図1-5. トマトの雌蕊特異的発現遺伝子 (PSGs) の開花日及び開花後4日目での発現 (続き)
( ) RNA PSGs
(Gibberellic acid, GA) D) 72 PSGs 1
(RPKM 0.5) GENE-E Peason correlation
D0, - ; D4p - 4 ( ); D4e - 4 ( ); d0
pro - ; d4e pro - 4 ( ); I GA ; II GA
図1-6. 雌蕊特異的GA応答遺伝子 (Pistil-specific GA-responsive genes; PS-GRGs)
SlDELLA (Gibberellic acid, GA)
(WT) procera (pro) RNA-seq RPKM
DBA days before anthesis ( ) ; DAA days after anthesis ( ); p ; e (
) GA ; GA
0 5 10 15
20 WT pro
0 1 2 3 4
WT pro
0 0.5 1 1.5 2
WT pro 0
1 2 3 4
WT pro
0 2 4 6 8
WT pro
0.00 5.00 10.00 15.00 20.00
WT pro
0 2 4 6
8 WT pro
0 10 20 30 40
WT pro
0 1 2 3 4 5
WT pro
0 2 4
6 WT pro
0 10 20 30 40
WT pro
0 2 4 6 8
WT pro
0 10 20 30
WT pro 0 10 20 30 40
WT pro
0 2 4 6 8
WT pro
0.00 1.00 2.00 3.00
WT pro
0 1 2 3 4
WT pro
0 2 4 6 8 10
WT pro
0 0.5 1 1.5
WT pro
0 5 10 15 20 25 30
WT pro
0 1 2 3 4
WT pro 0
2 4 6 8 10
WT pro
0 1 2 3 4 5
WT pro
0 0.2 0.4 0.6 0.8 1
WT pro
0 2 4 6
WT pro
0 2 4 6
WT pro
0 1 2 3 4 5
WT pro
0 5 10 15 20
25 WT pro
0 20 40 60 80 WT pro 0
0.5 1 1.5
WT pro
0 0.5 1 1.5
2 WT pro
0 0.5 1 1.5
WT pro
0 5 10 15 20
25 WT pro
Solyc04g014750 Solyc02g077170
Solyc09g056040
Solyc03g025240 Solyc07g053400 Solyc02g085290
Solyc02g067630 Solyc04g007310 Solyc01g008540
Solyc04g074890 Solyc05g052440
Solyc09g011280
Solyc06g070950/OPE1
Solyc06g048400 Solyc07g007520 Solyc07g032700 Solyc06g073100
Solyc11g005540 Solyc02g022860 Solyc01g081360
Solyc01g106730 Solyc04g058040
Solyc03g058330 Solyc05g005240/SlINO
Solyc01g095760
Solyc10g044690 Solyc07g064780 Solyc11g043160 Solyc09g089590
Solyc09g011290 Solyc08g015750
Solyc02g093540 Solyc11g072650
Solyc10g051370/OPE5
0 20 40 60 80 100 120
WT pro
RPKMRPKMRPKMRPKMRPKMRPKMRPKM
0 5 10 15 20
2DBF 6DPE 4DPAP 4DPAT 4DPGT
TPM
(Solyc09g011290)
0 5 10 15 20 25
2DBF 6DPE 4DPAP 4DPAT 4DPGT
TPM
(Solyc01g010600)
0 3 6 9
2DBF 6DPE 4DPAP 4DPAT 4DPGT
TPM
(Solyc10g017990)
NP NP
0 5 10 15 20
2DBF 6DPE 4DPAP 4DPAT 4DPGT
TPM
(Solyc09g011290)
0 5 10 15 20 25
2DBF 6DPE 4DPAP 4DPAT 4DPGT
TPM
(Solyc01g010600)
0 3 6 9
2DBF 6DPE 4DPAP 4DPAT 4DPGT
TPM
(Solyc10g017990)
NP NP
2DBF : 2 day before flowering 6DPE : 6 day post emasculation 4DPAP : 4 day post artifical pollination 4DPAT : 4 day post auxin treatment 4DPGT : 4 day post GA treatment NP : data not provided
