Ahmad Fathoni1,2, Ima Zainuddin3, Hervé Vanderschuren3 ...€¦ · • Southern blot analysis...
1
ØKey compound: Scopoletin - Accumulates rapidly during PPD process. - Oxidised resulting in blue-black discoloration of the roots which is observed as PPD. - Synthesised de novo and released from scopolin. Scopoletin and Scopolin are interconverted by scopoletin glucosyltransferase (GT) and scopolin-b-glucosidase (BG) (Figure 2). - Change scopoletin and scopolin content in cassava through RNAi and overexpression constructs. - BLAST known tobacco Scopoletin-GT gene and Arabidopsis Scopolin-BG gene on phytozome. - Identified and selected cassava gene(s): Scopoletin-GT (Cassava4.1_006629), Scopolin-BG (Cassava4.1_029389, cassava4.1_008910). ØIdentification of cassava homologous gene(s) ØPreparation of gene constructs (GATEWAY Cloning Tech.) LB Promoter* RB Terminator Full-length cDNA of Arabidopsis Scopolin-BG -RNAi construct LB Promoter* Sense fragment of cassava scopoletin-GT PDK intron Antisense fragment of cassava scopoletin-GT RB Terminator -Overexpression construct - *Promoter: Constitutive (35S) and Root-specific (PATATIN). - Expression vector: pCAMBIA 1305.1 Scopoletin O O OH OH HO O OH O O CH 3 O OH O O CH 3 Scopolin Scopoletin-glucosyltransferase Scopolin-beta-glucosidase PAL (Phenylalanine lyase) Figure 2. Interconversion of scopoletin and scopolin by scopoletin glucosyltransferase and scopolin ß-glucosidase. Figure 4. Maturation, development of transformed cassava FEC and regeneration of cassava transgenic plants. A. FEC on GD+C250+H5. B. Developing cotyledons on MSN+C250+H15. C-D. Cotyledons on CEM+C100 (week 1&6) . E-F. Cassava plantlet in CBM+C50 pot (week 1&5) A B C D E F ØAnalysis of transgenic cassava plants • GUS assay Positive control ; p1305.1::GUS A B C Positive control 35S::260 PAT::260 35S::629 PAT::629 Positive control 35S::260 PAT::260 35S::629 PAT::629 Figure 5. Histochemical Gus assay results. A. Transformed FEC, B. Developing cotyledons on CEM+C100 plate, C. Cassava leaves from transgenic plants. • Rooting test A B Figure 6. Rooting test of cassava transgenic lines and wild-type TMS60444 on CBM+C50+H10 (negative control). A. Week 0, B. Week 2. • PCR test-Hygromycin probe Figure 7. Gel electrophoresis of cassava transgenic lines (3-5) and wild-type TMS60444 as negative control (1-2). • Southern blot analysis Figure 8. Southern blot analysis. Lane 1. DIG ladder, Lane 2. Control pCAMBIA 1305.1, Lane 3. No sample, Lane 4. Sample 1 (negative), Lane 5. Sample 2 (positive, single insert) - Analyse Scopoletin and Scopolin in transgenic plants. - Evaluate PPD in cassava transgenic plants. This research is fully funded by The Ministry of Research and Technology, Indonesia. I would like to thank the Department of Biology and Biochemistry, University of Bath and The Biochemical Society for the Travel Grant and the Organizers of the Congress for the partial fellowship to attend this congress. Thank you everyone who has contributed to my work. Method Method Results Results Future Work Future Work Acknowledgments Acknowledgments 1,2 3 3 1 Ahmad Fathoni , Ima Zainuddin , Hervé Vanderschuren , Rod Scott 1 and John R. Beeching 1 Department of Biology and Biochemistry, University of BATH, BA2 7AY, United Kingdom 2 Research Centre for Biotechnology, Indonesian Institute of Sciences (LIPI), Indonesia 3 Department of Biology, Plant Biotechnology, ETH Zurich-LFW E56.1, Switzerland ØCassava ( Manihot esculenta C.) PPD - Roots become unpalatable and unmarketable. - Characterized by blue-black discoloration of the root (Figure 1B). Figure 1. A) Fresh roots B) 24 hours after harvest. ØAgrobacterium-mediated cassava FEC transformation Figure 3. Cassava Friable Embryogenic Callus (FEC). A. FEC under microscope. B. Transformed FEC on GD+C250+H5 plate. A B Introduction Introduction A B Results Results S10 The Role of The Interconversion of Scopoletin and Scopolin in Cassava Postharvest Physiological Deterioration (PPD)
Transcript of Ahmad Fathoni1,2, Ima Zainuddin3, Hervé Vanderschuren3 ...€¦ · • Southern blot analysis...
