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Genetic Transformation of the Phalaenopsis F3′5′H to OT Lily Robina

蝴蝶兰F3′5′H基因转化OT杂种百合Robina的研究



全 文 :书!"#$%&
,2016,36(4):0874-0880
犃犮狋犪犅狅狋.犅狅狉犲犪犾.犗犮犮犻犱犲狀狋.犛犻狀.
  !"#$:10004025(2016)05087407               犱狅犻:10.7606/j.issn.10004025.2016.05.0874
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:20160131;)*&%+(:20160426
,-./

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(31471905)
0123

./0
(1988-),1,2345678,9:;<=>?@#$ABCDE67。Email:1032239251@qq.com

FGHI

.JK

45

LM

N58OP

9:;<=>QRABCDE67
。Email:lyl6151@126.com
456犉3′5′犎 ,789犗犜:;
<=犚狅犫犻狀犪>?@
!"#

!$%
,
& ,()*

!"S>+TU% VWH$XY8$%(Z[\]^

S_`!"aW=bH$8$%cBdefZ[\]^

g!hi

712100)
A B:jklmABnopqrs,t67ursRobinajpq,vwxyz,u{|}~O8E€‚ƒ
„…†‡8ˆ#‰ˆŠ‹HjŒŽ

‘S’“”O•

–—˜™犉3′5′犎 ,vOšrsRobina›。œž
Ÿ

uˆŠ‹jŒŽ

 ¡¢3d,OD600j0.8,£¤10min,¥¡¢3d,¦š100μmol/LAS§lŒ¨w©
j12.78%;ªu€‚ƒjŒŽ, ¡¢2d,OD600j0.8,£¤10min,¥¡¢3d,¦š100μmol/LAS§l
Œ¨w©j12.22%。2BŒŽEw«¬­®¯°±²³j20mg/L。´µ#‰?¶·¸PCR†¹Œº
PCR»¼,no9½¾‰¿,SouthernÀÁ?·ÃÄÅlk6‰Œ,vrs›ÆÇpq,v犉3′5′犎,jÈÉ
·ÃÄnopqrsÊlk,Ë

CDE
:犉3′5′犎;Robinars;€‚ƒ„…;ˆŠ‹;QRŒ
FGHI$
:Q785;Q789 !JKLM:A
犌犲狀犲狋犻犮犜狉犪狀狊犳狅狉犿犪狋犻狅狀狅犳狋犺犲犘犺犪犾犪犲狀狅狆狊犻狊犉3′5′犎狋狅犗犜犔犻犾狔犚狅犫犻狀犪
LIUAiling,LIUYali,LOUQian,ZHANGHaiqin
(StateKeyLaboratoryofCropStressBiologyinAridAreas,MinistryofAgricultureKeyLaboratoryofHorticulturalPlantBiol
ogyandGermplasmInnovationinNorthwestChina,Colegeofforestry,Yangling,Shaanxi712100,China)
犃犫狊狋狉犪犮狋:Toobtainbluish犔犻犾犻狌犿spp.indirectbreeding,wescreenedthesuitablevarietyOTlilyRobina
forgenetictransformation.Here,bothembryoniccalusinducedfromfilamentandregeneratedbulbscales
ofOTlilyRobinawereusedasthetransformationmaterial.犃犵狉狅犫犪犮狋犲狉犻狌犿mediatedtransformationof
犘犺犪犾犪犲狀狅狆狊犻狊犉3′5′犎 wasstudied.Theresultsshowed:withthepreculture3d,OD600=0.8,infection
time10min,coculturedfor3d,100μmol/Lacetosyringoneconditions,thestabletransformationrateof
regeneratedbulbscalescouldreachtothehighest12.78%;however,embryogeniccaluspreculture2d,
OD600=0.8,infectiontime10min,cocultured3d,with100μmol/Lacetosyringoneconditions,which
thestabletransformationratewasthehighest12.22%.Inaltheconditions,thebesthygromycinresist
antscreeningconcentrationsalwayswas20mg/L.PCRandreversetranscriptionPCRassayshowed9pu
tativetransgenicplantswereobtained.Southernhybridizationanalysisfurtherproved6plantsthatthe
transgenicliliumflowerscarrybluegene犉3′5′犎.Theresultsprovidedtechnicalsupportandmaterialbasis
forthecontinuingdevelopmentofnovelbluish犔犻犾犻狌犿flowers.
犓犲狔狑狅狉犱狊:犉3′5′犎;lilyRobina;embryoniccalus;bulbscales;genetictransformation
  OTÌBrs Robina(犔犻犾犻狌犿狋犲狀狌犻犳狅犾犻狌犿o
riental×trumpet)ÍÎCrsÌÏ¿(orientalhy
brids)cÐÑÒrsÌÏ¿(trumpethybrids)EÌ
ÏB
[1]。
{ÓÔÕÖ

