免费文献传递   相关文献

Identification of GGB Mutant Caused by CRISPR/Cas9 in Arabidopsis

CRISPR/Cas9介导靶向敲除拟南芥GGB基因突变体的鉴定



全 文 :书!"#$%&
,2016,36(5):0857-0864
犃犮狋犪犅狅狋.犅狅狉犲犪犾.犗犮犮犻犱犲狀狋.犛犻狀.
  !"#$:10004025(2016)05085708               犱狅犻:10.7606/j.issn.10004025.2016.05.0857
%&(
:20160117;)*&%+(:20160418
,-./

()*+%,-
(31470289);./012)34567+58.9:(XJGRI2015084)
0123

;<=
(1989-),>,?@AB567,CDEFGHIJ7$%56。Email:15299175640@163.com

KLMN

OPQ

RB

S
),
TUV

CDEF#$IJ7$%56
。Email:xiaodongliu75@aliyun.com
犆犚犐犛犘犚/犆犪狊93456789:;
犌犌犅,<=>?@AB
!"#
1,
$%&
2,
 (1,)*+1,, -1,,./1
(1./WXY% W%Z,./WXY%WX7$[\]^_`a,bcde830052;2./fgWX[\hijk,./l2m
841000)
C D:犌犌犅nopqrs,t。uvwxyz{犵犵犫|}~€,†CRISPR/Cas9,t‡ˆ‰Š~。‹<Œ†ˆ‰Š~ŽW‘’†“H”•–—7˜yz{。™•,t
Sš犌犌犅,t†›œžIŸ ¡,?¢£^¤¥¦§4¨©,ª«¬1¨T©,†2­|}~®7。I¯™
—7˜yz{ª°±2­犵犵犫|}~²³´µ¶RTPCRIŸž·¸,|}~€j¹º»›¼½犌犌犅,t
¾¿

ÀÁwxv犌犌犅,tÂÃ|}~。™—7˜ª犵犵犫|}~ÄŧÆÇ、Èp¾˜ÉÊË­J¶†›µž
¾Á

̗7˜ÍÎ

yz{犌犌犅,t|}S,ÄŧÆÇ·ÏÐÑ,opÒÁ·«Ó,ÔÊË­J¶ÕÖ×¥Ø
}

56¾Á
,犌犌犅nÙ­ÚۆM$IJÜ­†ÝÞ¢,t,wx†|}~ußSEWM$jàá†犌犌犅âã
,t²³äåæç`èéêv¥Ž†ëì€

EFG

yz{
;CRISPR/Cas9;犌犌犅;,tÂÃ;§ÆÇ
HIJK$
:Q781;Q785;Q786 !LMNO:A
犐犱犲狀狋犻犳犻犮犪狋犻狅狀狅犳犌犌犅犕狌狋犪狀狋犆犪狌狊犲犱犫狔犆犚犐犛犘犚/犆犪狊9犻狀犃狉犪犫犻犱狅狆狊犻狊
LEIJianfeng1,XUXinxia2,LIYue1,DAIPeihong1,LIUChao1,LIUXiaodong1
(1ColegeofAgronomy,XinjiangAgriculturalUniversity,LaboratoryofAgriculturalBiotechnologyofXinjiangAgriculturalU
niversity,Urumqi830052,China;2CenterofBazhouAgriculturalTechnologyPromotion,Korla,Xinjiang841000,China)
犃犫狊狋狉犪犮狋:犌犌犅isanegativeregulatorofdroughtresistance.Inordertoobtainthe犃狉犪犫犻犱狅狆狊犻狊犵犵犫mu
tant,weusedAtU6promotertodrivetheexpressionof犃狋犌犌犅sgRNAandcorespondingCRISPR/Cas9
genomeedtingvectorwasconstructedandwastransferredinto犃狉犪犫犻犱狅狆狊犻狊by犃犵狉狅犫犪犮狋犲狉犻狌犿mediated
floraldip.Aftersequencingof犌犌犅inT2generation,twokindsofmutantswithadeletionof4basesand
anadditionof1base(T)werefoundatthetargetsite,respectively.SemiquantitativeRTPCRanalysis
resultsshowedthattheexpressionof犌犌犅genewasalmostnotdetectedinthe犵犵犫mutant,whichindica
tedthatthemutantswere犌犌犅knockoutlines.Throughmeasuringthewaterlossrate,droughtresistant
andseedyieldperplant,犵犵犫mutantexhibiteddecreasedwaterlossrate,improveddroughtresistance,but
unchangedseedyieldperplantcomparedtowildtype.Takentogether,theaboveresultsindicatedthat
犌犌犅isanidealcandidatetargetgeneforcropmolecularbreedingand犵犵犫mutantisusefulforfunctional
complementionof犌犌犅homologousgenesclonedfromcrops.
犓犲狔狑狅狉犱狊:犃狉犪犫犻犱狅狆狊犻狊;CRISPR/Cas9;犌犌犅;geneknockout;waterlossrate
  íîïðñò–óôníîõö÷Sóô†Ù
­øù

