phytoremediation and phytosensing of explosives: the target genes by: ariel moore
TRANSCRIPT
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Phytoremediation and Phytosensing of
Explosives: The Target genesBy: Ariel Moore
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BackgroundPlant metabolism of xenobiotics has three phases
Activation (transformation)
Conjugation
Compartmentation (elimination)
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Need to KnowTNT
2, 4, 6-trinitortoluene
RDXHexahydro- 1, 3, 5-trinitro-1, 3, 5-triazine
Toxic, mutagenic, and carcinogenic
PhytoremediationCost effective and soil stays in place causing less ecosystem
disruption
Phytosensing
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Structures
TNT RDX
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Materials and MethodsArabidopsis thaliana
Murashige and Skoog (MS) medium supplemented
RDX different milimolar concentrations
TNT different micromolar concentrations
DMSO in proportion to the RDX and TNT concentrations
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RNA Preparation
Grown on MS medium0.5 RDX2.0 TNTProportional amounts of DMSO
Total RNA extracted from all three growthsPurifiedAssessed with microarrays and RT-PCR
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AnalysisTwo-Color Microarray
Only for RDXThree biological replicates and dye
swap technique
Affymetrix MicroarraySame biological total RNA as two-color
to prepare cDNA24,000 genes and 22,500 probe setsGuanine cytosine robust multi-array
analysis algorithm and logs
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Analysis IIAmplification
Real-Time RT-PCR
Enzyme activation 2 min. 55 degrees Celsius
Denaturation 15 min 95 degrees Celsius
40 cycles of 15 seconds, 95 degrees C30 seconds, 55 degrees C30 seconds, 72 degrees C
Negative control without revers transcriptase
PCR products confirmed for size
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Results
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Two-Color ResultsMost highly upregulated
Leucine-rich repeat family protein (At4g33970)
Other upregulatedProtease inhibitor/lipid transferPutative mannitol transporterXyloglucan/xyloglucosyl transferase (At4g14130)Lipozygenase (LOX2;At3g45140)
Downregulated genesPutative cysteine proteasePutative protease inhibiorXyloglucan/xyloglucosyl transferase (At5g57560)
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Affymetric ResultsMost highly upregulated
Lipozygenase (LOX2;At3g45140)
Other upregulated genesABC transporterUridine diphosphate transferase family proteinPutative peroxidase
Most highly downregulatedUnknown expressed protein
Other down regulatedGene encoding for neurofilament protein-relatedCYTOCHROME P450
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Real Time RT-PCRCarried out to confirm the microarray results
6 genes were used
Molecular functions of differentially expressed genes in response to TNT and RDX were different.
Functional categorization for upregulated genes28% unknown function27% transcription factor activity12% transferase activity8% transporter activity11% protein binding
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DiscussionDetoxification of RDX
Several genes induced by RDXGenes upregulated (transferase and transporter)9 unknown genes in two-color and 20 from Affymetrix
Detoxification of TNTPlants readily uptake and accumulate TNT in the rootsGenes upregulated (transferase and transporter activities)30 unknown proteins functions upregulated
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CommonalityFew significant genes were upregulated or downregluated in
both explosives.
SpeculationDiffer chemically and have different metabolic pathways
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Future Applications and Studies
Need phytoremediation of both compounds
Identification of either TNT or RDX
Characterizing the
identified genes
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References "Landmines | Eric W. Barton - Man on a Mission." Eric W Barton Man on a Mission Landmines
Tag. N.p., n.d. Web. 05 Dec. 2012. <http://www.ericbartononline.com/tag/landmines/>.
Panz, Katarzyna, and Korneliusz Miksch. "Phytoremediation of Explosives (TNT, RDX, HMX) by Wild-type and Transgenic Plants." Science Direct. N.p., n.d. Web. 28 Nov. 2012. <http://www.sciencedirect.com/science/article/pii/S0301479712004240>.
Rao, Murali, and Et. Al. "Phytoremediation and Phytosensing of Chemical Contaminants, RDX and TNT: Identification of the Required Target Genes." Springer Linker. Springer-Verlag, 19 June 2009. Web. 29 Nov. 2012. <http://link.springer.com/article/10.1007/s10142-009-0125-z/fulltext.html>.
"Research Unit Microbe-Plant Interactions: Research » Research Groups » Root Ecophysiology » Research." Research Unit Microbe-Plant Interactions: Research » Research Groups » Root Ecophysiology » Research. N.p., n.d. Web. 05 Dec. 2012. <http://www.helmholtz-muenchen.de/en/amp-eng/research/research-groups/root-ecophysiology/research/index.html>.
Rylott, Elizabeth L., and Neil Bruce. "Plants Disarm Soil: Engineering Plants for the Phytoremediation of Explosives." Science Direct. N.p., Feb. 2009. Web. Nov. 28. <http://www.sciencedirect.com/science/article/pii/S0167779908002874>.