TPM : Number of transcripts per million clean tags 0
100 200 300 400
2DBF 6DPE 4DPAP 4DPAT 4DPGT
TPM
(Solyc03g007780)
0 200 400 600
2DBF 6DPE 4DPAP 4DPAT 4DPGT
TPM
(Solyc08g067640)
Invertase inhibitor homolog SlATHB13/23-like
SlCKX8
図1-7. インベルターゼインヒビター様遺伝子は着果に関連した発現パターンを示す
A) INVERTASE INHIBITOR HOMOLOG (Solyc09g011290) (Auxin
GA ) (Tang et al., 2015 ) 2DBF, 2 ; 6DPE, 6
6 ; 4DPAP 4 ; 4DPAT, 4 ; 4DPBT, 4 GA3 ;
np ; TPM, transcripts per milion cleanup reads B) GA ETHYLENE INSENSITIVE 3 (EIN3)
(Inv) (Suc) (Glc)
EIN3 INVERTASE INHIBITOR (INVINH) Inv GA
INVERTASE INHIBITOR HOMOLOG Inv Suc
GA GA INVERTASE INHIBITOR HOMOLOG
Vuc-Inv, ; CW-Inv,
A)
老化
Glc
EIN3
エチレン応答 CW-Inv Vuc-Inv
Suc
糖シグナル
INVINH
B)
糖シグナル GA
SlDELLA
転写抑制因子
CW-Inv Solyc09g011290
Vuc-Inv
糖シグナル GA
SlDELLA
転写抑制因子
CW-Inv Solyc09g011290
Vuc-Inv
胚珠の老化 胚珠の老化
着果の阻害 着果の阻害
[GAシグナル OFF] [GAシグナル ON]
56
着果
*
* * ** *
A)
C)
B)
図1-8.EGG CELL 1 (EC1) 様遺伝子の雌蕊特異的発現遺伝子の選抜
A) EC1 (Control) SAND EC1
(P) (A) (DBF) (DAF) B)
EC1 C) EC1
( ) ( ) D) EC1 E)
EC1 = (SD) DB A days before anthesis ( );
D)
Solyc11g005500 Solyc11g005540 Control Solyc05g010190
E)
0 2 4 6 8 10
WT pro
Solyc11g005540
0 5 10 15 20
WT pro
Solyc04g071640
0 2 4 6 8
10 WT pro
Solyc12g009850
0 2 4 6 8
WT pro
Solyc11g005500
0 20 40 60
80 WT pro
Solyc05g010190
RPKMRPKM
花粉管
卵細胞 助細胞
卵細胞の活性化
精細胞の活性化
細胞膜融合
さらなる受精の抑制
EC1分泌 胚珠
精細胞
図1-10. 第１章の解析のまとめ
SlDELLA (Gibberellic acid, GA) (PS-GRGs)
GA
SlDELLA GA procera
pat-2
PSGs : 108
遺伝子組織別トランスクリプトーム解析
細胞種別発現データ
胚珠及び種子優先的発現遺伝子が多数
procera
変異体発現データGAシグナルとの関連の評価
着果との関連性
開花後抑制：14
遺伝子恒常的に抑制：8
遺伝子 その他：12 遺伝子機能未知遺伝子が多数単離された
胚珠発達・受精関連遺伝子が procera 変異体で変動した 抑制的な制御を受ける遺伝子が６割以上であった
SlDELLA依存的なGAシグナルで 誘導される着果に関連したPSGs
58
2
SlDELLA (CDIs)
2 SlDELLA (CDIs) 2-1
1 108 (PSGs)
(GA) 34
GA GA
GA
GA DELLA 26S
DELLA (DELLA-INTERACTING PROTEINs DIPs)
DIPs ( 8)
GA GA DIPs
GA DIPs
DELLA DIPs 3
DELLA
DELLA GA GID1
DELLA DELLA
DELLA 26S (McGinnis et al., 2003; Wang
et al., 2009; Wang and Deng, 2011) DELLA
SUMO DELLA DELLA
GA (Dai and Xue, 2010; Qin et al.,
2014; Conti et al., 2014) SECRET AGENT (SEC) SPINDLY (SPY)
DELLA DELLA DIPs
(Zentella et al., 2016, 2017) SEC DELLA O-GlcNAcylation
DELLA DIPs (Zentella et al., 2016) SPY
DELLA O-fucosylation DELLA DIPs (Zentella et al.,
2017)
DELLA DIPs DELLA
DIPs DNA
DELLA
DNA DNA
