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Cloning and Expression of MeHB2 Transcription Factor in Cassava

木薯MeHB2转录因子的克隆与表达分析



全 文 :书!"#$%&
,2016,36(8):1522-1527
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HDZip-f犕犲犎犅2。犕犲犎犅2-fgh882bpijklmn,op293eq-r。stuvwxyz{|},
犕犲犎犅2opistgh Homeodomain、Leuzipper~ HDZip_Nxy€,‚ HDZipIIƒ9。„…†>‡ˆ
‰
,犕犲犎犅2Š‹Œ†、†~ŽH‘-fi’“”•–—。X˜™š›œPCR>‡ˆ‰,žŸ 、¡¢Ÿ
 
、ABA~ H2O2 £¤¥¦犕犲犎犅2-fiˆ§,¨ ©Ÿ ª«¬ˆ§。­®,犕犲犎犅2iˆ§œ¯°±²³Ÿ 
i¥¦

78ˆ‰
,犕犲犎犅2´UVµ@$¶·¸¹Hº»¼;½¾。
DEF

UV
;HDZip;犕犲犎犅2;bc;-fˆ§
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狅犳犕犲犎犅2犜狉犪狀狊犮狉犻狆狋犻狅狀犉犪犮狋狅狉犻狀犆犪狊狊犪狏犪
DINGZehong,FULili,TIEWeiwei,YANYan,XIAZhiqiang,WANGWenquan,HU Wei
(InstituteofTropicalBioscienceandBiotechnology,ChineseAcademyofTropicalAgriculturalScience,Haikou571101,China)
犃犫狊狋狉犪犮狋:Cassavacultivar‘Ku50’wasusedasexperimentmaterialsinthisstudy.A HDZipgene,
犕犲犎犅2,wasclonedfromleavesofcassava‘Ku50’byRTPCRmethod.Thisgenecontained882bpopen
readingframe(ORF)encoding293aminoacids.Proteinconserveddomainpredictionshowedthat犕犲犎犅2
proteincontainedseveraldomainssuchasHomeodomain,LeuzipperandHDZip_N,indicatingthisgene
belongedtomembersofHDZipII.Phylogeneticanalysisexhibitedthat犕犲犎犅2hadclosegeneticrelation
shipwithitshomologousgenesin犑犪狋狉狅狆犺犪犮狌狉犮犪狊,犘狅狆狌犾狌狊狋狉犻犮犺狅犮犪狉狆犪and犛犪犾犻狓狆狌狉狆狌狉犲犪.Quantita
tiverealtimePCRanalysisrevealedthatlowtemperature,osmotic,ABAandH2O2treatmentssignificantly
inducedtheexpressionof犕犲犎犅2,butsaltstressdepresseditsexpression.Inaddition,theexpressionof
犕犲犎犅2wasalsoinducedbyshadestress.Theresultsindicatedthat犕犲犎犅2playsimportantrolesin
abioticstressregulationincassava.
犓犲狔狑狅狉犱狊:cassava;HDZip;犕犲犎犅2;cloning;geneexpression
  UV(犕犪狀犻犺狅狋犲狊犮狌犾犲狀狋犪Crantz)¿¼;iÀÁ
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ê+vwi HD(Homeodomain)xy€~Leuzip
per(Zip),-。Ü. HDZipx×i(/ DNA
01~-fxy
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&
[5]。4eÂ&i HDZipst"hü*2i÷
Å
,´
#$@ÑÒÓ

345ƒ

@$Šµ@$Ÿ 
¶·67H8áµð¼;i¸¹C]
[67]。


HDZipⅡ&stŠš345ƒ:²³;6ö”。
犃狋犎犅2¿<(= HDZipⅡ>?ƒ9@Ê,´ AB
@ÑiC…DH
,犃狋犎犅2ê+ˆ§,E1š(R)~
F1š
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H
,犃狋犎犅2iˆ§œ°±R∶FRGH¥¦,JK
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ˆMWNOÑ
、`
aÞP

