免费文献传递   相关文献

Cloning and Expression Analyses of DlPPO1 from Dimocarpus longan Lour.

龙眼DlPPO1基因的克隆及其表达调控分析



全 文 :书!"#$%&
,2016,36(6):1098-1104
犃犮狋犪犅狅狋.犅狅狉犲犪犾.犗犮犮犻犱犲狀狋.犛犻狀.
  !"#$:10004025(2016)06109807               犱狅犻:10.7606/j.issn.10004025.2016.06.1098
%&(
:20160305;)*&%+(:20160426
,-./

()*+%,-
(31272149,31572088);./012+345(2015NZ00021)
0123

678
(1990-),9,:;,<=>?@AB$3CDE。
FGHI:JKL,:;,DEM,:;BNO,<=>?PQ#$B$3CRSTUVDE。Email:Laizx01@163.com
45犇犾犘犘犗1,6789:;<=>?@A
!"#

$%&

()

*+,

-./


./WX2% PQ#$B$YZDE[

.\350002)
B C:]DE^_`Vab3C,cdRTPCReRACE3C,f‘ghi’jklmcDNAnop,qrjks
tuvw,x
(狆狅犾狔狆犺犲狀狅犾狅狓犻犱犪狊犲,犘犘犗)y3z{|}犇犾犘犘犗1a、犇犾犘犘犗1be犇犾犘犘犗1cycDNA~€
(KM387405、KM516087e KM516088)e1‚DNA€犇犾犘犘犗1(KU837229)。犇犾犘犘犗1a、犇犾犘犘犗1be犇犾犘
犘犗1cy~ƒ„n1969、1960e1920bp,…†‡`yˆ‰Š‹ŒŽ1800bp‘599z’,“;],x
R”•

–—e˜™$šy犘犘犗,x`V›œ。B$Gž%ƒŸ ¡,犇犾犘犘犗1¢£¤¥¦§¨stuvwy
©ª¤¥¦«¬

cd­®¯°±²PCR3C³´犇犾犘犘犗1 µ¤@ ¡,¶jk·¸¹BºZ»,犇犾犘犘犗1>
¼½¸®¾Š¿ÀÁ µÂÃl¸®¾µÄŝ

ƴǶjk·¸¹B»È¾ÉʹË1=Hd
;犇犾犘犘犗1¶
jklm» µ²Å

ÇÌÍÎÏ

жÇÑÒÓÔÕ µ²œÖ

×ØeÙB$ÚÛÜÝÞy µƒŸ ¡

ßà“
(SA)、Öáâãä“åæ(MeJA)、NaCl、çèéêPEGÉëN犇犾犘犘犗1,xÀÁ µ,ìí µoîïð
ÇÉÊñRsšÙB$ÚÛòóºZ

DEF

jk

·ôõ¸ö¹B

stuvw

ÙB$ÚÛ

­®¯°±²PCR
GH@I$
:Q785;Q786 !JKLM:A
犆犾狅狀犻狀犵犪狀犱犈狓狆狉犲狊狊犻狅狀犃狀犪犾狔狊犲狊狅犳犇犾犘犘犗1犳狉狅犿犇犻犿狅犮犪狉狆狌狊犾狅狀犵犪狀犔狅狌狉.
TIANQilin,LINYuling,ZHENGQingyou,SURongfeng,LAIZhongxiong
(InstituteofHorticulturalBiotechnology,FujianAgricultureandForestryUniversity,Fuzhou350002,China)
犃犫狊狋狉犪犮狋:Basedonhomologycloningtechniques,weisolatedthe狆狅犾狔狆犺犲狀狅犾狅狓犻犱犪狊犲gene(3transcripts:
犇犾犘犘犗1犪,犇犾犘犘犗1犫,and犇犾犘犘犗1犮;Genbank:KM387405,KM516087,andKM516088)fromleaves
oflongan(‘Sijimi’cultivar)byRTPCRandRACE.ThefullengthcDNAsequenceof犇犾犘犘犗1a,犇犾犘
犘犗1犫,and犇犾犘犘犗1犮were1969bp,1960bpand1920bp,respectively,containinga1800bpopen
readingframe(ORF)whichencoded599aminoacids;犇犾犘犘犗1sharedhighhomologywith犘犘犗geneof
犔犻狋犮犺犻犮犺犻狀犲狀狊犻狊,犆犪狀犪狉犻狌犿犪犾犫狌犿,and犘狅狆狌犾狌狊犲狌狆犺狉犪狋犻犮犪,犲狋犮.Bioinformaticanalysisrevealedthatthe
deducedDlPPO1proteinwithconserveddomainssharedthetypicalcharacteristicsofthePPOfamily.
QPCRanalysisindicatedthatduringsomaticembryogenesis(SE)inlongan,theexpressionlevelof犇犾犘
犘犗1rosefromthestageofheartembryoandthenreachedthehighestatthestageofthecotyledonembry
o,whichsuggestedthat犇犾犘犘犗1mightplayimportantrolesduringthemiddleandlatestagesoflongan
SE.Itwasdetectedthat犇犾犘犘犗1abundantlyaccumulatedinlonganleaves,folowedbyflowerbuds;
therewaslowerexpressioninotherlongantissues.Afterexposuretophytohormonesandabioticstress,
theexpressionof犇犾犘犘犗1wasinducedbysalicylicacid(SA),methyljasmonte(MeJA),NaCl,mannitol,and
PEGtreatments.Consequently,itsuggestedthat犇犾犘犘犗1mightparticipateinabioticstressresponsiveness.
犓犲狔狑狅狉犱狊:longan;somaticembryogenesis;polyphenoloxidase;abioticstress,realtimequantitativePCR
  stuvw÷øùúûtw、ü’“w、ýþ“
weÿw™

