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Protoplast Isolation from Elephant Grass(Pennisetum purpureum ‘Huanan’)for Transient Gene Expression

华南象草原生质体分离及基因瞬时表达研究



全 文 :!23" !3#
 Vol.23  No.3
! " # $
ACTA AGRESTIA SINICA
   2015$  5%
  May.  2015
犱狅犻:10.11733/j.issn.10070435.2015.03.020
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:Q943.2;S543.9    >?@AB:A     >CD=:10070435(2015)03057109
犘狉狅狋狅狆犾犪狊狋犐狊狅犾犪狋犻狅狀犳狉狅犿犈犾犲狆犺犪狀狋犌狉犪狊狊(犘犲狀狀犻狊犲狋狌犿狆狌狉狆狌狉犲狌犿
‘犎狌犪狀犪狀’)犳狅狉犜狉犪狀狊犻犲狀狋犌犲狀犲犈狓狆狉犲狊狊犻狅狀
TANGRan,PENGXiaoqun,XIEXinming
(ColegeofAgriculture,SouthChinaAgriculturalUniversity,Guangzhou,GuangdongProvince510642,China)
犃犫狊狋狉犪犮狋:Elephantgrass(犘犲狀狀犻狊犲狋狌犿狆狌狉狆狌狉犲狌犿)isakindoftypicalC4clonalplant,whichgrowswel
insouthChina.Itiscommonlyusedasforagegrass,greenenergygrassaswelasnonwoodfiber.Young
leafsheathsofelephantgrasswereusedformesophylprotoplastsisolationinthisstudy.5factorsofcelu
laseconcentration,macerozymeconcentration,pH,mannitolconcentration,enzymolysistimewerestud
iedtooptimizeisolationconditionsbasedonL16(45)orthogonaltest.Andtheisolatedelephantgrasspro
toplastswereusedforgenetransientexpressionassays,aimingtolaythefoundationforgenefunctionala
nalysisinelephantgrass.Theresultsshowedthattheoptimumconditionsforelephantgrassmesophyl
protoplastsisolationwereintheenzymecombinationscontaining1.5%celulase,0.75% macerozyme,0.5
mol·L-1mannitol,pH5.8.Thedigestionwasconductedinthedarkunder28℃for4hwithgentlesha
king.Undertheseconditions,theprotoplastsyieldwas5.11×106cels·gFW-1(freshweight),andthe
vitalitywas91.08%.Varianceanalysisoforthogonaltestindicatedthattheenzymolysistimeisthemost
importantfactorinfluencingbothprotoplastyieldandvitality.Twoplasmidvectors,carriedgreenandred
fluorescentproteinrespectively,werethentransformedintopurifiedmesophylprotoplastsbyPEGmedia
tedmethod.Thegreenandredfluorescentsignalweredetectedinthetransienttransformedprotoplasts,
obtainedthehighesttransformationefficiencyof77.47%,andcotransformationefficiencyof8.79%.The
isolatedprotoplastscouldbeusedforgenetransientassayinelephantgrass.
犓犲狔狑狅狉犱狊:犘犲狀狀犻狊犲狋狌犿狆狌狉狆狌狉犲狌犿;Protoplast;PEGmediated;Transientexpression
EFGH
:20140606;IJGH:20140827
KLMN
:¸
¹_ö-.2Ê
(31272491)(30972138);Ü;UÑ.¥ÊË.-Ýß¿-Ì2Ê(20124404110011)ÀÁ
OPQR


