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TUNEL Detection of Cu2+ Induced Cell Death of the Phycobionts in Lichen Ramalina sinensis

TUNEL 法检测 Cu2+胁迫诱导的中国树花共生藻细胞的死亡



全 文 :书!"#$%&
,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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G H:]^_`abcdefghij:kNOlm=nopqrNOstouv,;wxyz{,|n}~
TUNEL€(PromegaMRoche‚ƒ„)f2mmol/LM4mmol/LCu2+de24hoghi…†‡j:kN
Oˆt‰Š‹ŒŽ

‘2
:(1)’“”•–—€˜™,ghij:kNOš›42M4mmol/L
Cu2+deœ˜žŸ, NOost¡¢£Cu2+¤¥o/¦M§¨©ªo«¬­2˜®¯;(2)42M4mmol/L
Cu2+de°±œ,ghij:kNOoTUNEL²³NO¡4Promega‚ƒ„g€´µ]50.30%M
31.21%,­4Roche‚ƒ„gj:kNOTUNEL²³¶·¸´µ]53.17% M36.88%,}~TUNEL€
¹º
。(3)U’“”•–—€oCu2+deghij:kNOš›»¼,½¾¿Ÿ¤¥oCu2+(2mmol/
L)def…†j:kNOolm=nÀÁÂÃÄNOˆtÅÆ,­¿¦¤¥oCu2+(4mmol/L)def…†j:
kÇ:¿ÈÉolmÊÂËSÌ´NOoÍs

ÎÏÐÑÒNOÓ¾NOˆt
。(4)}~‚ƒ„ÔÀnÕ¿Ÿ¤
¥Cu2+deÖ×o…†‡j:kNOˆto€;ØÙÚxyz{ÛÜnÕTUNELNOˆtuÝ€gÞß
à^¿¨áo

âã^qäåæz{oçèéê

ëìí

ïð^íîñÅ

IJK

ade

ghij:k

NOˆt
;TUNEL€
:Q255;Q944.5 !OPQR:A
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,westudiedtheceldeathofsymbiontalgainthehandslicedsectionsofthelichenthalus
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,thehandslicedsectionsoflichen
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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f…†‡Ç:ó
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[4]。
ghi
(犚犪犿犪犾犻狀犪狊犻狀犲狀狊犻狊Jatta)òó~…
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S<ÕcTöU
(Ascomycota),cTöV
(Ascomycetes),WX†Y(Lecanorales),hi+
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f:;goÉ-<=>–$?¿A
B

ò_€É-<>–o¨áÛÜ`ó
[58]。
ab8
9g½¾

ghifCu2+decÏAB³,g
hij:kfCu2+ode¤¥41~2mmol/L`
ª
;Cu2+¤¥UghiNOfogRÅ¥h4
£2˜oƒijklm
,Cu2+¤¥n¦, <Õj
okoõ¸j:kNOegÅ¥nS
[9]
aò:$‡/0oó~ÉHopiqþrs`
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=]9~tou´vU@9:¨wxwÅ

yw
ioaÞÀz{ÂË:$‡oalm
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|~
devcÂË:$‡ostGHÏ}~}~NO
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NOˆt

MÍs

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ÂËNO‚NOƒo„Í

)NOù…
$?Ó¾"†

­NOoÅƳstòô,v‡,
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NO)ďˆ:TowÅ
[1213]。
4‰9oNO
ˆt€}g
,TUNEL(TdTmediateddUTP
nickendlabeling)òuÝ€NOˆt2]A
B

Š‹

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;wŽ¸‘’“”t
zڕ϶·o"L³¸‘’%0DNA–—˜Ù
àˆtNODNA™šo3OH Ž›。TUNEL
€ôÕ Àœ³ž]YaÄ#$NOˆt€
oŸ y¡
[14]。
y¢£¤¥¦àôÉ-×o…†:$ÅƳstÏlo89&§



©íî|nPromegaMRocheNOˆt
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ªŽ«¬¤¥Cu2+§¨œgh
ij:kNOoˆt‰Š
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íîÃùÄ
ÅÆÇo…†´¹}‹Œ´¹È,É*hÕ
-20℃ ÊË。G©|ÌÉ1ü·ù4žíîùL。
1.2 犆狌2+78WX
ÍÎÏäÐÏÑÒoÓþÔ

