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,2016,36(4):0688-0695
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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TUNEL(PromegaMRoche)f2mmol/LM4mmol/LCu2+de24hoghi
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Ot
。
2
:(1),ghij:kNO42M4mmol/L
Cu2+de, NOost¡¢£Cu2+¤¥o/¦M§¨©ªo«¬2®¯;(2)42M4mmol/L
Cu2+de°±,ghij:kNOoTUNEL²³NO¡4Promegag´µ]50.30%M
31.21%,4Rochegj:kNOTUNEL²³¶·¸´µ]53.17% M36.88%,}~TUNEL
¹º
。(3)UoCu2+deghij:kNO»¼,½¾¿¤¥oCu2+(2mmol/
L)def
j:kNOolm=nÀÁÂÃÄNOtÅÆ,¿¦¤¥oCu2+(4mmol/L)def
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犜犝犖犈犔犇犲狋犲犮狋犻狅狀狅犳犆狌2+犐狀犱狌犮犲犱犆犲犾犇犲犪狋犺狅犳狋犺犲犘犺狔犮狅犫犻狅狀狋狊
犻狀犔犻犮犺犲狀犚犪犿犪犾犻狀犪狊犻狀犲狀狊犻狊
PATIGUYiming,MAIERDANAisikaer,WEINILAWufuer,PAZILATBahti
(TheLifeSciencesandTechnologyColegeofXinjiangUniversity,Urumqi830046,China)
犃犫狊狋狉犪犮狋:InordertounderstandthemechanismoftoxiceffectsofCu2+stressonthephycobiontsof犚犪犿犪
犾犻狀犪狊犻狀犲狀狊犻狊,westudiedtheceldeathofsymbiontalgainthehandslicedsectionsofthelichenthalus
whichweretreatedwith2and4mmol/LCuSO4for24hoursrespectivelybyusingtwokindsofTUNEL
reactionkit(PromegaDeadEndTM ColorimetricTUNELSystemandRoche犻狀狊犻狋狌Cel DeathDetection
Kit).Theresultsofthestudyshowed:(1)thephycobiontcelvitalitydecreasedsignificantlyunderCu2+
stressdetectedbyEvan’sBluestainingmethod.Moreover,therewasapositivecorrelationbetweenthe
celvitalityandtheCu2+ stressconcentrationandtime.(2)WhenPromegaDeadEndTM Colorimetric
TUNELSystemwasused,theTUNELpositivephycobiontcelswere50.30%and31.21%,respectively
under2and4mmol/LCu2+stressconditions.TheTUNELpositivecelsofphycobiontswere53.17%and
36.88%,respectivelywhenRoche犻狀狊犻狋狌CelDeathDetectionKitwasusedunderthesameCu2+ stress
conditions,theresultsofbothTUNELkitswassimilar.(3)Consideringtheresultsofphycobiontscelvi
talitywhichwasdetectedbyEvan’sBluestainingmethodunderCu2+stress,itcanbeconcludedthatap
optosis(programmedceldeath)of犚.狊犻狀犲狀狊犻狊phycobiontwereinducedunderlowerCu2+ concentration
(2mmol/L)treatment,buttheceldeathofphycobiontsweremainlycausedbythenecrosisandonlyfew
programmedceldeathofthephycobiontswasoccuredwhilethelichenwastreatedwithhigherCu2+con
centration(4mmol/L).(4)BothkindsofTUNELdetectionkitcanbeusedforthestudyofapotopsisof
lichenphycobiontsunderheavymetalCu2+stressconditions.Inaddition,thehandslicedsectionsoflichen
thaliwereusedinthisstudyinsteadoftheparaffinsections.Theexperimentprocedurewassimplifiedby
avoidingthecomplicatedexperimentalsteps.Thisresearchwilbeanimportantscientificreferenceforthe
furtherunderstandingsoflichenresponsestothevariousmetalstresses.