|U×Ø

ÙÚ±Û

Írs
›ÜÝÞßEà|áB
[2]。
rsRobinaE|âã
j3~5½,|äåqjæçèq,©²120~135
cm,8éêë100dìí,îïðñòEóôõ
ö

«÷ø²15~28℃,ù:úûüøýûþÈ
ÿ!"|

#$%&
[3]。
(8é)Y«÷
,Ro
binarsE|ä*+,-18~20cm,jrs›|Ü
ÝUEáB.Ã

/01

23{2rsà|›45
EÜZ616U

67{|q78Z:E9:

´;|
|q=>Ã*Í67E?[

u@E67›ÃAB‘EÍRCEABC•

D*
E67FGãHE|q

IÍJ;$BKL2BK
MN

RCABTOPQRSBT,vUVEWX

[4],
4uUUfYk|qEeH

Fû,vZþ
TO

,u[\RCABC•EWX

e]F^ã
EH|q
[5]。
_`a®
(delphinidin)ÍL2;#$›EÃB
|b®

c,ud#$e&pfq

ª犉3′5′犎(g
hi3′5′j,k)Ílm_`a®nol|b®
œp†|qEqrk
[6]。
rs›J;st犉3′5′犎
,vªst_`a®

4u)*u›v8fqwp
qErs|
[7]。
x\]^@ëFû´10½rsá
BEÜÝ?Â

‘yqqz?Â{|Ål«÷Œ
pq,vEyzáB

Ål|}›~€q®‚
ƒ

hi„‚©

…†pH ‡©E OTÌBrs
Robinaj«÷Œpq,vEyzáB[8]。@ë]^ˆú·¸kÆÇ犉3′5′犎 ,vž-‰zEŠ‹
Œ
[7],
x679:Í=>yzŽ†\]C•

F
ûS’“”O•§lŒ Robinars,nFû
PCRŒC•1»¼,noU‚ErsŒ,v#‰‘
;ÈÉ67

jpqrsEnoÊl,Ë

1 Ž†C•
1.1 N O
];201407Ž~201509Ž,2S_`
!"aW=bH$8$%cBdefZ["\]
^†VWH$XY8$%(Z[\]^m

u
‘;’“|<”•ERobinarsj]Ž,~O
|âE|}8€‚ƒ

‡8#‰

|}~O¡¢
,jMS+1.0mg/LPIC(–—l)+30g/L˜™+
3.0g/L#$š›。
]›d‘EœS’“
(犃犵狉狅犫犪犮狋犲狉犻狌犿狋狌
犿犲犳犪犮犻犲狀狊)“‰ÍLBA4404,OšEdžÍp1300
pPZPF3′5′HDFR,džE TDNA WŸ ÆÇ
犆犎犛|¡¢£¤@¥¤E—˜™犉3′5′犎 ,v,
E35SPro(¦§Ò犆犪犕犞35犛£¤@)¥¤E¨G
@E犇犉犚,v(—˜™犉3′5′犎 ,vž-E©ª,
v
),
u«¬­®¬ôŒ­k,v 犎犘犜Ⅱ(°®¯
Á,v
),
t‰zJx\]^p°

