ó¦
Tñ9ep~ë41˜,ÞJEFT“ª‰”ep,
=¾è MIO ep、Coreep6Insetedshiel
dingepp’。
D犘狆犘犃犔ó¦lj\‘󦦧=•g
h

Ylj67»J 犘犃犔 ¦§]»J犘犃犔
犎犃犔 ¦§,úe…W”…½6ä…W”…½T
Ð=ɏ®lGTITASGDLVPLSYIAG/³eL
½É&!

‘& AlaSerGlyñ5epnúe…
W”…½T%ðɏŸ­

YepL4Åtðcd
Kõñ=mQ

Ín9’ÞJ%ðT½Éep
 MIO,Ín°›%ðcd[38]。犘狆犘犃犔ó¦&°
PŸ…Ÿ­L210,V211,L2606 A261,j|%
041
!1# t˜˜?:R-犘犃犔®5d|‘ó¦Fep¦§SÃ
ðÉ  Ÿ ­ N264,G265,NDN(386388aa), H4006 HNQDV(490494aa)。
=3 犘狆犘犃犔st ºpÝfp8’°ð²
Fig.3 Predictionofsecondarystructureof犘狆犘犃犔proteinandbindingsite
=4 犘狆犘犃犔stúºpÝ´„
Fig.4 Threedimensionalstructures
simulationof犘狆犘犃犔protein
ü
:A:rp MIOep;B:pInsetedep;
C:_pCoreep
Note:A:BlueMIOdomain;B:Cyaninseteddomain;
C:Purplecoredomain
2.3 犘狆犘犃犔st¯NqÑY2)*ÒQ>r
<=NCBITBlastPD犘狆犘犃犔T…®Wlj
\‘@”¸D=•

efgh
,犘狆犘犃犔…®Wl
j»z犣犿犘犃犔、Q§犗狊犘犃犔、ss^(犃犲犵犻犾狅狆狊
狋犪狌狊犮犺犻犻犆狅狊狊.)犃狋犪犘犃犔、•äi(犅犪犿犫狌狊犪狅犾犱
犺犪犿犻犻)犅狅犘犃犔JtsˆQ,=¾n95%,93%,
90%,86%。LjTO,q»*JK犘犃犔…®Wl
jˆDÐ=

Ïd NCBITCDDD犘狆犘犃犔ó¦
\‘¦§üÙ

ef…˜
,犘狆犘犃犔ó¦\Júe…
W”…½ÉŸ­

AJä…W”…½TɏŸ­

»/0Í NCBIëϘ&_¯¡56Í6@K
GœWT犘犃犔…®Wlj,ÍnpJK犘犃犔…
®Wlj]^\ð{

‘&³´èéàJK

uàJ
K

vmJK

/mJKTYmXJK6÷mXJ
K

]^\ð{ef…˜

6@KGT犘犃犔 …®
Wlj/Y#=n3Yà¾(ë5,A,B,C),AàÚ
/mJKTYmXJK
,BàÚ/mJKT÷mX
JK
,CàèvmJK、èéàJK6uàJK1ä
’

ò÷mXJKYmXJKT*+ù5tä

ë
5)。»/01dT犘狆犘犃犔…®Wlj‘Xq
»*JK犘犃犔 …®Wlj¬éBà,6»z犣犿
犘犃犔…®WljT*+ù5Ýä。
j6@KGT犘犃犔…®Wlj]^\ð{n
ÿÏ

ÀÁf¡8Í犘犃犔…®Wlj\‘•
¸D

\ß=•犘狆犘犃犔…®WljTÐ=®l,
Æåf¡ß,»JEFTúe…W”…½Élj
TsÐ=´,

ë6)。¸Def…˜,6@KG
犘犃犔…®WljLŸASJߩغ,ILß}l
jSJsAÐ=

~úe…W”…½6ä…W”
…½Fqlj

ë6)。»/01kdTT犘狆犘犃犔
AÞJjS®l

gh犘狆犘犃犔 L§ÞJ‘X
犘犃犔 ó¦ˆ@T¦§。
3 ˆ‰
3.1 犘狆犘犃犔Nø2ÌÍfÑY>r
Xu?[39]Œ“ͨw(犑狌犵犾犪狀狊狉犲犵犻犪)&=5k
d犘犃犔®5TàŸlj,Ylj³»1M1935bp
T¼>(Éù

