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Cloning and Expression of ClHSP70 and ClHSP90 Gene from Chrysanthemum lavandulifolium

甘菊ClHSP70ClHSP90基因的克隆及表达分析



全 文 :书!"#$%&
,2016,36(7):1321-1330
犃犮狋犪犅狅狋.犅狅狉犲犪犾.犗犮犮犻犱犲狀狋.犛犻狀.
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(1982-),8,9:,;<=>?,@ABCDEFGHI=>。Email:changluo1983@126.com.
JKLM:NO*,=>?,@ABCDEFGHI=>。Email:conglinh@126.com
45犆犾犎犛犘706犆犾犎犛犘90,789:;<=>?
! ",#$%,& ,()*

"()*+%, ")PQ$-R=>ST

"100097)
@ A:U=>VWD(犆犺狉狔狊犪狀狋犺犲犿狌犿犾犪狏犪狀犱狌犾犻犳狅犾犻狌犿 )XYZ[\,J]RTPCR^_BWD`abcdSe
fghijklmnopq

rsX犆犾犎犛犘70t犆犾犎犛犘90。uvewxy,犆犾犎犛犘70pq ORFz{X2559
bp,|}852~p€,jk0‚ƒ„xy…†‡ˆ‰ HSP70jk NBDtSBDŠ‹ŒŽ;犆犾犎犛犘90pq
ORFz{X2094bp,|}697~p€,…† HATPaseŒŽt HSP90Š‹ŒŽ。Q$K%ewxy,W
DClHSP70‘’(犌犾狔犮犻狀犲犿犪狓)t“”(犖犻犮狅狋犻犪狀犪狋狅犿犲狀狋狅狊犻犳狅狉犿犻狊)HSP70jk†•–‰—˜™,犆犾犎犛犘90p
q|}‰p€uvš›œ
(犃犵犲狉犪狋犻狀犪犪犱犲狀狅狆犺狅狉犪)HSP90–žnŸ;Y ¡¢£¤x¥ewxy,¦42℃
§<¨© ª

WD«¬S犆犾犎犛犘70t犆犾犎犛犘90pqx¥­¦0.5h ®¯°±,1h¥²³‘´,2hµ¶·¸
¹
;¨
©bºx¥ewxy

WD¦42℃§<1hµ,犆犾犎犛犘70¦m»«S‰x¥¤®¯–¼½«t¾¿ÀÁb
º
;犆犾犎犛犘90¦m»›S‰x¥¤³–。=>Ây,犆犾犎犛犘70t犆犾犎犛犘90pqÆhijkÄÅ,ƐÇWD
hÈÉÊË]Ì

