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Cloning and Functional Analysis of MsHSP 17.7 Gene from Alfalfa

紫花苜蓿MsHSP17.7基因的克隆及功能分析



全 文 :!24" !1#
 Vol.24  No.1
! " # $
ACTA AGRESTIA SINICA
   2016$  1%
  Jan.  2016
犱狅犻:10.11733/j.issn.10070435.2016.01.017
0ðñò犕狊犎犛犘17.7Nø2ÌÍfÎ5>r
ˆ‰Š

‹Œ

0Ž

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’u¶


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:;100193)
78

pMó¦ÚßàwxžøtTÐяó¦

LJK9Ÿ°J|tlab&ª•«cd

n¡”_xìí
(犕犲犱犻犮犪犵狅狊犪狋犻狏犪L.)pMó¦®58|¦§,»/0<=@”dâ—kpMó¦®5 犕狊犎犛犘17.7,Y®5
³»477bp¼>(Éù,³)158M…®W,ó¦=m`n17.67kDa。lj¸D°P MsHSP17.7åñìí
(犕犲犱犻犮犪犵狅狋狉狌狀犮犪狋狌犾犪)6†f(犘犻狊狌犿狊犪狋犻狏狌犿)&T2Mu=mpMó¦@”ts,ˆQA=¾Ú93.38%,
83.13%。ä†ØºgW…˜,犕狊犎犛犘17.7L~&gWݍ,X6x“ã,L}&݃;^ 2©`PCR=•…
˜
,犕狊犎犛犘17.7csÛ、s)C"gW,L=ÁðC"œgW`<ð6h…。DT3oÚ®5&ZA(犃狉犪犫犻犱狅狆狊犻狊
狋犺犪犾犻犪狀犪)^ 2©`PCR=•…˜,Y®5Ls)6=Áðú5œ©§Y`gW,efgh犕狊犎犛犘17.7®5L§
ž¡s)=ÁðtlœTú5ŠMab

9:;

_xìí

pMó¦
;^
2©`PCR;È9Kú5;*+Úð
<=>?@
:S541    ABCDE:A     AFG@:10070435(2016)01012108
犆犾狅狀犻狀犵犪狀犱犉狌狀犮狋犻狅狀犪犾犃狀犪犾狔狊犻狊狅犳犕狊犎犛犘17.7犌犲狀犲犳狉狅犿犃犾犳犪犾犳犪
LIZhenyi,LONGRuicai,ZHANGTiejun,YANGQingchuan,KANGJunmei
(InstituteofAnimalScience,ChineseAcademyofAgriculturalSciences,Beijing100193,China)
犃犫狊狋狉犪犮狋:Heatshockprotein(HSP)isakindofubiquitousprotectiveproteinthatplaysanimportantrole
inplantdevelopmentandstressadaption.Inordertolearnmoreaboutexpressioncharacteristicsandfunc
tionofsmalHSPgene,犕狊犎犛犘17.7genewasclonedfromalfalfa(犕犲犱犻犮犪犵狅狊犪狋犻狏犪L.)throughhomolo
gy-basedcloning.犕狊犎犛犘17.7containsanopenreadingframeof477-bpencodingaproteinof17.67-
kDa.Theaminoacidsequenceshares93.38%and83.13%identitywithsHSPsin犕犲犱犻犮犪犵狅狋狉狌狀犮犪狋狌犾犪
andin犘犻狊狌犿狊犪狋犻狏狌犿.QuantitativeRT-PCRresultsshowedthatthemaximum mRNAexpressionof
犕狊犎犛犘17.7wasinstems,folowedbyleavesandflowers,andtheleastwasinroot.犕狊犎犛犘17.7wasin
ducedbyhightemperatureandhighsalinitystress,buttheexpressiondidnotchangobviouslyunderper
oxidestress.ThenthequantitativeRT-PCRindicatedthat犕狊犎犛犘17.7wasabundantlyexpressedunder
highsalinitystressandperoxidestressinT3transgenic犃狉犪犫犻犱狅狆狊犻狊.Insummary,resultsimpliedthat
犕狊犎犛犘17.7mightinvolveintheregulationofhighsalinitystressandperoxidestressresponse.
犓犲狔狑狅狉犱狊:犕犲犱犻犮犪犵狅狊犪狋犻狏犪;Heatshockprotein;QuantitativeRTPCR;Abioticstress;Genetictransfor
mation
HIJK
:20141219;LMJK:20160307
NOPQ