(1988),Å,«Tf›§,ÊËÌÍñ,mVÏÐ*]ñ&„…ÌÍ,Email:tangran@stu.scau.edu.cn;:6x×Author
forcorrespondence,Email:xiexmbs@scau.edu.cn
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Fig.1 Isolationof犘.狆狌狉狆狌狉犲狌犿leafsheathprotoplasts
•
:A:ñ·10dIÄ]Ë̄…,—–6äzÅz©ûñÀ2J‚IUS(tuÐ:3cm);B:£ÝáH\Þ0.6mol·L-1Æfê8
I2L+ù
;C:£Ý¢TUI£n§&;D:ûñÀ2Iœ„Mò(×40)
Note:A:Healthy10dayoldelephantgrassseeding,redarrowsindicatetheoptimalpartforprotoplastisolation(Scalebar:3cm);
B:Cutstripsofleafsheathsareimmersedin0.6mol·L-1mannitolbeforeenzymaticdigestion;C:Enzymaticmixtureisturbidafterdigestion;
D:Microphotographofprotoplasts(×40)
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Table1 Orthogonaltestofprotoplastisolationfrom犘.狆狌狉狆狌狉犲狌犿
Ë5U
TreatmentNo.
M(¥£ R10
Celulase
R10(W/V)/%
‚K£R10
Macerozyme
R10(W/V)/%
pH
DÆfê
DMannitol
/mol·L-1
£ÝŽh
Digestion
time/h
1 1.0 0.25 5.2 0.3 2
2 1.0 0.50 5.5 0.4 4
3 1.0 0.75 5.8 0.5 6
4 1.0 1.00 6.0 0.6 8
5 1.5 0.25 5.5 0.5 8
6 1.5 0.50 5.2 0.6 6
7 1.5 0.75 6.0 0.3 4
8 1.5 1.00 5.8 0.4 2
9 2.0 0.25 5.8 0.6 4
10 2.0 0.50 6.0 0.5 2
11 2.0 0.75 5.2 0.4 8
12 2.0 1.00 5.5 0.3 6
13 2.5 0.25 6.0 0.4 6
14 2.5 0.50 5.8 0.3 8
15 2.5 0.75 5.5 0.6 2
16 2.5 1.00 5.2 0.5 4
1.3 ÎÑÂ/oó)
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Table2 Theresultsoforthogonaltestonprotoplast
isolationfrom犘.狆狌狉狆狌狉犲狌犿leafsheaths
Ë5U
TreatmentNo.
ûñÀ2Í"
Protoplastyield/
×106cels·gFW-1
ûñÀ2!.
Protoplast
viability/%
1 0.43±0.06hH 94.90±1.71aA 
2 4.09±0.10bAB 91.27±0.80abAB
3 3.44±0.47bBC 84.86±2.37bcdefBC
4 2.72±0.25cCD 81.18±3.04cdefCD
5 1.92±0.24defDEF 81.07±1.89cdefCD
6 2.50±0.42cdCDE 83.19±1.93cdefBCD
7 4.87±0.12aA 88.48±1.49abcABC
8 1.84±0.22defDEFG 87.95±1.62abcdABC
9 3.75±0.17bB 87.34±1.45bcdeABC
10 1.27±0.24efgFGH 85.91±1.98bcdefABC
11 1.51±0.12efgEFG 80.65±2.30defgCD
12 1.98±0.06deDEF 78.46±1.49fgCD
13 1.21±0.21fgFGH 79.92±4.76efgCD
14 1.18±0.34fgFGH 73.61±2.55gD
15 0.85±0.06ghGH 85.66±2.83bcdefABC
16 3.90±0.17bB 80.65±0.74defgCD
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wx+őAü¥)ÉuhãR}9œ‹Éu
(犘<0.01)。Œ
Note:Aldataarethemeanvalueofeachtreatment±standard
error.Differentlowercaselettersdenotesignificantdifference(犘<
0.05)ineachcolumn;Differentcapitallettersdenotehighlysignifi
cantdifference(犘<0.01)ineachcolumn.Thesameasbelow
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(犘<0.01)。‚K£‡e、pH、
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u

Éu1)Ž,ûñÀ2!.vðzÑ,!›;Ñ
I£‡ej£ÝŽh‹£ÞûñÀ2!.Ié
)