犓犲狔狑狅狉犱狊:Cu2+stress;犚犪犿犪犾犻狀犪狊犻狀犲狀狊犻狊;apoptosis;TUNEL
òó~ôk¹Mõö¹j:Ç:o÷ø
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HoþÜ
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1ò)*#g23Ó
、
245o676:
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[13]。
8S9Ò
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)
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[4]。
ghi
(犚犪犿犪犾犻狀犪狊犻狀犲狀狊犻狊Jatta)òó~
:$
,
S<ÕcTöU
(Ascomycota),cTöV
(Ascomycetes),WXY(Lecanorales),hi+
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\o]^
,
f:;goÉ-<=>$?¿A
B
,
ò_É-<>o¨áÛÜ`ó
[58]。
ab8
9g½¾
,
ghifCu2+decÏAB³,g
hij:kfCu2+ode¤¥41~2mmol/L`
ª
;Cu2+¤¥UghiNOfogRÅ¥h4
£2oijklm
,Cu2+¤¥n¦, <Õj
okoõ¸j:kNOegÅ¥nS
[9]
aò:$/0oó~ÉHopiqþrs`
ó
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=]9~tou´vU@9:¨wxwÅ
,
yw
ioaÞÀz{ÂË:$oalm
[1011]。
|~
devcÂË:$ostGHÏ}~}~NO
oÅƳst
(
NOt
)
MÍs
。
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¨³wÅ
,
ÂËNONOoÍ
,
)NOù
$?Ó¾"
。
NOoÅƳstòô,v,
o
,
NO)Ä:TowÅ
[1213]。
49oNO
t}g
,TUNEL(TdTmediateddUTP
nickendlabeling)òuÝNOt2]A
B
、
、
÷o}
,
;w¸t
zÚ϶·o"L³¸%0DNAÙ
àtNODNAo3OH 。TUNEL
ôÕ À³]YaÄ#$NOt
o y¡
[14]。
y¢£¤¥¦àôÉ-
。
]¨
,
©íî|nPromegaMRocheNOt
uÝ
,
ª«¬¤¥Cu2+§¨gh
ij:kNOot
,^
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Ä,v°±oÅƳst
,
_²ghifCu2+d
eAB³ouvr stpq
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tÍs
),
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N89-
。
1 ÛÜM}
1.1 STUV
ghiÕ2014¶5·|)QRYZ[\¸
¹ºó»¼
,
½¾¦¥]1779m。89ÛÜ4“Q
RS%gQR¿ÀÁ
89gÂ
”
íîÃùÄ
ÅÆÇo
´¹}´¹È,É*hÕ
-20℃ ÊË。G©|ÌÉ1ü·ù4íîùL。
1.2 犆狌2+78WX
ÍÎÏäÐÏÑÒoÓþÔ
,
ÚCu2+¤¥]0
(
ÕÖ$
),2M4mmol/Lo×aØÙ(10mL)´
µÚÛ4ÓþÔùÑÒ
,
2ÉÚÜÝ
(
IÊËÞÓ
É4ÃßJ30minàá)oghi
JNÓ
þÔùÑÒ
,
4Ãß
(
â20℃)úÅ°±´µ§
9864b ./0·12,=:TUNELCu2+deÁÂoghij:kNOost
¨8、16M24hÉÞG,4ãäÚåCu2+de
§¨oghi
ÛÜxyzæ{
,
z{|
n«¬}j:kNOt
。
1.3 @ABCDYZ[\F]^
1.3.1 _!`abc456CDYZ z{n
0.05%(W/V)(n10mmol/LçèéÙ
êq
,pH7.0)10minÉ,Õú%pä
、
ëÅ
。
Þ10ìz{ÇíîNOÒi(â200
üNO
)
M gstNOÒi
,
Äï~íðO¡
。
NO¡ =(NOîÒ-stNOÒ)/NO
îÒ ×100%
1.3.2 犘狉狅犿犲犵犪犜犝犖犈犔CD\Fde56 z{
ñN4%9òóôõ,15min;nPBSñö5min
}÷
;