¬•­®¯/0°±²³
(973)—_xìí1F‰ˆ‰®5T¶*¦§=•(2014CB1387032);&()*+,:;ê
?\]/01®»*/)¼½´µ
(2014ywfzd2)“Å
5RST

¶\ø
(1989),—,šË/09,/0Òcn?-*+JG,Email:lizhenyily@163.com;<Ôc™ Authorforcorrespon
dence,Email:kangjmei@126.com
  _xìí(犕犲犱犻犮犪犵狅狊犪狋犻狏犪L.)cnHF?-
þLð÷Ü_,-P“

LÕìíYëGJé
Hl*C

ÙCwxTÙ_

‘¤`1Fcd¡ô
•UV

ä$ç

<=Ú®5¾¨›sìíŽ)

ø
‰

ø¥

ø•¸¬?øtþ’nPo()JGT
/0p­
[12],
5.

‰éøt®5T/0ÐDìí
*+;Ò|=mJG°›•«cd

pMó¦
(heatshockprotein,HSP)ÚßG,
-+Lé9K÷TÐÑó¦

LoØab

Ô{+"
6)H.Äl¾6?ÒÓ°›•«cd
[3],
‘
&JKpMó¦wxžš‰



)*

=Áð
6•¸¬?ŠMuŠ
[4]。
}Ïó¦=m`YuL=
n5M¬:HSP100,HSP90,HSP70,HSP606u
=mpMó¦
(Smalheatshockprotein,sHSP),‘
! " # $ !24"
&sHSPT=m`n15~24kDa,Lñ’200~800
kDa`N*šK,sHSPGà…,ßh=nH.
F6H.¨TClassI,ClassII6ClassIII¬,Ÿé
FN

2F36f]N&T7e3M¬。sHSP


,S

H.˜
Ë
”,
Ì«<=ÈúÒ⁺Pó¦eš

n_è
HSP70/HSP100œ’ó¦UîÃ,IsHSP6ž
ºPó¦T•ÂîÃ
[3],
5.u=mpMó¦LJ
K9Ÿ|ú5ŠM&ª•«cd
[5]。sHSPLJK
6@9Ÿ1,°›6@cd

LžEDä†

Ռ
Œ|Gm9Ÿ?9ù=Ä

Ltlú5œ<=í
–æTøt

ascd

asMu¨µpF?=
ÄÐÑJK‡P°J
[6],
LsÛ

¥&

š‰

)ú5
?tlÍ֜JKsHSPL‚itlDJK+’T

[7]。
þJ/0gh&ZA
(犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻
犪狀犪)&T犎犛犘18.1,犎犛犘17.46犎犛犘17.6犃®5
øtˆ‰

ò犃狋犎犛犘15.7®5‡PÍ֜6g
W

LsÛ6Áðú5C"œgW
[8]。
狊犎犛犘®5T=gWLJ:›sJKø,J
Š}i½Ú狊犎犛犘17.7®5Q§(犗狉狔狕犪狊犪狋犻狏犪)L
ÈÉ#gPø‰
[9],
_xìí 犕狊犎犛犘23®5L
“-&T=`gWLí–D)6+Tø

Uhp
M󦞁JKtlú5ŠMab
[10]。
áâ
(犛狅
犾犪狀狌犿犾狔犮狅狆犲狉狊犻犮狌犿)2F3u=mpMó¦®5
(犈犚-狊犎犛犘)T=`gWL›sDtKuTø,
nÈÚ®5Táâ6ÞJtKuø
[11]。
pMó
¦ø·þÀ/,-/0

I‰é_xìíu=mp
Mó¦®5Ti½ÿƒ

»/0Í_xìí&d
¡犕狊犎犛犘17.7®5,ÆDY®5TùðF、gW
8|¦§\‘¡8ð©/0

n\ß¿•
pMó¦®5Ltlú5œT=mcdÁ›œJ
MTž^

Æn_xìí=m*+;Ò|Ú®5
Â1GT“J›œ•«T‹®5

1 UVWXY
1.1 jkUVWjÏ

&ì1{’(犕犲犱犻犮犪犵狅狊犪狋犻狏犪L.cv.‘Zhong
mu1’)&ZA‘col0’(犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪)è
&()*+,:;ê?\]/01Ð+