݉2vÀ,£Ý2hŽ,+ÍÛ.S‡(‰
8q–&À
),
-"#£ÝŽhIùú

+ÍÛ.
œ…ùø

£Ý6hj8hI+ÍÛ.¬_›œ。
pHjÆfêI)ÉuãR

zcIpH ðjÆfê‡eJ*_6.0j
0.4mol·L-1。
q3 €klÁï*ÎÑÂ/Òå1rÓÔÕoqå,c
Table3 Intuitiveanalysesofeffectofdifferenttreatmentsonyieldandviabilityin犘.狆狌狉狆狌狉犲狌犿protoplasts
Éu
Level
M(¥£ R10
CelulaseR10(W/V)
‚K£R10
MacerozymeR10(W/V)
pH
DÆfê
DMannitol
£ÝŽh
Digestiontime
K1/×106cels·gFW-1 2.668±0.111aA 1.827±0.070cB 2.086±0.141bA 2.118±0.065bB 1.099±0.107dC
K2/×106cels·gFW-1 2.782±0.156aA 2.261±0.069bAB 2.211±0.087abA 2.163±0.057bAB 4.152±0.085aA
K3/×106cels·gFW-1 2.131±0.032bB 2.669±0.157aA 2.553±0.056aA 2.633±0.130aAB 2.284±0.187bB
K4/×106cels·gFW-1 1.787±0.183cB 2.611±0.093aA 2.517±0.141aA 2.455±0.087abA 1.832±0.134cB
犚犽/×106cels·gFW-1 0.995 0.842 0.457 0.515 3.053
L1/% 88.052±1.206aA  85.807±0.381aA  84.847±0.660aA 83.862±1.145aA 88.605±0.374aA 
L2/% 85.172±0.340abAB 83.496±1.167abA 84.115±0.383aA 84.945±0.711aA 86.932±0.727aA
L3/% 83.089±0.257bcBC 84.909±1.137abA 83.437±0.439aA 83.122±0.805aA 81.607±2.093bB
L4/% 79.957±1.978cC 82.058±0.791bA 83.872±2.775aA 84.342±1.929aA 79.127±1.331bB
犚犔/% 8.095 3.749 1.410 2.823 9.478
  •:K+ÅûñÀ2Í"f)ü¥‹ŒË5ÉuIvð;L+ÅûñÀ2!.f)ü¥‹ŒÉuIvð。犚犓 j犚犔 J*+ÅÍ"j
!.I9ã
Note:Kmeanvalueofprotoplastyieldatdifferentlevelsineachfactor;Lmeanvalueofprotoplastviabilityatdifferentlevelsineachfac
tor;犚犓and犚犔representrangesofyieldandviability,respectively
<2 €À.ñòÁï*ÎÑÂ/rÓoÔÕ
Fig.2 Effectsofdifferentenzymolysistimeson犘.狆狌狉狆狌狉犲狌犿protoplastsisolation(stainedbyevansblue)
•
:A:£Ý2h(Ë51,40×);B:£Ý4h(Ë52,40×);C:£Ý6h(Ë53,40×);D:£Ý6h(Ë54,40×)
Note:A:2henzymaticdigestion(TreatmentNo.1,40×);B:4henzymaticdigestion(TreatmentNo.2,40×);
C:6henzymaticdigestion(TreatmentNo.3,40×);D:8henzymaticdigestion(TreatmentNo.4,40×)
2.1.2 ½²³´Y¹@|qz{ Ý+4vÀ,‹
Œü¥<Ä]ûñÀ2Í"IÕÖîfœ‹ãR

|8

£ÝŽh

M(¥£j‚K£IÕÖ}Ø9œ
‹Éu
(犘<0.01),Æfêj£npH IÕÖ}Ø
œ‹Éu
(犘<0.05)。fíîË5ÉuIp×LM
å

£ÝŽh˜½ˆÄ]2LûñÀ2J‚Í"I
zmVÕÖür

M(¥£j‚K£~g

_mVÕ
Öür

æÆfêjpH <ûñÀ2J‚Í"IÕ
Ö¬
ÖãJK+›£ÝŽhjM(¥£<ûñÀ
2!.IÕÖ}Ø9œ‹Éu
(犘<0.01),æ‚K
£

ÆfêjpH <ûñÀ2!.IÕ֋œ‹。
575
! " # $ !23"
f?íî8£ÝŽh˜½ˆÄ]2LûñÀ2J
‚!.IzmVÕÖür

M(¥£˜mVÕÖ
ür

‚K£

ÆfêjpH <ûñÀ2!.xy
/âÕÖ

€0†JKjÖãJK€a

P

â!.Iû
ñÀ2Í"