U=>ŒÍXVµÎÏ=>Àpq0УÇpÑ

BCD

WD
;犆犾犎犛犘70;犆犾犎犛犘90;hÈÉ
EF>G$
:Q785;Q786 !HIJK:A
犆犾狅狀犻狀犵犪狀犱犈狓狆狉犲狊狊犻狅狀狅犳犆犾犎犛犘70犪狀犱犆犾犎犛犘90犌犲狀犲犳狉狅犿
犆犺狉狔狊犪狀狋犺犲犿狌犿犾犪狏犪狀犱狌犾犻犳狅犾犻狌犿
LUOChang,CHENDongliang,CHENGXi,HUANGConglin
(BeijingAcademyofAgricultureandForestrySciences,BeijingAgriculturalBiotechnologyResearchCenter,Beijing100097,
China)
犃犫狊狋狉犪犮狋:Basedon犆犺狉狔狊犪狀狋犺犲犿狌犿犾犪狏犪狀犱狌犾犻犳狅犾犻狌犿RNAseqdata,weobtainedtwocodeDNAsequences
named犆犾犎犛犘70and犆犾犎犛犘90usingRTPCR.Sequenceanalysisshowedthepredictedopenreading
frame(ORF)of犆犾犎犛犘70is2559bp,encodingaproteinof852aminoacids.Theconserverdomain
searchexhibitedthatNteminarcontainstypicalNBDdomainwhichbelongstoHSP70family.Thepredic
tedORFof犆犾犎犛犘90is2094bp,encodingaproteinof697aminoacids.Theconserverdomainsearch
showedthatNteminarcontainsATPasesuperfamilyand HSP90superfamilyconservativestructure.
BioinformaticsshowedthattheaminoacidsequencesofClHSP70hadhighconsistencywith犌犾狔犮犻狀犲犿犪狓
and犖犻犮狅狋犻犪狀犪狋狅犿犲狀狋狅狊犻犳狅狉犿犻狊HSP70,whileClHSP90hadhighconsistencywith犃犵犲狉犪狋犻狀犪犪犱犲狀狅狆犺狅狉犪
HSP90.RealtimePCRdemonstratedtheexpressionpatternsof犆犾犎犛犘70and犆犾犎犛犘90inleavesat42
℃.Theexpressionofthesetwogeneswerebothsignificantlyupregulatedafter0.5h,reachedthehigh
estlevelafter1h.Thentheexpressionofbothtwogenesdecreasedafter2h,andremainedatarelatively
lowerlevelafter4h.Indifferentplanttissues(root,stem,leaf)after1hat42℃,犆犾犎犛犘70washighly
expressedinmatureleaves,lowerinyoung,leaves,root,maturestems,youngstems,and犆犾犎犛犘90was
thehighestinmaturestems.Theresultsshowedthat犆犾犎犛犘70and犆犾犎犛犘90participatedtheprocessof
heattolerancein犆犺狉狔狊犪狀狋犺犲犿狌犿犾犪狏犪狀犱狌犾犻犳狅犾犻狌犿andlaidafoundationforanalyzing犆犾犎犛犘70and犆犾犎
犛犘90genefunction.
犓犲狔狑狅狉犱狊:犆犺狉狔狊犪狀狋犺犲犿狌犿犾犪狏犪狀犱狌犾犻犳狅犾犻狌犿;犆犾犎犛犘70;犆犾犎犛犘90;heatstress
  hijk(heatshockprotein,HSPs)Ò¦h
iÓÔ¸ÕÖ×Øx¥°±‰—ÙeÄÚjkÛ

¦#$ÊÜÝÞßàá1

âãä

å

æçè–ç
¿ éêëA‰Lì
[1]。HSPs‰íîQ$ïh
™ðñno

òóÒÂ

¦hÈɸ HSPs‰íîô
Võ–#ö‰ïh™

HSPsI÷ø¦ùúûü,¾dÀeý¤‘þ,
ôeX5÷:HSP110、HSP90、HSP70、HSP60t

þ HSPs’[2]。¦#$hijkÿ!S,HSP70³
XŠ‹t"#
,HSP70¦p€uv$–žnŸ,
…†%€Œl‚t&$Œl‚2~0‚,N(
Ƈˆ‰)45kD‰ ATPase‚Ž。=>xy,
HSP70¦hÈÉÓÔ¸0*Õ®¯Ö×,þ+,
HSP70B…¤¦hÈÉ2hµ°±³y®[3],EQ
(犃狉犪犮犺犻狊犺狔狆狅犵犪犲犪Linn.)犃犺犎犛犘70¦–çÈÉ
3hµx¥y®-–[4]。./0¦123[5]、“
”
[1]、
45
[6]
¿=>Sò6yÇ HSP70#$ïh
™no
。HSP90җ~7ATP89‰:;=>

