Expression of a Bifunctional Fusion of the Escherichia coli Genes for Trehalose6Phosphate Synthase a - PowerPoint PPT Presentation

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Expression of a Bifunctional Fusion of the Escherichia coli Genes for Trehalose6Phosphate Synthase a

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Title: Expression of a Bifunctional Fusion of the Escherichia coli Genes for Trehalose6Phosphate Synthase a


1
Expression of a Bifunctional Fusion of the
Escherichia coli Genes for Trehalose-6-Phosphate
Synthase and Trehalose-6-Phosphate Phosphatase in
Transgenic Rice Plants Increases Trehalose
Accumulation and Abiotic Stress Tolerance without
Stunting Growth1In-Cheol Jang,2 Se-Jun Oh,2
Ju-Seok Seo,2 Won-Bin Choi, Sang Ik Song, Chung
Ho Kim, Youn Shic Kim, Hak-Soo Seo, Yang Do Choi,
Baek Hie Nahm, and Ju-Kon KimDepartment of
Biological Science, Myongji University, Yongin
449728, Korea (I.-C.J., S.-J.O., W.-B.C.,
S.I.S., B.H.N., J.-K.K.) School of Agricultural
Biotechnology, Seoul National University, Suwon
441744, Korea (J.-S.S., H.-S.S., Y.D.C.)
Department of Food and Nutrition, Seowon
University, Chongju 361742, Korea (C.H.K.) and
Genomics and Genetics Institute, GreenGene
BioTech, Yongin 449728, Korea (Y.S.K., B.H.N.,
J.-K.K.)Presented by Laura Taborsky and Megan
Twomey
2
Trehalose- Plays an important role in stress
tolerance in plants and possibly in a variety of
other organisms such as insects, bacteria, algae,
fungi, yeast. Trehalose also serves a
carbohydrate reserve.
  • Glucose-6-P Trehalose-6-P Trehalose
  • (TPS) (TPP)
  • Trehalose-6-phosphate synthase (TPS) and
    Trehalose-6- phosphate phosphatase (TPP) are
    enzymes involved in the production of trehalose.

3
Goal To produce stress tolerant transgenic
plants by elevating trehalose production.
  • Problem Trehalose-6-P (T6P) accumulation
    causes phenotypic changes such as altered
    metabolism and growth defects.
  • In yeast, T6P has been shown to affect
    glycolysis by interacting with hexokinase.
    Although unknown in plants, T6P seems to have a
    similar effect on sugar signaling.
  • Solution Find a way to produce high levels of
    trehalose while minimizing T6P accumulation.
  • bring TPS and TPP closer together to speed up
    the reaction rate, increasing efficiency and
    decreasing T6P accumulation.

4
Why Rice?
  • All previous transgenic plants were transformed
    with TPS OR TPP, not both, AND were dicots.
  • dicots generally produce low levels of
    trehalose
  • Results Increased trehaloseIncreased drought
    tolerance but.resulted in growth defects.
  • Rice (monocot) appears to be more tolerant to
    trehalose.
  • Rice was treated exogenosly with trehalose
  • no growth defects.

5
The Plan Transform rice (Oryza sativa) with a
gene that encodes the bifunctional fusion of TPS
and TPP from E. coli TPSP.
  • 1. Make a chimeric gene-
  • Fuse E. coli genes that code for TPS and TPP
    together by PCR deleting the stop codon of the
    TPS gene.
  • The fused TPSP gene had a 3.5-4x higher
    catalytic efficiency than equimolar amounts of
    TPS and TPP alone. This resulted in less T6P
    accumulation.

6
  • 2. Make a transformation vector- Link the fused
    TPSP gene to a ubiquitin maize promoter Ubi1
    and bar and 3nos reporter genes.

3. Insertional mutagenesis- Use Agrobacterium
tumefaciens to mediate the transfer of the
Ubi1TPSP gene into rice seeds.
7
  • Did it work?
  • Transformants were verified by herbicide
    selection (bar) and the presence of the
    Ubi1TPSP gene was confirmed through Southern
    analysis.
  • 5 homozygous T2 lines
  • that had one or two copies
  • of TPSP were chosen for
  • further analysis.

8
  • Did transformants express Ubi1TPSP?
  • TPSP transcripts of leaf
  • tissues were verified
  • through Northern analysis.
  • Control check TPSP was shown not to
    significantly activate other stress inducible
    genes (Lip5 Dip1). Lip5 Dip1 were strongly
    induced by environmental stress factors.

9
Control check Trehalose accumulation in
transgenics was not due to lowered trehalase
activity. This was verified by comparison of
trehalose and trehalase activity in rice compared
with other transgenics.
10
Carbohydrate profiles Used high performance ion
chromatography (HPIC) to analyze the effects of
trehalose production on other carbohydrates in
leaf and seed tissues.
Results Where there were no changes in the
carbohydrate content of the leaves, there was a
significant reduction on sucrose content of the
seeds. Transgenic plants had trehalose levels of
up to 0.1 fresh weight (lots!) compared to
negligible control levels.
11
Interestingly, higher transcript levels of TPSP
did not mean higher trehalose levels.
Ubi1TPSP-2 showed lower TPSP transcript levels
than Ubi1TPSP-1, but trehalose levels were
similar.
12
Success?- T6P was not detected in Ubi1TPSP
transgenics even though trehalose levels were as
much as 200x higher than independently
transformed tobacco plants that had TPS or TPP
(Goddijn et al, 1997).
13
Did overproduction of trehalose alter growth
phenotypes of transformed plants?
NO!
Compared to control plants, transformants showed
no growth inhibition or changes in appearance.
14
Does trehalose overproduction mean higher
tolerance to environmental stresses?
Drought tolerance -After 10-12 days without
water, transformants had root and shoot growth
where the control plants were nearly dead due to
leaf damage and chlorophyll loss.
YES!!
15
Salt tolerance
YES!!
Shoot and root growth in Ubi1TPSP seedlings was
much faster than in controls 13 days after
germination (DAG).
16
Chlorophyll fluorescence was measured using a
flourometer to obtain Fv (variable fluorescence)
and Fm (maximal fluorescence) levels.
Environmental stresses decrease Fv/Fm ratios, so
this was used to measure the effects of
dehydration, salt, and cold stress on
transformants and controls.
Overall, Fv/Fm ratios were 15-19 higher in
transformed plants than in controls.
Transgenic plants are performing efficient
photosynthesis under stressful environmental
conditions.
17
Conclusions -The fused TPSP enzyme expressed in
the transgenic rice plants had a higher catalytic
efficiency. -The physical proximity of the two
enzymes increased the reaction rate and
production of trehalose while maintaining low
levels of T-6-P. -Overproduction of trehalose in
transgenic rice plants lead to a greater
tolerance of environmental stresses.
18
Potential Global Impact and Ethical
Concerns -Selectable markers could confer
antibiotic resistance? -Contamination of organic
crops and creation of super weeds? -GE plants
could produce toxins that damage soil
conditions? -Where does it end? PRO CON Inc
rease in crop yields can There is enough food in
the solve world hunger. world for every person
to consume 3,600 cal/day. Problem is
distribution not production
19
Any Questions?
Attack of the Frankenflowers!!!
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