Location: Commodity Utilization Research
Project Number: 6054-41000-113-005-I
Project Type: Interagency Reimbursable Agreement
Start Date: Mar 15, 2022
End Date: Mar 14, 2025
Objective:
Cyclopropyl fatty acids occur naturally in cottonseed oil, and these acids have been shown to improve fat metabolism and liver responses to obesity-inducing diets. Thus, these oils show great promise as active ingredients in biopharmaceutical products. However, very little is known regarding the biosynthesis of these acids or their tissue-specific or intracellular deposition. The concentration of these beneficial acids is also low in cottonseed. Therefore, we propose to identify promising lines of gene-edited cotton that accumulate enhanced levels of cyclopropyl acids. In conjunction with the co-investigator, oil from these lines will be used in mammalian fat metabolism studies can be perform. We will accomplish our goals through the following objectives:
(1) Complete the genotyping and phenotyping processes necessary to analyze gene-edited cotton lines that are currently under development. Determine all changes in seed and root lipid synthesis, including that of cyclopropyl fatty acid levels.
(2) Utilize high-throughput model organisms to characterize enzymes and other regulatory proteins that physically interact with cyclopropane synthase genes (which catatlyze the committed step of cyclopropyl fatty acid biosynthesis) to better understand the biosynthetic pathways involved.
(3) Assist co-investigator with studies aimed at testing mammalian liver and serum lipid levels and other metabolic markers from animals fed various cottonseed oil types containing varied fatty acid compositions.
Approach:
1) Complete the tissue culture and genotyping of first generation CRISPR-edited cotton lines being developed with collaborator (Chapman, Univ. of North Texas); examine seed fatty acid profiles and analyze whole plants for oil and other trait changes in seeds, roots, flowers, and other plant organs, to begin assessment of as-yet unknown physiological roles of unusual CPFAs found in some cotton tissues and organs; 2) Use rapid and high-throughput techniques in model organisms (yeast two-hybrid studies and bimolecular fluorescence complementation assays in bakers’ yeast and transient tobacco leaf assays, respectively) to identify and functionally characterize novel cotton fatty acid and seed oil metabolic enzymes and regulatory proteins, with particular focus on those that physically interact with the cyclopropane synthase (CPS) enzymes. 3) Begin the production process for novel second generation genome-edited cotton lines containing enhanced CPFA profiles, via creation of stable, transgene-free, CRISPR-based alterations to promising candidate genes identified in objective
2. During the breeding process, promising lines with substantial putative enhancements to seed CPFA profiles will be shared with co-investigator (C.P.) for mammalian metabolic studies.
Methods to be used for this project include molecular biology, biochemistry, plant transformation, mass spectrometry, gas chromatography, metabolomics, and functional genomics.