DELLA DNA
DIPs DELLA
DIPs GA
SlDELLA
SlDELLA GA
cDNA
(Y2H) SlDELLA
2-2
2-2-1 GID1
(Y2H) SlDELLA GA
GID1 AtGID1a
(At3g05120) AtGID1b (At3g63010) AtGID1c (At5g27320) SGN
(https://solgenomics.net/) BLASTP GID1
Geneious 10.2.2
2-2-2 SlDELLA
Yeast two hybrid (Y2H) 2 (X Y)
( 2-4) 2 Bait
( X) Prey ( Y)
Bait Prey Bait
(Saccharomyces cerevisiae)
GAL4 DNA (BD) Prey
GAL4 (AD)
(X:BD Y:AD)
( )
( )
2-2-2-1 Y2H
SlDELLA gDNA
(CDS) (FL)
PCR 2-2
SlDELLA-F1 SlDELLA-R1
In-Fusion ( 2-1) pCR8 (Invitrogen,
USA) pCR8-F1 pCR8-R2
PCR 1.0% DNA
Wizard® SV Gel and PCR Clean-Up System (Promega, USA)
pCR8 In-Fusion
Competent Quick DH5α (100 µg/l)
LB PCR
LB 37 °C/16 PureYield™ Plasmid Miniprep System (Promega, USA)
pCR8:SlDELLA^FL
Duke Tai-ping Sun Bait
pDEST32 Prey pDEST22
SlDELLA CDS 2 LR Clonase
II (Invitrogen USA) LR (Top10)
Bait LB (10 µg/ml )
Prey LB (100 µg/ml )
PCR PCR SlDELLA
pDE32:SlDELLA^FL pDE22:SlDELLA^FL
pDE32:empty pDE22:empty
2-2-2-2
( 2-4)
PJ69-4A YPAD 30°C 2 ~ 3
500 µl 0.9% NaCl (Yeast suspension mixture 100 mM LiAc, 0.5 x TE in water) (Transformation mixture 100 mM LiAc 40% PEG-3350 1 x TE in water) (
) ( 13000 rpm) 20 “
2 ml
10 700 µl 1.5 ml
10 ’
10 µl denatured, sheared salmon sperm DNA (10 µg/ µl) (Signma
USA) (Bait Prey) 400
ng 1000 ng 100 µl
1.5 ml 30°C 30
’ 88 µl DMSO (Dimethyl sulfoxide, Sigma)
42°C 15 45
30
15000 rpm 10 “ 1 ml 1×TE
15000 rpm 10 “
50-100 µl 1×TE 1 [SD-WL
(W) (L) ] 30°C 2 ~ 3
Bait Prey
(LEU2) (TRP1)
PJ69-4A 2
SD-WL 2
2 [SD-WLH (W)
(L) (H) ] 3 [SD-WLHA,
(W) (L) (H) ]
2-2-3 SlDELLA GID1 2-2-3-1 Yeast two hybrid
SlGID1s cDNA
CDS (FL) PCR
2-2
In-Fusion ( 2-1) 1.0 %
DNA Wizard® SV Gel and PCR Clean-Up System (Promega,
USA) EX-Taq HS (Takara, Japan)
3’ (dA) dA DNA
TA pCR8
(Competent Quick DH5α) PCR
37°C 16 PureYield™ Plasmid Miniprep System (Promega, USA)
pCR8:SlGID1b-1^FL pCR8:SlGID1b-2^FL pCR8:SlGID1ac^FL Prey
pDEST22
LR Clonase II (Invitrogen USA) LR
Competent Quick DH5α LB (100 µg/ml )
PCR
SlDELLA
Bait
pDE32:SlGID1b-1^FL pDE32:SlGID1b-2^FL pDE32:SlGID1ac^FL
GA procera (pro) procera2 (pro2)
SlDELLA GID1
gDNA SlDELLA
( 2-1) ( SlDELLApro SlDELLApro2
) SlDELLA
pCR8:SlDELLApro^FL pCR8:SlDELLApro2^FL Prey pDE22:SlDELLApro^FL pDE22:SlDELLApro2^FL
1 [SD-WLH (W)
(L) (H) ] 2 [SD-WLHA
(W) (L) (H) (A)
] 3 [SD-WLH+3-AT (W)