Ð

&
ì1{’GmÂé“D¹&°,R}Ÿ5cm­®
2Þd HoaglandEDá,¿³40½,ÆÂéÉÃ
Š‹“Dà

9ŸÍÖn

¦á16h、24℃/›Á8
h、20℃,ˆDŸA60%,¿r7dÛߓEDá;&
ZAGmÀ75%,å0.1%ê¥KÚ_,d"Þ
Y°Qz¼_Gé1/2MSêN“D®,RÈɟ
O4~6´X2ރEDÙ(EDÙ:-è:?Ä.=
3:1:1),R&ZA¼x2dé(®Þbµ。
gW/NpBI1216(®ÞGV3101©è»^
CËÐ+

d/NpEASYT3、Y0®ÞTrans1
T10c\H.、HiFi-PCRSupperMix1é:;
àâ¸9K¾¨JAºp
;RNA›f、aô¿ij
Ê_

sÚF]u›BÜß1é:;Ê2‡9K¾
¨JAºp
;¨
W2‡½
、T4å#½、Primerscrip
uÚNBÜß
、DnaseI,SYBR犘狉犲犿犻狓犈狓 TapII
1éTaKaRaºp,‘Xð+Î1$BÜ©Wd=
•Ú

1.2 jkXY
1.2.1  犕狊犎犛犘17.7ڎE€“slm”•¥Œ
 ž^&ZApMó¦®5犃犜-犎犛犘17.6犃l
j

ÏdBLASTLåñìípMó¦®5&×î
ˆQAtsTlj

}ÏkdTˆQlj²²ÊK
MsHSPF,MsHSP-R(g1)。›f‘&ì1{’
RNAÆuÚNkdcDNA,j.nÂPiíµT´
,

Ю5´,pEASYT3/Nå#ÆÚðd
Trans1T10c\H.,L»100μg·mL
-1
…3
KuTêNLB“D®37℃Í֜=Á“D,L
ddÆÃl

4Åabc3Ò(http://abc.cbi.pku.edu.cn)=•
®5lj

ŒjÏd WebLab&Tplotorf(v6.0.1)=
•cDNA¼>(Éù,\ߗk³)ó¦lj。j
ó¦ljn®¯

\‘jœ=•

ó¦å9ep=•
(garnier[v6.0.1]),ó¦ÏQ=•(pepwindow[v
6.0.1]),Ô{5SÃ(SignalP4.1),*H.©ŸSÃ
ProtComp9.0(http://www.softberry.com),lj
¸D
(DNAMAN6.0),<=ClustalW  MEGA5.1
ÕÖp MsHSP17.7\ð{。
1.2.2 犕狊犎犛犘17.7ڎ–{—aŒ j‘&ì
1{’_xìínBC‡ý,=¾›f}、~、X6x
RNAdéä†ØºgW=•;jSð9ŸÍ֜
25dTìíÈÉn‡ýD‘\‘ú5Óù,=¾Ð
ìíÈÉÂé37℃sۓDà\‘pMÓù,.×
An300mMNaCl30mM H2O2 Q“Í֜‘)ú5=Áðú5Óù

L0h,4h,8h,12h,
24h2›f~XRNA;<=ij`b×ÃRNAT
œW

òOD260/OD280TL1.8~2.0ã%,as
RNA×An0.35μg·μL
-1,
ÀuÚNš’cDNA
221
!1# ¶\ø?:_xìí犕狊犎犛犘17.7®5Td|¦§=•
!1 à;=¾j_xìíβ-犪犮狋犻狀(GenBank:
JQ028730.1)n2ž®5,犕狊犎犛犘17.7nµT®
5

²²iíŸAn200bp­®T8ºÊKac
tinF,actinR  qHSPF,qHSPR(g 1);<=
ABI7300Z‘©`PCRTÔdSYBRGreenIµý
â\‘ˆD©`=•

