presentasi hasil penelitian PFP alfa ASAHI
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Transcript of presentasi hasil penelitian PFP alfa ASAHI
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Dr. Sony Suhandono
Tati Kristianti, M.Si
Dewi Murni, S.Si
BINARY VECTOR CONSTRUCTION FOR
PYROPHOSPHATE-DEPENDENT
PHOSPHOFRUCTOKINASE (PFP)-SENSE AND ANTISENSE GENE
SEQUENCES
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INTRODUCTION
Sugarcane
(Saccharum officinarum L.)
Raw material ofsugarproduction
encouragethegrowth ofregionaleconomy
save foreignexchange
people's subsistence
providing jobs
Development of
sugarindustries
importantcrop in Indonesia
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sugarcontent of
the sugarcane
Sucrose metabolismaccumulation sucrose
that depend onthe
maturation ofthe stem
internodes
(Whittakerand Botha, 1997)
(Groenewald, 2006)
stem
internode
affected by theallocation ofcarbon
compound that produced by
photosynthesis onthecarbohydrate
metabolism
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catalyzethe phosphorylation of fructose 6-
phosphateto fructose 1,6-biphosphate
reversibly
using PPi as phosphate donor
Pyrophosphate -
dependen
fosfofructokinase(PFP)
Subunit(67 kDa) Subunit(60 kDa)
Binding site of fruktosa-2,6 bifosfat
Regulatordomain = Regulatingthe
activity of subunit
Catalyitic domain
Activated by fruktosa-
2,6-bifosfat
(Podestaet al., 1994)(Carlisleet al., 1990)
(Nielsenet al., 2004)
Composed of
negatively
correlated withsucrosecontentin
sugarcaneinternode
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PFP moleculearrangements
HomodimerHeterotetramer
catalyze
gluconeogenesis
increasinglevels of
sucrosein plants
catalyzethereactions
ofglycolysis
Silencing PFPE gene may give
significantcontributionto increase sugar
product
(Praag, 1997)
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gene silencingMechanism ofinduction ofgene
expressionby RNAi silencing
(Mansooret al.,2006)
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RNAi Konstruks
(Wesley etal, 2001)
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MATERIALS AND METHODSMATERIALS AND METHODSOF WORKOF WORK
We use synthetic PFPE gene based on sugarcanePFPE sequence from GenBank AB270695.1(Suhandono and Alamsyah, 2007)
GAATT_CCTGATGCTCTTTCTGCTGAGAAGTATTACTACTTTATTCGCCTGATGGGACGCAAGGCTTCTCATGTGGCATTGGAATGTGCTCTTCAGTCACATCCGAACATGGTCATCCTAGGAGAGGAGGTGGCTGCTTCCAAACTTACAATTTTTGATATTAC
GAAGCAGATTTGTGATGCGGTTCAGGCAAGGGCTGAAAAAGACAAGAACCATGGGGTTATTCTTATTCCTGAGGGCCTTGTGGAGAGTATTCCTGAACTATATGCTCTTCTTCAGGAAATCCATGGTCTCCGAATT_C
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Diagram ofDiagram of RNAiRNAiPFPPFPEE sensesense--antisenseantisense
constructionconstruction
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Plasmid Map of pJ204 and
sequence of PFP synthetic gene
GAATT_CCTGATGCTCTTTCTGCTGAGAAGTAT
TACTACTTTATTCGCCTGATGGGACGCAAGGCT
TCTCATGTGGCATTGGAATGTGCTCTTCAGTCA
CATCCGAACATGGTCATCCTAGGAGAGGAGGT
GGCTGCTTCCAAACTTACAATTTTTGATATTAC
GAAGCAGATTTGTGATGCGGTTCAGGCAAGGGCTGAAAAAGACAAGAACCATGGGGTTATTCTTA
TTCCTGAGGGCCTTGTGGAGAGTATTCCTGAA
CTATATGCTCTTCTTCAGGAAATCCATGGTCTC
CGAATT_C
Note:1 = 1 kb ladder
2 = pJ204 uncut
3 = pJ204 cut by EcoRI
Backbone pJ204(3004 bp)
Theresult ofrunning ofrestriction
pJ204 withEcoRIin 1% agarose
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Insertion of PFP genein pGEM7zf
Note:
1 = 1 kb ladder
2 = empty pGEM7Zf cut by EcoRI
3 6 = pGEM7Zf insert cut by EcoRI
7 = pGEM7Zf insert uncut
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Sequencing Results of PFP genein pGEM7Zf
EcoRIrestriction site
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Alignment of sequencingresults with
sequence of PFP syntheticgene
Note:
UpperSequent :theresult of sequencing
UnderSequent : sequent of PFP syntheticgene
EcoRIrestriction site
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BLAST(Basic LocalAlignmentSearchTool)
of PFP sequencingresults
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Construction ofConstruction ofRNAiRNAi PFPPFP sensesense--antisense inantisense in pHannibalpHannibal
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PFP geneConstructin pHannibal
a b
Pronouncement:
a. Crude PCR result of PFP sense-antisensein pHannibal
b. Plasmid Map of pHannibal+PFPE sense-antisense
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Conclusion: PFP genewas successfully constructed in pHannibalthroughthe XhoI-
KpnI (sense) and BamHI-XbaI (antisense) restriction siteto produce PFP
sense-antisenseconstruct sized 1452 bp. The success ofligationwas
confirmed by PCR screeningthat showed theband around 1500 bp.
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Saranuntuk penelitian selanjutnya:
Hasilekspresi dsRNA dariE. colidapat
diuji secarain-vitro padatanamantebu,
sebelum tanamantransgenik dibuat.
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THANK YOU
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SiRNAcandidates recommended by the program of BLOCK-IT RNAi Designer.
RESULTSAND DISCUSSION
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SiRNAcandidates recommended by the Program
Jura.wi.mit.edu / bioc / siRNAext / show_oligo.cgi
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Results juxtaposition siRNAcandidates recommended by
the program BLOCK-IT RNAi Designerand Invitrogen.
Keterangan:= BLOCK-iT RNAi Designerinvitrogenrecommendation= Jura.wi.mit.edu / bioc / siRNAext / show_oligo.cgirecommendation
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Raw material of sugarproduction.
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increase ofthe sucroseaffected by the
allocation ofcarboncompound that
produced by photosynthesis onthecarbohydrate metabolism.
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Diagram proses kontruksi RNAi
(sense-antisense PFP di pET-17b)
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Keterangan :
1 = ladder 1 kb
2 = insert hasil purifikasi
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Skema metabolisme senyawa
karbon padatumbuhan
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Pyrophosphate -dependen
fosfofructokinase (PFP) berperan dalam mengkatalis fosforilasi
fruktosa 6-fosfat menjadi fruktosa 1,6-
bifosfat secarareversibel danmenggunakan PPi sebagai donorfosfor
mengaturPPi sitosol
menjagakeseimbangancadanganheksosa/ triosa fosfat di dalam sel
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Fungsi Intron padakonstruk
Meningkatkan stabilitas konstruks di
dalam selE.coli
Memudahkankonstruksi plasmid.
Meningkatkanefisiensi silencing(sampai
100%). Proses splicingintron
memfasilitasi pembentukan duplekshpRNA di dalam kompleks spliceosome.
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