Engineering High Starch Corn Leaves - Poster
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
5PBUUFNQUUPPWFSDPNFUIFZJFMEQFOBMUZXFXJMMCMPDLTUBSDIEFHSBEBUJPOBU BTQFDJmDEFWFMPQNFOUBMUJNFQPJOUMBUFJOUIFMJGFDZDMFPGUIFQMBOU&BSMZJO QMBOU HSPXUI DBSCPO GSPN TUBSDI SFJOWFTUFE JO MFBWFT DBVTFT FYQPOFOUJBM HSPXUI 0ODF TVöDJFOU QMBOU NBTT JT QSFTFOU UP DPOUBJO UIF DBSCPO NFUBCPMJTNDBOCFTXJUDIFEUPDBVTFBDDVNVMBUJPOPGTUBSDI 5P EP UIJT XF XJMM VTF 3/" JOUFSGFSFODF 3/"J BHBJOTU UIF HMVDBO XBUFS EJLJOBTFFO[ZNF (8% BOEUIF4FYQSPUFJO5IFTF3/"JDPOTUSVDUTXJMMCF DPVQMFEUPBTFOFTDFODFJOEVDFEQSPNPUFS 4"( PSBOBMDPIPMJOEVDJCMF QSPNPUFS 'JH $POTUSVDUTGPSUIFWBSJPVTQSPNPUFSTBOEJOWFSUFESFQFBUT XFSFNBEFCZ#JP#BTJDJO.BSLIBN0OUBSJP$BOBEB XXXCJPCBTJDDPN
35S
alcR
UPOTESZNBUUFSIB
Sex 400bp
Intron
alcA
Sex 400bp
Intron
ALCR
'JHVSF)ZQPUIFUJDBM*ODSFBTFJO#JPGVFM:JFMEXJUIB1BSUJBMDPOWFSTJPO PG$FMMVMPTFUP4UBSDI *O NPTU QMBOUT USBOTJUPSZ TUBSDI JT QSPEVDFE JO UIF DIMPSPQMBTUT PG MFBWFT EVSJOHUIFEBZBOECSPLFOEPXOEVSJOHUIFOJHIU*OUIFQBTUZFBSTPVS MBC BOE MBCT BSPVOE UIF XPSME IBWF NBEF TJHOJmDBOU QSPHSFTT JO VOEFSTUBOEJOHUIFQBUIXBZPGUSBOTJUPSZTUBSDIEFHSBEBUJPO 'JH 8FJTFFU BM -V FU BM 4NJUI FU BM 8F DBO OPX NBLF FEVDBUFE EFDJTJPOTBCPVUXIFSFUPNPEJGZUIJTQBUIXBZJOPSEFSUPEJTSVQUUIFTUBSDI EFHSBEBUJPOQSPDFTTUIFSFCZJODSFBTJOHUIFBNPVOUPGTUBSDIJOUIFMFBWFT Chloroplast
Cytosol
Sucrose
Starch
GWD Sex4 B-Amylase
Maltodextrin
Sag12
Glucanotransferase
Maltose
Maltose
Glucose
Glucose
Heteroglycan
DEn zy m e
ALCR Ethanol added
Sex 400bp Sex 400bp
E
E
Dicer
Sex Sex
E
Target Sex mRNA for degradation
400 400
bp bp
RISC
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
'JSTU"SBCJEPQTJTUIFO$PSO 'JHVSF1BUIXBZPG5SBOTJUPSZ4UBSDI%FHSBEBUJPO 5IFSFBSFNBOZQPUFOUJBMUBSHFUTUPDBVTFTUBSDIBDDVNVMBUJPO8FUIJOLUIF HMVDBOXBUFSEJLJOBTF (8% FO[ZNFBOEUIFTUBSDIFYDFTT 4FY QSPUFJO BSFUIFCFTUUBSHFUTUPQSPWJEFUIFHSFBUFTUJODSFBTFJOUIFZJFMEPGTUBSDI XIJMFNJOJNJ[JOHEPXOTUSFBNDIFNJDBMBOETJHOBMJOHFõFDUTPGNBMUPTFBOE HMVDPTFBDDVNVMBUJPO 6TJOH"SBCJEPQTJTmSTUQSFTFOUTTFWFSBMBEWBOUBHFT 854UBSDI&YDFTT