2·Àƒ!"#

úûtuvweÿ
w

$%À&úûtuvwnstuvw
(poly
phenoloxidase,PPO),(Rÿw(pdiphenoloxa
dise)Laccase)¨ ¡*y+„[1]。PPOÍ,#-
.‘/¤012Ãy¤¥34

5v67t

87
t™s7tuv!9#

:;<¶=>#$

?@e
ô@»
。PPOA#$yB¹B§¨1=yCD,
EFGHIJKºZ
[2];
Ð/犘犘犗 ¶#$ÒÓe
LM»yƒN§¨®O«P›

E¶QR»
,犘犘犗
犅 ,x¶2sSÒÓyTUeVW» µ²,/
¶XYÒÓ» µ²/Z[#\y¹B µ²]
Ö

ж^_»
,犘犘犗,x¶¸Ï`e¸^» µ
ab
[12]。PPO É5vcdØe9#v0$yB
!

¥!¢e›fgÐhôõij)klm@mn
yop

¶#$qrst»u[1=Hd
[34];
qr
ÚÛ)mþ·ov™wxxØëN#$yBPPO


Ç»{|êÄã䓙×ØG}~¶#$
st€ò»yHd
[5]。
¶#$·
,miRNA Fº
Á‚‡òyƒ,x„…†#$ñR‡rÚÛyˆ
ò
,犘犘犗‰ÍjÄ miR1444aÁ…yƒ,x,ñR
àAA‡rÚÛyòóˆò
[6]。
Š‹„PPOyDEŒ!nŽ,sŽ‘’
“Ž‘c}@A

Ejk

”•

–—™
),
¶@­”
•¢–êÒӗ˜ºZ»™š¹B›œ

Ðstu
vwywF€ò͝ˆ›œy<=þx
[78]。
jk
@ž›œe@Ÿ) ™¡¢ÍN£jk¤È¥f
¾”
¦=jk@že@Ÿ™ÒÓy
stuvwz›DEŒ¨&§

E50℃ŽßÜÝ
10minɨ¨]Ö@žPPOz›,¢©œy@
žªt†²

ɨ¨]Ö¤Èjk@­t#$dJ
K

>Ы¬¤Èjk@­@ž›œy¹B
;PPO
w™`Yw{Rjk@­) y¹Be¹­®¯‡
¦
[910]。
s#$PPOyDE<=°»¶w%«
›À
[1112],
ÇBݱ†²³¡´

Š‹„s#$
µÇÍ@A犘犘犗 ,xŒrÄab[1316],¶#$
犘犘犗¶B¹BºZeqrÚÛ µÁ…±†·
¸yDE¹º»

¼½ƒ2qr–—犘犘犗,x
¹¾],x¶–—¿UÀB¹BºZ»¹Ë1=
Hd

ÉÊRXYÒÓyÁ>ƒv¨¦
[15];
Â(¢
™ƒ2qr”•犘犘犗,x,/³´Ä],x¶@
­¤È”•Ã¾@ž»ÀÁ µ

ÉÊĝPPOz
›>ÐÅƔ•@­¤ÈºZ»y@ž›œ
[16]。
#$¸ö¹BºZySTÁ…ÉfHnÇÈ#$¹
BºZ»½É¹BÊËꅆ±Ýy,ÌÍÎ

/
jk»犘犘犗 ,xyabê µÁ…±†ÏÐ&
§

xÑ}DEcdjk·¸¹BÒÓ
[17],
ƒ2

Ô
±,zˆ‰y犘犘犗,x,AǶjk·¸¹B
ºZ

³`ÒÓLM

fêqrÚÛÜÝÞºZ»y
ÕP µGփŸ

f¾nÇÈstuvwRjk
B¹BêqrÚۈò×Øy¦ÒÄÙÚÈ

1 ÛÜe·Ý
1.1 NO
f

ghi

ޚjkylmHn,xabyÛ
Ü

f

ß.Ã

ޚ
[17]
jk¸›àáÒÓ

â½
¸

¼½¸

ã佸eÃl¸fê

ghi

Þ
š
[18]
jky^

l

Îe@­™nd=³´,x¶
jkÒÓ³`ÔÕyÕP µÛÜ

A—˜18d
yjk¸›àáÒÓGÖãä“åæ
(MeJA)eß
à“
(SA)"š×ØÜÝ:200mLå½æ»Åç
40mLMS(èé20g/mL)귗˜,,귗˜,
ëÌìÅ,š×ØÂíáâëÌn
:0、25、50、75
e100μmol/L,3Ì1î,ïðñò、óô/õöœ
ôyjkàá2÷0.5gll‹ç—˜,»,øå½
æù=úû110r/min,25℃,ü24h。ÚÛ
xØÜÝ
:200mLå½æ»Åç40mLMS(èé
20g/mL)귗˜,,귗˜,ýìÅ NaCl
(150mmol/L)、çèé(150mmol/L)、PEG4000
(10%)eABA(10μmol/L),3Ì1îÈïðñò、
óô/õöœôyjkàá2÷0.5gll‹ç—
˜,»

øå½æù=úû110r/min,25℃,üï
—˜1、2、4、8、12、16e24hÈ,ƒ„þ°fÀÜÝ
ÛÜd=RNAÄðe±² µƒŸ。
1.2 PQ
1.2.1 犇犖犃RS犚犖犃TU:犮犇犖犃VW ¤d
ÿ!CTABÝ[19]ÄðDNA,¤dTriPure(Roche)
¿"#Äðª RNA,¤d GeneRacerKit(In
vitrogen)GÖ RTPCR €{|。qPCR ycDNA
0!¤dPrimeScript RTreagentKit(TaKaRa),
§··Ýñ$%¡&

