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Die quot;Flavr Savrquot; Tomate
           &
     Gene-silencing
Flavr Savr

                              • erstes gentechnisch verändertes Gemüse
                              • entwickelt 1988 von Calgene Inc. (USA)
                              • Paradebeispiel zu Sicherheitstests für Genfood
                              • 1994 auf dem Markt
                              • 1997 Produktion eingestellt

        quot;Anti-Matsch Tomatequot; - Reifungsprozeß unterbrochen
Smith CJS et al. (1988) Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes. Nature 334:724-726.
Sheey RE et al. (1988) Reduction of polygalacturonase activity in tomato fruit by antisense RNA. PNAS 85: 8805-8809.
Wieso Flav(ou)r Sav(ou)r Tomaten?
                     Flavr Savr   Normale


                                              Markt (1993)
  längere Reifung                           8 Mt (verarbeitet)
         =                                   0,5 Mt (frisch)
besserer Geschmack
                                            4 Milliarden US$/J
...reife Tomaten
Ethylen   Polygalacturonase     Abbau von Pektin
Polygalacturonase inaktivieren
Polygalacturonase (PG)
      (Tomate)



      PG mRNA



Anti-Sense RNA




• Abbau der PG mRNA
• PG-level = 6%

                                 A. tumefaciens - Binärer Vektor
Umfangreiche Sicherheitstests
1.) FDA (Food & Drug Administration): Kan-Resistenz ist unbedenklich




2.) Inhaltsstoffe unverändert
• Vitamine
• Proteine
• Mineralstoffe
• Toxine
  (Solanin, Tomatin)
Warum so wenig erfolgreich?

     Business-Träumereien            wissenschaftliche Realität

           • nicht schadensfrei transportfähig (unrentabel)

           • Tomatensorte hat: wenig Geschmack
                               geringen Ertrag
                               geringe Resistenz

           • Anti-GM-Food Proteste

1995-1997: Calgene von Monsanto (USA) aufgekauft
           Anti-Sense Konstrukt in höherwertige Sorten einbringen
GM-Tomaten in Großbrittannien
Zeneca (Syngenta): Tomaten mit verzögerter Reifung

    • Tomatenpüree mit hoher Viskosität
      (kein Erhitzen nötig)
    • eindeutig gelabelt
    • sehr populär (60% Marktanteil)
    • 1996-1999 am Markt
60 Tage
                Alternative Ansätze

                Ethylen-Synthese blockieren

                         Methionin
 Antisense
 ACC-Synthase
 78 Tage                                     quot;Co-Suppressionquot;
               S-Adenosyl-Methionin            quot;Antisensequot;
                            ACC-Synthase
     1-Amino-Cyclopropan-1-Carboxylsäure (ACC)
 Antisense
 ACC-Synthase
 78 Tage
 (+ Ethylen)
                          Ethylen


DNA Plant Technology Corp. (USA) - quot;Endless Summerquot; (1995)
Der Mechanismus hinter Flavr Savr:
           quot;Silencingquot;

• Produktion von Anti-Sense RNA


             • Sense-mRNA in der Zelle abgebaut



• Expression von Transgenen in Pflanzen
  (starke, konstitutive Promotoren)

             • keine Expression des Transgens
                                                  1990
             • endogenes Gen supprimiert
Gene-Silencing
Post-transcriptional gene silencing (PTGS)
                                             Transcriptional gene silencing (TGS)
         (Co-suppression; RNAi)




                                                                             1994
Produktion von dsRNA (1998)



• Transkription durch IR




• gegenläufige Transkription
  (sense-antisense)




• RNA-abhängige
  RNA-Polymerase (cRNA)
Welche Faktoren/Signale sind involviert?




                                 1


         3                             4
                                 2
     quot;Dicerquot;: RNaseIII
Wozu Gene-silencing?

                                   TGS
Inaktivierung von Retroviren, Retrotransposons und DNA-Transposons

             • Methylierung: Transkriptionelle Inaktivierung



                                 PTGS
                    Abwehr von Pflanzenviren

           • 90% ssRNA-Viren
           • dsRNA bei Replikation
           • Abbau des Templates für Replikation & Translation
           • Systemisches Signal
Transport viraler RNA durch Plasmodesmata
Virale Suppressoren des Silencing

• HC-Pro (helper component-proteinase)
     Tobacco etch potyvirus quot;maintenancequot; supprimiert
                                - keine 25 nt dsRNA produziert
                                - Methylierung geht z.T. verloren
                                - Reaktivierung von gesilencten Genen

• 2b Protein
      Cucumber mosaic virus (CMV)         quot;Initiationquot; supprimiert




• p25 Protein
    Potato virus X (PVX)        quot;systemisches Signalquot; supprimiert
                                - 3 Movement-Proteine: p8, p12, p25
Wie quot;silencequot; ich ein Gen?

                       dsRNA
Agrobacterium tumefaciens
       • sense & antisense, >300 bp
       • splicebares Intron zwischen sense & antisense

       • Vorraussetzung: transformierbare Pflanze



VIGS (Virus-induced gene silencing)
       • Potato virus X (PVX), Tobacco rattle virus (TRV)

       • Vorraussetzung: Wirtsspezifität
Methodische Ansätze zum Gene-Silencing

• Expression von Transgenen   - Induzierbare Expression
                              - Verwendung von Suppressoren