¿MÓù3M•*。RT-
PCRuŠÄl:95℃ 10min,95℃ 15s,60℃ 1
min,GHën40;FÛ”éfÄl:95℃15s,
60℃1min,95℃ 15s,60℃ 15s;ëÏ=•Ïd
SAS9.13Excel2010ÕÖ。
1.2.3 om³–{˜™pBI121犕狊犎犛犘17.7E¸
s”š›œE¾ ÏdÊKpBIFpBIRií
»J½‡Ÿ­犡犫犪I犅犪犿犎IT犕狊犎犛犘17.7®5
´,

ÐÚð_T®5´,pEASY-T3/Nå#
kdT3-犕狊犎犛犘17.7•äm,Ïd犡犫犪I犅犪犿犎
IA2‡½Y½‡T3-犕狊犎犛犘17.7pBI121
F]

ÀT4å#½16℃=Áå#,PCR=•|½‡
ð©JKQgW•ämpBI121犕狊犎犛犘17.7。
Ðp`TQgW•äF]pBI121-犕狊犎
犛犘17.7Úðd(®Þ GV3101,L28℃,180rpm
Í֜ÐÞá6JÍDë9Ÿ#
(OD600=0.6~
0.8)2débµ&ZAxl,ÐÞá5!_Ûé»
JSilwetL77(200μL·L
-1)
T1/2MSáN“D
®

(9Ÿòjòx%tT¾9F&ZA

A
e¼>Tx

7f

Ðx%º»éÏÄ`T1/2MS
“D®10min,º»2~3“,›Í“D24h_ÚÞ
dÉʋ“Dà9Ÿ

n¡"ÐÚð:Ý
,1/#
_«„bµ

Ý__—GmnT0o。ЗkTT0
o G m  é 1/2 MS ê N “ D ® (» J 50
μg·mL
-1
ó8Ku

LÚ®5&ZA

—kGm
nT1o,«„L"d—kT3oGm。
›fÚ®5&ZA®5äDNARNA,Ïd
35SF MsHSPRÊKð©Ú®5Jr,ÆÃl


jcDNAnÂP,ÏdAtactFAtactRi
í&ZAβ犪犮狋犻狀2ž®5,pBIFpBIRiíµ
T®5

ÍÚNQRð©Ú®5&ZA

1.2.4 ÚŽš›œž犕狊犎犛犘17.7E–{Œ 
jÚ犕狊犎犛犘17.7®5&ZAnBC‡ý,L Hoag
landEDáQ“Í֜“D28d,DÚ犕狊犎犛犘17.7
&ZA\‘)ú5
(300mMNaCl)=Áðú5(30
mMH2O2)Óù,L0,6,12,24,48h2›fX´
RNA,j&ZA9!ó¦β犪犮狋犻狀n2ž®5²²Ê
KAactF AactR,Ïd^2Z‘©`PCR=•
犕狊犎犛犘17.7®5L6@ú5œTgWxö。
`1 jk\,
Table1 Primerssequences
ÊKPrimername ljSequence(5'-------3') dï Usage
MsHSPF CCTCCCATAATCTTCCAACCAC 犕狊犎犛犘17.7®5d
MsHSPR CAAAAAACCATTGCCACACACG Geneamplificationof犕狊犎犛犘17.7
actinF CAAAAGATGGCAGATGCTGAGGAT _xìíβ犪犮狋犻狀2ž®5ií
actinR CATGACACCAGTATGACGAGGTCG β犪犮狋犻狀PCRamplificationinalfalfa
qHSPF CACCACATAATGGACCTCACAGAT ^2Z‘©`=•
qHSPR TGATGTCACCTGATTTCAACCCTG RTqPCR
pBIF TGCTCTAGAATGGATTTCAGGCTAATGGGT 犕狊犎犛犘17.7QgW
pBIR CGGGATCCAGCAACCTTAACCTCAATAGTC Overexpressionof犕狊犎犛犘17.7
35SF GAGCACGACACACTTGTCTACTCCA •ämð©
35SR CCTTTCCTTTATCGCAATGATGGCA Identificationofrecombinantplasmid
AtactF GAAGTCTTGTTCCAGCCCTCGTTTG &ZA2ž®5ií
AtactR GAACCACCGATCCAGACACTGTACT PCRamplificationinArabidopsis
  ü:œ³fú=og½‡Ÿ­
Note:Theunderlinesindicatedthesitesforrestriction
2 pq>r
2.1 0ðñò犕狊犎犛犘17.7Nø2ÌÍW>r
}Ïåñìí&pMó¦®5lj