1.2.2 XYZ[\犘犆犚]^ ^_ GenBank&
§yPPO’,“¢£€e.“€ƒ„(`
Vab)$DlPPO1F1e*)$DlPPO1R1G
99016¾              678,™:jk犇犾犘犘犗1,xyabêÇ µÁ…ƒŸ
Ö犇犾犘犘犗1ԃcDNA€y+,,^_+,r
ÄyԃcDNA €( RACE)$,d=+,
犇犾犘犘犗1,xcDNA3′-.e5′-.€;^ _q
ry3′-.e5′-.€()$,GÖ犇犾犘犘犗1
,xŠ‹ŒŽy+,

DE»[dy)$/ê
€Ð 1,)$01"23042,x+356
¨7890!
。PCR €òZ€n:94 ℃:œ›
3min;94℃œ›30s,57~59℃;<30s,72℃«
=1min,30~35z>‡;72℃«=6min。
1.2.3 /7_`7a% PCR+,y$1.0%
?@éABCDGÖ³´

Ey‚‘¤dBIOMI
GA89 DNAFGHvIþ¿"#GÖIþ,¤
dpMD18T Vector(TakaraBiotechnology))I
pEASYT5ZeroCloningKit(TransGenBiotech)G
ÖEymÎyab

J›abÃy´€01ÀK:
²B$3C¨789ˆ!

1.2.4 bYcde@A ¤d NCBIyBlaste
DNAMAN6.0Aqry,x~€GփŸ。
DlPPO134yB$Gž%ƒŸ,¤dfރŸY
§GÖ

34d,}Ýv›dyƒŸ
(ExPASyPro
tparam,http://web.expasy.org/protparam/),G
}L:´
(SignalP4.1Server,http://www.cbs.
dtu.dk/services/SignalP/),“ ô õ ± Õ : ´
(PSORT,http://psort.hgc.jp/),34dMN¤
¥y:´
(EMBnetTMpred,http://www.ch.em
bnet.org/software/TMPRED_form.html),O“
Տ:´
(NetPhos2.0Server,http://www.cbs.
dtu.dk/services/NetPhos/),34d¢£¤¥¦:
´
(NCBIProteinTools,http://www.ncbi.nlm.
nih.gov/Structure/cdd/wrpsb.cgi),34dPQ¤
¥:´
(PSIPREDProteinStructurePredictionServer,
http://bioinf.cs.ucl.ac.uk/psipred/),34dRQ¤
¥:´
(SWISSMODEL,http://swissmodel.expasy.
org/),ƒÃÒÓGvAy¥/(Mega5)。
1.2.5 fghijk犘犆犚@A ­®¯°±²
PCR(qPCR)€òñ$LineLai[20]ySTGÖ,U
L n LightCycler480(Roche Applied Science,
Switzerland)。€ò·Ò:2×SYBR PremixEx
TaqTM(TaKaRa)10μL,cDNA1μL,)$(DlPPO1
QF、DlPPO1QR)ý 0.8μL,ªV·W 20μL。
qPCR€òZ€:95℃:œ›30s;95℃œ›10s,
60℃;<30s,72℃«=10s,40z>‡;FºX
YZڃŸ´±+,€òy«P›

f犲犐犉4犃、
犈犉1犃e犉犲犛犗犇 nñ,x[20]。
<1 犇犾犘犘犗1,689R狇犘犆犚XYl<
Table1 Primersequencesforthegeneisolation
andqPCRof犇犾犘犘犗1
)$/
Primername
)$€
(5′→3′)
Primersequence(5′→3′)
DlPPO1F1 GCTGTTGAGCTCATGAAAGC
DlPPO1R1 CACAAARCTYCCKGCAAAMTC
DlPPO15RACE1 GCATAGCAAAGGTTGGGTCAC
DlPPO15RACE2 ATCACAATAGGCACAATGGACG
DlPPO13RACE1 TAGCTCTTGCAGCCGAGATAAAC
DlPPO13RACE2 CGAGTTTGCCGGGAGTTTTGTG
DlPPO1CF1 CCTTATTATAATCACAACCACAAC
DlPPO1CR1 GCACGAAACCAGAGACATTATC
DlPPO1QF TGTGGACGGTTTGGAAGAC
DlPPO1QR CAAGATTGGCGTTCTCATCA
2 ¤@RƒŸ
2.1 45犇犾犘犘犗1,6犮犇犖犃mnol7pq
fjklmcDNA nop,f DlPPO1F1e
DlPPO1R1n)$+,qr1137bpy€,
NCBIyBlast[AÆ´]€njk犘犘犗,x¢
£+ԃ€

\^_qry€( RACE)
$

qr犘犘犗,xy5′-.€e3′-.€,
¶Ç5′-.e3′-.€(«P›)$ DlP
PO1CF1eDlPPO1CR1GÖ ORF]^,u¿®
‘Ãeí_®‘ÄnATGeTGA,Š‹Œ
Ž
(ORF)n1800bp,‘599z’,“;犇犾犘
犘犗1a、犇犾犘犘犗1be犇犾犘犘犗1cy5′.ِ‘+
(5′UTR)‡`,ân 32bp,3′ِ‘+(3′
UTR)â³`,ƒ„n 137、128 e 88bp,/
polyA⃄n20、28e23bp。ø]€ÆN
y‘+’,“€¶NCBIGÖ[A,¹¾犇犾犘
犘犗1RÇÑ#$E”•、–—、` à™y犘犘犗,x
â`V