ú~nûü15¨ý†”þ,ÿ20¨ý†
!Ä",#$%&½íîõý()S4¨*,+
CaaX†´,*+°,-jCn´,*+,a..n
/01*,+

ÔXK.n23*+、4*+、5*
"6

7*+ÿ´,*+
[1]。
:89:¥3­íî
;<=~>Ìvíîïðñò–†óô

íî”þ
,•?;
、Ⅰ˜íî!Ä",•?;ªⅡ˜íî!
Ä",•?;

íî”þ,•?;ªⅠ˜íî!Ä
",•?;@nAαªβB¨C,‡D,BN#Ž
âÙ¨αC,,EβC,¼â。?yz{jíî”
þ,•?;†βC,n犈犚犃1,ÔⅠ˜íî!Ä"
,•?;†βC,n犌犌犅
[1]。
56 ¡犈犚犃1 ™
F$íî†ý()S4¨*,+CaaX¦GHŒ
Ò

Ô犌犌犅¹ºIïðñò–óô)SÙ£nJ*
+†íîõ

Ký()S4¨*,+nCaaL†
íîõ

L-n™Mý(uCaIL†íîõ†ó
ôNÒ)O
[2]。
56 ¡íîïðñò–óôÌo
pPQR?S.TU†VW

yz{犲狉犪1|}~o
pÒÁ·«Ó
[34]。
*Ô犲狉犪1|}~X¡YIHZ
Ü

Ô[\H]^_h`
[5]。
Ì犲狉犪1 |}~ÍÎ,
yz{犵犵犫|}~a?bcVd°™ABAe¬f
g

hyz{i½jpkl]
,犌犌犅,t|}S,m
n#ËbcVd

opÒÁ·«Ó

)]D†n

犵犵犫|}~?7o Ü°Ì—7˜ÍÎ×¥Á·p
ï
[1],
¾ÁqråsïÒtóôN–v ABALu
Kvj†Ù¨VwqrstJ

²ÔqrsvABA
?bcVdj†äå

7$Lx%IŸ·¸?Æy

z{

GH

|}

~€

‚ƒM$j„¥1¨…††、Ìyz{
犌犌犅O‡âã†,t。ˆ‰âã,tnŠú¥Ì
yz{犌犌犅⋆äå,åŠr‚MuM$opÜ
­†Ù­ÚÛ¢Œ,t

ˆD²ÙŽˆ‰,
t†äå

Ô)‘’r“†è”•)Mˆ‰âã,
tnŠåäåæçyz{犵犵犫|}~†¾˜。*
ÔAMyz{犵犵犫|}~56&–]^—˜,ÍV
|}~†­J9™§š å›

tœIåK-ž
Ÿ •w¡

,t‡ˆ‰[\n.X¡†Ù­56,täå
†]D[\¢ú

qr‚O£®7sµ,täå§
N|}~

åu7$~äå,t‡%56éê¤õ
†ëì€

:8q¥¦3­[\~W:§û¨+
;
(Zincfingernucleases,ZFNs)、TALE ¨+;
(Transcriptionactivatorlikeeffectornucleases,
TALENs),‚É2013©ª«†CRISPR/Cas9[W¬
[6]。
ˆ‰­¢¨+;@r‚?,t‡DNAs
µ£^°®7®¯°±

)SKSâã(%&
ÿâã]‡B­ DNA ó<Ÿ •_¡™®¯
DNA°±^†ó<。?)*²³´,âã]‡ó<
nÙ­µ¶ó<·ù

ÔSâã(%&ó<¸n
Ù­¼µ¶ó<·ù

ó<µDS-®¯DNA°
±¤†©,_ 7¦§ÿ«¬ÿØ}

EԒ¹,
t‡œº†µ^ˆ‰
[7]。ZFNsª TALENsˆB
9[\?_»¼M½j[\¾‡—Y

<‡¿
]^—o

HÀ—O
[8]。
Á©•

ÂÃÄ).†
CRISPR/Cas9,t‡ˆ‰[\Å¡š。CRISPR/
Cas9W¬ÆÇnȐ?oɲ–½jøD†Ù
­ÊQÒËÌW¬
[910]。2013©,CRISPR/Cas9W
¬ÄØÍDÙ­.†,t‡µ^ˆ‰[[1112]。
ÎÏ?,t‡ DNA °ÞЍDØ͆,t¢£
^

Ñҁ<ÙÓÔ¥¢œº†sgDNA,*SÌ
犆犪狊9,tÙ«K¾¿Š~•Õi~ÈÖ。?•
×X†sgRNA †Ø’´,K©,æçٙ,‹
Cas9íî¢Ú=½,t‡DNA†™Q£Û°,
Cas9ÜÝ:ŒDNAœº®7®¯°±,Ô°±†
DNAÄSâã(%&·ùót‡¢:Œ£^†ˆ‰