n100μLoøùtK(20g/mL)ÃßNO;
ú§¨20min;nPBSñö5min;ñN4%9òó
ôõ,5minɯN100μLDNase1èéÙûü
5min;ýþÙÉÿ¯100μLDNaseI(200U/
mL)tz10min;n!bc$ö4÷;PBS ñö
5min;¯ N10μL "#Ù-"#10min;¯
100μLTUNEL$k%BÙ,&(É4)§M
-g37℃$k1h;ñN20×SSC15min*+$
k
;
n PBSñö5min;ñN0.3%o H2O2J,
15min;nPBSñö5min,ÉÜ3÷;nDAB)
§ 10min。Leica p ä ,Ç í
TUNEL ² ³ N O . ë Å。- N O ¸
TUNEL²³NO,.üíîÉÜ3÷, Þ10ì
z{nÕÇí´/
。
ÄÅ02
,
íîg12
,²³fÅuM3³fÅu
。
1.3.3 犚狅犮犺犲犜犝犖犈犔CD\Fde56 z{ñ
N4%9òóôõ,15min;nPBSñö5min2
÷
;
n100μLoøùtk(20g/mL)ÃßNO;ú
§¨20min;nPBSñö5min;ñN4%9òóô
õ,5minɯN100μLDNaseIèéÙûü
5min;ýþÙÉÿ¯100μLDNaseⅠ(200U/mL)
tz10min;n!bc$ö4÷;PBSñö5min;¯
N10μL"#Ù-"#10min;¯ 100μLTUNEL
$k%BÙ
,
&(É4)§M-g37℃$k
1h;ñN20×SSC15min*+$k。4úpä
,
ÇíTUNEL²³NO.ëÅ。Ï4ú¶
·oNO¸]TUNEL²³NO。
2 U´/
2.1 fgh78i<=>?@ABYZ;jk
;wNOstòó~2Ø
、
ï5
o}
,
nÕNOsto~¹¿9
,
6
g³7
、
(Evan’sblue)=。g³7
©
,
Àzòv]j:kNO©8
ÏSio9:
,
j.«¨á
,
stNO;;¿<´=
。
4
9ÀnÕNOst¡ogòó
~¿>B
j:öko
,
nÕ
ùj
:kNOostj?@
,
stNO£³
,
NO«£
(
:
),
À2
ÁµNOM
stNO
。
A1MA2,¢£Cu2+de§¨©
ªo«¬
,
|¤¥§¨ghij:kNOh¡
ÔBC
。
g
,
ghij:kNOh¡4
2mmol/LCu2+§¨8、16、24h©1÷]78.43%、
64.36% M55.32%,44mmol/LCu2+§¨8、16、
24h©´µ]23.91%、6.06%M3.84%(A1)。
42mmol/LCu2+§¨24h©DóEogh
ij:kNOst
,
4¿¦¤¥o4mmol/L
Cu2+deÊSÌ´NOst。ÀF,ghij
:k42M4mmol/LCu2+deeàG
q
,
NOost¡UCu2+de¤¥M§¨
©ªHI»l
,
¢£Cu2+¤¥o/¦M§¨©ª
o«¬
,
j:kNOost¡2®¯
。
J
*zKNOost
,
yò«zK´=NOst
ouvtÍs
。
2.2 犘狉狅犿犲犵犪犜犝犖犈犔CD\Fde56lmn
]^<=>?@ABCD;EF
NOÅƳstÉHo:$ð%÷L`óòN
O¸ùo¸MDNAoNOÎHó°O
Ó¾C(©
,
Ç:oómPDNAo3′OH À
4¸Q¸t
(TdT)o=n,Ú
:$s¶·odUTPÙàDNAo3′,.U
A1 «¬¤¥Cu2+deghij:kNOh¡
Fig.1 Thephycobiontcelvitalityof犚.狊犻狀犲狀狊犻狊
underCu2+stress
096 ! " # $ % & 36R
a、d、g´µfŧ¨8h、16hM24h;b、e、h´µ]2mmol/LCu2+§¨8h、16hM24h;
c、f、i´µ]4mmol/LCu2+§¨8h、16hM24h
A2 «¬Cu2+de¤¥M©ªghij:kNOo()(10×40)
a,dandgstandfortreatmentundercontrolfor8h,16hand24h,respectively;b,eandhstandfortreatmentwith2mmol/LCu2+
for8h,16hand24h,respectively;c,fandistandfortreatmentwith4mmol/LCu2+for8h,16hand24h,respectively
Fig.2 Thecelviability(Evan’sbluestaining)of犚.狊犻狀犲狀狊犻狊phycobionttreatedwithdifferentCu2+
concentrationsandtime(10×40)
ÙSTwðt
(HRP,horseradishperoxi
dase)oOUVMs(streptavidin)÷³B,É>
