presentasi hasil penelitian PFP alfa ASAHI

8/7/2019 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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presentasi hasil penelitian PFP alfa ASAHI