³)645M…®W,=m`n70.4
kD,󦁑XJKTó¦ÞJsATˆQ。Æ

(犘犻犮狉狅狉犺犻狕犪犽狌狉狉狅狅犪)犘犃犔 ®5ljàŸn
2142bp,³»TÝY¼>(ÉùL儳)713M
…®W

=m`n77.766kD [10]。»BC’¦—k
¡犘狆犘犃犔®54293bp,¼>(Éù2613bp,³)
720M…®W,=m`n77.7kD,»JßM2»m,
2»m6e…mTæ÷ljٚ GT-AG£(。
YëJK犘犃犔®5€rÞJ1M2»m,IÏ


ƒëJK犘犃犔®5îJ2»m,~Z"¦
(犛犲犾犪犵犻狀犲犾犾犪犿狅犲犾犾犲狀犱狅狉犳犳犻犻)犘犃犔,nJ}JK
犘犃犔 »J 2 M$ 3 M2»m,~xX&ZA
犘犃犔3,犘犃犔4[40]。2»mL®5gW&ªabTc
d

<=L(P#&cd÷®5gWGó
¦

Ínëìó¦:
[4142]。
Ï/0

L—¨JK
&
,mRNA2»m67DÚNÞJ[\cd]Dœ
ÝmRNAÂtoØÞJ[\cd,§B›s®5T
gW
[43]。
I犘犃犔®52»mcdÁ¢6z),
3\‘\ßT/0

141
! " # $ !24"
=5 犘犃犔¯NqÑY)*ÒQÓ
Fig.5 Neighbourjoiningphylogenetictreeof犘犃犔aminoacidsequences
ü

ë&=<Óënbootstrap˜‡;0.05n*+ù5;´ {enKGàï|ó¦ÙN{
Note:Numbersabovebranchesdenotebootstrapvalue;thevalueof0.05representstheevolutiondistance;
speciesfulnameandproteinaccessionnumberwereinparentheses
241
!1# t˜˜?:R-犘犃犔®5d|‘ó¦Fep¦§SÃ
=6 c~,e犘犃犔¯NqÑYNт$
Fig.6 Aminoacidsequencealignmentof犘犃犔
ü

œ³fFüO犘犃犔TFqljó¦GTITASGDLVPLSYIAG;犘狆犘犃犔:R-(AGZ95689);犣犿犘犃犔:»z(DAA36916);
犘狏犘犃犔:c8y(AFY17067);犘狋犘犃犔:nzé(AAA84889);犖狋犘犃犔:“-(ABG75910);犃狋犘犃犔:&ZA(AAM12956);
犌犫犻犘犃犔:“{(ABU49842);犐犾犘犃犔Q|(AAW80637)
Note:Thelabelproteinsequencesareunderlined;犘狆犘犃犔:犘犲狀狀犻狊犲狋狌犿狆狌狉狆狌狉犲狌犿 (AGZ95689);犣犿犘犃犔:犣犲犪犿犪狔狊(DAA36916);
犘狏犘犃犔:犘犪狀犻犮狌犿狏犻狉犵犪狋狌犿 (AFY17067);犘狋犘犃犔:犘犻狀狌狊狋犪犲犱犪,(AAA84889);犖狋犘犃犔:犖犻犮狅狋犻犪狀犪狋犪犫犪犮狌犿 (ABG75910);
犃狋犘犃犔:犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪(AAM12956);犌犫犻犘犃犔:犌犻狀犽犵狅犫犻犾狅犫犪(ABU49842);犐犾犘犃犔:犐狊狅犲狋犲狊犾犪犮狌狊狋狉犻狊(AAW80637)
  Ls?JK&,犘犃犔®5ëj®5¬Tñ
âOP

Y¬³´2~6M’¯,LûKG&þ
ÀdO¡犘犃犔®5T®5¬[4447]。L}v~
(犈狆犻犿犲犱犻狌犿犫狉犲狏犻犮狅狉狀狌犿 )&kd¡3M犘犃犔®
5

‘&犘犃犔1ž|DFuš’,犘犃犔3ž|xu
Tš’
,犘犃犔267ž|DFu、xuTš’]
àw2Tš’ˆ‰

XÛgWTúŸAÚ6@
T
[29]。
Q§犘犃犔®5¬J11M犘犃犔®5,ò
c6@HlÍÖTab
[48]。
L&ZA&kd4M
犘犃犔®5,XÛLÜMúŸTgW`6@[49],gh
6@T犘犃犔®5ÞJ6@9Kɏ6gWúŸ。
»/0}Ï@”dTÒârkd¡1MR-
犘犃犔®5,<=9KÔ:+=•gh犘犃犔®5n
bDFuš’T1M®5。IÚdéab/0,
]3«d‘X¬’¯