ÚCu2+¤¥]0

ÕÖ$
),2M4mmol/LoגaØÙ(10mL)´
µÚÛ4ÓþÔùÑқ

2ÉÚÜÝ

IÊËÞÓ
É4ÃßJ30minàá)oghi…†‡JNÓ
þÔùÑқ

4Ãß

â20℃)úÅ°±œ´µ§
9864b        ./0·12,=:TUNEL€Cu2+deÁÂoghij:kNOost
¨8、16M24hÉÞG,4­ãäœÚåCu2+de
§¨oghi…†‡ÛÜxyzžæ{

z{|
n«¬}‹Œj:kNOˆtŽ

1.3 @ABCDYZ[\F]^
1.3.1 _!`abc456CDYZ z{n
0.05%(W/V)’“”•(n10mmol/Lç’èéÙ
êq
,pH7.0)–—10minÉ,Õú%˜p䜍
Ž

ëÅ

 Þ10ìz{ÇíîNOÒi(â200
üNO

M gstNOÒi

Äï~íðšO¡

šNO¡ =(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;ýþهÉÿ¯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  Ž  ,Ç í
TUNEL ² ³ N O . ë Å。- — N O ¸ ‘ ™
TUNEL²³NO,.üíîÉÜ3÷, Þ10ì
z{nÕÇí´/

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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;ýþهÉÿ¯100μLDNaseⅠ(200U/mL)
tz10min;n!bc$ö4÷;PBSñö5min;¯
N10μL"#Ù-„"#10min;¯ 100μLTUNEL
$k%BÙ

&(É4)§M-„g37℃$k
1h;ñN20×SSC15min*+$k。4ú˜pä
Ž

ÇíTUNEL²³NO.ëÅ。•Ï4ú¶
·oNO¸]TUNEL²³NO。
2 U´/
2.1 fgh78i<=>?@ABYZ;jk
;w–—€NOstòó~2؍

ï5
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nÕ€NOsto–—‚ƒ~¹¿9

6
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(Evan’sblue)=。g³7–—
©

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~¿>B…†j:öko–—ƒ

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stNO£—³•
—

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),
À2˜…ÁµšNOM
stNO

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ªo«¬

|¤¥§¨ghij:kNOhš¡
ÔBCžŸ

 g

ghij:kNOhš¡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+deœÊSÌ´NOst。ÀF,ghij
:kš›42M4mmol/LCu2+deœ˜eàG
q­žŸ

 NOost¡UCu2+de¤¥M§¨
©ªHI»l

¢£Cu2+¤¥o/¦M§¨©ª
o«¬

j:kNOost¡2˜®¯

–—J
*zK€NOost

yò«zK´=NOst
ouvˆt‚Ís

2.2 犘狉狅犿犲犵犪犜犝犖犈犔CD\Fde56lmn
]^<=>?@ABCD;EF
  NOÅƳstÉHo:$ð%÷L`óòN
O¸ùo¸M‡DNAoNO™š‚ÎHó°O›
Ó¾C(©

Ç:oómPDNAo3′OH ŽÀ
4¸Q¸‘’Ž“”t
(TdT)o=nœ,Ú
:$s¶·odUTP˜ÙàDNAo3′Ž,.U
A1 «¬¤¥Cu2+deœghij:kNOhš¡
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©ªœghij:kNOoš›Ž(’“”•–—)(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)
˜ÙSTwðt
(HRP,horseradishperoxi
dase)oOUVMs(streptavidin)÷Œ³B,É>
WUPOD X$wðY(H2O2)、Z[,\]^
(DAB)$kÇ:-—Ç$。ôÕI_o‚I4®
`oNOabcÏDNAo™š,v­cÏ3′OH


