Campania - ENEA UT-AGRI

09 Aprile 2014 FOOD FLAVOUR
Industria 2015
PL 30
Determinazione dei “markers trascrizionali”
per tipicità ed origine geografica.
Obiettivo: Sviluppo di una metodologia di profiling trascrizionale (analisi
microarray) per la determinazione di profili molecolari globali in genotipi di fragola
coltivata (Fragaria x ananassa) (var. candonga), per l’individuazione di geni chiave
associati all’espressione di caratteri legati alla tipicità e all’origine geografica.
Loredana Lopez -‐ Fabrizio Carbone UTTRI-‐GENER Laboratorio di Gene4ca e Genomica per l’Energia e L’Ambiente Responsabile Laboratorio: Gaetano Perro?a PL 30
Saggio sperimentale –Fase 1
Progettazione e sintesi del DNA chip array di Fragola
Sequenze di fragola
(Fragaria vesca,
Fragariaxananassa)
TIGR plant transcript
assemblies databases
Sintesi in situ
di 93.300 oligonucleotidi
CustomArray™ Technology
(CombiMatrix)
DNA chip array con 93.300 oligonucleotidi fragola-specifici
Loredana Lopez – UTTRI-‐GENER PL 30
Saggio sperimentale –Fase 2
Analisi dei dati
Saggio di profiling trascrizionale
Frutti di fragola
liofilizzati
Laser
per Cy5
Scanning
Emissione
Estrazione RNA totale
ProbeWeaver
Acquisizione immagini
Cy5
Amplificazione RNA
e marcatura
Normalizzazione intensità
spot
GeneSpring GX
Ibridazione
Analisi dati Identificazione geni
differenzialmente
espressi Chip con 93.300 sequenze
MapMan
Funzione metabolica dei
geni differenzialmente
espressi Loredana Lopez – UTTRI-‐GENER PL 30
Campionamento
4 Aree Geografiche:
NORD
Regioni
Veneto
SUD Trentino
Campania
Basilicata
Aziende
1
1
3
3
Repliche
3
3
3
3
3
3
9
9
Saggi
d'ibridazione
24 Chip-array
Loredana Lopez – UTTRI-‐GENER PL 30
Risultati
Analisi GS1: 8 campioni Basilicata A-‐B-‐C; Campania A-‐B-‐C; Veneto A; Tren:no A 17,702 UNIGENES Analisi ANOVA False Discovery Rate < 0.05 Mul;ple Test Correc;on: Bonferroni Score plot 4624 UNIGENES (26%) Differenzialmente espressi in almeno 1 degli 8 campioni PCA dei profili trascriHomici dei campioni di fragola Loredana Lopez – UTTRI-‐GENER PL 30
Risultati
Analisi GS2: 3 campioni Basilicata; Campania ; Nord 17,702 UNIGENES Score plot Analisi ANOVA False Discovery Rate < 0.05 Mul;ple Test Correc;on: Bonferroni 2964 UNIGENES (17%) Campania Basilicata Differenzialmente espressi in almeno 1 delle 3 aree geografiche Nord PCA dei profili trascriHomici dei campioni di fragola Loredana Lopez – UTTRI-‐GENER PL 30
Risultati
Metabolismo Overview Map 413/2964 geni Nord/Campania Nord/Basilicata Gene ID
Verde, down-‐regola4 Rosso, up-‐regola4 Secondary metabolism: Phenylpropanoids (17)