±1)。
1.2 P Q
1.2.1 RSTU>VWXY ²1.0mg·L-1–
—l~O8E€‚ƒ„…2.0g(³Z)à´,
µB¶100mL·l¸›,2¸T¦š40mLc
t€‚ƒ¹º»¼½®†˜™E…z¡¢,

·
¸¾¿¡¢

*Ȗ·l¸À;
(24±2)℃,100r/
minÁøÂÛ,Ä¡¢。@2ÅÆ3d¹º1Å,
{ÈÆ7d¹º1Å*ÇÈɦé。¹º‹,:Ê
9Ãl:ËÀ¶¾¿…›ÌÍÎ϶·l¸Ð`
È

ÑÒ `1/2ÓE Ô…,ÕÖH³E¡¢…。
Æ6dׂظ¾¿¡¢$E³Z,¼lƽ¾¿
¿E¦Ù‚

*¶Èɦé

no¾¿¡¢$

1.2.2 Z[\\]>^_ Ú²ÛÜÝLE8
#$ž-‰zES’““‰100μL ަǁ
50mg·L-1kan†25mg·L-1RifE25mL…z
YEB¡¢,›,28℃、180r/min“30hìí。
犆犎犛pro.|¡¢£¤@;Phalaenopsis犉3′5′犎 .—˜™ghi3’5’j,k,v;NOSter.NOSßÉ@;E35Spro.
¦§Ò犆犪犕犞35犛£¤@;Hyacinth犇犉犚 .¨G@àáhi„4âãk,v;犎犘犜Ⅱ.¬­®¬ôŒ­k,v
±1 džp1300pPZPF3′5′HDFRETDNAä9±
犆犎犛pro.Flowerspecificpromoter;Phalaenopsis犉3′5′犎.Phalaenopsisflavonoid3′5′hydroxylasegene;NOSter.NOSterminator;
E35Spro.Enhanced犆犪犕犞35犛promoter;Hyacinth犇犉犚.Hyacinthdihydroflavonol4reductasegene;犎犘犜Ⅱ.Hygromycin
phosphotransferasegene
Fig.1 TDNAschematicdiagramofplasmidp1300pPZPF3′5′HDFR
5785ë           ./0,Œ:—˜™犉3′5′犎 ,vŒOTÌBrsRobinaE67
–ÂòE“…À;50mLNåæ›,çèȇ–N
åæ À ; 4 ℃、5000r/min N å é ›,N å
15min,ê Ô…,ëìíÏš30mLZ¾
…
(MS+10mmol·L-1 MES+1.0mg·L-1PIC
+30g·L-1˜™),Z¾ïðÈÚ²2mL‘;“
… OD600‡¼l,Fûñò,m“…›Þ¦«‚Z¾
…uóô{ OD600j0.8。wÈ228℃、180r/min
Âۓ2~3h,Èu{Hjõ¤…。
1.2.3 89`a>bXY –¾¿¡¢ÈE€
‚ƒÀ; ¡¢, MS+1.0mg·L-1PIC+30g
·L-1˜™+3g·L-1#$š› ;–UˆÃöE
‡8#‰EˆŠ‹À; ¡¢, MS+1.5mg·
L-1NAA+30g·L-1˜™+3g·L-1#$š›
 