_³~ïz{

vÀÄ]ûñÀ2J
‚IzcNO_

M(¥£1.5%,‚K£0.75%,£
npH5.8,Æfê0.5mol·L-1,£Ý4h。’àº
pqIzcÖ³5WÄ]ûñÀ2J‚

Í"v}
5.11×106 A·gFW-1(‰1D),!ñ}91.08%。
q4 €klÁï*ÎÑÂ/Òå1rÓÔÕoVã,c
Table4 Varianceanalysisofeffectofdifferenttreatmentsonyieldandviabilityin犘.狆狌狉狆狌狉犲狌犿protoplasts
—R:?
Source
uÖj
犛犛
_Ýe
犇犉

Meansquare
犉ð
犉Value
犘ð
犘Value
M(¥£
Celulase
7.833 3 2.611 15.324 0.000
‚K£
Macerozyme
5.418 3 1.806 10.598 0.000
Í" Yield pH 1.899 3 0.633 3.716 0.021
Æfê
Mannitol
2.157 3 0.719 4.220 0.013
£ÝŽh
Digestiontime
60.990 3 20.330 119.312 0.000
M(¥£
Celulase
419.488 3 139.829 9.120 0.000
‚K£
Macerozyme
97.195 3 32.398 2.113 0.118
!. Viability pH 12.563 3 4.188 0.273 0.844
Æfê
Mannitol
21.367 3 7.122 0.465 0.709
£ÝŽh
Digestiontime
711.176 3 237.059 15.461 0.000
2.2 PEGR“oï*ÎÑÂ/­)
W1AUâ mCherry—–ô=ëÒI”2
pUTL2mCherry(É10μg):; PEGpŸ³}
\5Ä]ûñÀ28<Ž+}

fD=lŸœ
„`ôÛöØçÜI—–ô=ëÒ6U

}
\æ}77.47%(+5,‰3A)。ŒŽ,W1AU
âEGFPñ–ô=ëÒI”2 pAN580EGFP

É10μg)P¬ŒIÖ³}\ØÄ]ûñÀ2
8

Pî{}\>æI~¡ñ

-˜}\¢Iñ
–ô=6Uçôf‡GIûñÀ28öØ

}
\æçâ11.24%(+5,‰3B)。!›íînJ
‚IÄ]ûñÀ2vP¤Þ2üI<Ž+}Ì
Í



‹ŒI”2<ûñÀ2I}\>æ
‹Œ

q5 狆犝犜犔2犿犆犺犲狉狉狔狆犃犖580犌犉犘Áï*ÎÑÂ/;o­)1N
Table5 TransformationefficiencyofpUTL2mCherryandpAN580GFPto犘.狆狌狉狆狌狉犲狌犿protoplasts
”2
Vector
œ„`€B
Thefieldof
microscope
ûñÀ2AG
Thenumberof
protoplasts/cels
’ô=IûñÀ2G
Thenumberoffluorescent
protoplasts/cels
}\>æ
Transformation
efficiency/%
1 42 34
pUTL2mCherry 2 36 24 77.47±5.51aA
3 46 39
1 24 3
pAN580GFP 2 36 5 11.24±2.00bB
3 41 3
1 19 2
pUTL2mCherry+pAN580GFP 2 46 4 8.79±0.98bB
3 42 3
675
!3# p ö:XTÄ]ûñÀ2J‚Q2ü<Ž+}ÌÍ
<3 狆犝犜犔2犿犆犺犲狉狉狔狆犃犖580犌犉犘Â$¶ï*ÎÑÂ/;oðñqÑ
Fig.3 TransientexpressionofpUTL2mCherryandpAN580GFPin犘.狆狌狉狆狌狉犲狌犿protoplasts
•
:A:pUTL2mCherryI<Ž+};B:pAN580EGFPI<Ž+};C:pUTL2mCherryjpAN580EGFPI+};
Cherry:—–ô=ëÒ6U;GFP:ñ–ô=ëÒ6U;Bright:›;Merged:ènâ‰.I`ú
Note:A:TransientexpressionofpUTL2mCherry;B:TransientexpressionofpAN580EGFP;
C:CoexpressionofpUTL2mCherryandpAN580EGFP;Cherry:Signalofredfluorescentprotein;
GFP:Signalofgreenfluorescentprotein;Bright:Brightfield;Merged:Overlappingofalleftphotos
  <pUTL2mCherryjpAN580EGFP5WŒ
Ž},2A”2f}\ŽI‡e¬,15μg。
íö’,