'JHVSF8JMEUZQFBOEB4UBSDI"DDVNVMBUJOH.VUBOUPG"SBCJEPQTJT " NBKPS PCTUBDMF UP PWFSDPNF JT SFEVDJOH PS FMJNJOBUJOH UIF ZJFME QFOBMUZ PCTFSWFEJONPTUTUBSDIBDDVNVMBUJOHNVUBOUT mHVSFGSPN/JUUZMÊFUBM
5IFTUBSDINFUBCPMJDQBUIXBZJTXFMMDIBSBDUFSJ[FE ,OPDLPVUTPG(8%BOE4FYIBWFCFFODIBSBDUFSJ[FE :VFUBM ;FFNBOFUBM
5IFTFOFTDFODFJOEVDJCMFQSPNPUFS 4"(Q JTDIBSBDUFSJ[FE /PIBOE"NBTJOP
"GBTUMJGFDZDMFXJMMHJWFVTBOEFBSMZiQSPPGPGDPODFQUw "SBCJEPQTJTJTWFSZBNFOBCMFUPHFOFUJDNBOJQVMBUJPOBMMPXJOHVTUPmOFUVOF XIBUXFMFBSOGSPNJOJUJBMFYQFSJNFOUT $PODVSSFOU XJUI UIF "SBCJEPQTJT XPSL 3/"J DPOTUSVDUT UBSHFUFE UP UIF DPSO IPNPMPHVFPG(8%BOE4FYIBWFCFFONBEF5IFTFXJMMCFQMBDFECFIJOEUIF BMDPIPMJOEVDJCMFQSPNPUFS BOECFIJOEUIFDPOTUJUVUJWFQSPNPUFSPG6CJRVJUJO 5IFUSBOTGPSNBUJPOTBSFCFJOHDBSSJFEPVUCZ%S)FJEJ,BFQQMFSBUUIF6OJWFSTJUZ PG8JTDPOTJO.BEJTPO
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
5PBUUFNQUUPPWFSDPNFUIFZJFMEQFOBMUZXFXJMMCMPDLTUBSDIEFHSBEBUJPOBU BTQFDJmDEFWFMPQNFOUBMUJNFQPJOUMBUFJOUIFMJGFDZDMFPGUIFQMBOU&BSMZJO QMBOU HSPXUI DBSCPO GSPN TUBSDI SFJOWFTUFE JO MFBWFT DBVTFT FYQPOFOUJBM HSPXUI 0ODF TVöDJFOU QMBOU NBTT JT QSFTFOU UP DPOUBJO UIF DBSCPO NFUBCPMJTNDBOCFTXJUDIFEUPDBVTFBDDVNVMBUJPOPGTUBSDI 5P EP UIJT XF XJMM VTF 3/" JOUFSGFSFODF 3/"J BHBJOTU UIF HMVDBO XBUFS EJLJOBTFFO[ZNF (8% BOEUIF4FYQSPUFJO5IFTF3/"JDPOTUSVDUTXJMMCF DPVQMFEUPBTFOFTDFODFJOEVDFEQSPNPUFS 4"( PSBOBMDPIPMJOEVDJCMF QSPNPUFS 'JH $POTUSVDUTGPSUIFWBSJPVTQSPNPUFSTBOEJOWFSUFESFQFBUT XFSFNBEFCZ#JP#BTJDJO.BSLIBN0OUBSJP$BOBEB XXXCJPCBTJDDPN
35S
alcR
UPOTESZNBUUFSIB
Sex 400bp
Intron
alcA
Sex 400bp
Intron
ALCR