N/C
N/B
C/B
Gene ID
Cell wall.pectin*esterases (15)
N/C
N/B
C/B
ex678491
4-‐coumaroyl-‐CoA synthase family protein
-‐2.3
-‐1.6
1.5
ay324809 ta9040_57918
pectinesterase family protein
-‐3.4
-‐5.0
-‐1.4
NICOTINAMIDASE 2Cell ; catalytic/ nicotinamidase
Gene ID
wall degradation (13)
N/C-‐1.6 N/B-‐2.5 C/B-‐1.5
ay357183 co817421
pectinesterase
1.5
-‐1.5
PAL2; phenylalanine mmonia-‐lyase2.3(pectinase) ai795138
paolygalacturonase family protein
2.1 2.6
1.8 4.5 -‐1.21.7
ta10360_57918
pectinesterase f
amily p
rotein
-‐4.3
-‐5.9
-‐1.4
co378880
C4H; C
INNAMATE-‐4-‐HYDROXYLASE
-‐1.2
ay282613
polygalacturonase (pectinase) family protein 3.6
6.8-‐1.3 1.9-‐1.1
co816661
C4H; C
INNAMATE-‐4-‐HYDROXYLASE
-‐1.9
-‐1.5
ta12280_57918
pectinesterase f
amily p
rotein
1.0
-‐1.6
-‐1.6
co379133
pectate l yase family protein ( 6)
-‐1.6-‐2.9 -‐1.1
Gene Ita541_3747
D
Major CCINNAMATE-‐4-‐HYDROXYLASE
HO metabolism.synthesis.starch N/C-‐1.4-‐1.8 N/B
C/B
C4H; -‐2.8
ta13356_57918
pectinesterase f
amily p
rotein
-‐2.0
-‐2.0
-‐1.0
co380475
polygalacturonase (pectinase) family protein
-‐1.4
-‐1.0
1.4-‐1.5
ta9163_57918
C4H; CINNAMATE-‐4-‐HYDROXYLASE
-‐1.8
ay518344
glucose-‐1-‐phosphate adenylyltransferase
ta9598_57918
pectinesterase
2.0(pectinase) 1.3 family p-‐1.5
co381010
polygalacturonase rotein 1.5 2.4 -‐1.8 -‐2.3
4.1-‐3.2 1.7-‐3.5
Gene ID
Secondary metabolism: Flavonoids (6)
N/C N/B2.1 C/B1.8
dy667987
4-‐coumarate-‐CoA l igase
-‐1.2
co381913
pectate l
yase f
amily p
rotein
-‐1.5
-‐1.7
-‐1.1
co816930 pectinesterase
glucose-‐1-‐phosphate adenylyltransferase
-‐1.9
-‐1.9
ta9928_57918
1.9
1.4
-‐1.4 1.0
ta9431_57918
oxidoreductase, 2OG-‐Fe(II) oxygenase family protein
-‐3.9
2.0-‐1.9 7.8-‐2.7
ta10539_57918
4-‐coumarate-‐CoA l
igase
-‐1.4
dv438382
pectate l yase family 1.4
-‐1.1
Gene Ico380607
Ddv438579
Fermentation (6)
N/B
C/B
pectinacetylesterase family protein
-‐1.5 protein-‐1.9
-‐1.3 N/C
adenylyltransferase
1.1 1.5
-‐4.4
dv440593 glucose-‐1-‐phosphate caffeoyl-‐CoA 3-‐O-‐methyltransferase, putative
-‐1.3 -‐4.1
-‐1.8
-‐1.4
ta9432_57918
oxidoreductase, 2
OG-‐Fe(II) o
xygenase f
amily p
rotein
-‐3.7
1.6
5.8
dv440413 f amily polygalacturonase (pectinase) rotein
2.5
2.4
-‐1.1
co382107 dy668456
pectinacetylesterase rotein
-‐2.9
-‐3.9family p-‐1.3
ta9791_57918
L-‐lactate dehydrogenase, pputative -‐2.2
2.1
caffeoyl-‐CoA 3p-‐O-‐methyltransferase, putative
-‐1.5 -‐1.0
-‐2.4
-‐1.6
ex681803
ADP-‐glucose yrophosphorylase f
amily p
rotein
2.6
1.9
-‐1.4
dy668458
UDP-‐glucosyl t
ransferase f
amily p
rotein
4.6
3.4
-‐1.4
dv440645
pectate l yase family protein 1.7
-‐5.4