ñ–ô=6Uj—–ô=6UvPf
Ä]ûñÀ2I+ÍÀj+Ív8öØ

‰3
C),}\æ8.79%(+5),!›Ä]ûñÀ2#
vP¤Þ2üI}\ÌÍ

-˜}\>æSƒ

3 œ
3.1 ï*Ë1ÎÑÂ/,•/0o‡Ÿ
£n«j£ÝŽh˜ÕÖûñÀ2Í"j!
ñI·­ü¥
[29,32]。
¥zø_

£n‡eÑ

£Ý
ŽhH

æ£n‡eƒ

£ÝŽh­¬­?·


z‹ôˆ;24h[3334]。¥á,¤Þž&ûñÀ2J
‚I£â

M(¥£

‚K£

aÁ£

gh£jll
ü£
[31,3435]。
?ÌÍp×I£n«_M(¥£
j‚K£

ü_f2hIûñÀ2J‚8

M(¥£
j‚K£I«z_šª
[2,5,10,27],
-ÝދŒJ‚
þâ+ÍN€›j«nIãR

£I‡ej£ÝŽ
h#‹Œ
[2728,31]。
z©XTÄ]2LûñÀ2J
‚IM(¥£j‚K£‡eJ*_ 1.5% j
0.75%,£ÝŽh_4h,¶“8¸¡”4E(犜狉犻狋犻犮狌犿
犪犲狊狋犻狏狌犿 ‘Chinesespring’)ûñÀ2J‚I£¤"
j£ÝŽhOè
(1.5%M(¥£,0.70%‚K£,£
Ý5h)[24]。擳3”[h(犣犲犪犿犪狔狊‘Zong3’)û
ñÀ2f1.5%M(¥£,0.5%‚K£,£Ý7hI
FGJ‚zc
[24],
!›£I¤"j£ÝŽhf‹
Œ&`IãRvà

üNgC

<`ãRi2,f
‹Œ2übgh
[32]。
ß

²?¨

É¿
(犗.狊犪狋犻狏犪
‘Nipponbare’)IzcJ‚NO_:2%M(¥£,
0.7%‚K£,£Ý7h[24],æ“8L11”É¿(犗.狊犪
狋犻狏犪‘Zhonghua11’)f1.5%M(¥£,0.75%‚


£Ý5hIFGzc[5]。{M(¥£j‚
K£‡e;Ñ

Ä]ûñÀ2Í"j!.

£
ݎh˜Ä]ûñÀ2Í"j!.IzmVÕÖü
¥

£ÝŽh;H

ûñÀ2‹ô”ÃJ‚

I·Ô
Íñà"+ÍÛ.

Ž~ÕÖûñÀ2Í"Q!.

á§ÌÍ+›£n‡e;Ñ

6ûñÀ2·ŽhH
\Þ£n8

Ô£+åÎ&’q

+ÍzÛ

ÕÖû
775
! " # $ !23"
ñÀ2Í"
[29,3334]。
nP

_‰J‚ÑÀ"Iûñ
À2

µÑP

zà@eƒ£n‡e

P|"ŸH
£ÝŽh

_û°

0†JKjÖãJKv+›

Æfê‡ejpH
<Ä]ûñÀ2J‚>aIÕÖ¬
-˜‹ô
‹ÆfêjpHf()£Ýn[EojûñÀÀ
v:Eñ’Ž’”Ix¤
[28,34]。
àøGûñÀ2ù
ôf0.29~0.9mol·L-1IÆfê8î!,Pf0.55
~0.6mol·L-1IÆfê8é)SÑ!ñ[3233]。l
õíî€a+›

eÆfê‡e0.5mol·L-1Ž,Ä
]ûñÀ2Í"zÑ

¶0.6mol·L-1ãR‹›œ,
!›Ä]ûñÀ2f0.5~0.6mol·L-1Æfê8ù
ôé)SÑÍ"