'JHVSF)ZQPUIFUJDBM*ODSFBTFJO#JPGVFM:JFMEXJUIB1BSUJBMDPOWFSTJPO PG$FMMVMPTFUP4UBSDI *O NPTU QMBOUT USBOTJUPSZ TUBSDI JT QSPEVDFE JO UIF DIMPSPQMBTUT PG MFBWFT EVSJOHUIFEBZBOECSPLFOEPXOEVSJOHUIFOJHIU*OUIFQBTUZFBSTPVS MBC BOE MBCT BSPVOE UIF XPSME IBWF NBEF TJHOJmDBOU QSPHSFTT JO VOEFSTUBOEJOHUIFQBUIXBZPGUSBOTJUPSZTUBSDIEFHSBEBUJPO 'JH 8FJTFFU BM -V FU BM 4NJUI FU BM 8F DBO OPX NBLF FEVDBUFE EFDJTJPOTBCPVUXIFSFUPNPEJGZUIJTQBUIXBZJOPSEFSUPEJTSVQUUIFTUBSDI EFHSBEBUJPOQSPDFTTUIFSFCZJODSFBTJOHUIFBNPVOUPGTUBSDIJOUIFMFBWFT Chloroplast
Cytosol
Sucrose
Starch
GWD Sex4 B-Amylase
Maltodextrin
Sag12
Glucanotransferase
Maltose
Maltose
Glucose
Glucose
Heteroglycan
DEn zy m e
ALCR Ethanol added
Sex 400bp Sex 400bp
E
E
Dicer
Sex Sex
E
Target Sex mRNA for degradation
400 400
bp bp
RISC
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
'JSTU"SBCJEPQTJTUIFO$PSO 'JHVSF1BUIXBZPG5SBOTJUPSZ4UBSDI%FHSBEBUJPO 5IFSFBSFNBOZQPUFOUJBMUBSHFUTUPDBVTFTUBSDIBDDVNVMBUJPO8FUIJOLUIF HMVDBOXBUFSEJLJOBTF (8% FO[ZNFBOEUIFTUBSDIFYDFTT 4FY QSPUFJO BSFUIFCFTUUBSHFUTUPQSPWJEFUIFHSFBUFTUJODSFBTFJOUIFZJFMEPGTUBSDI XIJMFNJOJNJ[JOHEPXOTUSFBNDIFNJDBMBOETJHOBMJOHFõFDUTPGNBMUPTFBOE HMVDPTFBDDVNVMBUJPO 6TJOH"SBCJEPQTJTmSTUQSFTFOUTTFWFSBMBEWBOUBHFT 854UBSDI&YDFTT
'JHVSF8JMEUZQFBOEB4UBSDI"DDVNVMBUJOH.VUBOUPG"SBCJEPQTJT " NBKPS PCTUBDMF UP PWFSDPNF JT SFEVDJOH PS FMJNJOBUJOH UIF ZJFME QFOBMUZ PCTFSWFEJONPTUTUBSDIBDDVNVMBUJOHNVUBOUT mHVSFGSPN/JUUZMÊFUBM
5IFTUBSDINFUBCPMJDQBUIXBZJTXFMMDIBSBDUFSJ[FE ,OPDLPVUTPG(8%BOE4FYIBWFCFFODIBSBDUFSJ[FE :VFUBM ;FFNBOFUBM
5IFTFOFTDFODFJOEVDJCMFQSPNPUFS 4"(Q JTDIBSBDUFSJ[FE /PIBOE"NBTJOP
"GBTUMJGFDZDMFXJMMHJWFVTBOEFBSMZiQSPPGPGDPODFQUw "SBCJEPQTJTJTWFSZBNFOBCMFUPHFOFUJDNBOJQVMBUJPOBMMPXJOHVTUPmOFUVOF XIBUXFMFBSOGSPNJOJUJBMFYQFSJNFOUT $PODVSSFOU XJUI UIF "SBCJEPQTJT XPSL 3/"J DPOTUSVDUT UBSHFUFE UP UIF DPSO IPNPMPHVFPG(8%BOE4FYIBWFCFFONBEF5IFTFXJMMCFQMBDFECFIJOEUIF BMDPIPMJOEVDJCMFQSPNPUFS BOECFIJOEUIFDPOTUJUVUJWFQSPNPUFSPG6CJRVJUJO 5IFUSBOTGPSNBUJPOTBSFCFJOHDBSSJFEPVUCZ%S)FJEJ,BFQQMFSBUUIF6OJWFSTJUZ PG8JTDPOTJO.BEJTPO
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