¦eý?@$–žŠ‹

A~BŒŽ

SªŒŽtD× HSP90:;@‰C(
ŒŽbm
[78]。
EFܼ#$S HSP90jk‰=
>nܕG

123
[9]
tHI
[10]
S‰=>xy7²
hÈÉ 犎犛犘90x¥¤®¯°±,HSP90jk‰
íî¤y®õ–

JÀŒt0KL?—MN>



†=>6YHSP90¦#$Q{PH?ÌSé
êQoëA‰Lì

À0‰RSTUé#$VW
tQ<ęPQXY
[1112]。


C¼hÖ×tb
mˆx¥‰hijkô£[¼\]‰\]Ût^I
\]_S
,`
a«b<

cÛd
[1314]
¿

Øefê¨
©‰0

òô¦¨©‰_gSÕÖ×

ô¦¾

›

«

IýVèhiSjQ

qZ–çÈɸ#$
HSPspq‰x¥òðkgy®‰bºÄڙ。
DE
(犆犺狉狔狊犪狀狋犺犲犿狌犿×犿狅狉犻犳狅犾犻狌犿 Ramat.)
Ò^/Q”l#$

ém¼Sn

oÞ$­†üpq
r

¦SnEsQjSt†ëAu[

v/QjwE
ÒDEQj‰Éxyz



v/Qj]ÌSD
E¦k{|}~7²€–çÈÉ

טE‚ƒ
»„…†

qZ

LJDE¦7²hÈÉ ‰ïh
ˆ‰Ü¼rHŠlv/Qj‰DE‹Iû†ŒA

WD
(犆犺狉狔狊犪狀狋犺犲犿狌犿犾犪狏犪狀犱狌犾犻犳狅犾犻狌犿)XD+D
:Ž<QI
(2n=2x=18),ÒDE‘I’
—

Q{¼“”200~2800m•è–—,†•˜
™š™

ÀQ{9t›Eœ™qrDEn
Ÿ
[1516]。
l=>ƝWD­—@`abcdž

Ÿ
ìRTPCR-R,ef ²犆犾犎犛犘70t犆犾犎犛犘90
‰cDNAz{uv,ŸìY ¡¢£¤PCR-R
ewÇ2~pq¦–縨©§< ªt¨©bº
Ù[S‰x¥Ä™

X犆犾犎犛犘70t犆犾犎犛犘90pq
‰0¡£VèDEïhHIУpÑ

1 [\^_
1.1 LMNO;PQ
B")PQ$-R=>STbr¢S£¤15
öQ{H•—˜‰WDbriXYZ[。
¥¢
r¦§çž3¨X42℃,A3öLX—~ë©,eª
§<0、0.5、1、2t4h £¤$Ù/w«¬¨¼«¬
S­®
,¯
°Ï-80℃±§S²ì;42℃§<1h,
£¤#ö½«

m»«

½›

m»›

¾¨¼«¬S
­®
,¯
°Ï-80℃±§S²ì。
1.2 RS
1.2.1 T犚犖犃UV;45犆犾犎犛犘70W犆犾犎犛犘90
,7 9 :   ì # $ RNA þ ¤ õ ¤ ³ ´ µ
(MiniBESTPlantRNAExtractionKit,TaKaRa¶
·

õ¤WD«¬¸RNA,¹ìPrimeScriptTMⅡ
1stStrandcDNASynthesisKit(TaKaRa¶·)¥
ºõ¤RNA»`aXcDNA¯¼„À½ž。
BWD­—@`abcdS¾£犆犾犎犛犘70t
犆犾犎犛犘90pquvK,¾d¦NCBId¿$ƒ„
2~pq‰ ORF3ÀU$(x1)。PCR»Ê<Á
(50μL):2μLcDNAÂÃ(100mmol/L),0.5μL
PrimeSTARHSDNAPolymerase(2.5U/μL),
10μL5×PrimeSTARBuffer(Mg
2+plus),4μL
dNTPMixture(Ä2.5mmol/L),ñ»ÅU$Ä
2μLVè29.5μLddH2O。PCRÆ°ÓÔX98℃
2231 ! " # $ % &                   36Ç
<1 XYZ[B\M
Table1 Relatedprimerinthistest
U$÷ˆPrimertype U$sÈPrimerdescription U$uvPrimesequence(5′→3′)
E‰pqU$
Targetgeneprimers
ClHSP70F   TTATGCAGTAGCTTCAGGCTTATC
ClHSP70R   ATGAGCGTGGTTGGATTTGATC
ClHSP90F   TTAGTCGACTTCCTCCATCTTGC
ClHSP90R   ATGGCTGATACAGAAACGTTTGC
Y ¡¢£¤PCR
RealtimePCR
RTClHSP70F AGTCCCGTTTTGTGAATCTTGTG
RTClHSP70R ACAGTTAGTGATATTTATGCTGTCGAG
RTClHSP90F AACCTTCCCTTCCTCGTCTTTC
RTClHSP90R GAGTCGCAAGCTGGTGGTTCGTTCA
RTClActinF TGCTATTCAGGCTGTGCTTTCTC
RTClActinR TAACTTGTCCATCAGGCAATTCG
ƒY™5min;98℃Y™10s,58℃©™5s,72℃
ÉÊ15s,30~Ëß;72℃ÉÊ6min。PCRj$
Ì1%ÍÎÏÐÑÒÓ,ÔÕÖ@E‰¬}µ×Ø
²pGEMTEasyÙ<$,42℃hi`@‘ÚÛÜ
DH5αÝ7W\],?f Amp™™¾£t Xgal/
IPTGÞkß¾£。à¤káÜâ?fPCR¼„,
ã™äåæç$“Q$è̆鶷?f„u