[89]。
Q­@®
,HDZipⅡ>?ƒ9¯°ÌÍ、ê
©
、ABA~RSæ·i¥¦,ATUŠVWBX
YZÆþê#$[¶/
[10]。
\]´<(=
、^
_

`a~†bÈ$íHcõ›dÉee HDZip
>?ƒ9
[7]。


UVHh” HDZip-fi7
8¯fß

Jg-f´UVÿ¶Ÿ HiÐ6h2
ij

k78犎犅2),î¬01„ldmýi@$Bn%>‡,o
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©

pqr
、ABA、H2O2 ~²³Èíµ@
$¶·Ÿ Ø78¬ˆ§/

Ws} HDZip)
*f?犕犲犎犅2´UV¶·¸¹Hi>?t«u›
-v

1 Z[~^_
1.1 N O
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%NJK@$OP78Qþz

1.2 P Q
1.2.1 NORS=TU {UVíN|ƒÑ+Ç
}15cmiN~,€‚(3~4e/N~)ƒ„…
£†ÊÚiN~‡ˆ‚‰[Š

ê18.8cm,‹ŒZ
18.5cm,،Z14.8cm)H,1N~/Š。-u\]
ŽŠ‘T1∶1i’“”„l•×。UVí
#}10d–„l—D,v˜1D/Š。
í#60d–,€™ÑšÊÚiUV›DCWz
wZ[

T2œ6iUVWî

žŸ 5eœ6:
(1)]300mmol/Lpqr~200mmol/LNaCl>
¡î›D„l¢Üœ60、2~6hT:3、14、18~
24d;(2)]100μmol/LABA~10mmol/LH2O2
>¡î›D„l£¤¥¢Üœ60、2、6、10、24、48
~72h;(3)4℃žœ6UV0、2、5、15、48h–¦
§±¨ž7~14d。@–î‹©Qh˜—ªiUV
`a„l™Ë

«þRNA,„lqRTPCR>‡。
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iUVí#‚¨®
。2e­–€®Ñš”–ÊÚi


>¡þ™2`a

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、´
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iRNA,„lqRTPCR>‡。
1.2.2 犚犖犃VW=狇犚犜犘犆犚 ]RNAþ™w·
¸

¹Ü@…,Ohº»¼

þ™UV½RNA。@
–]RevertAidFirstStrandcDNASynthesisKit
(Fermentas»¼){½ RNA ;)*ƒ cDNA,
-20℃¾¿。]Primer6.0ŸÀÁ$,y‹@Â
@$ÂÃÄÅhº»¼×ƒ

ˆ1)。
]SYBRGreenIw·¸(TaKaRa»¼)„l
qRTPCR,ƝÇCÈÉ~;6’•´ Mx3005P
™š›œPCRÊ(Stratagene»¼)‹„l。Ëe
Ëx3Ì@$%¼§,Ɲ2-ΔΔCtÀÍ-föîˆ
§œ
[11]。
1.2.3 XYZ[\@A ]BLASTPÎÏPhyto
zomeÉ.Ðљ¬Ò$íŠ 犕犲犎犅2‘/–ê
i01

] ORFFinder(http://www.ncbi.nlm.
nih.gov/gorf/gorf.html)~ GENSCAN (http://
genes.mit.edu/GENSCAN.html)z{jklmn;
>1 ]^_G`+:aY
Table1 Primersusedinthisstudy
]Y
Purpose
Á$ÓÔ
Primername
Á$01
(5′→3′)
Primersequence
PCRÕ-
PCRcondition
犕犲犎犅2qRTPCRÁ$
qRTPCRprimerfor犕犲犎犅2
qMeHB2F AATGAATCCACCCACCACCC 95℃30s;95℃10s,55℃10s,72℃20s,40eÖæ
qMeHB2R TATGTGGATGGGGCAGAGGA 95℃30s;95℃10s,55℃10s,72℃20s,40cycles
犃犮狋犻狀qRTPCRÁ$[12]
qRTPCRprimerfor犃犮狋犻狀gene
qActinF TGATGAGTCTGGTCCATCCA 95℃30s;95℃10s,55℃10s,72℃20s,40eÖæ
qActinR CCTCCTACGACCCAATCTCA 95℃30s;95℃10s,55℃10s,72℃20s,40cycles
犕犲犎犅2-fâÑ×Ø
Fullengthamplificationof犕犲犎犅2
MeHB2F CTCAATCCAACGCCACACCT 95℃5min;95℃40s,55℃40s,72℃2min,35eÖæ
MeHB2R ACCAAATCACCCCTACACCC 95℃5min;95℃40s,55℃40s,72℃2min,35cycles
32518Ù              /01,:UV犕犲犎犅2)*f?ibcŠˆ§>‡
]ClustalX„l01”î;]MEGA5.2Ú-y5
„…†