Ç»R”•y,£›Å

µ94%。
¤¥¦ƒŸÆ´],xnjk犘犘犗,x,],x3
z{|}€¶ GenBank»a|,ƒ„b/n
犇犾犘犘犗1a、犇犾犘犘犗1be 犇犾犘犘犗1c,a|}ƒ„
nKM387405、KM516087eKM516088。f‘gh
i

jklmDNAnop,fDlPPO1CF1eDlP
PO1CR1n)$+,qr1‚ân2163bp
DNA € ,b / n 犇犾犘犘犗1(GenBank a | }
KU837229);犇犾犘犘犗1,xc†¨1z†Ã,
ân214bp。
2.2 bYcde@A
2.2.1 45 犇犾犘犘犗1rs,tuv@A ¤d
ExPASy»y34d,}ñSƒŸY§ProtParam
0011 ! " # $ % &                   36d
ADlPPO1,}ñSGÖ(e ¡,犇犾犘犘犗1‘
y34nf›34

§¨gߛ

/‘hC’,“S
²
(Arg+Lys)i2=‘jCy’,“S²(Asp+
Glu);DlPPO1ͳk±34。¤d SignalP4.1
Server:´ ¡DlPPO1³†G}L,³Íƒl3
4

¤dPSORTƒŸ*ðDlPPO134±Õ=l
ý·,dyÉʛÅ2

ƒmn0.892。¶“ôõ
±ÕƒŸy,ÌÀ

cdExPASy»yTMpredY
§GÖMN¤¥y:´*ð
,DlPPO1§¨MN¤
¥¦

ÉÊRN±ÕeMN{n¨¦

O“vՏ
:´*ð
,DlPPO134V†35zO“vՏ,Ç
»o’“yO“vՏ21z,p’“eü’“yO
“vՏƒ„n8ze6z,DlPPO134»o’
“y[q¡*2=p’“eü’“

ADlPPO13
4yPQ¤¥GÖ:´

ǻrʉdZ[s[q
Å2

Ðαtueβvw[s[q‡x;¶yY34
PQ¤¥y,ÌÀ

G,SAÇRQ¤¥GÖ:´

:´y¤@RPQ¤¥:´y¤z‡{

hdNC
BICDSearchY§GÖ34¢£+¦ƒŸ,¤@*ð
DlPPO1’,“€»173382+¦Rü’“w|(
}y.¼€â`V

§¨PPO34y©ª«¬,
†PPO1_DWLePPO1_KFDV"z¤¥¦(~1)。
2.2.2 45犇犾犘犘犗1,6wD 犕犻犮狉狅犚犖犃xy@A miRNAsHn,šÁ‚ƒÃ,ñR…†#$
yB¹BeqrÚÛòó

Œ¨DE ¡ miR
NAs¶#$Î、šÃ、l、^ eTU™½É/!e¹
B»i¹Ë1=Hd

/ miRNAsyÉfˆò×
ØG}Á…ñR…†ÇÑ miRNAsyB$0!,
¹ÉfFºòó€ÚÛ

m‚ov™qrÚÛÁ
…,x µ



DE miRNAsAƒ,xyÁ…
Hd

A=DEƒ,xEƒñR#$B¹BÁ…
ê‡rÚۈò¨1=CD

cd¶Ú„…psR
NATarget™[21]AÁ…犇犾犘犘犗1y miRNAsGÖ


¤@¶àA miRNAS_†»[A¹¾犇犾犘
犘犗1<¶àAmiR1444(ptcmiR1444)y‡ˆ‰†Õ


%¡¶jk犇犾犘犘犗1ÉÊ{<¶RptcmiR1444
#Š‰†Õ

jÄjk»#Š miRNA !My
Á…

2.2.3 z{|}~@A ÒÓGvƒŸ ¡,jk
DlPPO1R`+y”•(JF926153.1)PPO‹2œŠ,
RcŒ+#$–—
(JQ319005.1)、Ž+#$˜
(HQ634289.1)e+#$‘’(XM_006468155.
1)™yPPOyST‹2{œŠ(~2)。
2.3 45€b‚ƒG犇犾犘犘犗1,6<=„…
#$,x¶¸ö¹BºZy{|ߓœvÉn
ÇÈǶ#$¹BºZ»yÁ…±†ÄÙÚÈ

犇犾犘犘犗1,x¶jk·¸¹B³`®¾(¸ ›àá
ÒÓ

³ˆ~”­¤¥

⽸

¼½¸

ã佸e
Ãl¸

y‡A{|ߓGփŸ

¤@ ¡


›àáÒÓ®¾Ä¼½¸®¾y犇犾犘犘犗1,xy
{|ߓ,•‡AœÖ

Ð>¼½¸Š¿–—ÀÁ

ã佸®¾y{|ߓ÷nàáÒÓ®¾y4
˜

Ãl¸®¾µÄŝ

÷nàáÒÓ®¾y6˜

~3)。%¡¶jk·¸¹BºZ»,犇犾犘犘犗1,
x<=¶È¾™Ãl¸®¾ µ

2.4 犇犾犘犘犗1,67†‡ˆ‰u<=@A
nG,SÇÈ犇犾犘犘犗1,xyÒÓÕP› 
ƒšÀyS›J 1000ÌBootstrap1î]^»]‚y
ÉG✃[m
~2 DlPPO1ÒÓGvA
Numbersatthenodesrepresentthebootstrapvaluesbased
on1000replications
Fig.2 PhylogenetictreeforDlPPO1
~1 DlPPO134¢£¤¥¦:´
Fig.1 FunctionaldomainspredictionforDlPPO1
10116¾              678,™:jk犇犾犘犘犗1,xyabêÇ µÁ…ƒŸ
µ«

