BINARY VECTOR CONSTRUCTION FOR

PYROPHOSPHATE-DEPENDENT
PHOSPHOFRUCTOKINASE (PFP)-α
SENSE AND ANTISENSE GENE
SEQUENCES

Dr. Sony Suhandono

Tati Kristianti, M.Si
Dewi Murni, S.Si
 INTRODUCTION

Sugarcane
important crop in Indonesia
(Saccharum officinarum L.)

Raw material of
sugar production

 encourage the growth of regional economy

 save foreign exchange Development of
 people's subsistence sugar industries
 providing jobs
sugar content of affected by the allocation of carbon
compound that produced by
the sugarcane
photosynthesis on the carbohydrate
metabolism

accumulation sucrose
that depend on the Sucrose metabolism
maturation of the stem
internodes
(Whittaker and Botha, 1997)

stem
internode

(Groenewald, 2006)
  catalyze the phosphorylation of fructose 6-
phosphate to fructose 1,6-biphosphate
Activated by fruktosa- reversibly
2,6-bifosfat  using PPi as phosphate donor

(Carlisle et al., 1990) (Podesta et al., 1994)

Pyrophosphate
negatively correlated -dependen
with sucrose content fosfofructokinase
in sugarcane (PFP)
internode

Composed of

α Subunit (67 kDa) β Subunit (60 kDa)

Binding site of fruktosa-2,6 bifosfat

Catalyitic domain
Regulator domain = Regulating the
activity of β subunit
(Nielsen et al., 2004)
 PFP molecule arrangements
(Praag, 1997)

Heterotetramer Homodimer

catalyze the reactions of

glycolysis catalyze
gluconeogenesis
Silencing PFP gene may give
significant contribution to increase sugar
product
increasing levels of
sucrose in plants
 Mechanism of induction of gene expression
by RNAi silencing gene silencing

(Mansoor et al.,2006)
 RNAi Konstruks

(Wesley et al, 2001)

 MATERIALS AND METHODS
OF WORK
• We use synthetic PFP gene based on sugarcane
PFP sequence from GenBank AB270695.1
(Suhandono and Alamsyah, 2007)

G’AATT_CCTGATGCTCTTTCTGCTGAGAAGTAT
TACTACTTTATTCGCCTGATGGGACGCAAGGCT
TCTCATGTGGCATTGGAATGTGCTCTTCAGTCA
CATCCGAACATGGTCATCCTAGGAGAGGAGGT
GGCTGCTTCCAAACTTACAATTTTTGATATTAC
GAAGCAGATTTGTGATGCGGTTCAGGCAAGGG
CTGAAAAAGACAAGAACCATGGGGTTATTCTTA
TTCCTGAGGGCCTTGTGGAGAGTATTCCTGAA
CTATATGCTCTTCTTCAGGAAATCCATGGTCTC
CG’AATT_C
Diagram of RNAi
PFP sense-
antisense
construction
 Plasmid Map of pJ204 and sequence
of PFPα synthetic gene
G’AATT_CCTGATGCTCTTTCTGCTGAGAAGTATT
ACTACTTTATTCGCCTGATGGGACGCAAGGCTT
CTCATGTGGCATTGGAATGTGCTCTTCAGTCACA
TCCGAACATGGTCATCCTAGGAGAGGAGGTGG
CTGCTTCCAAACTTACAATTTTTGATATTACGAAG
CAGATTTGTGATGCGGTTCAGGCAAGGGCTGAA
AAAGACAAGAACCATGGGGTTATTCTTATTCCTG
AGGGCCTTGTGGAGAGTATTCCTGAACTATATGC
TCTTCTTCAGGAAATCCATGGTCTCCG’AATT_C

Backbone pJ204(3004 bp)

The result of running of restriction

pJ204 with EcoRI in 1% agarose
Note:
1 = 1 kb ladder
2 = pJ204 uncut
3 = pJ204 cut by EcoRI
 Insertion of PFPα gene in pGEM7zf

Note:
1 = 1 kb ladder
2 = empty pGEM7Zf cut by EcoRI
3 – 6 = pGEM7Zf insert cut by EcoRI
7 = pGEM7Zf insert uncut
Sequencing Results of PFPα gene in pGEM7Zf

EcoRI restriction site

 Alignment of sequencing results with
sequence of PFPα synthetic gene

EcoRI restriction site

Note:
Upper Sequent : the result of sequencing
Under Sequent : sequent of PFPα synthetic gene
BLAST (Basic Local Alignment Search Tool)
of PFPα sequencing results
 Construction of
RNAi PFPα sense-antisense in pHannibal
PFPα gene Construct in pHannibal

a b

Pronouncement :
a.Crude PCR result of PFPα sense-antisense in pHannibal
b.Plasmid Map of pHannibal+PFP sense-antisense
Conclusion :
 PFP α gene was successfully constructed in pHannibal through the XhoI-
KpnI (sense) and BamHI-XbaI (antisense) restriction site to produce PFPα
sense-antisense construct sized 1452 bp. The success of ligation was
confirmed by PCR screening that showed the band around 1500 bp.
Saran untuk penelitian selanjutnya:

 Hasil ekspresi dsRNA dari E. coli dapat

diuji secara in-vitro pada tanaman tebu,
sebelum tanaman transgenik dibuat.
THANK YOU
 RESULTS AND DISCUSSION
SiRNA candidates recommended by the program of BLOCK-IT ™ RNAi Designer.
SiRNA candidates recommended by the Program
Jura.wi.mit.edu / bioc / siRNAext / show_oligo.cgi
 Results juxtaposition siRNA candidates recommended by
the program BLOCK-IT ™ RNAi Designer and Invitrogen.

Keterangan :
= BLOCK-iT™ RNAi Designer invitrogen recommendation
= Jura.wi.mit.edu / bioc / siRNAext / show_oligo.cgi recommendation
 Raw material of sugar production.

 increase of the sucrose affected by the
allocation of carbon compound that
produced by photosynthesis on the
carbohydrate metabolism.
 Diagram proses kontruksi RNAi
(sense-antisense PFP α di pET-17b)
Keterangan :
1 = ladder 1 kb
2 = insert hasil purifikasi
Skema metabolisme senyawa
karbon pada tumbuhan
Pyrophosphate -dependen
fosfofructokinase (PFP)
 berperan dalam mengkatalis fosforilasi
fruktosa 6-fosfat menjadi fruktosa 1,6-
bifosfat secara reversibel dan
menggunakan PPi sebagai donor fosfor
 mengatur PPi sitosol
 menjaga keseimbangan cadangan
heksosa/ triosa fosfat di dalam sel
Fungsi Intron pada konstruk
 Meningkatkan stabilitas konstruks di
dalam sel E.coli
 Memudahkan konstruksi plasmid.
 Meningkatkan efisiensi silencing (sampai
100%). Proses splicing intron
memfasilitasi pembentukan dupleks
hpRNA di dalam kompleks spliceosome.