-‐9.4
dy669772
pectinacetylesterase amily protein
-‐1.1
-‐2.6
ta9008_57918
caffeoyl-‐CoA 3f-‐O-‐methyltransferase, putative -‐2.9
-‐1.5 -‐3.0
-‐2.2
-‐1.5
dv439286
pyruvate d
ecarboxylase f
amily p
rotein
-‐3.8
1.3
ex687113
UDP-‐glucosyl t
ransferase f
amily p
rotein
5.2
5.1
-‐1.0
polygalacturonase (pectinase) family roteinN/B
1.3
1.1-‐1.5
co816963
starch ex682543
synthase
-‐1.11.2 C/B
-‐1.6
Gene IDex661508
Secondary metabolism: Wax (f4)
N/Cp-‐1.2
pectinacetylesterase amily p
rotein
-‐1.4
-‐1.7
ay285922
cinnamoyl-‐CoA r
eductase 1
1.8
1.9
1.0
cx661806
aldo/keto reductase, putative
-‐1.6
-‐1.5
1.1
ta10720_57918
polygalacturonase family protein
-‐2.0
-‐2.31.1
ta10152_57918
pyruvate decarboxylase amily protein ta11349_57918
alpha-‐amylase/ ifsoamylase 36.4(pectinase) 1.8 1.2 1.8 1.1-‐1.9
4.0
2.2
co380204
membrane bound O-‐acyl transferase / w1ax synthase-‐related 1.5 -‐1.2 -‐2.2
1.6
ta10698_57918
cinnamoyl-‐CoA rrotein
eductase 1.7
-‐1.0
ay357182
pectinesterase f
amily p
5.5
ta9907_57918 oxidoreductase
5.0
8.4
1.7
polygalacturonase (pectinase) family protein
-‐1.1
-‐2.4
-‐2.3
co381406
3-‐chloroallyl ata10721_57918
ldehyde -‐4.9
-‐1.1
4.6
co378812
CINNAMYL ALCOHOL DEHYDROGENASE 2/ z4.6
inc i on binding
-‐4.1
1.2
dy668259
3-‐KETOACYL-‐COA SYNTHASE 9 dehydrogenase
5.0 1.2 -‐5.01.1
ay357184
pectinesterase
-‐1.2HpOMOLOG -‐2.5
ta9959_57918 pectate l yase family rotein-‐2.9
-‐3.9
-‐4.7
ex661527
ELI3-‐1 (
ELICITOR-‐ACTIVATED G
ENE 3
-‐1) z
inc i
on b
inding
-‐1.8
-‐1.7
1.1
ta10191_57918
3-‐KETOACYL-‐COA SYNTHASE 5/fatty acid e longase 3.4
-‐1.1
co381719
3-‐chloroallyl aldehyde ehydrogenase/ -‐1.2
1.0
ex662828
pectinesterase family pd
rotein
-‐2.1 oxidoreductase
1.6 3.7 3.3 -‐1.3
ta9645_57918
ELI3-‐2 (
ELICITOR-‐ACTIVATED G
ENE 3
-‐2
1.5
1.4
-‐1.1
ta12899_57918
CER1 protein, putative 1.8
-‐1.01.0
ta10249_57918
pectinesterase
-‐1.7
-‐4.4 1.8 -‐2.6 -‐4.3
ta13187_57918
3-‐chloroallyl aldehyde dehydrogenase
4.4
Loredana Lopez – UTTRI-‐GENER Campania/Basilicata PL 30
Risultati
Gene ID
Light Reactions (32)
N/C N/B C/B
co817381
LHB1B2; chlorophyll binding 3.5 3.6 1.0
cx661207
CAB3 (CHLOROPHYLL A/B BINDING PROTEIN 3)(PS II)
6.6 5.7 -‐1.2
ex659419
LHB1B1; chlorophyll binding 1.4 1.3 -‐1.1
ex666515
LHB1B2; chlorophyll binding 1.4 1.4 1.0
ex668563
LHCB3 (LIGHT-‐HARVESTING C
HLOROPHYLL B
-‐BINDING P
ROTEIN 3
)
2.0 1.7 -‐1.2
Fotosintesi ex686440
LHB1B2; chlorophyll binding 1.5 1.4 -‐1.1
ta504_3747
CAB3 (CHLOROPHYLL A32/2964 /B BINDING PgROTEIN 4.6 4.0 -‐1.1
eni 3)
ta8707_57918 CAB3 (CHLOROPHYLL A/B BINDING PROTEIN 3)
4.8 3.8 -‐1.3
