N-acylethanolamines (NAEs) are fatty acid derivatives that occur naturally in plant and animal systems. In mammals, they regulate physiological functions, including neurotransmission, immune responses, vasodilation, embryo development and implantation, feeding behavior, and cell proliferation. NAEs are metabolized by fatty acid amide hydrolase (FAAH), which belongs to the amidase signature family. It is hypothesized that putative FAAH functions as the catalyst in the metabolism of N-acylethanolamine in tomato plants. To test the hypothesis, FAAH protein homologs were identified in tomato via in silico analysis. Among the six homologs identified, FAAH1 and FAAH2 were selected for further validation. This study is focused on 1) in silico analyses of SlFAAH2, 2) quantification of transcript levels for SlFAAH2, 3) determination of FAAH activity at various developmental stages of tomato, and 4) isolation of and synthesis of SlFAAH2 cDNA for cloning. Putative SlFAAH2 showed high homology to Arabidopsis FAAH1. Transcript levels, as measured by qPCR using RNA extracted from various developmental stages, were highest at 0 days and lowest at 4 days. Enzyme activity at certain developmental stages coincided with SlFAAH2 transcript levels. In order to confirm that putative SlFAAH2 encodes for an enzyme that hydrolyzes NAEs, SlFAAH2 gene was isolated from total RNA of tomato, cDNA was synthesized by reverse transcription and the gene was amplified by PCR for further cloning in a heterologous expression system for biochemical characterization. To gain better molecular and biochemical understanding of FAAH and determine its broader functions, it is pertinent to characterize FAAH in other plant species.
| Identifer | oai:union.ndltd.org:ETSU/oai:dc.etsu.edu:honors-1352 |
| Date | 01 May 2016 |
| Creators | Stuffle, Derek A |
| Publisher | Digital Commons @ East Tennessee State University |
| Source Sets | East Tennessee State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Undergraduate Honors Theses |
| Rights | Copyright by the authors., http://creativecommons.org/licenses/by-nc-nd/3.0/ |
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