(L) (H) 3-Amino-1,2,4-triazol (3-AT)
] Bait Prey
SD-WL 30°C 2 ~ 3
1 ml 0.9 % NaCl
10 OD600
OD600 = 0.25 0.05 1.2 µl
30 °C 3 4
2-2-4 cDNA CDIs
Yeast two hybrid (Y2H) DELLA
(Candidates of DELLA-interactor, CDIs) SlDELLA DELLA
GRAS GRAS
LHRI VHIID LHRI PFYRE SAW ( 4) DELLA
(1 – 588 aa) Bait
( 2-5), (Creative Biolabs USA) SlDELLA SAW (496 – 573 aa) PFYRE (390 – 495 aa) Bait
cDNA ( 1)
Y2H Matchmaker GAL4-based two-hybrid system (Clone-tech) Bait
Prey pGBKT7 pGADT7
SD-Leu-Trp-His-Ade (SD-WLHA) X-gal assay
CDS CDIs
(Hibrigenics, France) SlDELLA GRAS (193 – 588 aa) Bait
cDNA ( 2)
Y2H ULTImate Y2H SCREEN (Hibrigenics, France)
2-2-5 RNA-sequence CDIs
(Tomato Genome Consortium, 2012; Ezura et al.,
2017) CDIs (RPKM )
2-2-6 SlDELLA CDIs
2-2-6-1 Y2H
CDI CDS TA In-Fusion
pCR8 PCR
2-2 ( 2-1)
CDS 1.0% DNA
Wizard® SV Gel and PCR Clean-Up System (Promega, USA) SAMS1
SAMS2 SAMS3 SAMS4 CDI1 CDI2 CDI3 CDI4 CDI5 CDI6 CDI7 CDI8 CDI10
SlCNR13/CDI16 TA 2-2-3
TA CDI11 CDI12 CDI13 CDI14 CDI15
CDI17 In-Fusion CDI5 cDNA
(LEFL1036CB09) CDI9
2-2-2-1 pCR8
, CDIs PCR In-Fusion 5 µl
(Top10 Competent Quick DH5α) (Toyobo, Japan)
LB (100 µg/ml )
TA In-Fusion
M13 Forward
PCR 1
37°C 16 PureYield™ Plasmid Miniprep System
(Promega, USA) M13 F
M13 R
pCR8:SAMS1^FL pCR8:SAMS2^FL pCR8: SAMS3^FL pCR8: SAMS4^FL pCR8:CDI1^FL pCR8:CDI2^FL pCR8:CDI3^FL pCR8:CDI4^FL pCR8:CDI5^FL pCR8:CDI6^FL pCR8:CDI7^FL pCR8:CDI8^FL pCR8:CDI10^FL pCR8:CDI11^FL pCR8:CDI12^FL pCR8:CDI13^FL pCR8:CDI14^FL pCR8:CDI15^FL
2-2-2-1 Bait
pDE32:SAMS1^FL pDE32:SAMS2^FL pDE32:: SAMS3^FL pDE32::
SAMS4^FL pDE32::CDI1^FL pDE32::CDI2^FL pDE32::CDI3^FL pDE32::CDI4^FL pDE32::CDI6^FL pDE32::CDI7^FL pDE32::CDI8^FL pDE32::CDI10^FL pDE32::CDI11^FL pDE32::CDI12^FL pDE32::CDI13^FL pDE32::CDI14^FL pDE32::CDI15^FL
2-2-6-2
2-2-2-2 SlDELLA CDI SAMS
SlCNR13 SlDELLA
SD-WLH
CDI Bait
pDE22:empty SD-WLH
CDI Bait pDE22:SlDELLA^FL
SD-WLH
3-AT
2-2-6-3
Bait Prey SAMSs
SlCNR13
70% 15
ml
1 cm 30
20 30
4
SD-TL
0.9% NaCl 10000 rpm 30 “
DW OD600=10 1 ml 250 µl
10000 rpm 30 “ 100 µl DW 0.2 N
NaOH 5 10000 rpm 30 “
2x Laemmli 50 µl 5 100 10000 rpm
1 “ ( 20 )
1×Tris-glycine 10×Tris-glycine (250 mM Tris pH8.3, 1.92 M glycine, 1% SDS) 10
5 µl (Precision Plus Protein Dual color standards; 10 - 250 kD) 1.5 µl