ì
SegmanuvêØëÔ܄uŒÍ?fêØ,eª
ì 犆犾犎犛犘70t犆犾犎犛犘90‰ORFz{uv。
1.2.2 犆犾犎犛犘70W犆犾犎犛犘90,78]M^_`
>? ŸìNCBIcdžSBlastèÃÜ犆犾犎犛犘70
t犆犾犎犛犘90pquv?fíÜt©m™ew(ht
tp://blast.ncbi.nlm.nih.gov/Blast.cgi);î ì
Finderë Ô (http://www.ncbi.nlm.nih.gov/
gorf/gorf.html)?f›°ïðñew;¦ NCBI
ConservedDomainDatabase(http://www.ncbi.
nlm.nih.gov/Structure/cdd/wrpsb.cgi)S?fj
kۊ‹ŒŽƒ„

J]ExPASyProtParamë
Ô
(http://web.expasy.org/protparam)ƒ„jk
‰eý¤è¿Òò
;SMART¦óëÔÜjkی
Ž?fewƒ„

ŸìSignalP(http://www.
cbs.dtu.dk/services/SignalP/)¦óewjkۉ
Kôõ

öìClustalXëÔ?fp€uv^ë
íÜ
[17],
Ÿì MEGA5.0ëԍ2?@÷(Neigh
borJoining)[18],¯ ?fBootstrap¼„。
1.2.3 犆犾犎犛犘70W犆犾犎犛犘90,7<=>? ì
#$RNAþ¤õ¤³´µ(MiniBESTPlantRNA
ExtractionKit,TaKaRa¶·)õ¤42℃¨© ª
(0、0.5、1、2、4h)§<µ‰WD«¬ RNA,øÌ
42℃§<1hµ¨©bºÙ[(½«、m»«、½
›

m»›

¾

‰RNA。ìPrimeScriptTMⅡ1st
StrandcDNASynthesisKit(TaKaRa¶·)¥ºõ
¤RNA»`aXcDNA。¾d犆犾犎犛犘70t犆犾犎
犛犘90pq‰z{uv,ìPremier5.03À Real
timePCRU$(x1),ùì犃犮狋犻狀pqLXcú。
Ɲ¡¢£¤³´µSYBR PremixEXTaqTM
Ⅱ(TilRNaseH Plus,TaKaRa)Âyû?füL:
SYBRPremixEXTaq(2×)10μL,$、¸ ýU$
(10μmol/L)Ä0.4μL,ROXReferenceDye(50
×)0.4μL,cDNA ÂÃ (100 mmol/L)1μL,
ddH2O7.8μL,¸ <í20μL。»ÊÌuX95℃ƒ
Y™10min,95℃Y™15s,60℃þÿ1min,40~
ËßµL!‡"ó

A~#‹­ë© 3 $,¦
ABIStepOnePlusTM RealtimePCR %(&n ABI
¶·

$?f

ùì2-ΔΔCt_ÀnÜx¥¤。
2 ŒÍew
2.1 犆犾犎犛犘70W犆犾犎犛犘90,79:
¾dWD`abcdUnigeneblastx()ŒÍ
¾£犎犛犘70t犎犛犘90uv,¦NCBIS?f›°
ïðñew¯3ÀÄڙU$

VWD«¬cDNA
XÂÃÆ°²犆犾犎犛犘70t犆犾犎犛犘90|}ñuv,
Æ°j$Ì1%ÍÎÏÐÑÒÓew,2~pqÆ°
Ó*{žeªX2500bpt2000bp+,(-1),
¬}̄uµñ»ÅêØ ²pq‰z{

ewŒ
Íxy
,犆犾犎犛犘70…†1~2559bp›°ïðñ,
32317.            6 7,¿:WD犆犾犎犛犘70犆犾犎犛犘90pq‰äåèx¥ew
|}852~p€(- 2);犆犾犎犛犘90…†—~
2094bp›°ïðñ,|}697~p€(-3)。
2.2 犆犾犎犛犘70W犆犾犎犛犘90,7]M^_`>?
Ì Protparatam ¦ ó è Ã (http://web.ex
pasy.org/protparam/)ew,ŒÍ®/(x2)ClH
SP70jk‰¸•­0H™X-0.444,ClHSP90j
k‰¸•­0H™X-0.604,­X1H™jk;¸
‰*2Ò3‰p€íë­4–¼*ñÒ3‰p
€