ß[\¼M͙àÊ

á
MDÇâ

:89ã?ä­#$$­

å|}

æç

Æy

èéªz{ƒ

jDätÂÃ:Œ,t
[6,1314]。
Ç56‹ŽCRISPR/Cas9,t‡ˆ‰[\,µ^
ÂÃ犌犌犅,t,wx.†yz{犵犵犫|}~,֛ê
-opÒª­J®¶

u`è-žM$犌犌犅âã,
t†äåéêæç•,t†i~€

1 €ª·”
1.1 PQRS
AtU626::sgRNAŠ~[15]、GV3101Wg
i³ÈÖ

#$¾¿Š~p1300„u./WXY%
WX7$[\]^_`aëR

yz{犆犪狊9¾¿Š
~
[15]
Aj+%Z°ì7í+%56Z#$7Ú
7³56Åîï

Ø$=Dɛœ„A°ìðñ7
$[\¥òóôµD

1.2 TU
1.2.1 狊犵犚犖犃 VWX?YZ   ,M 犃狋犌犌犅
(At2g39550),t‡œº,õö犆犪狊9÷?¢Ú£
^

ø”¢£^†£ÛªGCù¶ÑÒsgRNA(¾
1)。¢œºKúû:95℃ü5sýâ0.5℃þ
20℃,ÿÕ½ã犅犫狊Ⅰ;܆AtU626::sgRNAŠ
858 ! " # $ % &                   36!
V1 [\]H^_@`abc
Table1 Primersequencesusedinthisstudy
Ø$"#
Primer
ϼ
(5′→3′)Sequence
$ÚForward PÚReverse
ÀÁPurpose
犌犌犅2C GATTGTCTCTGCAGGAGAATTCTA AAACTAGAATTCTCCTGCAGAGAC 犌犌犅¢œºÑÒThedesignof犌犌犅targetsequence
犵犵犫2test ACTCACTACCGGAGAACGA TCAGAATAGCCAAAGCACAG 犵犵犫
|}£^»› Detectionof犵犵犫muta
tion
犪犮狋犻狀2 GCACCCTGTTCTTCTTACCG AACCCTCGTAGATTGGCACA r%cDNA &(‡ AdjusttheconcentrationofcDNAtemplate
犃狋犌犌犅 AATCGTCTTACACTCGCTCAT TTGGCTTGTTAGTCCTACCTT 犃狋犌犌犅,t¾¿¶pﻛ Detectionof犃狋犌犌犅geneexpression
)

´*+u犈犮狅RⅠ;Ü£^
Note:Underlinedsequencesare犈犮狅RⅠrestrictionenzymesite
,1 sgRNAªCas9#¾¿Š~
Fig.1 ConstructionofsgRNAandCas9expressionvector
~ÍQ†£Û°
,T4 %&;%&-,•–犜狉犪狀狊1
T1 gi³ÈÖ,.¡Êàᛜ/µÔ¥犌犌犅¢
œº†sgRNA¾¿Š~。
1.2.2 defghX?YZ 0Ž;Ü%&†·
ù<sgRNA ª犆犪狊9#¾¿Š~,Ž 犓狆狀Ⅰª
犎犻狀dⅢ®;Ü AtU626::sgRNA¾¿Š~,12
AtU626::sgRNAÅÓ(600bp),%&â‹ã犓狆狀
Ⅰª犎犻狀dⅢ;܆犆犪狊9¾¿Š~,•–犜狉犪狀狊1
T1 gi³ÈÖ,;Ü/µ†õ3í"u AtU6::
sgRNACas9(,1)。*S‹sgRNAª犆犪狊9#¾
¿Š~犈犮狅RⅠ;Ü AtU6::sgRNACas9õ3,12:Œ
ÅÓ
(5.9kb),%&â‹ã犓狆狀Ⅰª犈犮狅RⅠ;܆
#$¾¿Š~p1300õ3。•–犜狉犪狀狊1T1 gi³
ÈÖ

Ýކ4Òàáã;Ü/µ$S

¡10μL
AtU6::sgRNACas9p1300õ3•ÕWgi
³GV3101,28℃5Û672d,.¡4ÒàáMLB
67,

ù50μg·mL
-1Kanª25μg·mL
-1Rif)
67þ™87oÉ

ŽM´ÙŽ9:

1.2.3 9:;ijgh ‹<Œ†Ô¥犃狋犌犌犅
¢œº† AtU6::sgRNACas9p1300W;R
<10μL&­MLB67,(ù50μg·mL
-1 Kan
ª25μg·mL
-1Rif)jN–。28℃,180r/min=
>6720h?@。*SA1∶100~B΋N–
<&­½15mLLB67,j,M28℃,180r/min
=>67

Cþ<† OD600≈1.8~2.0],4000
×gDk10min2E~,FG°H<,]IM.
JÙᆕ–<
(1/2MS<~67,ù5%KL,
0.02%SilwetL77),þÞ(‡ OD600≈0.6~0.8。
0Ž“H”
[16]
•–yz{Col0,M­JDNS2
wT1 š­J,jO7d。‹T1 š­JPQ¤Ú,R
­?ù¥30μg·mL
-1
STU†1/2MS67,
°
,4℃VÛ72h,*S25℃(16hW67,8hX6
7

YÞ4ÒZ

Ö¬Ò4ÒZ#Ë8¶

1.2.4 9:;犵犵犫=>?kl ‚•,tyz{
,t‡DNAu&,Ž Primer5.0[\ÑÒy
z{犵犵犫|}£^»›Ø$(¾1)。PCR]«犃狋
犌犌犅,t,‚—7˜yz{u™^。PCRPQ_u
:94℃`}Ò3min;94℃}Ò30s,60℃ úû
30s,72 ℃ab45s,35¨cdSM72 ℃ab
10min,Ž1%e/Lfg²³hi»›。™—7
˜ª|}~PCR®$²³犈犮狅RⅠ;Ü/µ,37℃
3h,™×¥P–†PCR®$²³12,%&½j(
Š~BZero(犜狉犪狀狊犵犲狀)°,.Êàᛜ,›œ
žÌ犃狋犌犌犅,t†>kœº²³œºÎ™IŸ。
)S