• Gezielte Suppression von Genen   - quot;knock-outquot;-Phänotyp
                                   - Virus-Resistenz
nächste Woche:
quot;Advanced Backcross Breedingquot;

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Flavr Savr

  • 1. Die quot;Flavr Savrquot; Tomate & Gene-silencing
  • 2. Flavr Savr • erstes gentechnisch verändertes Gemüse • entwickelt 1988 von Calgene Inc. (USA) • Paradebeispiel zu Sicherheitstests für Genfood • 1994 auf dem Markt • 1997 Produktion eingestellt quot;Anti-Matsch Tomatequot; - Reifungsprozeß unterbrochen Smith CJS et al. (1988) Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes. Nature 334:724-726. Sheey RE et al. (1988) Reduction of polygalacturonase activity in tomato fruit by antisense RNA. PNAS 85: 8805-8809.
  • 3. Wieso Flav(ou)r Sav(ou)r Tomaten? Flavr Savr Normale Markt (1993) längere Reifung 8 Mt (verarbeitet) = 0,5 Mt (frisch) besserer Geschmack 4 Milliarden US$/J
  • 4. ...reife Tomaten Ethylen Polygalacturonase Abbau von Pektin
  • 5. Polygalacturonase inaktivieren Polygalacturonase (PG) (Tomate) PG mRNA Anti-Sense RNA • Abbau der PG mRNA • PG-level = 6% A. tumefaciens - Binärer Vektor
  • 6. Umfangreiche Sicherheitstests 1.) FDA (Food & Drug Administration): Kan-Resistenz ist unbedenklich 2.) Inhaltsstoffe unverändert • Vitamine • Proteine • Mineralstoffe • Toxine (Solanin, Tomatin)
  • 7. Warum so wenig erfolgreich? Business-Träumereien wissenschaftliche Realität • nicht schadensfrei transportfähig (unrentabel) • Tomatensorte hat: wenig Geschmack geringen Ertrag geringe Resistenz • Anti-GM-Food Proteste 1995-1997: Calgene von Monsanto (USA) aufgekauft Anti-Sense Konstrukt in höherwertige Sorten einbringen
  • 8. GM-Tomaten in Großbrittannien Zeneca (Syngenta): Tomaten mit verzögerter Reifung • Tomatenpüree mit hoher Viskosität (kein Erhitzen nötig) • eindeutig gelabelt • sehr populär (60% Marktanteil) • 1996-1999 am Markt
  • 9. 60 Tage Alternative Ansätze Ethylen-Synthese blockieren Methionin Antisense ACC-Synthase 78 Tage quot;Co-Suppressionquot; S-Adenosyl-Methionin quot;Antisensequot; ACC-Synthase 1-Amino-Cyclopropan-1-Carboxylsäure (ACC) Antisense ACC-Synthase 78 Tage (+ Ethylen) Ethylen DNA Plant Technology Corp. (USA) - quot;Endless Summerquot; (1995)
  • 10. Der Mechanismus hinter Flavr Savr: quot;Silencingquot; • Produktion von Anti-Sense RNA • Sense-mRNA in der Zelle abgebaut • Expression von Transgenen in Pflanzen (starke, konstitutive Promotoren) • keine Expression des Transgens 1990 • endogenes Gen supprimiert
  • 11. Gene-Silencing Post-transcriptional gene silencing (PTGS) Transcriptional gene silencing (TGS) (Co-suppression; RNAi) 1994
  • 12. Produktion von dsRNA (1998) • Transkription durch IR • gegenläufige Transkription (sense-antisense) • RNA-abhängige RNA-Polymerase (cRNA)
  • 13. Welche Faktoren/Signale sind involviert? 1 3 4 2 quot;Dicerquot;: RNaseIII
  • 14. Wozu Gene-silencing? TGS Inaktivierung von Retroviren, Retrotransposons und DNA-Transposons • Methylierung: Transkriptionelle Inaktivierung PTGS Abwehr von Pflanzenviren • 90% ssRNA-Viren • dsRNA bei Replikation • Abbau des Templates für Replikation & Translation • Systemisches Signal
  • 15. Transport viraler RNA durch Plasmodesmata
  • 16. Virale Suppressoren des Silencing • HC-Pro (helper component-proteinase) Tobacco etch potyvirus quot;maintenancequot; supprimiert - keine 25 nt dsRNA produziert - Methylierung geht z.T. verloren - Reaktivierung von gesilencten Genen • 2b Protein Cucumber mosaic virus (CMV) quot;Initiationquot; supprimiert • p25 Protein Potato virus X (PVX) quot;systemisches Signalquot; supprimiert - 3 Movement-Proteine: p8, p12, p25
  • 17. Wie quot;silencequot; ich ein Gen? dsRNA Agrobacterium tumefaciens • sense & antisense, >300 bp • splicebares Intron zwischen sense & antisense • Vorraussetzung: transformierbare Pflanze VIGS (Virus-induced gene silencing) • Potato virus X (PVX), Tobacco rattle virus (TRV) • Vorraussetzung: Wirtsspezifität
  • 18. Methodische Ansätze zum Gene-Silencing • Expression von Transgenen - Induzierbare Expression - Verwendung von Suppressoren • Gezielte Suppression von Genen - quot;knock-outquot;-Phänotyp - Virus-Resistenz