xkX€™jk

B5£Ác6„ClHSP70X
¨5£jk
,ClHSP90X5£jk。J]:7Œ
ƒ„PkClHSP70…†40.73%‰α89,5.87%
M.DL5000;1.犆犾犎犛犘70;2.犆犾犎犛犘90
-1 WD犆犾犎犛犘70t犆犾犎犛犘90pqÆ°ŒÍ
Fig.1 PCRamplificationresultsof犆犾犎犛犘70and
犆犾犎犛犘90genefrom犆.犾犪狏犪狀犱狌犾犻犳狅犾犻狌犿
¸:óÙeXN(NBDŒŽ(A)tN(Š‹‰ATPaseŒŽ(B);;á<=ÙeX–žŠ‹‰犆犾犎犛犘90p€uv(B);
>á<=ÙeXC(]Û?ˆÄ@Kô‰pu(B);.x/ABC}ý
-2 WD犆犾犎犛犘70(A)t犆犾犎犛犘90(B)pq‰%D€uvèÀ|}‰p€uv
UnderlinerepresentsNBDdomainofNterminusdomain(A)andtheATPbindingdomainofNterminusdomain(B);shadedgray
representshighlyconservedaminoacid犆犾犎犛犘90signaturesequencemotifscharacteristic(B);shadedblackrepresentstheCterminus
cytosolicisoformspecificsignaturemotif(MEEVD)(B);.Stopcodon
Fig.2 Thenucleotideanddeducedaminoacidsequencesof犆犾犎犛犘70(A)and犆犾犎犛犘90(B)in犆.犾犪狏犪狀犱狌犾犻犳狅犾犻狌犿
4231 ! " # $ % &                   36Ç
NtHSP7015.“” (XP009624163.1);GmHSP7014.‘’(XP003546366.1);AtHSP70.123(NP850984.4);
BoHSP7014.WÞ(XP013591718.1)
-3 WDClHSP70ÀÁ$I HSPs^uvíÜ
NtHSP7015.犖犻犮狅狋犻犪狀犪狋狅犿犲狀狋狅狊犻犳狅狉犿犻狊(XP009624163.1);GmHSP7014.犌犾狔犮犻狀犲犿犪狓(XP003546366.1);
AtHSP70.犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪(NP850984.4);BoHSP7014.犅狉犪狊狊犻犮犪狅犾犲狉犪犮犲犪var.狅犾犲狉犪犮犲犪(XP01591718.1).
Fig.3 MultiplealignmentofClHSP70withHSPsfromotherplantspecies
<2 5a犆犾犎犛犘70W犆犾犎犛犘90bcde8Qfgh
Table2 PhysicalandchemicalcharacterizationofClHSP70andClHSP90heatshockproteinsfrom犆.犾犪狏犪狀犱狌犾犻犳狅犾犻狌犿
jk™ÛProteincharacter ClHSP70 ClHSP90
¸p€cENumberofaminoacids/aa 852 697
jkeýÛ¤ Molecularweight/kD 94.1 79.9
jkeýFFormula C4133H6584N1136O1302S34 C3544H5649N917O1124S26
¸*2Ò3‰GpcTotalnumberofnegativelychargedresidues(Asp+Glu) 135 138
¸*ñÒ3‰GpcTotalnumberofpositivelychargedresidues(Arg+Lys) 103 106
ÎH!IcAliphaticindex 78.91 82.50
¨5£ÁcInstabilityindex 43.08,¨ 5£Unstable 37.55,5£Stable
¸•­0H™Grandaverageofhydropathicity -0.444 -0.604
52317.            6 7,¿:WD犆犾犎犛犘70犆犾犎犛犘90pq‰äåèx¥ew
‰β†Jt36.85%‰KˆÇ";ClHSP90L…†
51.51%‰α89、6.31%‰β†Jt24.82%‰K
ˆÇ"
。SignalP¦óew ClHSP70t ClHSP90
­¨…Kôõuv