Ïd@”
dâ—kŸn686bpTcDNA lj,L123~
599%»J477bp¼>(Éù,L³)158M…®
W

ë1),þ›æ GenBank(lj{n KJ621408),
Æýïn犕狊犎犛犘17.7。
9KÔ:+=•…˜
,犕狊犎犛犘17.7³)ó¦
=m`n17.67kDa,ùy?`­n5.858,ó¦l
j‘XKGpMó¦H.FClassII¬ó¦ˆ
QAts
;ProtCompÕÖSÃY®5L§©Ÿé
H.F
;garnierSŘ MsHSP17.7ó¦Tå9
ep³´α;(56.3%),Áoà(26.1%),ï£
("é
(18.3%)6Ú6ep(10.6%);pepwindow
ó¦ÏQ=•…˜

Yë…®WI®nWë

1jSà MsHSP17.7Ú5SŘ
,MsHSP17.7ó¦ïÔ{5。
321
! " # $ !24"
=1 犕狊犎犛犘17.7犮犇犖犃Ð>ÑYfg¯NqÑY
Fig.1 FragmentofcDNAsequenceof犕狊犎犛犘17.7anditsdeducedaminoacidsequence
  lj¸Def…˜,MsHSP17.7MYm
XJKpMó¦ˆQAts

òf*JKˆQAs
é‘XKG

ë2),jœ7GJKTˆQA=¾n:
åñìí
(犕犲犱犻犮犪犵狅狋狉狌狀犮犪狋狌犾犪)93.38%、†f(犘犻狊狌犿
狊犪狋犻狏狌犿)83.13%、Yf(犌犪犾狔犮犻狀犲犿犪狓)74.10%、&Z
A56.63%、ÿ!(犞犻狋犻狊狏犻狀犻犳犲狉犪)72.89%、»z(犣犲犪
犿犪狔狊)65.66%、“-(犖犻犮狅狋犻犪狀犪狋犪犫犪犮狌犿)74.70%。}
ÏJKH.FClassIIpMó¦ljLã,LN?»
JÐ=ljRDAKAMAATPADV(Ð=ŽI),C?³
´90M…®W­®TÐ=Ž(alphacrystalindo
main,ACD),~œë1˜Ð=ŽII、Ð=ŽIII,C?+
L`†…WÐ=ljPPPEPKKP[1213]。
=2 犕狊犎犛犘17.7¯NqaÑY‚$>r
Fig.2 MultiplesequencealignmentofMsHSP17.7withotherplantsHSPs
ü

›pg˜100%T…®Wˆ@;Ápg˜75%ˆ@;rpg˜50%ˆ@;³fú=g˜Ð=lj,Òù2g˜`†…WÐ=lj
Note:Blackrepresentsalaminoacidsarethesame;pinkindicatesthat75%ofthesequencesareconserved;blueindicates
that50%ofthesequencesareconserved.Underlineindicatesconservativemotif,polyprolinemotifsareboxedatthecarboxylendofproteins
  ~ë31˜,MsSHP17.7åñìí、†fT
sHSP@”ts,YmXJKYf、“-、ÿ!
sHSP@”sé÷mXJK»z、u^、Q§
sHSP(ë3)。
421
!1# ¶\ø?:_xìí犕狊犎犛犘17.7®5Td|¦§=•
=3 犕狊犎犛犘17.7)*ÒQÓ>r
Fig.3 Neighborjoiningphylogenetictreefor
犕狊犎犛犘17.7andsHSPsfromotherplantspecies
ü
(Note):MsHSP17.7(_xìí,犕犲犱犻犮犪犵狅狊犪狋犻狏犪,A0A060CW40);
MtHSP(åñìí,犕犲犱犻犮犪犵狅狋狉狌狀犮犪狋狌犾犪,G7J8C7);
PsHSP17.1(†f,犘犻狊狌犿狊犪狋犻狏狌犿,P19242);
GmHSP17.9(Yf,犌犪犾狔犮犻狀犲犿犪狓,P05477);
Nthsp17.6(“-,犖犻犮狅狋犻犪狀犪狋犪犫犪犮狌犿,A0A077DBK4);
VvHSP(ÿ!,犞犻狋犻狊狏犻狀犻犳犲狉犪,A5B8Y3);
ZmHSP17.8(»z,犣犲犪犿犪狔狊,P24632);
OsHSP18.0(Q§,犗狉狔狕犪狊犪狋犻狏犪,Q5VRY12);
AtHAP17.7(&ZA,犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪,O81822);
TaHSP16.9(u^,犜狉犻狋犻犮狌犿犪犲狊狋犻狏狌犿,Q41560)
2.2 犕狊犎犛犘17.7Nø`ÔhÏ>r
^2©`PCR…˜,۔éf¶÷ß,Uh
iíTµT®58ºt`