¬Ò犵犵犫|}~?Å¥•–Jj†|}


1.2.5 犃狋犌犌犅,PlantRNA(犜狉犪狀狊犵犲狀)é¡êmn¼MŽoé¡Z
pu20d†—7˜Ì|}~#ËÄņRNA,*
9585É          ;<=,ƒ:CRISPR/Cas9‘’¢ÚÂÃyz{犌犌犅,t|}~†/µ
SŽP•×êmn
(TransScriptⅡ FirstStrand
cDNASynthesisSuperMix,犜狉犪狀狊犵犲狀)=DcDNA。
‚cDNAu&,犃犮狋犻狀2,tuq>™^。0Ž
犈犪狊狔犜犪狇;(犜狉犪狀狊犵犲狀)]«,PCRPQ_\:94℃
`}Ò3min;94℃}Ò30s,60℃úû30s,72℃
ab30s,27¨cd;72℃ab10min。A^r%
q>,t†&¶

²³犃狋犌犌犅 ,t¢£^:Œ
Åӆ]«
。PCRPQ_\:94℃`}Ò3min;
94℃}Ò30s,60℃úû30s,72℃ab30s,35¨
cd
;72℃ab10min。)SŽ1%e/Lfgh
i»›PCR®$。Å¥Ø$r¾1。
1.2.6 9:;m?nopqrlB 0Ž#]
”
[17]
Η—7˜yz{ª犵犵犫|}~#ËD~Ä
Å͙ùƶpï

s¡Zpu30d†—7˜yz
{ª犵犵犫|}~#Ët°Y,I¯Ž#¶t²³#
]

üu1h#¶-J]›µ-§ÆÇ,ÑÛ3¨
]<

ÄŧÆÇ
(%)=(Ævõ¶w-]^^§
ÆSõ¶
)/
Ævõ¶×100%。8”%ÚÌIŸ0
ŽExcel2013[\;0Ž DPSv7.05[\†LSD
)è·Ïp8”²³pï·ÏÒ»`

1.2.7 stVuABvwxyz{lB ‹Zp
u20d†—7˜ª犵犵犫|}~#˲³jp‚É
jpS<ƤÚ

x^yפÚ8S¾˜pï

Ö¬
Ò#ËRNÇ

z{|¤Ú†â}o~Ù¹†—7
˜ª|}~#Ë­JDNS

0Ž#]”›µÊË
­J¶

ÑÛ3¨]<,6|}~­J¶nŠÌ—
7˜­J¶R?pï

2 žÌIŸ
2.1 犃狋犌犌犅=>|}~
?yz{犃狋犌犌犅 ,t‡œº536bp¤Ù¨
PAM(theprotospaceradjacentmotif)£^8,ÑÒ
v2_ou24bp、Ô¥犈犮狅RⅠ;Ü£^†¢œº
DNA(GATTGTCTCTGCAGGAGAATTCTA ª
AAACTAGAATTCTCCTGCAGAGAC),ߢœ
ºãúû<ÒÿÕ½ã犅犫狊Ⅰ;܆ AtU626::
sgRNAŠ~°(,2)。
2.2 犆犚犐犛犘犚/犆犪狊9,<#€X?YZ
‹<Œ†Ô¥犌犌犅 ,t¢œº† AtU6::
sgRNACas9†]‡,tÅÓ(5.9kb)%Õ#$¾
¿Š~p1300,ã;Ü/µ,;ܝžÌ`É;ÜÅ
ÓYè̀

¾Á¢ÚÂÃyz{犌犌犅 ,t†
CRISPR/Cas9,t‡ˆ‰Š~92.3 犃狋犵犵犫=>?kl
Ž30μg·mL
-1
STUYÞ T1 ­J,#YÞ
X45 ¨ T1 š•,tËW。uv`è<†
CRISPR/Cas9,tˆ‰~WnŠú¥¢£Q,å
Š?`ÉÑ҆¢£^¤®7DNAœº†ˆ‰,
™—7˜ª•,t T2 š#Ë犌犌犅 ,t¢œº²
³vIŸ

