全 文 :!23" !3# ,
Vol.23 No.3
! " # $
ACTA AGRESTIA SINICA
2015$ 5%
May. 2015
犱狅犻:10.11733/j.issn.10070435.2015.03.021
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fXL\]
(犕犲犱犻犮犪犵狅狊犪狋犻狏犪)8ëìq犕犢犅22üPíé_ 犕狊犕犢犅2,犕狊犕犢犅22üä@÷834bp,ä1N·e_
278Aü2½IøÚJ。:;DNA~«
W|¶pBI121öO,nóÕ&+}2pBIMsMYB2。:;
Lu¾ð³ôKðâWmk¥^ñI}2üvTä6
,
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¥I2üéïØvTä2ü«8PçvP+}
,
nóôK犕狊犕犢犅22ü+}IvTä}2ü6。
89:
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XL\]
;犕狊犕犢犅22ü;vTä;};<,-=
:Q943.2 >?@AB:A >CD=:10070435(2015)03058006
犆犾狅狀犻狀犵犪狀犱犜狉犪狀狊犳狅狉犿犪狋犻狅狀狅犳犕狊犕犢犅2犌犲狀犲犳狉狅犿犕犲犱犻犮犪犵狅狊犪狋犻狏犪犔.
FENGZirong1,SUNYan1,CHAOYuehui2,YUANZhu1,LUJixiao1
(1.GrasslandResearchInstituteofChinaAgriculturalUniversity,Beijing100093,China;
2.TheColegeofForestry,BeijingForestryUniversity,Beijing100083,China)
犃犫狊狋狉犪犮狋:Agenenamed犕狊犕犢犅2wasclonedfromalfalfabyPCRtechniquewith834bpcodingdomainse
quenceencoding278aminoacids.The犕狊犕犢犅2genewasinsertedintoplasmidpBI121byDNArecombi
nationtechnology;andaplantexpressingvectorpBIMsMYB2wasconstructed.Theplantsoftransgenic
犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪withkanamycinresistancewerescreenedthroughfloraldipmethodandanalyzedby
PCRandRTPCR.TheresultsshowedthatthetargetgenewasinsertedintothegenomesofArabidopsis
andexpressed.Therefore,thetransgenicArabidopsisthalianaplantsexpressing犕狊犕犢犅2geneweresuc
cessfulyobtainedinthisstudy.
犓犲狔狑狅狉犱狊:犕犲犱犻犮犪犵狅狊犪狋犻狏犪L.;犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪;犕狊犕犢犅2gene;Transformation
EFGH
:20140521;IJGH:20140716
KLMN
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(1990),á,Èq§,úËÌÍñ,ÏÐ*]G;j`rñÍÌÍ,Email:pipishan@yeah.net;:6x× Authorfor
correspondence,Email:ctsoffice@163.com
MYB}ür&}ür¹^8Izà
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,
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、
D¥6Uäj®Ú¸ø
ÖID]
[13]。
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|n¯IMYBG",vJ_Ç¥MYB
ëÒ
(singleMYB)、2Aå MYBëÒ
(R2R3MYB)j 3 A å MYB ë Ò
(R1R2R3MYB)3A&a。MYBÑeé
I
,
A8â50~53Aü2½Ê
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&MòID,8â¥5(IHI
[4]。
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L.)8,I,vPD]J¶L&¥ñ&n
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DNAIëÒ,iýØ ABAID][56]。g