2
(25±2)℃÷Äøùì ¡¢, ¡¢‹K
j0、1、2、3†4d。
1.2.4 cdefXY ‘ úfûòEõ¤…´
úû ¡¢E2BŒyz·¸õ¤,£¤‹Kj
10min,õ¤È–ŒyzÀ;¥¡¢, ¥¡¢,
¥¡¢‹K0、1、2、3、4†5d。€‚ƒE¥¡¢
,j MS+1.0mg·L-1 PIC +10mmol·L-1
MES+(0、50、100、150、200)μmol·L
-1 AS+30g
·L-1˜™+3g·L-1#$š›;‡8ˆŠ‹E¥
¡¢,j MS+1.5mg/LNAA +10mmol·L-1
MES+(0、50、100、150、200)μmol·L
-1AS+30g
·L-1˜™+3g·L-1#$š›。
1.2.5 g\hiejkXY –¥¡¢ÈEŒ
yzÀ;500mg·L-1cefE#“ñ›ü“ý{
10min,#“ñþÿ2~3ÅÈÀ;Úñ! "#,
µ;¯°¡¢, 

€‚ƒ¯°¡¢,j
:MS+
1.0mg·L-1PIC+30g·L-1˜™+3g·L-1#$
š›+200mg·L-1carb+(0、5、10、15、20、25)mg
·L-1 hpt;‡8ˆŠ‹E¯°¡¢,j MS+
1.5mg·L-1NAA+30g·L-1˜™+3g·L-1#
$š›+200mg·L-1carb+(0、5、10、15、20、25)
mg·L-1hpt,ÆM2ê2Ø)¡¢, ¹º1Å。
l1 mno?@>pq
Table1 Primersequencesusedinthisresearch
$$Þ×
Primername
$$%&
(5′-3′)
Primersequence
犉3′5′犎F ATGTCCATCTTCCTCATCGCAACCC
犉3′5′犎R AAACTCTCCTTTATCAAACAACCCC
犇犉犚F GGATCCATGGAGATGGAGAAGGGGC
犇犉犚R TCTAGACTAGCGCGAAGCAATGTGAACC
犎犘犜ⅡF TACACAGCCATCGGTCCAGA
犎犘犜ⅡR CGCAAGGAATCGGTCAATACAC
1.2.6 rstu ¯°¡¢225℃、16h(ø
ùì·¸
,40dÈ,Œyz),2Ø)E¯°¡
¢,?Fˆ*

2¯°¡¢,›éFEˆ*

Œ
µÇ8œ¡¢,
(1/2MS+0.5mg·L-1IBA+
0.1% AC+30g·L-1˜™+3g·L-1#$š›)。
8œÈEˆ*¹É¡¢

d{8é^¦§Ö

u Ø½ý{³²Œyz40~60½,ƽ
ý{Z+3Å。ƽý{2¯°¡¢3½ŽÈ·¸


CñoÇE¬­®µ*,

ñò§lŒ¨

§lŒ¨=¬­®µ*,/Œyz,)。
1.2.7 89rs>犘犆犚vw -²µ#‰EÌ
.$‹

B‘ CTAB•/²,v„ DNA。u/²
Ers$‹EDNAj01,œ2‰z›3šE犉3′
5′犎、犇犉犚†犎犘犜Ⅱ ,v4ñ$$·¸»¼。{
›犉3′5′犎 5¦‹Ÿé²j1550bp,犇犉犚5¦‹
Ÿé²j 1110bp,犎犘犜Ⅱ 5 ¦ ‹ Ÿ é ² j
554bp。4ñ$$(6ž1)。–džp1300pPZP
F3′5′HDFRHj¾´(,7Œ,v#‰EDNA
Hj8´(

¹9z¿°²25μLz¿,¹9þ
%j
:94℃ :3min;94℃:30s,55℃;
<45s,72℃=>1min,30½?);72℃‡=>
2min。·¸@A™š›BC\],oÇPCRœ。
1.2.8 8,7rs犚犜犘犆犚vw d‘Omega
DE/²rs PCR ¾#‰†FŒ#‰G
RNA,Fû@A™š›BC·¸RNAEH²«±
²»¼

