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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