¢
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L.)[7]、Ê]Ð(犃狀狋犻狉狉犺犻狀狌犿犿犪犼狌狊L.)&[8]、
ZJLÐ
(犘犲狋狌狀犻犪犺狔犫狉犻犱犪Vilm)&[9]、vTä
(犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪)[1011]j41_Ð(犘犺狔
狊犮狅犿犻狋狉犲犾犾犪狆犪狋犲狀狊)&[12]8,。
& MYB2}ür¹^8I¥A4&
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。
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!3# r~:XL\]犕狊犕犢犅22üIëìQ}HoerenFU[13]IÌÍ+,犃狋犕犢犅2:;f
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6;D]<ñ&4#
。
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,MYB2}ür#ø~ÌÍ。
2008$,Yu[20],犕犢犅22üfòo÷O!"J
\n{I;S8~Vx¤
,
Y{NëÒj|
ýå?2üâ>I.µD!ür
。2011$,3¸´
[21]
:;<Æû
(犛犪犮犮犺犪狉狌犿狊犻狀犲狀狊犻狊Roxb)MYB2}
ürIÌÍ+
,
Æû犕犢犅22ü÷_991bp,
|8Y¯570bpÛÓ>Pä189Aü2½,Y
¯ MYBIóô。:;?ñJK,ÆûI
犕犢犅22ü¶|ý&ß[hfuæ«n、Ñy
j!ñSÝÖðâvÑI¬Ùñ
。
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L\]8IóôÏç
,
?íîPXL\]_þ
â
,
ëìq 犕狊犕犢犅22ü,öOØ+}2pBI121
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1.1 ST
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(犕犲犱犻犮犪犵狅犛犪狋犻狏犪
‘ZhongmuNo.1’)` r_8¸®÷-./À)*
+,ÌÍn]÷ÌÍÞéî
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²RNA¦í)®、À
ÁÂDNAªÓí)®、Àq4¦í)®_
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;PCRMixQ"ð
âUltraSYBRMixture_Ì_ØÙñ&-â@
r
;DNA Marker、犜犪狇 DNA l£、ëì2
pEASYT1_ÀõÊñ&-â@r;
DNaseA、.}í)®、T4DNAöO£、)`@O
ñåK£_TaKaRar;àcýòDH5α,
_ÀÈñ&-â@r
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éî
;
|ýí)v°¤5µJ©6¸ÍJK²
。
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1.2.1 ÂrµF H˸¸1ñ&
6789
(NationalCenterofBiotechnologyInformation,NCBI)
¦RI}M\]犕犢犅22ü(GenBankÏU:XM
_003591311)uæ,¼¥
MYB2r¤Þ2üëìQ+}2Õ;H&+
}2pBI121Iuæ,¼¥<:¤d&,pBIfj
pBIr,ð2uæß+1nÅ。|8^-¶I_@Oñ
åK£犡犫犪Ij犛犿犪II£KSÝ。
q1 ùº¨©
Table1 Thesequencesofprimers
é9
Name
uæ
Sequence
MYB2f5’CCCTCTAGAATGGATACTAATTACAAAACC3’
MYB2r5’GGGCCCGGGTAAATCATCAGCCAATTGTTGC3’
1.2.2 ÃÄÅÆ 犕犢犅2ºÇ@ÈÉ £¤²
RNA¦í)®¦XL\]² RNA,:;
RNaseAìü2ü«DNAEð¢,.}_!1
JcDNA。P!1JcDNA _´x,MYB2fj
MYB2r_d&,5W PCR ù。ùSu_:
95℃añ10min;95℃ñ30s,55℃[i30s,
75℃?ß1min,30A1;75℃?ß5min;4℃
yéî
。PCRÍ&í0.8%üÀÁÂö,
£¤ÀÁªÓí)®²\¢
,
öOØëì2
pEASYT1,}\àcýò+ÍDH5α,
f¯â50mg·L-1WÕk¥ILB"«2,37℃
"«;Ï
,
<Çëìc
,
5Wc2PCRö,S
u_
:95℃añ3min;95℃ñ30s,55℃[i
30s,72℃?ß90s,30A1;72℃?ß5min;4℃