d‘PremeScriptRTreagentkitDE
(TaKaRaIJ)·¸¹ŒºsmcDNAK。ucD
NAj01´犉3′5′犎 ,v·¸PCR5¦?Â。
1.2.9 8,7rs犛狅狌狋犺犲狉狀xyHz °²PCR
»¼j¾EŒ,vµ#‰/²L‚DNA,d‘
‘M;NOEDIGHighPrimeDNALabelingand
DetectionStarterKitIDE·¸SouthernÀP


udžp1300pPZPF3′5′HDFRj01,d
‘犎犘犜Ⅱ犉 †犎犘犜ⅡRj$$,smE‹Ÿ·
¸QRfûST

°²犈犮狅犚IkàûU,*È·¸
@A™š›BC

‡ú:

›†È‘©VŒ­•–
› EWጭÇXYZ 

·¸ÌÏ

ÿZ

[。
2 œc?Â
2.1 犚狅犫犻狀犪<={|89a}>~9
2.1.1 bXY€89‚ƒ>„… ´rsE
2BŒyz?¶·¸0~4d ¡¢(±2)。œ
žŸ]ú ¡¢*µ·¸õ¤E€‚ƒ†ˆŠ
‹^ÜÝ_`a8b

±cI,a),µd~O¨
678 ! " # $ % &                   36e


ˆŠ‹ ¡¢ 3d‹§lŒ¨w©,j
12.78%; ¡¢4dȵd~O¨f"gìh。
ª€‚ƒ ¡¢2d§lŒ¨w©,j12.22%。
,6]úû ¡¢Ers€‚ƒ†ˆŠ‹*µc
“…µijƒkÇÌÍ

];µdE8


Kûé

ÌÍlŒ]mn

E];S’“£¤

2.1.2 fXY€89‚ƒ>„…  ¡¢
È

´rs€‚ƒ†ˆŠ‹·¸¥¡¢

J±3
,uoF

€‚ƒ†ˆŠ‹^Í¥¡¢3d§l
Œ¨w©

?¶Í12.23%†11.94%。{›€
‚ƒ¥¡¢‹Koû3dŒ¨pqìh,ªˆ
Š‹EŒ¨ìhrs

¥¡¢‹K6é

ŒŽ
6]`ü“

4uŒ¨Ejhü

2.1.3 犃犛†‡89‚ƒ>„… 2£¤†¥¡
¢Eûþ›^ù:¦šÃl‚E AS,¦š AS8
;/©S’“犞犻狉,vt,犞犻狉,v(virulence
genes)olkS’“£¤!u[910],;ª,u/©
Œv¨

J±4,uwF,ASj100μmol/L
‹

xIŒ¨w©

€‚ƒj11.67%,ˆŠ‹
j11.11% 。D*‚ƒ„…†ˆŠ‹Ew«AS±
²»¼

I͈Š‹2AS<100μmol/L‹,§lŒ
¨ü;€‚ƒ

2AS>100μmol/L‹,§lŒ
Ÿ[©;€‚ƒ

2.1.4 ˆ‰Š†‡jk 2Œûþ›°‘¬­
®·¸¯°\]

úû2½ŽE¯°,œžŸ(ž
2),y]¦š¬­®‹,ˆŠ‹†€‚ƒE?¨
?¶Í88.89%†91.33%,23¬­®±²EE¦
¦

?¨]zhü

y¬­®±²j20mg·L-1
‹

{#z|}~`b

4uˆŠ‹†€‚ƒE
¬­®E¯°±²Ålj20mg·L-1。
2.2 8,7rs>tu
2BŒyzEno(±cⅠ,b~i),úû¯
°¡¢45dÈ,FŒE€‚ƒ†ˆŠ‹€b
‚