]ExPASyProtParamÚ-(http://web.
expasy.org/protparam/)>‡stui>?œ~
Ûª

]CDDÉ.Ð(http://www.ncbi.nlm.nih.
gov/cdd/)„lvwxy€>‡。
2 x܊>‡
2.1 犕犲犎犅2,8:;<
Ü.UVHŠ<(=犃狋犎犅2(AT4G16780.1)
ê+‘icDNA 01(Manes.01G084500.1)Ÿ
À-f(/Á$

TUVÝÞí Ku50`acD
NAWßà„l PCR ×Ø,{0–ÑáÊeÑ
1191bpia~(â1),{S-fãÓW 犕犲犎犅2。
ú°±128bpi5′UTR、181bpi3′UTR ~
882bpijklmn,S01op293eq-r。
Blastx܈‰,犕犲犎犅2ŠPhytozomeUV(ÝÞx
í
:AM506)É.ÐH»äi01öå/ê§99%。
úû@—žh6eæ-iç(,¬H4eWµ%
èé

â2)。z{stui>?œW32859.0Da,
M.DNAmarker;1.PCR×ØÝ$
â1 犕犲犎犅2-fPCR×ØxÜ
M.DNAmarker;1.ProductofPCR
Fig.1 PCRamplificationresultof犕犲犎犅2
A.éêr01;B.q-r01
â2 犕犲犎犅201”î
A.Nucleotidesequence;B.Proteinsequence
Fig.2 Sequencealignmentof犕犲犎犅2
4251 ! " # $ % &                   36ë
â3 犕犲犎犅2stuxy€>‡
Fig.3 Proteindomainanalysisof犕犲犎犅2
âHü>ìœÉíˆ}¼§1000ÌBootstrapí,±XîWq-rï*X
â4 犕犲犎犅2Š¬Ò HB2-fi„…†>‡
ThenumberateachnoderepresentstheBootstrapvaluederivedfrom1000replicates,andtheaccessionnumber
ofeachsequenceisgiveninthebracket
Fig.4 Phylogeneticanalysisof犕犲犎犅2andotherHB2genes
6ðÛªW7.54。stuvwxyz{|},
犕犲犎犅2opistgh Homeodomain(cd00086)
~Leuzipper(smart00340)xy€,ˆ‰bc±i
犕犲犎犅2-f‚ HDZip>?。­®,´ Nñ¯
ÒMd HDZip_N(pfam04618)xy€(â3),„Ê
ýˆ‰犕犲犎犅2‚ HDZipⅡƒ9。
2.2 犕犲犎犅2,8bcd@A
ò] MEGA5.2Ú-î犕犲犎犅2ŠÆ*¬Ò$
íi‘/–êiq-r01„l”î

y5•ó
„…†

â4)。x܈‰,Jg01Çڋ¤Tô
&W2õ:´ IõTC4 $íWöˆ,°±÷?、øù
ú

ûü
、`
a~êý
;´ Ⅱõ°±tþÿ!þ、<
(=

¤¤†

"#

‹Œ†

UV

†~Ž

¬
HUVŠ‹Œ†
(XP_012070041.1)、†(Potri.
001G155100.1)~Ž(SapurV1A.0886s0110.1)
i’“”•–—