yéî
。
WñëìØÀÈ¥>µñ&
â@ru
。
1.2.3 krPÊÓz{ £¤DNA ManÉO<
ua5WJK
,
Pa|ä@Sò
。
£¤
DNA Man j MEGA 5.1 É O < ë ì q I
犕狊犕犢犅22ü5WJK+|?ñ,PÕ5
\。
f ExPaSyPE (http://web.expasy.org/
compute_pi/)aÝjJr"。:;SignalP
4.1Server(http://www.cbs.dtu.dk/services/
SignalP/)a|6UÚFG。£¤PlantPLocR
ð
(http://www.csbio.sjtu.edu.cn/bioinf/plant/)
a|&+ÍSFG
。
1.2.4 Jrs¸$@Ë+ p¯âl
犕狊犕犢犅2uæIàcc6,¦Àq。P¯â
¥ I 2 ü I pEASYMYB2 j & + } 2
pBI121_<Ä,¤ 犡犫犪Ij犛犿犪I5Wó£K,
37℃.2h。£KÍ&í;ÀÁÂö¢,:
185
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;ÀÁªÓí)®²\¥I.ù
,
¤T4DNA
öO£16℃öO6h,P}\Øàcýò
+Í8
,
Pf¯â50mg·L-1WÕk¥ILB
"«2
。
Yc
,
PpBIfjpBIr
_d&
,
5Wc2PCRö。pPCRöñ
c6
,
¦Àq
,
:;犛犿犪Ij犡犫犪I@OñåK£
5W£K@
。
1.2.5 ÌÍÎ{,FÏA £¤Àq4¦í)®
¦pBIMsMYB2Àq,¤CaCl2Zê³}\Ö³
¬®c GV31018。<}\I®c5W
PCRöJK,£¤LuH\³,<ËÞL#I
BñbvTä5WË5
,
ð2zÊ_
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¤éOÁ2I
¯âpBIMYB2&+}2ÀqI®ccn
<éí·qñLuIvTä65W¾ð
,
ÉH
\2min,îâ:vé)3e, =Ë51d¢
~®ÓòاRxÿR8"«
。
Óô}2üvTä`r
,
í75%I~Ï¡10
min¢,vyÝfC¯â50mg·L-1Wmk¥1/2
MS"«2,Ô`r,pôÛlñIv
Tä}%CYB8°"«
。
pðâWmk¥^
ñvTä2.
,
¦2ü« DNA,P2ü« DNA
_´x
,
£¤ MYB2fj MYB2r_d&,5W
PCRö;pñvTä}2ü&,¦²
RNA,.}_cDNA ¢,PcDNA _´x,£¤
MYB2fj MYB2r_d&,5WPCRö。PCR
Í&í1%üÀÁÂJK。
2 mnU,c
2.1 öö÷ø犕犢犅2Koª«
PX L \ ] cDNA _ ´ x,P MYB2f,
MYB2r_d&,5WPCRù,PCRÍ&í
üÀÁÂÅnóôK1NÉ_800bpà4
IDNA NU(1)。W.ùöOØëì2
pEASYT1P}\àcýò+Í,íc2
PCRö,p¯â¥INUIÇëìc2ñ&
ru
,
uaÅ
:
nóôK¯âï
犕狊犕犢犅22üä@,ÿD2ß,/âñ%ä
ß
,
WulIÀqíé_pEASYMsMYB2。
2.2 Ѻ}~,,c
ëìKØI2ü÷_834bp,|¯â1A
ïIä@
,
ä1N·e_278Aü2½IøÚ
J
。
<ôKI MsMYB2ü2½uæ5Wñ&67
<1 öö÷ø犕狊犕犢犅2K犘犆犚æ
Fig.1 PCRamplicationof犕狊犕犢犅2genefrom
犕犲犱犻犮犪犵狅狊犪狋犻狏犪
(Note):M:Trans2KTMPlusⅡDNA{Á;M:Trans2KTMPlusⅡ
DNAMarker;1:犕狊犕犢犅22ü;1:犕狊犕犢犅2gene
.JK+
,
ÏXL\]8JqI MsMYB2¶
Ï|ý&`8JqI MYBëÒ,ðâSÑI
?ñj¬ÙIé@
(
2、3)。fExPaSyPE
(http://web.expasy.org/compute_pi/)a
ÝjJr"
,
J*_
:5.90/31.88KDa。íSignalP
4.1Server(http://www.cbs.dtu.dk/services/
SignalP/)a,îf6UÚ(4)。&+ÍS
aÅ
,MsMYB2SÞ+Íë8。
2.3 ºqÑL/à
¦ÀqpEASYMsMYB2Àq,¤犡犫犪Ij
犛犿犪I5Wó£K,ªÓ.ù¢öOCpBI1212
,
}\àcDH5αýò,<Çcc2
PCR¢,íÀÁÂöKÀ:¯ â¥I~«rI
c2vôùq¶¥I2üà4¥IDNA NU
(
5)。¦PCRöñcnÀq,¤犡犫犪Ij
犛犿犪I5Wó£K,£KÍ&í1%üÀÁÂ
JKKÀ
:
f800bpËîf¥NDNANU,¶_¬
2üà4¥
(
6),+&+}2Õnó,
PJ*íé_pBIMsMYB2。
2.4 #ú)1»m
P2 ü « DNA _ ´ x,£ ¤ MYB2f j
MYB2r_d&,5WPCRö,PCRÍ&í
üÀÁÂJKKÀ
:
npI136ðâWm^ñI
vTä6