ìí®5β犪犮狋犻狀L
6@ä†|6@ú5œgW`¸t¨©

Lcn2
ž®5

}ψD©`ºâ2-ΔΔCt(Ct:-ií¤K
TZ‘Ô{Wd²©Tñ21À=TiíGH
ë

\‘ëÏ=•
[14],
j_xìí}

~

X6xn
/0DR
,^
2©`PCR…˜,犕狊犎犛犘17.7®5
L6@ä†&©JgW

L~&gW`…†sé‘
Xä†

X6x“ã

}&݃

ë4)。
=4 xÕ}¢Ï`Ô>r
Fig.4 Expressionanalysisof犕狊犎犛犘17.7indifferenttissues
ü
:og犕狊犎犛犘17.7®5gWQR‘X
䆈¸Øºú…†
(犘<0.01)
Note:indicatessignificatediferenceatthe0.01levelcomparedwithothers
<=RTqPCR=•¡犕狊犎犛犘17.7®5cs
Ûú5

)ú5=Áðú5œTgW8

ˆDé
ËÛ
(25℃),sÛ(37℃)ú5œ,犕狊犎犛犘17.7®5
gW`gØês
,4h2ÚDÒT68.7±,ÀsÛ
ú52%Tíî

®5gW„œóI
,12h_g
W`<ðغ6…†

ë5A),è.eÿsÛú5D
®5ÞJC"cd

L)ú5œ

Y®5gW`©s
éDÒ

„PjSê_œUSêTóI

L8hO
P!ßM¶

ÚDÒT4.8±,24h2WdÝs
ÚDÒT6.0±(ë5B),UhµT®5)ú5T
asÁJûYTˆ‰

L=Áðú5œ

Y®5
gW`D҈¸îJh…<ð

ë5C),UhµT
®5c=Áðú5TUVtu

=5 犕狊犎犛犘17.7{·z{、›z{、¥¦Qz{P2`ÔÖ_
Fig.5 QuantitativeRTPCRanalysisof犕狊犎犛犘17.7inalfalfaunderhightemperature,saltandperoxidestress
ü
:A37℃sÛú5;B200mMNaClú5;C15mM H2O2ú5,6@uU;Çg˜Øº…†(犘<0.05)
Note:Aunderhightemperaturestress;Bunder200mMNaClstress;Cunder15mM H2O2stress.
Differentlowercaselettersindicatesignificantdifferenceatthe0.05level
521
! " # $ !24"
2.3 ×Nø„ØÙ2Ú³
Ïd®5•ä¾¨p¡JKQgW/N
pBI121-犕狊犎犛犘17.7,’ ¦ Ð µ T ® 5 g ƒ
CaMV35S–Ú!mljã_。Y½‡ð©…˜þ
—k•äF]

½‡_»JñMÍÜ

‘&u´,L
477bp­®(ë6),ÀÃl@:]hþ—k‡"l
jTpBI121犕狊犎犛犘17.7•äm。
=6 狆犅犐121犕狊犎犛犘17.7ÌÛ2HÜÚ³
Fig.6 EnzymeanalysisofpBI121犕狊犎犛犘17.7
ü
:M:DNAmarkertrans2000plusII;
1:plasmidofpBI121犕狊犎犛犘17.7;
2:Y½‡pBI121犕狊犎犛犘17.7F]
Note:M:DNAmarkertrans2000plusII;
1:plasmidofpBI121犕狊犎犛犘17.7;
2:DoubledigestionofpBI121犕狊犎犛犘17.7
  »BCÏd(®Þbµxlâ—kÚðU9T
&ZAGm