ta8943_57918 LHCB3 (LIGHT-‐HARVESTING CHLOROPHYLL B-‐BINDING PROTEIN 3) 2.1 1.8 -‐1.2
co816858
PSBR (photosystem II subunit R) 2.7 2.0 -‐1.4
co817826
OEE1 (PS II OXYGEN-‐EVOLVING COMPLEX 1)
-‐1.9 -‐2.4 -‐1.3
cx661668
PSBP-‐2 (photosystem II subunit P-‐2); calcium i on binding 1.4 1.1 -‐1.3
cx661875
OEE1 (PS II OXYGEN-‐EVOLVING COMPLEX 1)
2.1 1.9 -‐1.1
Nord/Campania Nord/Basilicata ex663556
PSBW (PHOTOSYSTEM II REACTION CENTER W)
2.8 3.0 1.1
ex669715
PSBR (photosystem II subunit R)
2.4 1.8 -‐1.4
ta12137_57918 YCF9 | e ncodes PsbZ (subunit of photosystem II)
2.3 2.1 -‐1.1
ta8765_57918 PSBR (photosystem II subunit R)
3.3 2.8 -‐1.2
ta9811_57918 photosystem II family protein
2.3 2.2 -‐1.0
co816950
LHCA2; chlorophyll binding -‐2.2 -‐3.2 -‐1.4
cx661765
LHCA2; chlorophyll binding -‐2.5 -‐2.7 -‐1.1
ex672152
CAB4 (LIGHT-‐HARVESTING CHLOROPHYLL-‐PROTEIN COMPLEX I SUBUNIT 1.2 A4)-‐1.9 -‐2.3
ta8946_57918 CAB4 (LIGHT-‐HARVESTING CHLOROPHYLL-‐PROTEIN COMPLEX I SUBUNIT 1.6 A4)
-‐1.4 -‐2.2
co818102
PSAE-‐1 | PSAE-‐1 (PSA E1 KNOCKOUT)
4.2 4.1 -‐1.0
dy672550
YCF6 | hypothetical protein 2.1 2.0 -‐1.1
dy673904
PETC (PHOTOSYNTHETIC ELECTRON TRANSFER C)
-‐1.4 -‐2.2 -‐1.5
dy668423
ATP synthase protein I -‐related 1.3 1.0 -‐1.3
ta10156_57918 ATP SYNTHASE DELTA-‐SUBUNIT GENE
-‐1.4 -‐1.8 -‐1.3
co379005
ATPase F subunit
5.4 3.4 -‐1.6
co817901
DRT112; copper i on binding / e lectron carrier -‐3.5 -‐1.3 2.7
ta8787_57918 DRT112; copper i on binding / e lectron carrier 1.5 1.4 -‐1.1
Campania/Basilicata co381453
NADP adrenodoxin-‐like ferredoxin reductase -‐2.1 1.5 3.0
Loredana Lopez – UTTRI-‐GENER PL 30
Risultati
Nord/Campania Nord/Basilicata Risposta a Stress Bio4ci/Abio4ci 644/2964 geni Campania/Basilicata Loredana Lopez – UTTRI-‐GENER PL 30
Risultati
Trascrizione Overview Map 174/644 geni Nord/Campania Gene C2H2 zC/B
inc finger family (13)
N/C N/B C/B
Gene ID
WRKY domain transcription factor family (13)ID N/C N/B
co381242
zinc f
inger (
C2H2 t
ype)
-‐1.4 -‐1.3 1.0
dv439173
WRKY50
-‐1.2 1.6 1.9
Gene ID
AP2/EREBP, A
PETALA2/Ethylene-‐responsive e
lement b
inding p
rotein f
amily (
9)
N/C
N/B
C/B
co817418
Z
INC-‐FINGER P
ROTEIN 1
4.8 5.7 1.2
dv439455
WRKY3
-‐2.0 1.0 2.0
dy667186-‐1.6 -‐2.1
zinc finger -‐2.3 -‐1.7 1.4
dv440456
WRKY21; calmodulin binding -‐1.3 (AN1-‐like) family protein
ZAT10-‐like -‐2.7 N/B
1.4 C/B
3.8
dy673518
WRKY75transcription factor, Gene 1.1 zinc 1.7 finger 1.5 protein cx662118
AP2 domain-‐containing putative
-‐1.8
-‐1.6 1.1
IDex665093DNA.synthesis/chromatin structure.histone (6) N/C
ex666957
zinc f
inger p
rotein
1.5
1.1
-‐1.3
ex665677
