Download Identification and Localization of Carbon Concentrating

Identification and Localization of Carbon Concentrating Components
in Chlamydomonas reinhardtii
INTRODUCTION
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Most crops, including wheat, rice and soybean use C 3
photosynthesis
C4 and CAM photosynthesis overcome the tendency
of RuBisCO to wastefully fix oxygen rather than
carbon dioxide (photorespiration) while C3 does not
KEY QUESTIONS
RESULTS
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PIPELINE EFFICIENCY
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What proteins are essential to the functioning of the CCM in
Chlamydomonas?
Which proteins should be transformed into C3 plants to improve
photosynthetic efficiency?
How can we optimize the High Throughput Tagging Pipeline?
PCR
67%
Cloning
95%
METHODOLOGY
http://www.citruscollege.edu/lc/archive/biology
CHLAMYDOMONAS REINHARDTII
• Photosynthetic apparatus similar to land plants
• Unicellular
• Heterotrophic and autotrophic
photosynthetic apparatus
• Partially sequenced genome
http://protist.i.hosei.ac.jp
CARBON CONCENTRATING MECHANISM
• CCM increases the concentration of carbon dioxide
available to the initial carboxylase of the Calvin cycle,
RuBisCO
• Benefits include an increased tolerance to low
concentrations of inorganic carbon, reduced
photorespiration and a greater tolerance to
water stress
• Multiple genome saturated mutant screen
• Touchdown PCR
• Products were run through Gel Electrophoresis
Identification
and
Amplification
Maurino, Veronica G., et al., 2010
• Synthetic detours naturally occurring in cyanobacteria
were installed in Arabidopsis thaliana, to bypass
photorespiration
McGrath, Justin M., et al., 2014
• Models predict the benefits of engineering a full
cyanobacterial CCM into a C3 leaf
• Increased:
o Leaf photosynthesis by 25%
o Soybean yield by 15%
o Water use efficiency by 20%
REFERENCES
Eckardt, Nancy A. "Gene Regulatory Networks of the Carbon-Concentrating Mechanism in Chlamydomonas
reinhardtii." The Plant Cell Online 24.5 (2012): 1713-1713.
Fang, Wei, et al. "Transcriptome-wide changes in Chlamydomonas reinhardtii gene expression regulated by
carbon dioxide and the CO2-concentrating mechanism regulator CIA5/CCM1." The Plant Cell Online 24.5
(2012): 1876-1893.
Hom, Erik FY, and Andrew W. Murray. "Niche engineering demonstrates a latent capacity for fungal-algal
mutualism." Science 345.6192 (2014): 94-98
Kebeish, Rashad, et al. "Chloroplastic photorespiratory bypass increases photosynthesis and biomass
production in Arabidopsis thaliana." Nature biotechnology 25.5 (2007): 593-599.
Maurino, Veronica G., and Christoph Peterhansel. "Photorespiration: current status and approaches for
metabolic engineering." Current opinion in plant biology 13.3 (2010): 248-255.
Wang, Yingham, and Deqiang Duanmu. "Carbon dioxide concentrating mechanism in
Chlamydomonas reinhardtii: inorganic carbon transport and CO2 recapture."
pringer Science and Business Media (2010): n. pag. Print.
95%
• Purified products were run against
samples of known concentration to
determine DNA concentration
Purification
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Localization
65%
ELECTROPORATION
EFFICIENCY
467 DNA fragments were
successfully amplified
83 fully amplified genes
were transformed into
E.coli
52 genes were localized
efficiently
LOCALIZATION
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PCR PRODUCTS
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Cre04.g229300_F, a
suggested RuBisCO Activase
coding protein
The concentrated green pocket
indicated that the gene was
localized in the pyrenoid
In total, 83 genes were
localized, and 60% were
identifiable
• Gel purified genes were cloned in frame
with yellow fluorescent protein, CrVenus
• Ampicillin resistance
• Transformed E.coli by heat shock
Gibson
Assembly
Image provided courtesy of
Dr. Luke Mackinder
Transformation of CCM into C3 crops could increase
crop yield
REVIEW OF LITERATURE
Transformation
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PCR ALTERATIONS
• Plasmids were extracted then cut with the restriction enzyme Eco-RV to
confirm successful cloning
Plasmid
Sequencing
• Cassettes were incubated in a 16oC water bath for 5 minutes
• DNA was added, and a shock was administered at 800V and 25 uF
Electroporation
and Plating
6% DMSO
CONCLUSIONS
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• Transformation plates were screened for
strong Venus expressing colonies using the
Typhoon Imaging System
• Confocal imaging determined subcellular
localization
Localization
DISCUSSION
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4.5% DMSO
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High Throughput Tagging Pipeline can be successfully
utilized to amplify and localize putative photosynthetic
genes of the CCM in the alga Chlamydomonas
reinhardtii
Electroporation efficiency is negatively affected by
increased time transformation cassettes were left in a
16oC water bath
Failed PCR primers can be recovered by increasing
DMSO levels and decreasing extension times
82 genes will be imaged and analyzed at a later date
Complementation vectors will be constructed with different resistance markers
Increased availability of information to other labs will assist in all realms of C.reinhardtii research
It is estimated that there will be about 11 billion people living on the earth by 2100
New innovations need to be made to increase food production
Transforming a viable CCM into crop plants augments photosynthetic productivity,
increasing crop yield
www.pflanzenforschung.de