Àø9u“L

Ý.—kT3oÚ
®5r]
。PCR𩅘,ÀÁgT4Mr]©
§iíOµTÍÜ

ÃlefA…˜µT®5l
jß#

n¾9FJrö§iíOµTÍÜ

ë
7A),8]h 犕狊犎犛犘17.7®5þWšd&ZA
®5ä&
;RTPCR𩅘,¾9FJrriíO
2ž®5β-犪犮狋犻狀ÍÜ,öiíOµT®5ÍÜ,n
4rdJr\§iíO2ž®5,LiíOµT
®5

ë7B),efgh犕狊犎犛犘17.7®5LÚ®5
&ZA&’¦ÚNgW

8ð©þ—k4MÚ®
5&ZAr]

2.4 犕狊犎犛犘17.7×Nø„ØÙ<2`Ô>r
<=^2©`PCR=•¡ 犕狊犎犛犘17.7Ls
)6=Áðú5œTgW8

~ë8A 1˜,L
300mMNaCls)ú5œ,犕狊犎犛犘17.7®5ÚN
QRL6h2ö°9<ð,L12,24,48h2©sé

,12h2ÝY,ÚDÒT7.2±,Uh 犕狊犎
犛犘17.7Ls)ú5œ<ðh…;L=Áð(30mM
H2O2)ú5œ(ë8B),犕狊犎犛犘17.7®5ÚNQR
„jSê_œUêsTóI
,6hWdDÒT3.3
±_¼½œ
,24h2DÒïh…غ,À_,
ês

L48h2®5ˆDgWWdÝY,ÚDÒ
T3.7±,UhÚ犕狊犎犛犘17.7®5&ZA&µT
®5L§ž¡s)ú56=Áðú5ŠMuŠ

=7 ×Nø„ØÙ2Ú³
Fig.7 Identificationoftransgenic犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪
ü
:AnPCR×Ã;BnTRPCR×Ã,
M:DNAmarkertrans2000plusII;
WT:¾9F&ZA;CK+:pBI121犕狊犎犛犘17.7F];
1~4:Ú®5&ZA
Note:A:PCRanalysis;B:RTPCRanalysis;
M:DNAmarkertrans2000plusII;
WT:Wildtype犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪;
CK+:PlasmidofpBI121犕狊犎犛犘17.7;
1~4:Transgenic犃狉犪犫犻犱狅狆狊犻狊
3 ˆ‰Wp‰
  sHSPLJK9Ÿ°JtlKŠ=Ä&°›
•«cd
[15],
ÆòcÈ9Kú5C"gW
[16],
/0
gh狊犎犛犘®5T=`gWLJ:›s>x(犖犲
犾狌犿犫狅狀狌犮犻犳犲狉犪)[12]、»z[17]、u^[18]?ŽsÛ|‘
Xøt§¥

»Šd¡_xìíu=mpMó¦
®5犕狊犎犛犘17.7,Y®5åñìí 犕狋犎犛犘、†
f犘狊犎犛犘17.1、Yf犌犿犎犛犘17.9ˆQAts,L
MsHSP17.7ó¦lj&°P¡Ð=ŽI、Ð=Ž
II、Ð=ŽIII6†…WÐ=lj,.BondinoT
/0efˆ&š
[13]。
}Ï9KÔ:+=•


犕狊犎犛犘17.7ÑàdH.FsHSPClassII¬。
JŠYi½&ZAH.FCII犎犛犘17.6®5rg
WéxÁ
,犎犛犘17.7 rgWéGm6xÁ[19],
犕狊犎犛犘17.7L_xìí~&gW`ÝY,Æò»
ŠrD_xìí}