AǶjk³`ÒÓÔÕ
(^ 、
l

ÎÏ

!
Î

X@

!@



@ŸešÃ

y‡A µ²
GփŸ

¤@ ¡
,犇犾犘犘犗1,x¶lm» µ²
ŝ

¶!λ{¨»²<¶

¶jkÇÑÒÓÔÕ
 µ²œÖ

~4)。%¡¶jkB¹BºZ»,
犇犾犘犘犗1,x<=¶lmeλ µ。
2.5 犇犾犘犘犗1,67Š‹Œ:ŽŒ
#$PPOEƒòAwx‡ryœvêǀò
±†yDE§¨1=CD

Öáâ MeJA ëN
犇犾犘犘犗1y µ,Нáâ MeJA‰†犇犾犘犘犗1y
 µ

%¡犇犾犘犘犗1,x§¨œîžyã䓈ò
±†

Z[SAÜÝáâyĝ,犇犾犘犘犗1,x µ
EC.ñòª¸›àáÒÓ;ICpEC.³ˆ~¸›”­¤¥;
GE.⽸;HE.¼½¸;TE.ã佸;CE.Ãl¸
~3 jk·¸¹BºZ»犇犾犘犘犗1y‡A µ²
EC.embryogeniccalus;ICpEC.incompletecompactproembryogenic
cultures;GE.globularembryos;HE.heartshapedembryos;
TE.torpedoshapedembryos;CE.cotyledonembryos.
Fig.3 Relativeexpressionof犇犾犘犘犗1atdifferentstages
oflonganSE
R.^;L.l;FB.ÎÏ;F.!Î;YF.X@;MF.!@;
P.@Ÿ;PC.@ž;S.šÃ
~4 jk³`ÒÓÔÕ犇犾犘犘犗1y‡A µ²
R.Roots;L.Leaves;FB.Floralbuds;F.Flowers;
YF.Youngfruits;MF.Maturefruits;P.Pulp;
PC.Pericarp;S.Seeds.
Fig.4 Relativeexpressionof犇犾犘犘犗1indifferent
tissuesoflongan
²‰·Ÿ¾–—À y¡¢

%¡SAA犇犾犘犘犗1
§¨hÁ…Hd

~5)。³`ÙB$ÚÛy®€ 
µƒŸ ¡
,NaClo£y¤ÚÛeçèéo£y¥
¦ÚÛÜÝÞ

Z[ÜÝ®Øy«犇犾犘犘犗1,x
ª· µ²À 

Ð PEG o£€ÚÛ{ëN
犇犾犘犘犗1 µ,%¡犇犾犘犘犗1ñRA¤ÚÛ、¥¦
ÚÛe€ÚۙsšÙB$ÚÛºZ

Ç»A¤
ÚÛyˆòÅn*§

Ñw
,ABAÜÝÞ犇犾犘犘犗1
,xy µß“‡A“k

%¡犇犾犘犘犗1³•¨
ëJ=ABA×ØG}~(~6)。
3 © z
3.1 45犇犾犘犘犗17‘’“”
B$Nøôõƒª!‘¨³`š#34dyô
õL

¹Ëý)³`y«Ê

34dy“ôõ±Õ
ƒŸÉfnDEÇ«ÊÄÙÚÈ

-=PPO¬=
.,x‘yd·w

,x¶ôõd» µÈ­
{nÂd·ƒÕÐ¹ËHd
[22],
Ð}DE:´y
DlPPO1§¨œ®¯MN¤¥y«,h{0],
~5 wV×ØÜÝÞ犇犾犘犘犗1
mRNAy‡A µ²
Fig.5 Relativeexpressionof犇犾犘犘犗1inresponseto
exogenousphytohormones
~6 ÙB$ÚÛxØÜÝÞ犇犾犘犘犗1y‡A µ²
Fig.6 Relativeexpressionof犇犾犘犘犗1inresponseto
abioticstress
2011 ! " # $ % &                   36d
xyHd«¬

d·9Í,š9ƒÃ

R°0Hd
»y°€òyCÃT°±¨¦

}DE»:´
DlPPO1ÉʱÕ=lý·,d»y#²·À,Æ
´DlPPO1¨ÉÊñRlý·»d·9y½!,É
Êبˆjk°0HdyCÃT°±€ò

}DE»DlPPO134y¤¥«¬{0³´¦
=PPOy&§。DlPPO1§¨PPO34y©ª«
¬

†PPO1_DWLePPO1_KFDV"z¤¥¦,`
®¹†¨"z¢£y12õ¶+CuAeCuB8
¤0Տ
[23]。
·w犇犾犘犘犗1¶jk»ÉÊ<¶R
ptcmiR1444s#Šy‡ˆ‰†Õ。
3.2 犇犾犘犘犗17†‡<=ˆu
#$犘犘犗¶³`ÒÓ»y µ§¨«P›,
E¸@犘犘犗¶X¹、Xl、κ、•ž™ÒÓ»i
¨ µ

¶Xl» µ²ÅÐ¶Îº»y µ²
ÅÖ
[24];
–—»XYÒÓLMy犘犘犗{|ߓ
=!ÒÓ
[15];
”•犘犘犗¶Îel» µ²Å,
¶@Ÿ» µ²ÅÖ
[16]。
»U@A»¨¦=犘犘犗
¶#$¼€ÒÓÔÕyÕP› µDE