ÛÄ]ûñÀ2!.æw

Æfê
‡eLMf0.3~0.6mol·L-1ghùô£|()S
Ñ!ñ

ü_Æfê)‡eÉuhÿœ‹ãR

á§
Ì͒,

P2.J‚IûñÀ2Iz©Æfê‡e
šËÞ0.4~0.6mol·L-1gh,¶?Ì̀a¥Š。
ßvTä
(犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪)2.ûñÀ2J‚
Iz©Æfê‡e_0.4mol·L-1[9],VL(犌狅狊狊狔狆犻
狌犿犺犻狉狊狌狋狌犿)0.5mol·L-1[27],4E0.4mol·L-1[24],
\]
(犕犲犱犻犮犪犵狅狊犪狋犻狏犪)0.55mol·L-1[28],[h0.4~
0.6mol·L-1[2,24],É¿0.4~0.6mol·L-1[5,10,24]。
pH;Ñ6;ƒÔ£Àvp\Éu÷Ñ,4—+̀
›

Ïæz{ûñÀ2MòIé)
[28]。
£ÝnpH¥
zP5.6~6.0_ª[9]。epH=5.8Ž,Ä]IûñÀ
2Í"j!.zÑ

NC

þâ:?QaË5

£Ýn8IiC*ú
)°±<ûñÀ2IJ‚>a#⥈ÕÖ

ß
þâf£Ýáí;X

ƒy6Ñ[EoønHË5

ôÛâ>4¦ÑûñÀ2J‚>æ
[5,3233]。
iâÌ͒,f£Ýn8*ú©eIž&D¥

ß
ÏÐ3û½(indole3aceticacid,IAA)、jk¥
(gibberelin,GA)ô‡5ûñÀ2J‚,*úl
ûÝlmÓZ
(polyvinylpyrrolidone,PVP)ô*·
ûñÀ2.[9],
<‘°±vPfš¢Ä]ûñÀ
2J‚íî85Wní

3.2 xtï*ÎÑÂ/ÆÇK‡ðñqÑ
PEGpŸIž&ûñÀ2I<Ž}\>æý
ø`ü¥ÕÖ

ßPEG‡e、PEG`a(PEG4000
6PEG6000)、Àqà4Q²e、ûñÀ2G"

[6,10,24]。
¥zø_PEGI‡e|Ñ,x¤Žh|
·

ûñÀ2}\>æ|Ñ
[27]。


ÝÞPEG<
ûñÀ2⥈¡Dx¤

nP}\S„MIûñ
À2Ž

­Š©eƒ PEG ‡e,P†‡x¤Ž
h
[9]。
?ÌÍJàÇr2ž&2.ûñÀ2}\Ö
³

£¤40%PEG4000,x¤15min,ôKIzÑ
}\æ}77.47%。
á§ÌÍ+›

½ˆ<Ž}\æI·­˜Àq
‡ejûñÀG"Itu
[2],
çŠtuf‹Œ&`
8ãRSà
[9,27]。
eûñÀ2G"¥ˆŽÿ

Àq
|4

}\>æ|Ñ

PÉ¿ûñÀ2_u
,2.7kb
IÀq}\>æ90%P†,4.5kbIÀq}\>æ
_75%,eÀqàÞ12kb,}\>æŽ~ƒ,p
Þ25%j30%gh[5,10,36]。Í¿W2Aà4¬Œ

É5kb),‡e¬(É10μg),U⋌ô=ëÒ
I”2J*}\52×106 AÄ]ûñÀ28,€a
’,
,pUTL2mCherryI}\>æ_77.47%,æ
pAN580EGFPI}\>æçâ11.24%。!›ü
‰Àq‡ejà4C

ÀqI`a<<Ž}\>æ
vô#âSàÕÖ

-ð2ýOâÔ5¥zÌÍ

Ä]ûñÀ2J‚Í"zÑv}5.11×106
A
·gFW-1,fG"†vP2Ü2ü<Ž}\IV
y

fz}\íî{›Ä]ûñÀ2v¤Þ2ü<
Ž+}ÌÍ

-}\>æiâ¦ÑIÑh

H*˜
}\>æ

f?ÌÍ8çâ8.79%。_‰¬2I£
¤Ä]ûñÀ2ÌÍ2üóô

­Š³Í§ÕÖ
}\>æI)`ü¥

ü‰PEG‡e、PEGx¤Ž
h

ûñÀ2G"