q-r01öå+>¡§±
84%、78.5%~77.5%。
2.3 犕犲犎犅2>?ef@A
Wd78犕犲犎犅2-fîüíµ@$Ÿ iÐ
6

$ûò]qRTPCR^_%{d¬´UV`a
Hiˆ§œ

â5)。´žŸ œ6Ø,犕犲犎犅2i
ˆ§œ´2hh&‹(,5h§±|)^*,@–
´15h&ØÙ,´ ¦§¨ž7d–§±+ê^
*

´pqrœ6–2h~18d,犕犲犎犅2iˆ§
œ,‰|é…

E´24d|)ØG。´NaClœ6
Ø
,犕犲犎犅2iˆ§œ£|)ØÙd,¬HT14~
18d+W‰|,>¡ØÙd86.8%~74.6%。´
ABAœ6Ø,犕犲犎犅2iˆ§œ´2h,‰|é…,
6h|)ØG,@–´10hØÙ,Œ-72hˆ§^
*+ê

´ H2O2 œ6Ø,犕犲犎犅2iˆ§œ´2h
|)ØÙ
,´ 6h|)ØG,@–./ØÙ,´ 24
~48h§±+ꈧ^*。JgxÜ0>ˆ‰,´
ž

¡¢Ÿ 
、ABA ~ H2O2 œ6Õ-Ø,犕犲
犎犅2-fiˆ§^*1|)¥¦,¨ ©Ÿ GHÃ
ª«犕犲犎犅2-fiˆ§。


%{d 犕犲犎犅2-fî²³œ6iÐ6

â5)。Šîö”,²³œ6– 犕犲犎犅2´²³
j`Hiˆ§œh&‹(

E´´Êaµâ³j
`~¶`H£|)Ë(

3‰ 犕犲犎犅2iˆ§œ
¯°±²³œ6i¥¦

52518Ù              /01,:UV犕犲犎犅2)*f?ibcŠˆ§>‡
É.]

£í±456甈},2P789ˆ}öœ6Ø2˜—ª(¥õ:)@—ç(|)(犘<0.05)
â5 犕犲犎犅2´2Ÿ Øiˆ§ß;
Dataareshownasmean±standarddeviation,anddifferentlowercaselettersrepresentsignificantdifferenceamongdifferenttime
points(ortissues)underthesametreatment(犘<0.05)
Fig.5 Expressionpatternsof犕犲犎犅2geneunderdifferentstresstreatments
3 < ð
HDZip¿#$HÊ&¼;i)*f?,´ #
$@ÑÒÓ

345ƒ

@$Šµ@$Ÿ ¶·6
7H8áµð¼;i¸¹C]
[67]。HDZip)*f
?¿Êe=Çi-f>?
,´
<(=
、^
_
、`
a~
†$íH&>¿´33~63e HDZipƒ9[7]。
Šß;#$ö”