ž·¸—7˜†PCR®$ã犈犮狅RⅠ
;ÜS_®7528bpª208bpB_Ô。Ô?•,
t#Ëj垢œº|} 7?犈犮狅RⅠ;Ü£^
¤

‚_’¹犈犮狅RⅠ;Ü£^ă„,EÔ犈犮狅R
Ⅰ…”†¯ª;Ü:Œ£^,)Þë‡v736bp†Æ
M.1kbDNAŒ)
,3 AtU6::sgRNACas9p1300,tˆ‰Š~†;Ü/µ
M.1kbDNAmarker
Fig.3 IdentificationofAtU6::sgRNACas9p1300gene
editingvectorbyrestrictionenzymedigestion
ˆ‰Šœº¾¸yz{U6ƒ„J;‹‰ŠYI¾¸yz{犌犌犅,tsgDNAœº;´ *+¾¸犌犌犅,t¢œº。
,2 yz{U6ƒ„JÉ犌犌犅sgDNAœº
The犃狉犪犫犻犱狅狆狊犻狊U6promoterishighlightedinlightshadow;DarkshadingindicatesthesgDNA
scaffoldfor犌犌犅;犌犌犅targetsequenceisunderlined
Fig.2 The犃狉犪犫犻犱狅狆狊犻狊U6promoterand犌犌犅sgDNAscaffoldsequence
068 ! " # $ % &                   36!
M.1kbDNA;A.0ŽPCR/RE†·”»›|}~(1ª2i–
I¯u4ҙ^—7˜yz{PCR;ÜS®$ªPCR;Ü8®
$hi,
;3i–u犵犵犫|}~PCR]«®$;4~10i–I¯u
犵犵犫|}~PCR;ÜS®$);B.ŒÙ³u—7˜yz{犌犌犅,
tϼ

Žœº¾¸Ô¥犈犮狅RⅠ;Ü£^†¢œº,Žœº¾
¸PAM£^(NGG),‘M¾¸|}~,©,†ÿՎ‘+’u¾
¸

©,†¦§Ž
‘’u¾¸;C.—7˜犌犌犅›œ=,;D.|}
~犌犌犅;4›œ=,;E.|}~犌犌犅,+T›œ=,
,4 yz{犵犵犫|}~»›
M:1kbDNAMaker;A.PCR/REassayswereperformedtodetect
mutations(Lanes1and2representundigestedanddigestedPCR
productofwildtype犃狉犪犫犻犱狅狆狊犻狊respectively;Lanes3represent
PCRamplificationproductofthe犵犵犫mutant;Lanes4-10represent
digestedPCRproductsofthe犵犵犫mutations);B.Thewildtype犌犌犅
sequenceisgivenattheabove,thetargetsequenceswith犈犮狅RⅠ
restrictionsitehighlightedinblueandthePAM(NGG)highlighted
inred.Mrepresentsmutation,‘+’representsinsertionbases,‘’
representsdeletionbases;C.TheSangersequencingresultforwild
type犌犌犅;D.TheSangersequencingresultfor犌犌犅:4;E.The
Sangersequencingresultfor犌犌犅:+T
Fig.4 Detectionof犃狉犪犫犻犱狅狆狊犻狊犵犵犫mutants
PCR:†_Ô(,4,A)。™×¥Ä犈犮狅RⅠÜ°†
PCR®$›œ。›œž¾Á:?¢£^¤»›½
2­åØ«’@“Ø}†|}~,Ù­u¦§4¨
©,†|}

í"u犌犌犅:4;zÙ­n«¬1¨T
©,†|}

í"u犌犌犅:+T(,4,B)。Ì-ž”
˜|}~Ù«²³¬ÒIŸ

ž ¡ 7œºØ
}†|}#ËΕu84.4%(38/45)。
2.4 犃狋犌犌犅,uv²ÙŽ»›|}~j犃狋犌犌犅,t?•×
Æj°†¾¿}–

0Ž´µ¶RTPCR·”IŸ
v—7˜Ì|}~#Ëj犃狋犌犌犅†¾¿Æj。
ž

¾5)·¸,—7˜€j犌犌犅,t¾¿Æj—
O

Ô2­|}~€j¹º»›¼½犌犌犅 †
¾¿

2.5 犃狋犵犵犫=>?pqrlB
jpn’¹#$rŽÆI–¦†CDtU

Ô§ÆÇn#ËnŠÈp†]D7ÚûŒ
[18]。
8
­56ž¾Áyz{犵犵犫,tTDNAÿ՗=
|}~D~ÄŧÆÇ·ÏâM—7˜yz{
[1]。
Ç56›µv.犵犵犫|}~#ËD~§ÆÇ,ž
·¸

Ìyz{犵犵犫,tTDNAÿ՗=|}~
†¾˜Í˜
,犵犵犫|}~§ÆǙ·ÏâM—7˜
yz{

,6)。
2.6 犃狋犵犵犫=>?t‚VuAB
jp¤Ú8

—7˜yz{ª犵犵犫|}~#Ë
7o Ü,ÇÙ¹

ˆÌ8­
[1]
56žÍâ

j
p¤Ú23dS,—7˜#Ë\v 7š›,|}~
,5 犃狋犌犌犅,t¾¿¶IŸ
Fig.5 Expressionanalysisof犌犌犅in犃狉犪犫犻犱狅狆狊犻狊
,6 yz{ÄŧÆǛµ
Fig.6 Thewaterlossrateof犃狉犪犫犻犱狅狆狊犻狊leaves
1685É          ;<=,ƒ:CRISPR/Cas9‘’¢ÚÂÃyz{犌犌犅,t|}~†/µ
¼â蜐‘¾¸¼â¤ÚR?·Ïpï
(犘 <0.05)
,7 <ÆSRNǛµ
Fig.7 Determinationofsurvivalrateafterrehydration
Differentlettersindicatesignificancedifferencesamong
differenttreatmentsforthesameparameter(犘<0.05)
,9 yz{ÊË­J¶›µ
Fig.9 Determinationofseedweightperplant
in犃狉犪犫犻犱狅狆狊犻狊
#ËÖ×¥ 7š›