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|812AôÛùq¥I2üà4
DNANU,æd}2üjñ<àvôôK
DNANU(7);PcDNA _´x,£¤ MYB2f
j MYB2r_d&,5WPCRö,PCRÍ&í
üÀÁÂJKKÀ
:
npI126PCRö
ñ}2üvTä6
,
|88AôÛùq¥I2
üà4 DNA NU,æ|846ôùq¥I
DNANU(8),Pa+nóôK}
犕狊犕犢犅22üvTä}2ü6,æç 犕狊犕犢犅2
2üôÛf}2üvTä8nó+}
。
285
!3# r~:XL\]犕狊犕犢犅22üIëìQ}<2 犕狊犕犢犅2ÐÄÂUfº#ÐÄo¨©Á
Fig.2 ComparisonofaminoacididentitiesoftheMsMYB2proteinfromalfalfaandotherdifferentplants
<3 犕狊犕犢犅21#ÐÄÆ),c
Fig.3 PhylogeneticanalysesofMsMYB2andorthologs
<4 犕狊犕犢犅2ÐÄ}=ûüm
Fig.4 SignalpeptidepredictionofMsMYB2protein
<5 狆犅犐犕狊犕犢犅2Iýþc犘犆犚,c
Fig.5 PCRanalysisofEschherichiacoliharboringpBIMsMYB2
:M:Trans2KTMPlusⅡDNAMarker;1~9:pBIMsMYB2c2PCR
Note:M:Trans2KTMPlusⅡDNAMarker;
1~9:PCRamplicationofpBIMsMYB2
<6 狆犅犐犕狊犕犢犅2Àÿ!®
Fig.6 EnzymeanalysisofpBIMsMYB2
:M:Trans2KTMPlusⅡDNAMarker;
1:pBIMsMYB2ó£K
Note:M:Trans2KTMPlusⅡDNAMarker;
1:pBIMsMYB2digestedwith犡犫犪Iand犛犿犪I
3
f&I}D,8
,犕犢犅¹^_LH2üóô
IãR:¨JrýO¦R¥Av2I´b
,
õö
f×&jkc8óôS4
,
-fÛL&8犕犢犅2ü
I¹^´JàæçøF[1,2223]。
& MYB2}
ür¹^8I¥A4&^
,
&8犕犢犅2I
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Fig.7 PCRanalysisoftransgenicArabidopsisplants
:113:}2üvTä;CK:ñ<à;M:Trans2KTMPlusIIDNAMarker
Note:113:TransgenicArabidopsis;CK:negativecontrol;M:Trans2KTMPlusIIDNAMarker
<8 K#ú犚犜犘犆犚»m
Fig.8 RTPCRanalysesoftransgenicArabidopsisplants
:112:}2üvTä;M:Trans2KTMPlusIIDNAMarker
Note:112:TransgenicArabidopsis;
M:Trans2KTMPlusIIDNAMarker
?íînóÏXL\]8ëì犕狊犕犢犅22
ü
,
PÕ2
。
:;ñ&67.JK,
,
犕狊犕犢犅22üäëÒ¶|ý&ëÒðâSÑ
I?ñ
。
íSignalP4.1Server(http://www.
cbs.dtu.dk/services/SignalP/)a,îf6U
Ú
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<ÞÆûIÌÍPQ~¶
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<Þ&SV
(犃狊犻犪狋犻犮犮狅狋狋狅狀)IÌÍa¬ã。
&+ÍSaÅ
,MsMYB2SÞ+Íë8,
<¶~ÿ
[16]
<ÞvTäIÌÍa¬ã
。pBI
MsMYB2+}2IÕ;S8,:;PCR
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,
vPf¥I2üÛÓ>âøJ*d¬犡犫犪
Ⅰj犛犿犪Ⅰ£KSÝ,éöO;S8_¬ÖI
lñ
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üjñ<àvôôKDNANU,+nó
ô K } 犕狊犕犢犅2 2 ü v T ä 6,æ ç
犕狊犕犢犅22üôÛf}2üvTä8nó+}。
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>?
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!3# r~:XL\]犕狊犕犢犅22üIëìQ}theDNAbindingdomainofthemyboncoproteins[J].Molec
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