¶¶šÃ
½!׳y¸ö¹B½Îy µÁ…DE²Ï¨&
§

/jk犘犘犗 ,xy µÁ…DE¾nO4。
jk¸›àáÒÓy{|ÒS_
[25]
»¿¨¹¾
犘犘犗,xyÀ|,-ÑÉÁSÆ´,犇犾犘犘犗1ÉÊ
¶jk¸ö¹Bþ µ²œÖ)IfÇѳ·™
½î<¶

FºA犇犾犘犘犗1,xy¶jk·¸¹
BºZyÕP µGփŸ¹¾
,犇犾犘犘犗1>·¸
¹B»¾Š¿WÂ

ÄÃl¸®¾y‡A µ²‡
Aþ*§ÀÁ

%¡犇犾犘犘犗1¶jk·¸¹BÈ
¾Š¿¹Ë¦ÃHd

ÉÊnñRX¸¹BêȾ
řºZ»yt#uvecdؽ!ºZÅÆÇ
˜$deʲ
。犇犾犘犘犗1¶jk·¸¹BºZ»y
 µÁ…±†¾­=ÈÈGÖG,SÉçDE

Ò
Ó«P› µƒŸ ¡
,犇犾犘犘犗1¶jklm» 
µ²Å

ÉÊRǓôõ±Õ=lý·,d¨¦

Ç̶ÎÏ µ²œ

ÉÊþxn‡Aœy
PPOz›¨c=Îσv[26];Ð犇犾犘犘犗1,xy
¶ÇÑÒÓÔÕÊËr µ

ÉÐÇ µ§¨ÒÓ
«P›

/jk»Éʹ<¶ÇÑ!Mf­¾PPO
¶jk³`ÒÓÔÕyuv¹þ€òÁ‚

3.3 犇犾犘犘犗1•”–\ŽŒ
×ض#$òAýšB$eÙB$ÚÛòó»
¹Ë1=Hd

ŒÌãä“
(JA)eßà“(SA)É
Hn#$Ímˆò[­yG}ƒÃ„×z#$st
±†
[27];JA e SA ƒ„R#$ëN›ÒÓ͛
(ISR)eÒÓqr›Í› (SAR)¨ ¦[28];#$·
ABAÍòóB$ÚÛeÙB$ÚÛy1=G}ƒ
Ã

sqr‚…Þdž² 

>Ðĝ#$)Î
Íq›fòAwx‡r
[29]。
¨DEÏÐ

#$×Ø
G}yBy®Øe®â¶Ñ±#$mpZâ»u[
¦ÃHd
[30]。
}DE»
,犇犾犘犘犗1y µjÄÖ
áâ MeJA、SA、¤ÚÛ、¥¦ÚÛe€ÚÛyë
N

Ç»¤ÚÛyëNHdÅn*§

Ð ABA A
犇犾犘犘犗1¿¨*§yëNHd,ÉÐ犇犾犘犘犗1É
ʳ•¨ëJ= ABAyqrÚÛG}{N~,
ÐÉÊÍFºJA)SAyqrÚÛG}{N~
Á…#$AÚÛy͛

·w

àA miR1444ÉÊ
FºHd=犘犘犗,xñRÒ@àAwx‡rÚÛ
yòó
[6,31];
}DE¶àA miRNAS_†»:´
ƒŸÄ犇犾犘犘犗1{<¶àA miR1444‡ˆ‰†Õ