ØKey compound: Scopoletin- Accumulates rapidly during PPD process.- Oxidised resulting in blue-black discoloration of the roots
which is observed as PPD.- Synthesised de novo and released from scopolin.
Scopoletin and Scopolin are interconverted by scopoletin
glucosyltransferase (GT) and scopolin-b-glucosidase (BG) (Figure 2).
- Change scopoletin and scopolin content in cassava through RNAi and overexpression constructs.
- BLAST known tobacco Scopoletin-GT gene and Arabidopsis Scopolin-BG gene on phytozome.
- Identified and selected cassava gene(s): Scopoletin-GT (Cassava4.1_006629), Scopolin-BG (Cassava4.1_029389, cassava4.1_008910).
ØIdentification of cassava homologous gene(s)
ØPreparation of gene constructs (GATEWAY Cloning Tech.)
LB
Promoter*
RB
TerminatorFull-length cDNA of Arabidopsis Scopolin-BG
-RNAi construct
LB
Promoter* Sense fragment of cassava scopoletin-GT
PDK intron Antisense fragment of cassava scopoletin-GT
RB
Terminator
-Overexpression construct
- *Promoter: Constitutive (35S) and Root-specific (PATATIN).- Expression vector: pCAMBIA 1305.1
Scopoletin
O O
OH
OH
HO
OOH
O
O
CH3
O OHO
O
CH3Scopolin
Scopoletin-glucosyltransferase
Scopolin-beta-glucosidase
PAL(Phenylalanine lyase)
Figure 2. Interconversion of scopoletin and scopolin by scopoletin glucosyltransferase and scopolin ß-glucosidase.
Figure 4. Maturation, development of transformed cassava FEC and regeneration of cassava transgenic plants. A. FEC on GD+C250+H5. B. Developing cotyledons on MSN+C250+H15. C-D. Cotyledons on CEM+C100 (week 1&6) . E-F. Cassava plantlet in CBM+C50 pot (week 1&5)
A B C
D E F
ØAnalysis of transgenic cassava plants• GUS assay
Positive control ; p1305.1::GUS
A
B
C
Positive control 35S::260 PAT::260 35S::629 PAT::629
Positive control 35S::260 PAT::260 35S::629 PAT::629
Figure 5. Histochemical Gus assay results. A. Transformed FEC, B. Developing cotyledons on CEM+C100 plate, C. Cassava leaves from transgenic plants.
• Rooting test
A B
Figure 6. Rooting test of cassava transgenic lines and wild-type TMS60444 on CBM+C50+H10 (negative control). A. Week 0, B. Week 2.
• PCR test-Hygromycin probe
Figure 7. Gel electrophoresis of cassava transgenic lines (3-5) and wild-type TMS60444 as negative control (1-2).
• Southern blot analysis
Figure 8. Southern blot analysis. Lane 1. DIG ladder, Lane 2. Control pCAMBIA 1305.1, Lane 3. No sample, Lane 4. Sample 1 (negative), Lane 5. Sample 2 (positive, single insert)
- Analyse Scopoletin and Scopolin in transgenic plants.- Evaluate PPD in cassava transgenic plants.
This research is fully funded by The Ministry of Research and Technology, Indonesia. I would like to thank the Department of Biology and Biochemistry, University of Bath and The Biochemical Society for the Travel Grant and the Organizers of the Congress for the partial fellowship to attend this congress. Thank you everyone who has contributed to my work.
MethodMethod
ResultsResults
Future WorkFuture Work
AcknowledgmentsAcknowledgments
1,2 3 3 1Ahmad Fathoni , Ima Zainuddin , Hervé Vanderschuren , Rod Scott 1and John R. Beeching
1Department of Biology and Biochemistry, University of BATH, BA2 7AY, United Kingdom2Research Centre for Biotechnology, Indonesian Institute of Sciences (LIPI), Indonesia
3Department of Biology, Plant Biotechnology, ETH Zurich-LFW E56.1, Switzerland
ØCassava (Manihot esculenta C.) PPD
- Roots become unpalatable and unmarketable.
- Characterized by blue-black discoloration of the root (Figure 1B).
Figure 1. A) Fresh roots B) 24 hours after harvest.
ØAgrobacterium-mediated cassava FEC transformation
Figure 3. Cassava Friable Embryogenic Callus (FEC). A. FEC under microscope. B. Transformed FEC on GD+C250+H5 plate.
A B
IntroductionIntroduction
A B
ResultsResults
S10 The Role of The Interconversion of Scopoletin and Scopolin in Cassava Postharvest Physiological Deterioration (PPD)
