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Phytoremediation and Phytosensing of
Explosives: The Target genesBy: Ariel Moore
BackgroundPlant metabolism of xenobiotics has three phases
Activation (transformation)
Conjugation
Compartmentation (elimination)
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
Structures
TNT RDX
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
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
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
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
Results
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)
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
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
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
CommonalityFew significant genes were upregulated or downregluated in
both explosives.
SpeculationDiffer chemically and have different metabolic pathways
Future Applications and Studies
Need phytoremediation of both compounds
Identification of either TNT or RDX
Characterizing the
identified genes
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>.