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Identification Of Novel Growth Regulators In Plant Populations Expressing Random Peptides

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Title:
Identification Of Novel Growth Regulators In Plant Populations Expressing Random Peptides
Series Title:
19th Annual Undergraduate Research Symposium
Creator:
Mourad, Nadia
Language:
English
Physical Description:
Undetermined

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Subjects / Keywords:
Center for Undergraduate Research
Center for Undergraduate Research
Genre:
Conference papers and proceedings
Poster

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Abstract:
The need for new herbicidal compounds, that selectively target certain plants as opposed to others, is steadily increasing due to the demand for environmentally friendly chemicals within agriculture, and the increasing of weeds’ chemical resistance. This project utilizes rapidly screened populations of Arabidopsis thaliana in libraries where individual plants have been transformed to induce a small, novel, random cyclical peptide (RCP) that has the potential to disrupt the plant’s growth and development. This allows for the screening for lethal phenotypes of multiple plants or peptides. These phenotypes are assumed to have been caused by the insertion of the RCP. These molecules, constructed from a degenerative DNA oligonucleotide sequence PCR reaction, could be essential for the creation of new herbicides. Currently, around 25,000 seedlings have been selectively screened on minimal media and Kanamycin, since the vector contained a Kanamycin resistance gene. It was confirmed that the plant contains the RCP insert with microscopic fluorometric detection of GFP that was also included in the vector. Almost 400 seedlings that were underperforming, vitrified, and GFP-positive were extracted for the peptide DNA. The next step is to re-clone these peptides using Agrobacterium into healthy plants and confirm reproducible lethal phenotype. ( en )
General Note:
Research authors: Nadia Mourad, Liliam Martinez-Bello, Kevin Folta - University of Florida
General Note:
Faculty Mentor: The need for new herbicidal compounds, that selectively target certain plants as opposed to others, is steadily increasing due to the demand for environmentally friendly chemicals within agriculture, and the increasing of weeds’ chemical resistance. This project utilizes rapidly screened populations of Arabidopsis thaliana in libraries where individual plants have been transformed to induce a small, novel, random cyclical peptide (RCP) that has the potential to disrupt the plant’s growth and development. This allows for the screening for lethal phenotypes of multiple plants or peptides. These phenotypes are assumed to have been caused by the insertion of the RCP. These molecules, constructed from a degenerative DNA oligonucleotide sequence PCR reaction, could be essential for the creation of new herbicides. Currently, around 25,000 seedlings have been selectively screened on minimal media and Kanamycin, since the vector contained a Kanamycin resistance gene. It was confirmed that the plant contains the RCP insert with microscopic fluorometric detection of GFP that was also included in the vector. Almost 400 seedlings that were underperforming, vitrified, and GFP-positive were extracted for the peptide DNA. The next step is to re-clone these peptides using Agrobacterium into healthy plants and confirm reproducible lethal phenotype. - Center for Undergraduate Research,

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University of Florida
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Copyright Nadia Mourad. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.