5nL@ßKG&


DFuš’TJM犘犃犔 ®5。‰éR-犘犃犔
®5¬+LTL§

E3\߂ƒ/0

3.2 犘狆犘犃犔st2pÝfÎ5ð²
犘狆犘犃犔ó¦è720M…®Wp’,À=NCBI
TBlastP¸D,»z犣犿犘犃犔@”ts。‘å
9ep䒁YëJKT犘犃犔 ó¦ß#。D
犘狆犘犃犔ó¦ñ¢epTSÞҬs(犘犲狋狉狅狊犲犾犻
狀狌犿犮狉犻狊狆狌犿 )犘犮犘犃犔ó¦[50],°P犘狆犘犃犔ó¦
ÞJEFT

ª‰

ep

L)LD
(犚犺狌狊犮犺犻狀犲狀
狊犻狊)犚犮犘犃犔ó¦[11]6€^(犉犪犵狅狆狔狉狌犿狋犪狋犪狉犻
犮狌犿)犉狋犘犃犔ó¦[51]&A»J.ep。犘狆犘犃犔
ó¦ÞJŸ…Ÿ­

j|Ÿ…ɏŸ­

L­Íè
(犇犲狀犱狉狅犫犻狌犿犮犪狀犱犻犱狌犿 )犇犮犘犃犔ó¦[52]6pä
w
(犖犲狉犻狌犿犻狀犱犻犮狌犿)犖犻犘犃犔ó¦[53]&AÞJ@
:TŸ­ep

gh¡犘狆犘犃犔ó¦ÚÞJúe…
W”…½ÉTó¦

Calabrese?é犚犺狅犱狅狊狆狅狉犻犱犻狌犿狋狅狉狌犾狅犻犱犲狊
犘犃犔 Tñ¢epgh[54],犚狋犘犃犔 n@”>`N,
¿M*®ñM¾T*®BEˆå

ñ’

ª

‰

1jíY¡úä*®Tˆöcd

¤9¡1M
>`K
。犘犃犔 6犎犃犔 ÞJsA@”,犎犃犔 T
Xbfep=•°P%ðT<`BÜÆÈ
DHA,n Ú 3,5å U5“ k ®4H‚ o42
(MIO)[55]。
犘狆犘犃犔ó¦lj67»J犘犃犔¦§]»J
犘犃犔犎犃犔¦§,ÞJLyase_I_LikeQ¬W”…
½ÉŸ­
。Lyase_I_LikeQ¬èLyaseclassI¬


ä…W”…½6úe…W”…½ä’
。Lyase
classI¬½Ì«Ú\‘àQβKõT%ðuŠ,ä
…W”…½6úe…W”…½%ðŸ…TuŠ
[56]。
•lj¸t°P犘犃犔¦§ŽT…®Wl
jtnÐ=
,N?TغÝY。…®Wlj]^ð{Tp=•gh
,犘狆犘犃犔…®Wlj»z
犣犿犘犃犔 JìÝäT*+ù5,‘Xq»*JK
犘犃犔JìtsT@”,¬éEFTq»*JK
犘犃犔,@2Uh犘犃犔…®WljT=ð=āJ
K\ð=ÄÚß#T

eÃ犘犃犔cnúe(oØ
ïÂT!ßM‰½L\ð=Ä&У¡2BT*
+¨©6çðó@

4 p‰
»/0—k¡R-犘狆犘犃犔®5lj4293bp
341
! " # $ !24"

ÙN{ KF447872.1),‘&³)ŽŸAn2163
bp,2»m1744bp。犘狆犘犃犔®5³)¡720M…
®W

=m`Yun77.7kD。犘狆犘犃犔 ó¦ÞJ
GTITASGDLVPLSYIAGúe…W”…½Ð=É
®l

]»J犘犃犔犎犃犔 ¦§,j|úe…W
”…½T%ðɏŸ­ AlaSerGlyñ5ep,g
h»/01dOT犘狆犘犃犔®5nEFTúe…
W”…½®5

”•AB
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