ÉÐjk»ÉÊ{<¶ miR1444#Š!MFº
Á…jkPPOyz>fòAwx‡rœv。ª×,
EÓ犇犾犘犘犗1¶jk·¸»EƒÁ‚#$ôõf
òAÚÛ

EƒòóqrG}{N™,ҁ¡¢¹
­=G,SDE

–—!J

[1] Â Ô.^_犘犘犗 ,xœPê犘狆狅犃1,xsɛR¸Õ4
â¦Ö›DE
[D].×!àØ:!"WX+32%,2012.
[2] NEWMANSM,TANTASAWATP,STEFFENSJC.To
matopolyphenoloxidaseBisspatialyandtemporalyregulated
duringdevelopmentandinresponsetoethylene[J].犕狅犾犲
犮狌犾犲狊,2011,16(1):493517.
[3] FUERSTEP,ANDERSONJV,KENNEDYAC,犲狋犪犾.In
ductionofpolyphenoloxidaseactivityindormantwildoat
(犃狏犲狀犪犳犪狋狌犪)seedsandcaryopses:adefenseresponsetoseed
decayfungi[J].犠犲犲犱犛犮犻犲狀犮犲,2011,59(2):137144.
[4] MAHANILS,ATTAJARUSITJ,STOUTMJ,犲狋犪犾.Over
expressionoftomatopolyphenoloxidaseincreasesresistanceto
commoncutworm[J].犘犾犪狀狋犛犮犻犲狀犮犲,2008,174(4):456466.
[5] MAYERAM.Polyphenoloxidasesinplantsandfungi:Going
places?A review[J].犘犺狔狋狅犮犺犲犿犻狊狋狉狔,2006,67(21):
23182331.
[6] ÙÚÛ,ÜÝÞ,pßà,™.miR1444añRÒ@àAáÚÛ
yˆò
[J].»+%:Bb+%,2012,42(10):850860.
CUIXN,YUANLC,XUXJ,犲狋犪犾.miR1444aisinvolved
intheresponseof犘狅狆狌犾狌狊狋狉犻犮犺狅犮犪狉狆犪tozincstress.[J].
犛犮犻犲狀狋犻犪犛犻狀犻犮犪犞犻狋犪犲,2012,42(10):850860.
[7] â ã,Ž Ý,™.”•、jk¤ÈwF›œ€òv%±Ý
30116¾              678,™:jk犇犾犘犘犗1,xyabêÇ µÁ…ƒŸ
DEG­
[J].’·Wä%&,2012,43(10):15611568.
SUNJ,LIL,犲狋犪犾.Chemicalmechanismadvancesofenzymatic
browningreactioninpostharvestlycheeandlonganfruits[J].
犑狅狌狉狀犪犾狅犳犛狅狌狋犺犲狉狀犃犵狉犻犮狌犾狋狌狉犲,2012,43(10):15611568.
[8] åæ½,ç è.–—ªst†²êstuvwz›Rҗ
›œy¦Ò
[J].»W%F&,2010,26(22):5457.
ZHANGZHX,HONGP.Theconnectionbetweenconcentration
oftotalpolyphenol,activityofpolyphenoloxidaseandbrowningof
explantsin犆犪狀犪狉犻狌犿犪犾犫狌犿 (Lour.)Rauesch[J].犆犺犻狀犲狊犲犃犵狉犻
犮狌犾狋狌狉犪犾犛犮犻犲狀犮犲犅狌犾犾犲狋犻狀,2010,26(22):5457.
[9] éêë,XìF,XQí,™.ŽÜÝ«¬¤Èjk@­@ž
›œêÇRt#$dJKy¦Ò
[J].¾JîÞ+3,2014
(5):218224.
ZHAOYF,LINHT,LINYF,犲狋犪犾.Effectofheattreat
mentonbrowningdelayingandphenolicsmetabolisminperi
carpofharvestedlonganfruit[J].犕狅犱犲狉狀犉狅狅犱犛犮犻犲狀犮犲犪狀犱
犜犲犮犺狀狅犾狅犵狔,2014,(5):218224.
[10] ïÃã,ðæñ,™.jk@Ÿ) Rԃuv¹þw`Y
wœvy¦Ò
[J].Ž‘H$%&,2014,(7):14341438.
CHENZJ,WUZHX,犲狋犪犾.Relationshipbetweenlonganpulp
breakdownandthechangesofsomeoxidoreductaseisozymes[J].
犆犺犻狀犲狊犲犑狅狌狉狀犪犾狅犳犜狉狅狆犻犮犪犾犆狉狅狆狊,2014,(7):14341438.
[11] ANDERSONJV,MORRISCF.Purificationandanalysisof
wheatgrainpolyphenoloxidase(PPO)protein[J].犆犲狉犲犪犾
犆犺犲犿犻狊狋狉狔,2003,80(2):135143.
[12] NISHIMURA M,FUKUDAC,MURATA M,犲狋犪犾.Clo
ningandsomepropertiesofJapanesepear(犘狔狉狌狊狆狔狉犻犳狅犾犻犪)
polyphenoloxidase,andchangesinbrowningpotentialduring
fruitmaturation[J].J狅狌狉狀犪犾狅犳狋犺犲犛犮犻犲狀犮犲狅犳犉狅狅犱犪狀犱犃犵
狉犻犮狌犾狋狌狉犲,2003,83(11):11561162.
[13] Žòó,Žôõ,ö÷,™.øùstuvw,xCDS+
yabê µ
[J].@A%&,2008,25(4):577580.
LIG,LIHX,XUDQ,犲狋犪犾.Cloningandexpressionof
polyphenoloxidasegeneofCDSfromYaliPear[J].犑狅狌狉狀犪犾
狅犳犳狉狌犻狋狊犮犻犲狀犮犲,2008,25(4):577580.
[14] ú?,Xûü,JKL.-Îûstuvw,xyabê
Ç·¸¹BºZ»y µƒŸ
[J]../WX2%%&:)*
+%ý
,2014,43(6):602608.
GAOYQ,LINYL,LAIZHX.Cloningandexpressionof
犘犘犗geneduringsomaticembryogenesisin犆犪犿犲犾犾犻犪狀犻狋犻犱犻狊
狊犻犿犪Chi.[J].犑狅狌狉狀犪犾狅犳犉狌犼犻犪狀犃犵狉犻犮狌犾狋狌狉犲犪狀犱犉狅狉犲狊狋狉狔
犝狀犻狏犲狉狊犻狋狔(NaturalScienceEdition),2014,43(6):602608.
[15] ¼ ½,JKL,Xûü,™.–—stuvw,x(犘犘犗)a
bêÇ¿UÀ2·¢<ºZ»y µƒŸ
[J].Ž‘H$%
&
,2014,35(4):738745.
RONGX,LAIZX,LINYL,犲狋犪犾.Cloningandexpression
ofpolyphenoloxidase(犘犘犗)geneduringdifferentprocessof
preservationin犻狀狏犻狋狉狅ChineseOlive(犆犪狀犪狉犻狌犿犪犾犫狌犿)[J].
犆犺犻狀犲狊犲犑狅狌狉狀犪犾狅犳犜狉狅狆犻犮犪犾犆狉狅狆狊,2014,35(4):738745.
[16] WANGJ,LIUB,XIAOQ,犲狋犪犾.Cloningandexpressiona
nalysisoflitchi(犔犻狋犮犺犻犆犺犻狀犲狀狊犻狊Sonn.)polyphenoloxidase
geneandrelationshipwithpostharvestpericarpbrowning[J].
犘犾狅犛狅狀犲,2014,9(4):e93982.
[17] LAIZ,CHENC,ZENGL,犲狋犪犾.Somaticembryogenesisin
longan[犇犻犿狅犮犪狉狆狌狊犾狅狀犵犪狀Lour.][M]//.In:SomaticEm
bryogenesisinWoodyPlantsSpringer,2000:415431.
[18] LINY,LAIZ,TIANQ,犲狋犪犾.Endogenoustargetmimics
downregulatemiR160 mediationofARF10,16,and17
cleavageduringsomaticembryogenesisin犇犻犿狅犮犪狉狆狌狊犾狅狀犵犪狀
Lour[J].犉狉狅狀狋犻犲狉狊犻狀犘犾犪狀狋犛犮犻犲狀犮犲,2015,6(e219).
[19] ïòG,þÿ!,JKL,™.",xÒDNAyÄðRHv
[J].#!Wä2%%&,2004,26(3):329333.
CHENGX,LVLX,LAIZX,犲狋犪犾.Extractionandpurifi
cationofgenomicDNAinNai(犘狉狌狀狌狊狊犪犾犻犮犻狀犪Lindl.var.
犮狅狉犱犪狋犪)[J].犃犮狋犪犃犵狉犻犮狌犾狋狌狉犪犲犝狀犻狏犲狉狊犻狋犪狋犻狊犑犻犪狀犵狓犻犲狀狊犻狊,
2004,26(3):329333.
[20] LINY,LAIZ.ReferencegeneselectionforqPCRanalysis
duringsomaticembryogenesisinlongantree[J].犘犾犪狀狋犛犮犻
犲狀犮犲,2010,178(4):359365.
[21] DAIX,ZHAOPX.psRNATarget:aplantsmalRNAtar
getanalysisserver[J].犖狌犮犾犲犻犮犃犮犻犱狊犚犲狊犲犪狉犮犺,2011,39
(suppl2):W155W159.
[22] SOMMERA,NEEMANE,STEFFENSJC,犲狋犪犾.Im
port,targeting,andprocessingofaplantpolyphenoloxidase
[J].犘犾犪狀狋犘犺狔狊犻狅犾狅犵狔,1994,105(4):13011311.
[23] LIAOZ,CHENR,CHENM,犲狋犪犾.Molecularcloningand
characterizationofthepolyphenoloxidasegenefromsweet
potato[J].犕狅犾犲犮狌犾犪狉犅犻狅犾狅犵狔,2006,40(6):907913.
[24] $%,&ØÎ,çD.‘()’¸ @stuvw,x犕犱犘
犘犗yabR µƒŸ[J].#$BÝ%&,2013,(8):803810.
MACQ,BAISH,DAIHY.Cloningandexpressionanaly
sisofpolyphenoloxidasegene(犘犘犗)identifiedfrom‘Gala’
apple[J].犘犾犪狀狋犘犺狔狊犻狅犾狅犵狔犑狅狌狉狀犪犾,2013,(8):803810.
[25] LAIZ,LINY.Analysisoftheglobaltranscriptomeoflon
gan(犇犻犿狅犮犪狉狆狌狊犾狅狀犵犪狀Lour.)embryogeniccalususingIl
luminapairedendsequencing[J].犅犿犮.犌犲狀狅犿犻犮狊,2013,14
(2):178189.
[26] *++,é,-,# .,™.Ú/0ÎσvºZy½É1
2êԃJKy$ewz›yœv
[J].!"WX+32%
%&