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Printing: large Customizing the Content: The placeholders in this formatted for you. placeholders to add text, or click an icon to add a table, chart, SmartArt graphic, picture or multimedia file. T from text, just click the Bullets button on the Home tab. If you need more placeholders for titles, make a copy of what you need and Smart Guides will help you align it with everything else. Want to use your own pictures instead of ours? No problem! Just right Change Picture. Maintain the proportion of pictures as you resize by dragging a corner. Identification of Novel Growth Regulators in Plant Populations Expressing Random Peptides Folta,K Martinez Bello L. & Mourad, N. M. Horticultural Sciences Department, University of Florida, Gainesville ABSTRACT The need for new herbicidal compounds, that selectively target certain plants as opposed to others, is steadily increasing due to the demand for environmentally friendly chemicals within agriculture, and the increasing of chemical resistance This project utilizes rapidly screened populations of Arabidopsis thaliana where individual plants have been transformed with a library of a small, novel, random cyclical peptides (RCP) that have the potential to disrupt the growth and development These molecules, constructed from a degenerative DNA oligonucleotide sequence PCR reaction, could be essential for the creation of new herbicides Currently, around 25 000 seedlings have been selectively screened on minimal media with Kanamycin, since the vector contains a Kanamycin resistance gene Confirmation of RCP insertion into the plant is done in a two step procedure First, seedlings are screened under a fluorometric microscope for detection of GFP Then, the DNA is extracted from the GFP positive seedlings and the transgene presence and sequence is confirmed by PCR and Sanger sequencing Almost 100 underperformers, vitrified, and GFP positive seedlings that carry different RCP peptides have been identified to date The next step is to re clone these peptides into healthy plants using Agrobacterium tumefaciens and confirm reproducible underperforming phenotype Finally, a specific mechanism of action for the peptides in the plant must be found along with expanding the population to identify new targets OBJECTIVES To discover potential small cyclical peptides that, when inserted into Arabidopsis disrupts growth and development for the development of novel herbicides Hight Throughput strategies like Chemical Genomics allow to screen for a great number of compounds thus obtaining multiple candidates for further study Confirm underperforming phenotype due to random peptide insertion using fluorometric detection and PCR amplification METHODS 1. A library is assembled made up of 6 amino acid RCPs that are constructed from a degenerative DNA oligonucleotide sequence PCR reaction. 2. Library is recombined into the pK7WG2D vector and transformed into E.coli. 3. Agrobacterium tumefaciens GV3101 is transformed with the binary vectors using Gateway Recombination Cloning Technology. 4. Wild type (Col 0) Arabidopsis is transformed via floral dip of the Agrobacterium, and F2 seeds collected from these mutants. 5. F2 mutant seeds are plated on minimal media (MS) and Kanamycin. Each Batch was 18 plates with 0.1g of total Arabidopsis seed. 6. Seedlings that displayed underperforming phenotypes (pale pigmentation, stunted growth, etc.) were chosen from each plate to confirm GFP containing insert under a UV light microscope. 7. DNA extraction, PCR confirmation, and preparation for Sanger Sequencing. 8. The same sequence is then re introduced into new transgenic lines to test for recapitulation of the original phenotype (Phase 2). 9. Larger KAN resistant seedlings are transplanted in soil at 20 C under 16 hour light / 8 hour dark conditions. Plants exhibiting phenotypes were tagged and monitored for atypical growth throughout their development. METHODS CONT. RESULTS RESULTS CONT. DISCUSSION AND CONCLUSIONS The goal of this work was to test the hypothesis that overexpression of cyclical small random peptides could unveil new candidates for chemistries that modify plant biology. These trials have produced dozens of new candidates that interfere with discrete plant processes. The goal now is to increase the number of plants to be screened, screen more conditions for peptide dependent effects, and identify the mechanisms where the characterized peptides are integrating into plant biology (Phase 3). Phase 3 may include immunolocalization of the peptide, in situ peptide quantification, and epitope tagging to identify interactors in the cell. In this report we present a new way to potentially identify novel molecules that could modulate important processes in plants. The peptides identified may then be used to impart their effects in transgenic plants or potentially even when applied in drenches or sprays. The structure of the peptides may be a basis of drug discovery, leading to new compounds representing novel growth regulators, herbicides or developmental modulators. References Zhilong Bao, Maureen A. Clancy, Raquel F. Carvalho, Kiona Elliott, Kevin M. Folta 2017. Identification of Novel Growth Regulators in Plant Populations Expressing Random Peptides. Plant Physiology Oct 2017, 175 (2) 619 627; DOI: 10.1104/pp.17.00577 Transformation and isolation of transgenic Arabidopsis plants GFP positive underperformers To date, 6 Batches (>25,000 seeds) have been selectively screened. Over 500 GFP positive seedlings were isolated, and almost 100 of those have been confirmed to contain the RCP insert via PAGE and have been sent for sequencing. It is these peptide candidates that will be taken forward into Phase 2 re cloning. Discovery of underperformers / new peptides Confirmation of candidates Functional characterization of candidates Mechanism of action of peptide Transplanting of KAN resistant seedlings to soil PAGE of PCR products from DNA isolation of potential peptides Mutant 485 Mutant 363 Mutant 375 Arabidopsis underperformers in soil after 2 weeks after transplanting. Red arrows indicate severe phenotype that continued in soil. DNA was extracted from these plants. An approximately 500 bp DNA fragment containing the random peptide open reading frame and part of the pK7WG2D vector sequence was amplified using primers PEP F and PEP R and sequenced. The image on the left shows a PAGE showing a high amount of positive PCR products from Batch 6, mutants 453 492. Positive bands were cleaned and sent for sequencing.