ž¦Æ]^†ao

—7
˜#ËÂÃjŸ ¡

Ô|}~#˾˜Í™—¢

jp¤Ú28dS²³£¤<Æ,#ËRNǛµ
ž·¸

—7˜€AM§Æ‡Ô ¡

|}~
€<ÆSYä8å$.7o
,犌犌犅:4|}~RN
Çu86.67%,犌犌犅:+T|}~u51.11%(,7),
¾˜°Ìâ}¤Ú†—7˜€pï·Ï

,8)。
2.7 犃狋犵犵犫=>?wxyz{lB
ÊË­J®¶n¥$#Ë7®å›†]Dû
Œ

¾¿†$rs,tK._Ø«,t¦§ÿN
tu¿Ô’¹®¶ÐÑ
[1920]。犵犵犫opÒÁ
·«Ó
,¨
*7o Ü×¥i½Š©

EÖ¼š¾­
J®¶×¥}–

K›µDNS犵犵犫|}~Ì
—7˜yz{ÊË­J¶

ž·¸
,犵犵犫|}~#
ËÊË­J¶Ì—7˜Ö×¥Á·pï

2w†­
J¶,ÇÙ¹

ÀÁyz{犌犌犅,t|}S,-­
J®¶Ö×¥i½ªá

,9)。
3 « ¬
  jpnòá#$7o†ÎDS7$kltU,
쬆ܭ­Ó®¾¯°ˆ¨±²

0Ž•,t·
”

¶¾¿o³$rs,tn:8)ã´¥£

™
A.jp¤Ú8;B.jp¤ÚS;C.<ÆS
,8 jp¤Ú8Syz{†¾˜µ¶
A.Beforethedroughttreatment;B.Afterthedrought
treatment;C.Rehydration
Fig.8 Observationphenotypeof犃狉犪犫犻犱狅狆狊犻狊
beforeandafterdroughtstresstreatments
n)·¸nŽ†o³Ü­Ÿ 

*Ô?_¹½j
ÕX¡vºä±²

ÎÏ»¾¿†,t

AM#ª
᡼

..’¹,t¦§

ÍVÒ²¼åoɽµ
ëì
[19];
-¾AMˆ‰,t¾¿

ã.nM
$$.†7o Üi½ªá

’¹®¶ÐÑ
[20]。
K
nnŽ¿’˜ƒ„J•…„o³,t†¾¿

E,
t¦§’¹†ëì½µÒ±²ÀÁ*R?

)]D
†nqÂÅ6ÜX†.í@Ô¥•,tDI

-
Åԕ†7$Ķұ²ÅYtòávM$.í
†ÆÇhiªQŽ

?#$~qÃv:8·¸nŽ†o³$rs,
t{

ÀR?ىqrs,t

ˆ‰,t|}

äå
268 ! " # $ % &                   36!
™§S

#$†opҙYY«Ó

å犈犚犃1[3]、
犃犅犎1[21]、犛犃犇1[22]、犌犌犅[1]ª 犇犛犜[23]ƒ,- j
犈犚犃1ª犌犌犅n>Ìíîõïðñò–óô†B
¨]D,t

ˆ­#$óôm²víîõÌȂÉ
íîÌíîG^†æM
[24]。
56 ¡犲狉犪1†|}
~?bcVd°™ ABAe¬fg,-opÒÁ·
«Ó
[34]。
œ{?Éʪèé°犈犚犃1†|}@¾
¡Xˌ†opÒ
[2526]。
z{?ÌÍ
[2728]
ª|
}
[24,29]
j™/µX°±3­>Ìvíîïðñò
–†íî<=~

ˆ¾Áíîïðñò–óôrå
?#$jO‡ëÎ

Ì犲狉犪1|}~ÍÎ,犵犵犫|}
~opҙÁ·«Ó

E?7o Ü°Ì—7˜Í
ÎÖ×¥Á·pï
[1],
tœ犌犌犅rånM$o³I
JÜ­†Ù­ÚÛ,t¢Œ

uv·Ï‘’t`èGH

|}

~€

‚

Æy

z{ƒM$jÌyz{犌犌犅O‡âã,t†
äå

Ç56Ž CRISPR/Cas9,t‡ˆ‰[

²³v¢ÚÂÃyz{犌犌犅,t†56。ž
·¸

?45˕,t4Ò#Ëj,X¡犌犌犅,tœ
ºØ}†¥38Ë,T1 š†|}£ÇO¿84.8%,
·¸Xß,tˆ‰[\†O£Ò

E38Ë T1 š
jYÞwxv2­¥£†犌犌犅,tÂÃ|}~,Ù
­¦§4¨©,,zÙ­n«¬1¨ T©,,-
ž@n®7,t?Ð|}

)ޒ¹ä嵶™§

ä妧†,t

-mRNA†•×ÿ½µÒ_R?
Ñ]¦Ò

’¹mRNAÆjÅâ。™.wx†犵犵犫
|}~²³†´µ¶RTPCR»›ž¾Á,|}
~€j™¹º»›¼½犌犌犅,t†¾¿,ž¾
Á.wx†yz{|}~n犌犌犅,tÂÃ|}~。
²ÙŽ†§ÆǪop҆›µ™Ì犌犌犅,tT
DNAÿÕ|}~†»›žÙ¹。犌犌犅MuÙ¨
opqrs,tDێMWM$IJܭ