òMNOŒ

p€uví܌ͮ/

WDClHSP70‘
’
(犌犾狔犮犻狀犲犿犪狓)t“”(犖犻犮狅狋犻犪狀犪狋狅犿犲狀狋狅狊犻犳狅狉
犿犻狊)HSP70jk†•–‰—˜™,eªX78%t
76%,À$Ґ123(犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪)、W
Þ
(犅狉犪狊狊犻犮犪狅犾犲狉犪犮犲犪var.狅犾犲狉犪犮犲犪)©X74%(-
3);WD ClHSP90š›œ(犃犵犲狉犪狋犻狀犪犪犱犲狀狅
狆犺狅狉犪)HSP90†®¯‰—˜™,–¥93%,À$Ò
“”X92%,PQ(犛狅犾犪狀狌犿犾狔犮狅狆犲狉狊犻犮狌犿 )、
123—˜™eªX91%t90%(- 4)。Ây
ClHSP70tClHSP90Æhijkÿ!–žŠ‹
䀁

Š‹Žƒ„ŒÍ

-5)xyClHSP70j
kC(3~691aaXHSP70jkÿ!Š‹‚Ž,782
~818aaX—}æ©R™‚}(lowcomplexity
region,LCR)。N ( 9~381aa Ò % € Œ l ‚
(nucleotidebindingdomain,NBD)(-2,A),UŒ
l‚p€uv–žŠ‹

ƌl¯H‡ ATP
‰S™ST

T ATP‰ŒlH‡]Ì8UêT
Ü^õ‰1t1
,34~219aaX&$Œl‚(sub
stratebindingdomain,SBD)。
ClHSP90jkیS…† N(–žŠ‹‰
ATPaseŒŽ29~178aa(-5),Æ ATPaseS
™
,ClHSP90jkÛ184~697aa。OÝ,ClHSP90
NtHSP80like.“” (XP009594054.1);AaHSP90.š›œ (ABX76302.1);SlHSP901.PQ (NP001234436.1);
AtHSP812.123 (NP200414.1)
-4 WDClHSP90ÀÁ$I HSPs^uvíÜ
NtHSP80like.犖犻犮狅狋犻犪狀犪狋狅犿犲狀狋狅狊犻犳狅狉犿犻狊(XP009594054.1);AaHSP90.犃犵犲狉犪狋犻狀犪犪犱犲狀狅狆犺狅狉犪(ABX76302.1);
SlHSP901.犛狅犾犪狀狌犿犾狔犮狅狆犲狉狊犻犮狌犿 (NP001234436.1);AtHSP812.犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪(NP200414.1);
GmHSP902.犌犾狔犮犻狀犲犿犪狓 (NP001236599.1)
Fig.4 MultiplealignmentofClHSP90withHSPsfromotherplantspecies
6231 ! " # $ % &                   36Ç
p€uvSV`aHSP90ÿ!5~ÄÅúWuv:
NKEIFL RELISNSSDALDKIR、LGTIARSGT、
MIGQFGVGFYSAYLVA、IKLYVRRVFI、GIVD
SEDLPLNISRE,C(XpuX MEEVD,6„U
jkX\]Ûhijk

-2,B)。
ÁY?@÷ewxy
,犆犾犎犛犘70pq123
犃狋犎犛犘70 (NP850984.4)、W Þ 犅狅犎犛犘7014
(XP013591718.1)‰oÁ•.,§¼©—eZ(-
6,A)。犆犾犎犛犘90©XD+‰š›œ‰犃犪犎
犛犘90(ABX76302.1)oÁ³.,š›œ HSP90ø
Z6ƐÈÉÊË

À$ғ”

PQ

þ[

12
3

WÞ¿‰犎犛犘90÷pqoÁ•\ (-6,B)。
2.3 犆犾犎犛犘70W犆犾犎犛犘90,7<=>?
Y ¡¢£¤ PCR ewŒÍxy,WD¦
42℃¨© ª§<ÓÔ¸,«¬S犆犾犎犛犘70t
犆犾犎犛犘90pqx¥¤­†®¯°±,ÀS犆犾犎
犛犘70t犆犾犎犛犘90¦§<0.5h x¥¤]›‘y
®-–

eªXܝ‰5.8t5.1Ž,1h x¥¤
¥²³–

eªXܝ‰12.2t9.7Ž,2t4hµ
犆犾犎犛犘70t犆犾犎犛犘90pqx¥¤†º¸¹(-
7)。Ây犆犾犎犛犘70t犆犾犎犛犘90¼hÖ׈x
¥‰hijk
,^
WD7²–çÈÉ 