Àq‡e¶à4PC

i­ŠÍ
§ûñÀ2c;nI[Eo
、pH、c;Žhü¥
<}\>æIÕÖ
[9,27,33]。
4 m
£ÝŽh˜Ä]ûñÀ2J‚IzmVÕÖü
r

æpHjÆfê‡e<Ä]ûñÀ2J‚IÕ
ÖS4

eM(¥‡e_1.5%,‚K£‡e_
0.75%,pH_5.8,Æfê‡e_0.5mol·L-1,
£ÝŽh_4hŽ,J‚IûñÀ2Í"}5.11×
106 A·gFW-1,!.}91.08%。Àq`avô
ÕÖ#ûñÀ2I<Ž}\>æ

Ä]ûñÀ2v
¤Þ2üI}\ÌÍ

žŸ>?
[1] HisanoH,NandakumarR,WangZY.Geneticmodification
ofligninbiosynthesisforimprovedbiofuelproduction[J].In
Vitro&DevelopmentalBiologyPlant,2009,45(3):306313
[2] CaoJ,YaoD,LinF,犲狋犪犾.PEGmediatedtransientgeneex
875
!3# p ö:XTÄ]ûñÀ2J‚Q2ü<Ž+}ÌÍ
pressionandsilencingsysteminmaizemesophylprotoplasts:
Avaluabletoolforsignaltransductionstudyinmaize[J].Ac
taPhysiologiaePlantarum,2014,36(5):12711281
[3] MarionJ,BachL,BelecY,犲狋犪犾.Systematicanalysisofpro
teinsubcelularlocalizationandinteractionusinghighthrough
puttransienttransformationof犃狉犪犫犻犱狅狆狊犻狊seedlings [J].
ThePlantJournal,2008,56(1):169179
[4] k,Oo,ÂfÌ,.É¿犗狊03犵190202üIëìQ&+
͈SJK
[J].‚„®÷à..¸,2014,45(1):2933
[5] YangJW,FuJX,LiJ,犲狋犪犾.Anovelcoimmunoprecipitati
onprotocolbasedonprotoplasttransientgeneexpressionfor
studyingproteinproteininteractionsinrice[J].PlantMolecu
larBologyReporter,2014,32(1):153161
[6] SheenJ.Signaltransductioninmaizeand犃狉犪犫犻犱狅狆狊犻狊meso
phylprotoplasts[J].PlantPhysiology,2001,127(4):1466
1475
[7] DaveyMR,AnthonyP,PowerJB,犲狋犪犾.Plantprotoplasts:
Statusandbiotechnologicalperspectives[J].Biotechnology
Advances,2005,23(2):131171
[8] §º,èä´.ž&ûñÀ2IOÁ¶!.öÌÍ5“[J].
ñ&„…:¸
,2009(S1):4650
[9] ­Ží.vTäûñÀ2J‚QÉ¿ž½£2ü犗狊犕犐犖犘犘
<Ž+}ÌÍ
[D].·,:ZT®÷à.,2011
[10]ZhangY,SuJ,DuanS,犲狋犪犾.Ahighlyefficientricegreen
tissueprotoplastsystemfortransientgeneexpressionand
studyinglight/chloroplastrelatedprocesses[J].PlantMeth
ods,2011,7(1):30
[11]CockingEC.Amethodfortheisolationofplantprotoplasts
andvacuoles[J].NatureProtocols,1960(187):962963
[12]YooSD,ChoYH,SheenJ.Arabidopsismesophylproto
plasts:Aversatilecelsystemfortransientgeneexpressiona
nalysis[J].NatureProtocols,2007,2(7):15651572
[13]UekiS,LacroixB,KrichevskyA,犲狋犪犾.Functionaltransient
genetictransformationof犃狉犪犫犻犱狅狆狊犻狊leavesbybiolisticbom