WRKY33transcription -‐1.7
1.8
3.0
dv439744
AP2 domain-‐containing f
actor
-‐1.4
-‐1.4
1.0
Gene ID
bHLH,Basic family (7)
N/C 1.2
N/B 1.9
C/B 1.6
co380618 Helix-‐Loop-‐Helix HTA11; DNA binding zinc finger -‐3.7
-‐1.1
3.5
ex673472
WRKY21; calmodulin binding
-‐1.7 -‐1.9
-‐1.1 protein C2H2, CZF2, ZAT6 dy672665
AP2 domain-‐containing transcription factor bHLH fco818047
amily pex674925
rotein
co382079
family protein 2.7 1.1
3.5 1.1
1.3 1.0
HTB4; DNA binding 9.1 11.5 1.3
ta10661_57918
zinc f
inger (
C2H2 t
ype) f
amily p
rotein
-‐2.2
2.9
6.3
ex676292
W
RKY7; c
almodulin b
inding
-‐1.7
-‐1.0
1.7
ex662697
RAP2.1 (related to AP2 1); DNA binding / transcription factor 1.56) 1.7
dy672027
cx661406
PIL6 (PHYTOCHROME INTERACTING FACTOR 3-‐LIKE 3.1 5.02.6 4.9
-‐1.2 -‐1.0
histone H1/H5 family p-‐1.1
rotein
ta11397_57918
P
MZ; z
inc i
on b
inding -‐1.4
2.2
ex687371
WRKY33
-‐3.3
1.0
3.5
ta10197_57918
helix-‐loop-‐helix (bHLH) family protein
-‐1.6 -‐1.5 1.1 3.0
ex676181
RAP2.2; DNA binding / transcription factor basic -‐2.4 -‐1.2
dv440238
HTA9 (ZAT12 HISTONE H2A (PRESPONSIVE ROTEIN 9) TO 1.9
1.62.8 1.2
ta11554_57918
|NA RHL41 HIGH LIGHT 1.2
41) 1.3
-‐2.6
7.2
ta10088_57918 WRKY11; calmodulin binding ILR3 -‐2.4
1.3
3.2
ta10893_57918
(
iaa-‐leucine r
esistant3); D
b
inding 1.5 1.3
ta11061_57918 DREB1C (C-‐REPEAT/DRE BINDING FACTOR 2)ex661043
-‐3.4 -‐2.3 1.5
HTB4; zinc D
NA finger b2.0
inding 1.1
1.2
1.2
ta10841_57918 WRKY40
-‐1.7
1.2
ta12086_57918
(
C2H2 t
ype) f
amily p
rotein -‐1.6
-‐1.6
-‐1.0
ta11007_57918 CIB1 (CRYPTOCHROME-‐INTERACTING BASIC-‐HELIX-‐LOOP-‐HELIX 1.0
Gene ID ta11944_57918
MYB domain family (3)binding
N/C3.0N/B
C/B
DREB2A; DNA 2.1DNA 1.5
-‐1.4 1) -‐1.3 1.9-‐1.3 2.6
GAMMA-‐H2AX |( C2H2 H2AXA; binding ta10905_57918 WRKY11; calmodulin binding ta9620_57918
-‐2.0
1.5 finger ta13478_57918
zinc type) family protein -‐1.2 1.2 1.4
ta12326_57918
bHLH071 (
beta H
LH p
rotein 7
1); D
NA b
inding
7.7
7.9
1.0 1.5
AP2 omain-‐containing ranscription factor -‐1.9
-‐1.8 protein
1.1
af401220 ta9387_57918
MYB3 (MYB DdOMAIN PROTEIN 3t); DNA binding / transcription factor
-‐1.1family ta10997_57918
WRKY27
3.1 zinc 3.5 1.5
1.1 (1.4
ta8811_57918
finger AN1-‐like) -‐2.2 -‐1.6 1.3
ta9902_57918
(PHYTOCHROME-‐INTERACTING FACTOR7); DNA binding
-‐2.1 -‐2.3 -‐1.1
AP2 domain-‐containing transcription factor, PpIF7 utative
-‐1.9
co381319 ta9610_57918
PC-‐MYB1; Dta13471_57918
NA binding / WRKY48
transcription coactivator
1.0
-‐3.3 -‐1.0
3.2 p1.8
ta9857_57918
zinc 1.7
finger rotein C2H2, -‐1.9
CZF2, Z1.0
AT6 -‐3.1 1.3 4.1
ta13032_57918 MYB92 (MYB DOMAIN PROTEIN 92); DNA binding / transcription factor