¦û_ ª
có0*ƐhÈÉ`Ê?f‘¤x¥

°±#ö
  ¾d犆犾犎犛犘70t犆犾犎犛犘90–ç§<¨© 
ª‰x¥ab

¥WD#ö42℃§<1h,eª¤
#ö‰½«

m»«

½›

m»›

¾õ¤RNA,Y
 ¡¢£¤PCRewŒÍxy,犆犾犎犛犘70pq¦
m»«S‰x¥¤®¯–¼ÀÁÙ[

À$Ò½«

¾

m»›

½›
。犆犾犎犛犘90pq¦m»›S‰x
¥¤®¯–¼ÀÁÙ[

½›Sx¥³æ

½«

m
»«

¾’ª‰x¥cÚ¨y®

-7)。Ây¦–
çÖ׸犆犾犎犛犘70t犆犾犎犛犘90‰x¥ø¦y®
‰bº_gÄڙ

© 犆犾犎犛犘70犆犾犎犛犘90’
ªx¥H•³–‰Ù[ò¨n©

6„$d2÷h
ijk¦#öS‰£[tef0†º¨©

-5 WDClHSP70tClHSP90Š‹ŽewŒÍ
Fig.5 PredictionofconserverdomainofClHSP70andClHSP90from犆.犾犪狏犪狀犱狌犾犻犳狅犾犻狌犿
9òcex/BootstrapZ6Sp¼1000$멉ôKž;aôc|ôXp€faô
-6 hijk犎犛犘70(A)t犎犛犘90(B)pq|}‰p€ÁY?@÷
Bootstrapvaluesareshownatnodesbasedon1000replications;TheaccessionNo.isgiveninbrackets
Fig.6 PhylogenetictreeforHSP70(A)and犎犛犘90(B)basedonalignmentofaminoacidsequencesfromotherspecies
72317.            6 7,¿:WD犆犾犎犛犘70犆犾犎犛犘90pq‰äåèx¥ew
¨©þgehx/©—pq¨©§< ªt¦¨©bºS‰cÚ®¯
,犘<0.05
-7 WD«¬S犆犾犎犛犘70t犆犾犎犛犘9042℃–ç§<¨© ªt¦¨©bºS‰x¥
Differentlettersrepresentsignificantdifferenceamongdifferenttreatmenttimesanddifferenttissuesunder42℃ (犘<0.05)
Fig.7 Relativeexpressionlevelof犆犾犎犛犘70and犆犾犎犛犘90geneindifferenttreatmenttimesanddifferenttissues
under42℃hightemperaturestress.
3 i E
–çÒ#$QrSjSkTlm‰nQ$š
à

hijk
(HSPs)Ò\]7²–çßà„ÀÁÈ
ÉßàÖ× jQ‰—÷0ˆjkÛ

ôVLX
eý=>Ɛ\]c/lmjkۉ†J

±è
、`
îVèjkÛY™µ‰©™
[19],
°˜\]ÜÈÉß
à‰oï1Bpõ–\]‰ŠÊ01
[20]。
DEÒ
Snq‘sE

oÞr‘xE

DE



tuv

wxy

’—

j¤z{

KêncDE(|}‰
P~

Àqrí¦¨€Æ‘

J€–ç=`ÇÀ
j¤t‹Û

qZ

=>hijk 犎犛犘狊pq‰Œ
t0

ܼõ–DEïh™

rHï–ç‹I†
{\‚

l=>ŸìWD­—@`abcd

BWD«
¬Säå ²犆犾犎犛犘70t犆犾犎犛犘90pq,lYZ
SŠ‹‚ƒ„ewxy
,ClHSP70…†‡ˆ‰NBD
ŒŽtSBDŒŽ,À¦ HSP70 ATPŒl
H‡tPƒeý=>0 éêëALì

d&
„
,HSP90^õN(ŒŽ`…1~ATPŒl‚
Ž

¦ HSP90efQ$0 éêëALì,…%
Q$\]Û HSP90‰C(…†Š‹‰ MEEVDp
u
[21],
0*TPR(tetratricopeptiderepeat)ŒŽ
‰jk†ª¯Œl