ªŒEj8µd

±cⅠ,j†k)。
8Eµdé²éÇ3~5cm‹,àì·¸‡¡
¢

d‘©™¡¢,,ud{8é^¦§Ö

±c
Ⅰ,l)。µ*éUÈ,ƒ,u·¸»¼†­„。
2.3 89rs>犘犆犚vw
no187‰rs¬­®µ*,2酰8é
§ÖEˆ*·¸PCR»¼,?¶´犉3′5′犎、犇犉犚
†犎犘犜Ⅱ,v·¸5¦,n·¸@A™š›BC
\]

wß815‰rs*5¦odžEøÇ,{›
9‰EPCRœ(ì(±5),‡Ÿ{V,vôsÇ
rs*›

±5)。
±2  ¡¢‹K´rs€‚ƒ†ˆŠ‹§l
Œ¨E\ˆ
Fig.2 Stabletransformationrateoflilyembryonic
calusandbulbscaleindifferentpreculturetime
±3 ¥¡¢‹K´rs€‚ƒ†ˆŠ‹§l
Œ¨E\ˆ
Fig.3 Stabletransformationrateoflilyembryonic
calusandbulbscaleindifferentcoculturetime
±4 AS±²´rs€‚ƒ†ˆŠ‹§l
Œ¨E\ˆ
Fig.4 Stabletransformationrateoflily
embryoniccalusandbulbscaleindifferent
acetosyringoneconcentration
2.4 89rs>犚犜犘犆犚vw
´PCR»¼no†EøÇE15‰rs*‡·
¸RTPCR»¼,wß89‰F&犉3′5′犎 ,vE
†EøÇ

±6),‡Ÿpq|,v犉3′5′犎 2rs#
‰›·¸kŒºž-

7785ë           ./0,Œ:—˜™犉3′5′犎 ,vŒOTÌBrsRobinaE67
l2 <=tu‹ŒŽRSTU>ˆ‰Š‘’S“”
Table2 Thesensitivitiesofregeneratedbulbletsandembryoniccalusfromlilytohygromycin
¬­®±²
Concentrationof
hygromycin/(mg·L-1)
‡8ˆŠ‹Regeneratedbulblets €‚ƒEmbryoniccalus
{#z,
No.ofexplants
]ld?¨
Frequencyof
adventitiousbuds/%
{#z,
No.ofexplants
]ld?¨
Frequencyof
adventitiousbuds/%
0 60 88.89±2.48a 50 91.33±2.33a
5 60 73.89±1.91b 50 75.33±5.14b
10 60 43.89±3.52c 50 44.67±3.09c
15 60 5.56±2.49d 50 8.00±1.98d
20 60 1.67±1.74de 50 0.67±1.12de
25 60 0±0.00e 50 0±0.00e
  Ê:ž›,2ºž3ÅZ+]E“‰³‡±¯Š‹”;¼&,2ȯ]¼ˆŒŽIžä2犘=0.05ñ‰¢[。
  Note:Datainthetableare“average±standarderror”ofthererepeattests.Datainthesamecolumnwithdifferentlowercaselettershave
significantdifferencesat犘=0.05level.
A.犉3′5′犎;B.犇犉犚;C.犎犘犜Ⅱ;M.Marker;
P.dž¾´(;WT.FŒ#‰;1~9.Œ,v#‰
±5 rs¬­®µ#‰E犉3′5′犎、犇犉犚†犎犘犜Ⅱ
,vEPCR»¼
A.犉3’5’犎;B.犇犉犚;C.犎犘犜Ⅱ;M.Marker;P.Positive
control;WT.Nontransformed;19.Transformedplantlets
Fig.5 PCRdetectionoftransformedplantletsforpresence
of犉3′5′犎gene,DFRgeneand犎犘犜Ⅱgene
2.5 89rs>犛狅狌狋犺犲狉狀xyHz
jk‡·ÃÄ»¼†E,v͑ŒšÇrs,
v„›
,´
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[3] ™»¼,꽩,¾¿¯,Œ.ÀÙrsEµ?8{Á¯ÂS
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狀犲狊犲犃犵狉犻犮狌犾狋狌狉犪犾犛犮犻犲狀犮犲犅狌犾犾犲狋犻狀,2005,21(3):240242.
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