~

X

x\‘¡gW=•

öD
xÁ6Gm\‘=•

1jefrˆDéBC&ž
|TJKä†n#

621
!1# ¶\ø?:_xìí犕狊犎犛犘17.7®5Td|¦§=•
=8 {›W¥¦Qz{P犕狊犎犛犘17.7×Nø„ØÙ<2`Ô>r
Fig.8 QuantitativeRTPCRanalysisoftransgenic犃狉犪犫犻犱狅狆狊犻狊with犕狊犎犛犘17.7underhighsalinityandperoxidestress
ü
:ALs)ú5;BL=Áðú5,6@uU;Çg˜Øº…†(犘<0.05)
Note:Ahighsalinitystress;Bperoxidestress.Differentlowercaselettersindicatesignificantdifferenceatthe0.05level
  LsÛú5œ,>x 犖狀犎犛犘17.5[12],u^
狊犎犛犘26[18],u­?@犘狆犎狊狆16.4[20]?®5gW
`ês

»/0&
,犕狊犎犛犘17.7®5AcsÛú5
C"

5.eÃY®5L§žsÛú5ŠMab

狊犎犛犘 ®5LcGtlC",&ZA&TM
狊犎犛犘®5(犎犛犘17.4犆犐,犎犛犘17.6犃犆犐,犎犛犘17.
6犅犆犐,犎犛犘17.6犆犆犐)õ¡csÛC"e,]cÐq
ú5

Áðú5

_efÇb?UV
[19],
=Áð½N
u=mpMó¦®5犃狋犎犛犘15.7cÁðú5C
"
[21]。
»/0&
,犕狊犎犛犘17.7®5gWcs)ú
5C"

.&ZAH.FCII®5犃狋犎犛犘17.6犃
ÚNcÐqú5C"efˆÙ
[22];
L=Áðú5
œ

µT®5gW`<ð6h…

L§c=Áðú5
UVtu

.&ZAH.FCII®5犃狋犎犛犘17.
6犃 ÚN6cÁðú5C"efß#[22],犕狊犎
犛犘17.7¬éH.FCIIsHSP¬,L§&ZA
犃狋犎犛犘17.6犃®5gW8+LˆQ。LÁð
ú5œ

JKN2íT O-2 6 H2O2 ÐQOÁð
U½|‘XɏÁzõ]^Tzõ§¥
[21],
5.L
§RM犕狊犎犛犘17.7®5Y`gW。
tlLʪJKN2ó¦<

½!É

ô=
Áð|=ÁðKí?uŠ
[23],
pMó¦<=Ü[
<ó¦`¯

Ðѽɏ

zõºPó¦

У
TœW?9K+¦§ÐÑJKÎc-±
[24]。
p
Mó¦®5T=`gWLJ:›sJKøt

Jiang?Ð%%犚犮犎犛犘17.8®5ڃ&ZA,Lí
–DsÛ

)*

Ðqú56š‰Tøt§¥
[25]。
Zhang?Ðiöx(犘狉犻犿狌犾犪犿犪犾犪犮狅犻犱犲狊)pMó¦
®5犘犳犎犛犘17.1ڃ&ZA,L)ú5œ,Ú®
5&ZAÈÉ+ÉÝts

†…W»`ts

ˆD»
Q`ts

gPOŽ)
[26],
&ZA犃狋犎犛犘17.6犃
®5T=`gWAL햑Ž)*§¥
[22]。
Áð
ú5œ

JKN2TO-2 6 H2O2 44é=Áð
U½|‘XɏÁzõ]^Tzõ§¥
[21],
pMó
¦<=ÐÑ@}ó¦TœWn%#žzõɏ
Á

»/0&

Ls)ú5=Áðú5œ

Ú®5
&ZA&犕狊犎犛犘17.7®5cdC"Y`gW,e
ÃY®5L§žs)=Áðú5ŠMab

I
Y®5AL§+LÚN_ab

ÚNQRêsÆÈ
ߩʪˆŠó¦gW`í
[22],
5.Y®5T¦
§JR\ß=•C]

»/0Í_xìí&dkd犕狊犎犛犘17.7T
cDNAlj,³´477bp¼>(Éù,³)158M
…®W

Y®5L~&gWݍ

ÆcsÛ6s)
C"gW

L=ÁðC"œgW`<ð6h…

L
s )  = Á ð ú 5 œ

Ú ® 5 & Z A &
犕狊犎犛犘犡犣17.7Y`gW,Y®5L§Ls)=
Áðú5ŠMab&°›•«cd

”•AB
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821