WUPOD X$wðY(H2O2)、Z[,\]^
(DAB)$kÇ:-Ç$。ôÕI_oI4®
`oNOabcÏDNAo,vcÏ3′OH
d
,
@ÑzKe
。
PromegaTUNEL,g
hij:k2~§¨u(2M4mmol/LCu2+)M²
³fÅuoz{gÔÓ¾«¬¼f TUNEL²³
¶·oNO¸
(
A4)。42mmol/LCu2+de,
ghij:kNOg TUNEL²³oNO
g50.30%(A3),Þ®¤¥
deNOost¡ò44.68%,ÀF¤¥Cu2+
de
(2mmol/L)ÀzGHÖ×j:kNOoÅÆ
³st
(PCD),t。¬©,4¿¦¤¥ Cu2+
(4mmol/L)d e ,g h i j : k N O o
TUNEL²³NO¡]31.21%(A3),1U¬
A3 «¬¤¥abcdeghij:kNOo
TUNEL(Promega)
Fig3 TheresultsofTUNELreactionof犚.狊犻狀犲狀狊犻狊
phycobiontunderCu2+stress(Promegadetectionkit)
GCu2+¤¥de(NOst¡
ò96.16%)Hß9。h1i=[9]89&§,úKj
cpä©¿¦¤¥oCu2+de(4mmol/L)
1964b ./0·12,=:TUNELCu2+deÁÂoghij:kNOost
a.3³fÅ(CK1);b.²³fÅ(CK2);c.2mmol/LCu2+§¨u;d.4mmol/LCu2+§¨u
A4 «¬¤¥Cu2+deghij:kNOoTUNEL(Promega)(10×40)
a:Negativecontrol(CK1);b:Positivecontrol(CK2);c.Treatmentwith2mmol/LCu2+for24hours;d:
Treatmentwith4mmol/LCu2+for24hours
Fig.4 TUNELreactionof犚.狊犻狀犲狀狊犻狊phycobiontunderCu2+stress(Promegadetectionkit)(10×40)
a:3³fÅ(CK1);b:²³fÅ(CK2);c:2mmol/LCu2+§¨u;d:4mmol/LCu2+§¨u
A5 «¬¤¥Cu2+deghij:kNOoTUNEL(Roche)(10X40)
a.Negativecontrol(CK1);b.Positivecontrol(CK2);c.Treatmentwith2mmol/LCu2+for24hours;
d.Treatmentwith4mmol/LCu2+for24hours
Fig.5 TUNELstainingof犚.狊犻狀犲狀狊犻狊phycobiontunderCu2+stress(Rochedetectionkit)
296 ! " # $ % & 36R
A6 «¬¤¥abcdeghij:kNOo
TUNEL(Roche)
Fig.6 TheresultsofTUNELreactionof犚.狊犻狀犲狀狊犻狊
phycobiontunderCu2+stress(Rochedetectionkit)
{Ö×NOrNOoÍ
。
v¨
,
klmB´
/n]
,4mmol/LCu2+deÎÏÐÑÒNOÀ
z{;wÃÄNOotÂËNOost
,
SÌ
´NOostÀzòv]Ís«òNOot
/Ë
。
2.3 犚狅犮犺犲犜犝犖犈犔CD\Fde56lmn]
^<=>?@ABCD;EF
opNOtuÝòYa[\nÕ
Ä#$NOtoqA¥@¦
、
rs¿t2M
oó~ï5o
,
Î0óé$kÉöu
À
,
«0HnPODvÙ,w"tNOùÇ
:oDNAo¶·$ò4ú
。RocheTUNELCu2+
deghij:kNOsto
(
A5、A6)
:
3³fÅg¤à4ú
TUNEL²³NO(A5,a);²³fÅug80.97%
oNOgÓ¾TUNEL²³¶·(A5,b);íî§
¨u
(2M4mmol/LCu2+de§¨)TUNEL²³
¶·NO´µg53.17%M36.88%(A5,c、d)。
1óUPromega@»º。À
F
,RocheÞÀnÕabcdeÖ×og
hij:kNOto
,
y1ó}?¯ï
5
、
,
y®}Îz4íîÃzä4úpäo
°±{z)n
。
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Oost
,
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(
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grammedceldeath,PCD)。NOÅƳstò:
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Gluchsman489}¹Ä$o¾©I~/
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#$
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、
õö
、
k¹=ò¯h4NOoÅƳs
tBé^[\³No89
,
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$oNOt89
[1718]。
k¹=#$NOÅ
Ƴsto89×é¿
。1997¶,Brussaard=
&§4
、
çde©ZpNkÓ¾NO)Ø
,
NOostòÏÆo
[19]。1998¶,Burgas=1
p½Ok
(犜.狑犲犻狊狊犳犾狅犵犻犻)Mpk(犇.狋犲狉狋犻狅犾犲犮
狋犪)frsdeo÷ø$kr÷øøùto
=½¾
,
kM:koNOstÞòó~)Ä
ðwÅ
[20]。2004¶,Berman=8¡½¾,Óþ©
ªw¬o¢ð£k
(犜狉犻犮犺狅犱犲狊犿犻狌犿sp.IMS101)
g45%oNOÓ¾NOg;,yNO4û
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Nö Anabaena4&deoNOÅƳs
t
[2122],
rNO:k犇狌狀犪犾犻犲犾犾犪狋犲狉狋犻狅犾犲犮狋犪[23]
M zúB=nop:$oNOÅƳ
st89Þ»¦&§
[2425]。2006¶,~:$§
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(犆犺犾犪犿狔犱狅犿狅狀犪狊狉犲犻狀犺犪狉犱狋犻犻)]woó
©NOúB=nk¹4«¬devs´µoN
OÅƳst¾2
,
=:$ÞÀzªcϹ
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÷L
[27]。
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(
k
)
j
:do\B
,
<Õ
“
Zr
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:$
。
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,
gg10%oj:kOPÏ
Sio«¬®£j:ko
¯õöj:ö
(
g
o90%),
ùoj:ökò)*#
g67j:õöfk¹Ï±°:o±²
。2011
¶
,Dilek=÷&§^³"´(UV)ÀÁÂ
j:ökNOoÅƳst
,
nPromegaNOt
uÝ89^ UVBÁÂo
犘犺狔狊犮犻犪
狊犲犿犻狆犻狀狀犪狋犪 ost,.µÓ³"´ÁÂo
ùj:kot÷LUÄ$NOoÅƳst@»
º
,TUNELÀnÕ
$~³"´ÁÂo
DNA89[28],¢£¤¥¦àôÉ-
。
ghiòó~f:;goÉ-<@ABo
3964b ./0·12,=:TUNELCu2+deÁÂoghij:kNOost
,
ò:;¨áoµ:$
。
kl4ab89
g½¾
,Cu2+de¤¥nS,ghij:kNO
h¡nM
[9]。
g
,
¤¥Cu2+(<1mmol/L)
fghiNOf,©%´µ
,
NO
、
NO
rNOùo´¶
、
9:4û
;
¢£Cu2+¤¥®
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,
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,
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,
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,
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,
9:UNO?%4ó
×
,
,¶{I½¾
,´
Z%
,´
¶d
;
Cu2+
¤¥4mmol/L©,SÌ´NOf4¿eà
Í
。
ghif Cu2+ de¤¥od³4 1~
2mmol/L`ª,4mmol/LCu2+Ú{Ö× j:k
NOoSist
[9]。
]^óé³N^
:$
É-
©íînTUNEL8
9^«¬¤¥abcde§¨o
j:kNOo
t
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