bardment[J].NatureProtocols,2009,4(1):7177
[14]ReichTJ,IyerVN,MikiBL.Efficienttransformationofal
falfaprotoplastsbytheintranuclearmicroinjectionofTiplas
mids[J].NatureBiotechnology,1986,4(11):10011004
[15]‰pp,–1,–ÓÑ,.ëÒÀJrô=¶ÌÍQ|­¤
®5“
[J].JK\.,2006,34(9):13411347
[16].[µ.Pgfp_¸I2üÌÍëÒ&+͈S[J].ñ&„
…:¸
,2006(2):1113
[17]VermeerJEM,VanMunsterEB,VischerNO,犲狋犪犾.Pro
bingplasmamembranemicrodomainsincowpeaprotoplastsu
singlapidatedGFPfusionproteinsandmultimodeFRETmi
croscopy[J].JournalofMicroscopy,2004,214(2):190200
[18]AliGS,PrasadKVSK,HanumappaM,犲狋犪犾.Analysesof
invivointeractionandmobilityoftwospliceosomalproteinsu
singFRAPandBiFC[J].PLoSOne,2008,3(4):e1953
[19]§™,3[Ø,ÔÏn.\]ùÔ«¶ûñÀ2–‚¶"«
[J].]4.¸,2010,18(2):258262
[20]Ä?,3[Ø,§™,.J];ùÔ«¶ûñÀ2J‚NOI
ÌÍ
[J].]4.¸,2011,19(2):288293
[21]Ý®›,Šœ,­e²,.ø$ñô?j]IÍôjñò>J
[J].ñò.¸,2008,28(5):23292342
[22]3â-,pö,Ý®›.XTÄ]J6›Oñ·9|ñ&"J
{I¬·ñ
[J].ñò. ¯,2011,30(9):18751880
[23]TangR,ZhangXQ,LiYH,犲狋犪犾.Cloningand犻狀狊犻犾犻犮狅a
nalysisofacinnamylalcoholdehydrogenasegenein犘犲狀狀犻狊犲
狋狌犿狆狌狉狆狌狉犲狌犿 [J].JournalofGenetics,2014,93(1):145158
[24]ýq,¦¯¼,ìº,.[h、4E、É¿ûñÀ2OÁNOo
[J].ñ&RS.¸,2013,29(2):224234
[25]ÃÒ=,Ãr´,1/,.ÑsLüùÔ«¶I‹ŸQûñÀ
2J‚
[J].8¸®.:¸,2009,25(2):100104
[26]§X˜,‰Vu,‰te.ž&ÌÍ
[J].ñ&RS.¸,2007,23(3):367374
[27]3už,;W«,–¯µ,.VLvhûñÀ2J‚Q¥{2
ü<Ž+}2ùIÕ1
[J].x&.¸,2014,40(2):231239
[28]­—™,–Ê,‰)),.£Ý<\]r2ûñÀ22J‚>
aIÕÖ
[J].]4.¸,2008,16(1):5053
[29]›wÓ,­4/,Ò4›,.]4HIŸªÏⅡUûñÀ2"
«Qž6!ñ
[J].]4.¸,2010,18(1):103107
[30]K®,·.´.]xyûñÀ2J‚¶²\ÌÍ[J].0Tà.

,2010,32(12):97101
[31]§Uö,Âí=,UÊz,.„Ë!×{ûñÀ2IJ‚¶"
«
[J].0„ž&.¸,2013,33(2):254260
[32]3[Ø,ÔÏn. LjXL\]ûñÀ2J‚"«NOIº
p
[J].0„ž&.¸,2014,34(1):184192.
[33]|f‡./\ûñÀ2J‚Q}IÌÍ[D].=!:r‚®
ֈ.
,2012
[34]3±,3µÊ,§ê¼,.€þÆqòåûñÀ2"«!ñ
ž62ùIo\ÌÍ
[J].0„ž&.¸,2012,32(12):2438
2443
[35]–3«,ÔÏn.ÇPØ L\]ùÔ«¶ûñÀ2–‚¶"
«NOIo[J].8¸]4.¸,2013,35(3):1217
[36]BartR,ChernM,ParkCJ,犲狋犪犾.Anovelsystemforgenesi
lencingusingsiRNAsinriceleafandstemderivedprotoplasts
[J].PlantMethods,2006,2(1):1321

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