@ÑzKe–—

Promega‚ƒ„TUNEL€˜™,g
hij:k2~§¨u(2M4mmol/LCu2+)M²
³fÅuoz{gÔÓ¾«¬¼f TUNEL²³
¶·oNO¸

A4)。42mmol/LCu2+deœ,
ghij:kNOg TUNEL€²³oNO
g50.30%(A3),’“”•–—Þ˜™®¤¥
deœNOost¡ò44.68%,ÀFŸ¤¥Cu2+
de
(2mmol/L)ÀzGHÖ×j:kNOoÅÆ
³st
(PCD),ˆt。¬©,4¿¦¤¥ Cu2+
(4mmol/L)d e œ,g h i j : k N O o
TUNEL€²³NO¡]31.21%(A3),1U¬
A3 «¬¤¥abcdeœghij:kNOo
TUNEL€(Promega€‚ƒ„)
Fig3 TheresultsofTUNELreactionof犚.狊犻狀犲狀狊犻狊
phycobiontunderCu2+stress(Promegadetectionkit)
GCu2+¤¥deœ’“”•–—(NOst¡
ò96.16%)HŸß9。h1i=[9]89&§,úKj
c˜p䜍Ž©¿¦¤¥oCu2+de(4mmol/L)
1964b        ./0·12,=:TUNEL€Cu2+deÁÂoghij:kNOost
a.3³fÅ(CK1);b.²³fÅ(CK2);c.2mmol/LCu2+§¨u;d.4mmol/LCu2+§¨u
A4 «¬¤¥Cu2+deœghij:kNOoTUNEL€(Promega€‚ƒ„)(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+deœghij:kNOoTUNEL€(Roche‚ƒ„)(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 «¬¤¥abcdeœghij:kNOo
TUNEL€(Roche€‚ƒ„)
Fig.6 TheresultsofTUNELreactionof犚.狊犻狀犲狀狊犻狊
phycobiontunderCu2+stress(Rochedetectionkit)
{Ö×NOƒrNOo„Í



klmB´
/n]
,4mmol/LCu2+deœÎÏÐÑÒNOÀ
z{;wÃÄNOoˆtÂËNOost

­SÌ
´NOostÀzòv]Ís­«òNOoˆt


2.3 犚狅犮犺犲犜犝犖犈犔CD\Fde56lmn]
^<=>?@ABCD;EF
  opNOˆtuÝ€‚ƒ„òYa[\nÕ
Ä#$NOˆt€oqA¥@¦

rs¿t2M
oó~ï5o‚ƒ„

Î0ó閗$kÉöu
ÀŽ

«0HnPOD“vÙ,w"ˆtNOùÇ
:oDNA™šo¶·$ò4úú˜p䍎
。RocheTUNEL‚ƒ„€Cu2+
deœghij:kNOsto

A5、A6)
˜™

3³fÅg¤Žà4ú

TUNEL²³NO(A5,a);²³fÅug80.97%
oNOgÓ¾TUNEL²³¶·(A5,b);íî§
¨u
(2M4mmol/LCu2+de§¨)TUNEL²³
¶·NO´µg53.17%M36.88%(A5,c、d)。
1óUPromega‚ƒ„€@»º。À
F
,Roche‚ƒ„ÞÀnÕabcdeÖ×og
hij:kNOˆto€

­y1ó}?¯ï
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Gluchsman489}Ž¹Ä$o¾‘©I~/
ÓNOÅƳsto’“É

+%(f ”:$
#$

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õö

k¹=ò¯h4NOoÅƳs
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狋犪)f˜rsdeo÷ø$kr÷øøùto‘
ž=½¾

ŸkM:koNOstÞòó~)Ä 
ðwÅ
[20]。2004¶,Berman=8¡½¾,Óþ©
ªw¬o¢ðˆ£k
(犜狉犻犮犺狅犱犲狊犿犻狌犿sp.IMS101)
g45%oNOÓ¾NOƒg;‘,yNO4û
.¤ÏCaspasetš³®¥oPCD¾‘[20]。É,
•Nö Anabaena4&deœoNOÅƳs
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r‹NO:k犇狌狀犪犾犻犲犾犾犪狋犲狉狋犻狅犾犲犮狋犪[23]
M ”z‹ŒúB=no•–p:$oNOÅƳ
st89Þ»¦&§
[2425]。2006¶,–~:$§
¨†k
(犆犺犾犪犿狔犱狅犿狅狀犪狊狉犲犻狀犺犪狉犱狋犻犻)]w‘oó
©‹NOúB=nk¹4«¬devs´µœoN
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Ÿ=:$ÞÀzªcϹ
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