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Phytoremediation and Phytosensing of Explosives: Gene Targets

Explore the plant metabolism phases for TNT and RDX, phytoremediation advantages, and gene expression analysis. Identification of target genes using Arabidopsis thaliana and RNA preparation methods discussed with results.

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Phytoremediation and Phytosensing of Explosives: Gene Targets

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  1. Phytoremediation and Phytosensing of Explosives: The Target genes By: Ariel Moore

  2. Background • Plant metabolism of xenobiotics has three phases • Activation (transformation) • Conjugation • Compartmentation (elimination)

  3. Need to Know • TNT • 2, 4, 6-trinitortoluene • RDX • Hexahydro- 1, 3, 5-trinitro-1, 3, 5-triazine • Toxic, mutagenic, and carcinogenic • Phytoremediation • Cost effective and soil stays in place causing less ecosystem disruption • Phytosensing

  4. Structures RDX TNT

  5. Materials and Methods • Arabidopsis thaliana • Murashige and Skoog (MS) medium supplemented • RDX different milimolar concentrations • TNT different micromolar concentrations • DMSO in proportion to the RDX and TNT concentrations

  6. RNA Preparation • Grown on MS medium • 0.5 RDX • 2.0 TNT • Proportional amounts of DMSO • Total RNA extracted from all three growths • Purified • Assessed with microarrays and RT-PCR

  7. Analysis • Two-Color Microarray • Only for RDX • Three biological replicates and dye swap technique • Affymetrix Microarray • Same biological total RNA as two-color to prepare cDNA • 24,000 genes and 22,500 probe sets • Guanine cytosine robust multi-array analysis algorithm and logs

  8. Analysis II • Amplification • Real-Time RT-PCR • Enzyme activation 2 min. 55 degrees Celsius • Denaturation 15 min 95 degrees Celsius • 40 cycles of • 15 seconds, 95 degrees C • 30 seconds, 55 degrees C • 30 seconds, 72 degrees C • Negative control without revers transcriptase • PCR products confirmed for size

  9. Results

  10. Two-Color Results • Most highly upregulated • Leucine-rich repeat family protein (At4g33970) • Other upregulated • Protease inhibitor/lipid transfer • Putative mannitol transporter • Xyloglucan/xyloglucosyltransferase (At4g14130) • Lipozygenase (LOX2;At3g45140) • Downregulated genes • Putative cysteine protease • Putative protease inhibior • Xyloglucan/xyloglucosyltransferase (At5g57560)

  11. Affymetric Results • Most highly upregulated • Lipozygenase (LOX2;At3g45140) • Other upregulated genes • ABC transporter • Uridinediphosphatetransferase family protein • Putative peroxidase • Most highly downregulated • Unknown expressed protein • Other down regulated • Gene encoding for neurofilament protein-related • CYTOCHROME P450

  12. Real Time RT-PCR • Carried 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 genes • 28% unknown function • 27% transcription factor activity • 12% transferase activity • 8% transporter activity • 11% protein binding

  13. Discussion • Detoxification of RDX • Several genes induced by RDX • Genes upregulated (transferase and transporter) • 9 unknown genes in two-color and 20 from Affymetrix • Detoxification of TNT • Plants readily uptake and accumulate TNT in the roots • Genes upregulated (transferase and transporter activities) • 30 unknown proteins functions upregulated

  14. Commonality • Few significant genes were upregulated or downregluated in both explosives. • Speculation • Differ chemically and have different metabolic pathways

  15. Future Applications and Studies • Need phytoremediation of both compounds • Identification of either TNT or RDX • Characterizing the identified genes

  16. 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 KorneliuszMiksch. "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:ÂResearch » Research Groups » Root Ecophysiology » Research." Research Unit Microbe-Plant Interactions:ÂResearch » 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>.

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