)*+%ý
,2014,(4):171178.
ZANGSS,ZHAOZL,JIANGS,犲狋犪犾.Morphologyand
changesinmetabolitesduringfloralbuddifferentiationofchili
pepper[J].犑狅狌狉狀犪犾狅犳犖狅狉狋犺狑犲狊狋犃&犉犝狀犻狏犲狉狊犻狋狔 (Natural
ScienceEdition),2014,(4):171178.
[27] RIVASSANVICENTEM,PLASENCIAJ.Salicylicacidbe
yonddefence:itsroleinplantgrowthanddevelopment[J].犑狅狌狉
狀犪犾狅犳犈狓狆犲狉犻犿犲狀狋犪犾犅狅狋犪狀狔,2011,62(10):33213338.
[28] DOORNBOSRF,GERAATSBP,KURAMAEEE,犲狋犪犾.
Effectsofjasmonicacid,ethylene,andsalicylicacidsignaling
ontherhizospherebacterialcommunityof犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻
犪狀犪[J].犕狅犾犲犮狌犾犪狉犘犾犪狀狋犕犻犮狉狅犫犲犐狀狋犲狉犪犮狋犻狅狀狊,2011,24
(4):395407.
[29] CUTLERSR,RODRIGUEZPL,FINKELSTEINRR,犲狋
犪l.Abscisicacid:emergenceofacoresignalingnetwork[J].
犃狀狀狌犪犾犚犲狏犻犲狑狅犳犘犾犪狀狋犅犻狅犾狅犵狔,2010,61(1):651679.
[30] MURLAJ,PAULK,犲狋犪犾.Theoutcomesofconcentration
specificinteractionsbetweensalicylateandjasmonatesignalingin
cludesynergy,antagonism,andoxidativestressleadingtocel
death[J].犘犾犪狀狋犘犺狔狊犻狅犾狅犵狔,2006,140(1):249262.
[31] SHANFAL,YINGHSUANS,CHIANGVL.Stressre
sponsivemicroRNAsin犘狅狆狌犾狌狊[J].犘犾犪狀狋犑狅狌狉狀犪犾犳狅狉犆犲犾犾
牔 犕狅犾犲犮狌犾犪狉犅犻狅犾狅犵狔,2008,55(1):131151.

!"

#$%
)  
4011 ! " # $ % &                   36d