Nord/Basilicata 6.4
6.2 -‐1.0
Campania/Basilicata Loredana Lopez – UTTRI-‐GENER PL 30
Risultati
Score plot Loading plot Loredana Lopez – UTTRI-‐GENER PL 30
Risultati
Loading plot Cluster alto: 216 geni UP Campania N N Cluster sinistro: 532 geni UP Sud C C B Cluster destro: 491 geni UP Nord B N C B Cluster basso: 80 geni UP Basilicata N C B Loredana Lopez – UTTRI-‐GENER PL 30
Geni up-‐regola4 in Campania Cluster alto: 216 geni UP Campania Nord Campania Basilicata Regolazione 48/216 geni (22,2%) Stress Bio4ci/Abio4ci 47/216 geni (21,7%) Metabolismo 28/216 geni (13%) Loredana Lopez – UTTRI-‐GENER PL 30
Geni up-‐regola4 in Basilicata Cluster basso: 80 geni UP Basilicata Nord Campania Basilicata Regolazione 11/80 geni (14%) Stress Bio4ci/Abio4ci 24/80 geni (30%) Metabolismo 14/80 geni (18%) Loredana Lopez – UTTRI-‐GENER PL 30
Stress ossidativo
Cluster alto: 216 geni UP Campania Nord Campania Cluster basso: 80 geni UP Basilicata Basilicata Nord Campania Basilicata Loredana Lopez – UTTRI-‐GENER PL 30
Stress ossidativo
Cluster alto: 216 geni UP Campania Nord Campania Basilicata Loredana Lopez – UTTRI-‐GENER PL 30
Geni up-‐regola4 al Nord Cluster destro: 491 geni UP Nord Nord Campania Basilicata Regolazione 68/491 geni (14%) Stress Bio4ci/Abio4ci 68/491 geni (14%) Metabolismo 51/491 geni (10%) Loredana Lopez – UTTRI-‐GENER PL 30
Nord Geni up-‐regola4 al Sud Cluster sinistro: 532 geni UP Sud Campania Basilicata Regolazione 96/532 geni (18%) Stress Bio4ci/
Abio4ci 137/532 geni (25%) Metabolismo 72/532 geni (14%) Loredana Lopez – UTTRI-‐GENER PL 30
Stress Bio4ci/Abio4ci Cluster destro: 491 geni UP Nord Nord Campania Stress Bio4ci/Abio4ci 68/491 geni (14%) Basilicata Nord Cluster sinistro: 532 geni UP Sud Campania Basilicata Stress Bio4ci/
Abio4ci 137/532 geni (25%) Loredana Lopez – UTTRI-‐GENER PL 30
Conclusioni
I risultaO oHenuO hanno dimostrato che con la tecnologia DNA microarray è possibile avere una discriminazione geografica della fragola. Sono staO idenOficaO 2964 geni la cui espressione è influenzata dalla diversa localizzazione geografica. Tra i 2964 geni differenzialmente espressi sono staO individuaO numerosi potenziali marcatori trascrizionali associaO all’espressione di caraHeri legaO alla Opicità e all’origine geografica della fragola. Nord
Sud
CAMPANIA
- Fattori Trascrizione
- Fotosintesi
- Flavonoidi
BASILICATA
-  Parete cellulare
-  HSP
- Stress ossidativo
- Stress Biotici/Abiotici
- Fenilpropanoidi
Loredana Lopez – UTTRI-‐GENER

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