‚DÓ
q†SšÍVҲ彵ëì

7o Ü¼_i½
ªá

®¶¼ÐÑ

yz{犵犵犫|}~#ËÊË­
J¶›µž·¸
,犵犵犫|}~7o Ü,Ç̗
7˜Ù¹

2w†­J¶™Ì—7˜€…·Ïp
ï


,犌犌犅,tŽMM$IJÜ­,r噼
_Š©WM$†7o Ü

’¹®¶ÐÑ

z{$
åCRISPR/Cas9[\ÆÚű,Žß[\W¬
r‚·ÏtwxÔÕ¢,t|}†#$€

*S
KaaٚÖ×ÿ)ׂr‚‘Øü•,tDI
Ì|}¢,tID,
EÔwx…•,tDI†|
}.€

Ô[|},tÉ-ÍQÒ²™åÙÚ|
}~ً½µëì
[30]。
۰ű

Ç56ŽCRISPR/Cas9,t‡
ˆ‰[\wxyz{犵犵犫|}~,²ÙŽ`èv
犌犌犅rånÙ­ÚۆM$IJÜ­†¢,t,u
IŸ-qM$犌犌犅âã,t†äåéêvÚۆ
æç•,ti~€

ƒ„!L

[1] JOHNSONCD,CHARYSN,CHERNOFFEA,犲狋犪犾.Pro
teingeranylgeranyltransferaseIisinvolvedinspecificaspects
ofabscisicacidandauxinsignalingin犃狉犪犫犻犱狅狆狊犻狊[J].犘犾犪狀狋
犘犺狔狊犻狅犾狅犵狔,2005,139(2):722733.
[2] ANDREWSM,HUIZINGADH,CROWELLDN.TheCaaX
specificitiesof犃狉犪犫犻犱狅狆狊犻狊proteinprenyltransferasesexplain
era1andggbphenotypes[J].犅犕犆犘犾犪狀狋犅犻狅犾狅犵狔,2010,10
(1):111.
[3] PEIZM,GHASSEMIAN M,KWAKC M,犲狋犪犾.Roleof
farnesyltransferaseinABAregulationofguardcelanionchan
nelsandplantwaterloss[J].犛犮犻犲狀犮犲,1998,282(5387):287
290.
[4] CUTLERS,GHASSEMIANM.Aproteinfarnesyltransfer
aseinvolvedinABAsignaltransductionin犃狉犪犫犻犱狅狆狊犻狊[J].
犛犮犻犲狀犮犲犕犪犵犪狕犻狀犲,1996,273(5279):12391241.
[5] YALOVSKYS,KULUKIANA,YoungCA,犲狋犪犾.Func
tionalrequirementofplantfarnesyltransferaseduringdevelop
mentin犃狉犪犫犻犱狅狆狊犻狊[J].犜犺犲犘犾犪狀狋犆犲犾犾,2000,12(8):1267
1278.
[6] CHENK,GAOC.Targetedgenomemodificationtechnologies
andtheirapplicationsincropimprovements[J].犘犪狀狋犆犲犾犾犚犲
狆狅狉狋狊,2014,33(4):575583.
[7] ZHOU H,LIUB,WEEKSDP,犲狋犪犾.Largechromosomal
deletionsand heritablesmal geneticchangesinduced by
CRISPR/Cas9inrice[J].犖狌犮犾犲犻犮犃犮犻犱狊犚犲狊犲犪狉犮犺,2015,42
(17):1090310914.
[8] SHANQ,WANGY,LIJ,犲狋犪犾.Genomeeditinginriceand
wheatusingtheCRISPR/Cassystem[J].犖犪狋狌狉犲犘狉狅狋狅犮狅犾狊,
2014,9(10):23952410.
[9] BHAYAD,DAVISON M,BARRANGOUR.CRISPRCas
systemsinbacteriaandarchaea:versatilesmalRNAsfora
daptivedefenseandregulation[J].犃狀狀狌犪犾犚犲狏犻犲狑犌犲狀犲狋犻犮狊,
2011,45(1):273297.
[10] WIEDENHEFTB,STERNBERGS H,DOUDNAJA.
RNAguidedgeneticsilencingsystemsinbacteriaandar
3685É          ;<=,ƒ:CRISPR/Cas9‘’¢ÚÂÃyz{犌犌犅,t|}~†/µ
chaea[J].犖犪狋狌狉犲,2012,482(7385):331338.
[11] CONGL,RANFA,COXD,犲狋犪犾.Multiplexgenomeengi
neeringusingCRISPR/Cassystems[J].犛犮犻犲狀犮犲,2013,339
(6121):819823.
[12] MALIP,YANGLH,ESVELTK M,犲狋犪犾.RNAguided
humangenomeengineeringviaCas9[J].犛犮犻犲狀犮犲,2013,339
(6121):823826.
[13] LIANGZ,ZHANGK,CHENK,犲狋犪犾.TargetedMutagen
esisin犣犲犪犿犪狔susingTALENsandtheCRISPR/CasSys
tem[J].犑狅狌狉狀犪犾狅犳犌犲狀犲狋犻犮狊犪狀犱犌犲狀狅犿犻犮狊,2014,41(2):
6368.
[14] CHRISTOPHERB,VLADIMIRN,ZACHARYB,犲狋犪犾.
Efficientgeneeditingintomatointhefirstgenerationusing
theclusteredregularlyinterspacedshortpalindromicre
peats/CRISPRAssociated9system[J].犘犾犪狀狋犘犺狔狊犻狅犾狅犵狔,
2014,166(3):12921297.
[15] FENGZY,ZHANGB,DING W,犲狋犪犾.Efficientgenome
editinginplantsusingaCRISPR/Cassystem[J].犆犲犾犾犚犲
狊犲犪狉犮犺,2013,23(10):12291232.