UV-fÿê+?×

¬-v6ð
78¯öî@A

\]UVH HDZip>?ƒ9i
É\h2ij

UVHh” HDZip>?-fi&
>ìe>hº
[12]。
´k78H

$ûbcdÊeU
V HDZip-f(犕犲犎犅2)iâÑcDNA01。s
tuxy€z{ˆ‰
,犕犲犎犅2gh HDZip>?v
wxy€

Ô Homeodomain~Leuzipperxy€


<-fxy‹B
,犕犲犎犅2Š<(=犃狋犎犅2Ê
Ë
[6],
쁂 HDZipⅡ>?ƒ9。ô&>‡ˆ‰,
úŠ‹Œ†

†~Ži‘-fiöå/–ê

犃狋犎犅2¿´Êe´I¾#$H1ÒMi°
R∶FR¥¦i-f[8]。犃狋犎犅2;%~Cˆ§#¯
>¡ˆMd”D@ÿ–E~–ÑiØFG

HMI
ÿiL³;6ˆÿ
[8]。
k78犃狋犎犅2i‘-f犕犲犎犅2,o78dS-f´²
³œ6Øiˆ§œé…

ÔzÙiJË
,犕犲犎犅2
iˆ§œ´²³œ6

R:FR)Ø1GH¥¦d。
¨ƒ

JíKš´ƒLi`aHMN‰|

Š<(=
H&>i犃狋犎犅2ˆ§ß;–Wöå[13]。
78ˆ‰
,HDZipⅡ-f°±ÌÍ、ê©È
í¶·Ÿ i¸¹

Ô´<(=H
,犎犃犜2 ~
犎犃犜22iˆ§œ°ÌÍ¥¦‹¸[14];´ ^_H,
犗狊犎犗犡11、犗狊犎犗犡19~犗狊犎犗犡27iˆ§œ´Ì
ÍÕ-؋¸¥Ø¸
[10];´
§O#$P]ú
(犆.
狆犾犪狀狋犪犵犻狀犲狌犿)H,犆狆犎犅2-f´ÜHiˆ§œ°
±^>~ABA¥¦[15]。k78HqRTPCR>‡
ˆ‰
,犕犲犎犅2iˆ§œ°±ž、pqr、ABA~
H2O2 ¥¦,E°±©Ÿ œ6ª«,3‰S-f´
Èíµ@$¶·Ÿ Õ-ØUŠ-fˆ§¸¹

¤
6251 ! " # $ % &                   36ë
TCWUVÿ/Óí–W¼;iå€-f

Jg7
8xÜ{h5‚dQ HDZip)*f?犕犲犎犅2´
UV¶·¸¹HiC]

W„Êý78~Q‡ HD
Zip>?¬Òƒ9i÷ÅþzUR。
gh!J

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notypicapproachestodroughtincassava:review[J].犉狉狅狀
狋犻犲狉狊犻狀犘犺狔狊犻狅犾狅犵狔,2013,4:93.
[2] SANTABA,DOSRS,COSTACN,犲狋犪犾.Molecularclo
ningandcharacterizationofacassavatranslationalycontroled
tumorproteingenepotentialyrelatedtosaltstressresponse
[J].犕狅犾犲犮狌犾犪狉犅犻狅犾狅犵狔犚犲狆狅狉狋狊,2014,41(3):17871797.
[3] HILLOCKSRJ,THRESH J,BELLOTTIA.Cassava:
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Publishing,2002:1766.
[4] STi,U V,WXY,.2008Z[!UVž\S]^
:6î_¤
[J].[!JKLM,2009,(1):2122.
LUSQ,PANH,MACX,犲狋犪犾.Freezingsituationofcassa
vaandemergencyresponseatGuangxiin2008[J].犌狌犪狀犵狓犻
犜狉狅狆犻犮犪犾犃犵狉犻犮狌犾狋狌狉犲,2009,(1):2122.
[5] ARIELFD,MANAVELLAPA,DEZARCA,犲狋犪犾.The
truestoryoftheHDZipfamily[J].犜狉犲狀犱狊犻狀犘犾犪狀狋犛犮犻犲狀犮犲,
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[6] `ab,cïd,c%e.#$ HDZip)*f?78„³
[J].…f@$%?g,2009,31(4):514520.
QINYF,LIDD,LIXB.ProgressinHDZiptranscription
factorsofplant[J].犆犺犻狀犲狊犲犑狅狌狉狀犪犾狅犳犆犲犾犾犅犻狅犾狅犵狔,2009,
31(4):514520.
[7] h i,cjk,lÇm,.#$ HDZip)*f?i@$
%ց
[J].no,2013,35(10):11791188.
WANGH,LIGB,ZHANGDY,犲狋犪犾.Biologicalfunctions
ofHDZiptranscriptionfactors[J].犎犲狉犲犱犻狋犪狊,2013,35(10):
11791188.
[8] STEINDLERC,CARABELLIM,BORELLOU,犲狋犪犾.Phy
tochromeA,phytochromeBandotherphytochrome(s)regu
late犃犜犎犅2geneexpressioninetiolatedandgreen犃狉犪犫犻犱狅狆
狊犻狊plants[J].犘犾犪狀狋,犆犲犾犾牔犈狀狏犻狉狅狀犿犲狀狋,1997,20(6):759
763.
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