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Phytoremediation and P hytosensing of Explosives: The T arget genes

Phytoremediation and P hytosensing of Explosives: The T arget genes. By: Ariel Moore. Background. Plant metabolism of xenobiotics has three phases Activation (transformation) Conjugation Compartmentation (elimination). Need to Know. TNT 2, 4, 6-trinitortoluene RDX

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Phytoremediation and P hytosensing of Explosives: The T arget genes

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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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