200 V (15 mA/ ) 15 200 V (20 mA/ ) 60
( )
PVDF (Hybond P. Amersham) ( 6×8.5 cm) 100%
30 Transfer (25 mM Tris pH8.3
192 mM glycine, 20% v/v methanol) Transfer
15
(+) PVD F
(-)
Transfer DW Electrode module
” V, A ( . 200 V/36 mA/30 min)
DW CBB
Blocking 2
4 1 x PBS (0.1% Tween20) Blocking
5% 5% 5 ml + 1
5 µl (1:3000 ) 1 DW 2
1 x PBS (0.1% Tween20) 10 3
, 5% 5 ml + 2 0.5 µl (1:10000 )
1 DW 2 1 x PBS (0.1% Tween20)
10 3 SuperSignal West Dura SuperSignal West
Pico (Thermo Fisher, USA)
Stable Peroxide Solution Luminol/Enhancer Solution
5 X
2-3
2-3-1 SlDELLA GID1 DELLA
SlDELLA GA GIBBERELIN
INSENSITIVE DWARF1 (GID1) GA
GID1 3
GID1 GID1b GID1ac
2 GID1b [SlGID1b-1
(Solyc09g074270) SlGID1b-2 (Solyc06g008870)] 1 GID1ac [SlGID1ac (Solyc01g098390)] (Voegele et al., 2011; 2-1)
GID1 (Østerlund, 2001)
GA DELLA (Murase et al., 2008; Shimada
et al., 2008) 3 GA (
2-2) GID1 GA
SlGID1b-1 SlGID1b-2
SlGID1ac (
[Tomato Genome Consortium, 2012; Zhang et al., 2017; Ezura et al., 2017], 2-3)
3 GID1 GA
SlDELLA
Yeast two hybrid (Y2H) ( 2-4)
Y2H 2
Y2H GAL4 DNA (BD)
(AD)
GAL4 ( )
BD
DELLA
(de Lucas et al., 2008) Y2H SlDELLA
SlDELLA Bait
Prey
( 2-5 ) SlDELLA Prey
Bait ( 2-5
) SlDELLA Prey
DELLA Bait
(Fukazawa et al., 2014)
SlGID1b-1 SlGID1b-2 SlGID1ac Bait GA
GA SlDELLA GID1 DELLA
GA ( GA ) GA
( GA ) GA
DELLA GA C
(Lid) Lid (Murase et
al., 2008; Hao et al., 2013) SlDELLA SlGID1s SlGID1ac GA
DELLA ( 2-7B) GID1b
(SlGID1b-1 SlGID1b-2) GA DELLA GA
( 2-7B) GIDb
GA GID1ac (Griffiths et
al., 2006; Nakajima et al., 2006) Yamamoto (2010) AtGID1b
( 2-1A) AtGID1a AtGID1c GA
GA
GA (2
GmGID1b-3 BrGID1b-GmGID1b-3) GID1ac GA
GID1
GA DELLA
GID1ac SlGID1ac SlDELLA GA
SlGID1
( 2-1B)
DELLA GID1 DELLA
(Ariizumi et al., 2008) SlGID1ac GID1b
(SlGID1b-1 SlGID1b-2) 1/2.5 1/43.6 GID1ac
GA SlGID1ac
GID1b
GID1 DELLA
SlDELLA (SlDELLApro SlDELLApro2) SlGID1s
GA procera (pro) VHIID
DELLA (DIPs) GA
(Jones et al., 1987; Bassel et al., 2008; Carrera et al., 2012; Levine et al., 2015) procera2 (pro2)
SAW GA pro2 pro
GA pro pro2
( 2-6) GA GID1
SlDELLA SlDELLA
(Ariizumi et al., 2008) pro pro2
GRAS GID1
(Hirano et al., 2010) SlDELLA
(SlDELLApro SlDELLApro2) SlGID1s
SlDELLAWT SlDELLApro2 GA
GID1b SlGID1ac
( 2-3C) SlDELLApro SlGID1
DELLA SAW DELLA
SLR1 SAW [slr1-3 (W609stop)