[16] CLOUGHSJ,BENTAF.Floraldip:asimplifiedmethod
forAgrobacteriummediatedtransformationof犃狉犪犫犻犱狅狆狊犻狊
狋犺犪犾犻犪狀犪[J].犘犾犪狀狋犑狅狌狉狀犪犾,1998,16(6):735743.
[17] ZHOUS,HU W,DENGX,犲狋犪犾.Overexpressionofthe
wheataquaporingene,TaAQP7,enhancesdroughttoler
anceintransgenictobacco[J].犘犾狅犛犗狀犲,2012,7(12):
e52439.
[18] YUEY,ZHANG M,ZHANGJ,犲狋犪犾.Overexpressionof
theAtLOS5geneincreasedabscisicacidlevelanddrought
toleranceintransgeniccotton[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,2012,63(10):37413748.
[19] GELVINSB.Theintroductionandexpressionoftransgenes
inplants[J].犆狌狉狉犲狀狋犗狆犻狀犻狅狀犻狀犅犻狅狋犲犮犺狀狅犾狅犵狔,1998,9
(2):227232.
[20] YANGSJ,VANDERBELDB,WANJX,犲狋犪犾.Narrowing
downthetargets:towardssuccessfulgeneticengineering
ofdroughttolerantcrops[J].犕狅犾犲犮狌犾犪狉犘犾犪狀狋,2010,3(3):
469490.
[21] HUGOUVIEUXV,KWAKJM,SCHROEDERJI.An
mRNAcapbindingprotein,ABH1,modulatesearlyabscisic
acidsignaltransductionin犃狉犪犫犻犱狅狆狊犻狊[J].犆犲犾犾,2001,106
(4):477487.
[22] HUGOUVIEUXV,MURATAY,YOUNGJJ,犲狋犪犾.Lo
calization,ionchannelregulation,andgeneticinteractions
duringabscisicacidsignalingofthenuclearmRNAcapbind
ingprotein,ABH1[J].犘犾犪狀狋犘犺狔狊犻狅犾狅犵狔,2002,130(3):
12761287.
[23] HUANGXY,CHAODY,GAOJP,犲狋犪犾.Apreviously
unknownzincfingerprotein,DST,regulatesdroughtand
salttoleranceinriceviastomatalaperturecontrol[J].犌犲狀犲狊
犪狀犱犇犲狏犲犾狅狆犿犲狀狋,2009,23(15):18051817.
[24] CROWELLDN,HUIZINGADH.Proteinisoprenylation:
thefatofthematter[J].犜狉犲狀犱狊犻狀犘犾犪狀狋犛犮犻犲狀犮犲,2009,14
(3):163170.
[25] WANGY,YINGJ,KUZMAM,犲狋犪犾.Moleculartailoringof
farnesylationforplantdroughttoleranceandyieldprotection
[J].犜犺犲犘犾犪狀狋犑狅狌狉狀犪犾,2005,43(3):413424.
[26] MANMATHANH,SHANERD,SNELLINGJ,犲狋犪犾.Vi
rusinducedgenesilencingof犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪 gene
homologuesinwheatidentifiesgenesconferringimproved
droughttolerance[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,
2013,64(5):13811392.
[27] QIAND,ZHOUD,JUR,犲狋犪犾.Proteinfarnesyltransferase
inplants:molecularcharacterizationandinvolvementincel
cyclecontrol[J].犘犾犪狀狋犆犲犾犾,1996,8(12):23812394.
[28] YANGZB,CRAMERCL,WATSONJC.Proteinfarne
syltransferaseinplantsMolecularcloningandexpressionof
ahomologofthebetasubunitfromthegardenpea[J].
犘犾犪狀狋犘犺狔狊犻狅犾狅犵狔,1993,101(2):667674.
[29] YALOVSKYS,LORAINEAE,GRUISSEM W.Specific
prenylationoftomatoRabproteinsbygeranylgeranyltypeII
transferaserequiresaconservedcysteinecysteinemotif[J].
犘犾犪狀狋犘犺狔狊犻狅犾狅犵狔,1996,110(4):13491359.
[30] FENGZY,MAOY,XUN,犲狋犪犾.Multigenerationanalysis
revealstheinheritance,specificity,andpatternsofCRISPR/
Casinducedgenemodificationsin犃狉犪犫犻犱狅狆狊犻狊[J].犘狉狅犮犲犲犱
犻狀犵狊狅犳狋犺犲犖犪狋犻狅狀犪犾犃犮犪犱犲犿狔狅犳犛犮犻犲狀犮犲狅犳狋犺犲犝狀犻狋犲犱
犛狋犪狋犲狊狅犳犃犿犲狉犻犮犪,2014,111(12):46324637.

!"

#$%
)  
468 ! " # $ % &                   36!