Bpö HSP90Pƒ¨©‰L
ì

l=>SClHSP90N(ŒŽ`… ATPase
Œl[ò
,C(…† MEEVDpu,‡ˆ[22]、P
Q
[23]
t123
[24]
S‰=>—˜

Ò\]Û HSP90。
p€uvíÜewxy
:ClHSP70‘’t“”
HSP70jk†•–‰©m™,ClHSP90š›œ


“”

PQ¿#$ HSP90†êû–‰©m™。
BÁY?@÷ôV g
,ClHSP70123、WÞ
HSP70;m—÷,ClHSP90©¼D+#$‰š
›œ;X—b
,¯
“”

PQ HSP90;m—‘
eZ

qZ
,犆犾犎犛犘70t犆犾犎犛犘90eª¼#$
犎犛犘70pqÿ!t犎犛犘90pqÿ!m?。
–çÈɸ

hijk7²Ö×Øx¥¤y®
°±

© ¦ñk‰ßà¸X‰Š\]‰ñkî`
HSP70t HSP90ôVbmˆx¥。l=>S,
犆犾犎犛犘70t犆犾犎犛犘90¦25℃ ­†x¥,Øx
¥nÜ5£

=>xy
,^
c HSPs¦7–çÈÉ
µ_ ªcó0Ö×lm

hi§<3~5minc,
HSPsmRNA‰¤°±,20min ô¼„²/lm
‰HSPs,Kêhi ª‰É{,HSP‰lm0*
Š‹cþ 

l=>S

WD犆犾犎犛犘70t犆犾犎
犛犘90¦42℃§< Àlm@A¦1hcŒm¯¥
²³–
,2hµ›‘¸¹,4hŽ¸¹。12
3S 犎犛犘70ÿ![25]t‡ˆ犔狊犎狊狆90[22]¦7²h
i§< ‰x¥ÂF÷Ÿ

Kêhi ª°±nÜ
x¥¤¸¹‰qô0ÒqXhipq‰{ ªx
¥ÜQ$<¨Ÿ

#$ŠÊ–çß൥Àx¥¸
8
[26]。
¾d?\]£[

#$ HSP70@AeXr
‘÷

eªX[¼\]ÛHSP70、cÛdHSP70、ó
<HSP70t«b<HSP70。HSP90@A£[¼
\]Û

ò†GÙeø¦¼cÛd

�\]S

=>xy
,HSPs¦hÈɸ‰x¥
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™

lYZS
,42℃hÈÉ1h,犆犾犎犛犘70t犆犾犎
8231 ! " # $ % &                   36Ç
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¯cڙ

ÀS犆犾犎犛犘70¦m»«S‰x¥¤³
–

’“犈犆狆犎犛犘701pq[27]、45hijk
70[28]¦Ä~bº_gS‰x¥ew—˜,6„ô0
À?\]£[@Ae”¦«¬S†o

nܼ½
«tÀÁbºÙ[

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,^
#ö7²–çÈÉ 

{
–TU髬—˜

«áSb¿xˆÄÅ
,犆犾犎
犛犘70¦m»«¬S‰–x¥xyÀ¦#$`ʖ
çÈÉt¶‡#$<7²‰h™š]ÌSéëAL
ì
。犆犾犎犛犘90¦m»›S‰x¥¤³–,¦«¬S
x¥¤•æ

›œ¿¦PQS‰=>ŒÍ÷
Ÿ
[23],
Ây HSP90¦#ö7²hÈɵ0¦^~
bºÙ[Ɛ#$–çÈɉÊË


,HSP90
žÇƐ–çÈɉÊ˒Ý

À¦#$Q{PH
?ÌSéêQoëA‰Lì

ÂF#$123
[2931]
S‰=>xy HSP90üpƐÇ#$‰Q{PH


lYZS犆犾犎犛犘90¦m»›t½›S‰x
¥cÚ³Xy®

6„Upqô0Ɛ›ŸeQb
ºno8USj

WDhijkpqܖçÈɉ
x¥ÂF=>XDEïh™=>õ ÇeýpÑ

¯XŸìpqèÌõ–DEïh™tïhIÛ¡m
./Êìõ 
o¼ 犆犾犎犛犘70 t
犆犾犎犛犘90¦DESâ£föÀ0¤¥¦µ¥§
?—M=>

ij!H

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ronmentalstressinChlorelaspeciesfromcontrastinghabitats
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