slr1-4 (W620stop)] [slr1-7 (T617P)] DELLA
(Hirano et al., 2012) SAW GAF1 ARR2
(Fukazawa et al., 2014; Marín-de la
Rosa et al., 2015) DELLA
2 GA (GA )
SLR1 SAW G576V
DELLA OsGID1
(Hirano et al., 2010) pro2 2 565
(R) R621A OsGID1 SLR1
(Hirano et al., 2010) SAW
SlGID1 DELLA pro2
SlDELLA-GID1 DELLA
pro2 SlDELLA SlGID1
GA DIPs
SlDELLA
2-3-2 cDNA CDIs
1 6 PFYRE 14 SAW
( 2-4, 2-9) 2 GRAS
37 ( 2-4, 2-9)
7 (A B)
2-3-3 CDIs SlDELLA
1 2
SlDELLA ( 2-5)
2-3-3-1 1 SlDELLA
SAMS4 CNR13
SAMS SlDELLA
S-adenosylmethionine synthase (SAMS) (PAs) S-adenosyl methionine (SAM)
4 SAMS (SAMS1- SAMS4) SAM DNA
DNA
DNA
(Xiao et al., 2006) SlDELLA SAMS GA
SAMS SlDELLA
SAMS1 SAMS2 SAMS3 SAMS4 Bait SlDELLA Prey Y2H
( 2-10)
ADE2
1 SAW
(79 ) SAW SAMS
( ; Match-maker ;
ProQuest.) Match-maker system
(Rajagopala et al., 2009; Caufield et al., 2012)
C SlDELLA GRAS (SlDELLA-GRAS) Bait
SMAS1-SAMS4 Prey Y2H
( 2-11) SlDELLA-GRAS
( 2-11)
SAMS3 SAMS4 SlDELLA-GRAS
SAMS3 SAMS4 ’ SlDELLA-GRAS
HIS3 SAMS
SAMS METHIONINE
ADENOSYLTRANSFERASE 2A (MAT2A) DNA
(Katoh et al., 2011; Kera et al., 2013) ( 2-12A)
SAMSs DNA (SAMSs Bait
2-10) SlDELLA-GRAS
(SAMS Prey 2-11) SAMSs
( 2-12B) Y2H
SAMSs SlDELLA Y2H
’
CNR13 SlDELLA
SlDELLA SlCNR13 SlCNR13 Cell number regulator
(CNR) FW2.2/SlCNR2
FW2.2/SlCNR2 30 QTL
fw2.2
(Frary et al., 2001) FW2.2/SlCNR2
CASEIN KINASE II (CKII)
(Cong and Tanksley, 2006) DELLA SLR1 CASEIN
KINASE I (CKI) Early Flowering 1 (EL1)
(Dai and Xue, 2010) SlDELLA SlCNR13
SlDELLA
SlDELLA SlDELLA SlCNR13
3-AT SlCNR13
( 2-13A, B) SlDELLA SlCNR13
FW2.2/SlCNR2 CNR
(Cong and Tanksley, 2006)
2-3-3-2 2 SlDELLA
SlDELLA 17 CDIs SlDELLA
CDI3 CDI5 CDI7 (
2-14, 15, 16) CDI11 CDI12
Split Ubiquitin Yeast two hybrid (Dünkler et al., 2012) Split Ubiquitin based Membrane Yeast Two hybrid (Snider et al., 2010) Y2H
2-3-5 2
2 , SlDELLA GA
1) SlDELLA GA SlGID1 SlDELLA
2) SlDELLA ( 2-20)
1) SlDELLA 3 SlGID1 GA GA
GID1ac GA
DELLA
GA GID1
1) 2) SlDELLA
3 (CDI3, CDI5, CDI7) 3 CDI5
図2-1. GA受容体GIBBERELLIN INSENSITIVE DWARF 1 (GID1) ファミリーの系統樹
(Gibberellic acid, GA) GID1 GA GI D1ac
GID1b 1 GI D1ac 2 GI D1b GA
( ) GA DELLA ( Yamamotoet al., 2010) At,Arabidopsis
thaliana; Os,Oryza satiba; Gm,Glycine max; Br,Brassica rapa; VIT,Vitis vinifera; Sk,Selaginella kraussiana
*
*
*
dicot GID1b group
dicot GID1ac group