Recipient Organization
USDA-ARS, GENETICS AND PRECISION AGRICULTURE UNIT
810 HIGHWAY 12 EAST
MISSISSIPPI STATE,MS 39762
Performing Department
Agricultural Research Service
Non Technical Summary
Vegetable oils have been used for millennia as a major human and animal nutritional component. Cottonseed oil is a key vegetable oil, and has been used as an ingredient in margarines, frying oils, spreads, salad dressings, and many other food and feed applications. Though its primary fatty acid composition is not markedly different that many other plant oils, cotton is one of a small number of plants that produces novel cyclopropyl fatty acids (CPFAs) in its seeds and roots. Recent studies compared the blood lipid profiles of mice and human subjects that had been fed high-fat diets rich in various types of oils, including safflower, olive, and cottonseed. Strong lipid-lowering effects conducive to increased heart health were seen only in the cottonseed oil-rich subjects. CPFAs were identified as the likely active component. As such, cottonseed oil shows great promise as natural biobased 'biopharmaceutical' that could compete with statins and other drugs as a treatment for high cholesterol. Thus, new research objectives have been undertaken to identify the biochemical pathways and key genes that produce cotton CPFAs, towards the longer-term goal of breeding and engineering new cottonseed varieties that contain altered quantities and profiles of CPFAs.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
The ultimate goal of this proposal is to generate novel cotton genotypes that have altered seed oil profiles to enhance the competitiveness, utility and marketability of U.S. cotton as a feedstock and as a source for novel value-added products. The specific objectives of the project are as follows:1) complete the tissue culture and genotyping of first-of-their-kind cyclopropane synthase (CPS) gene CRISPR-edited cotton lines being developed in collaboration with Kent Chapman, Univ. of North Texas, examine their seed fatty acid profiles, and analyze whole plants for qualitative and quantitative traits in seeds, roots, flowers, and other plant organs. This is the first step in the assessment of the as-yet unknown chemical and physiological roles of unusual CPFAs in cotton; 2) use rapid and high-throughput model organisms (such as bakers' yeast and transient tobacco leaf assays) to identify and functionally characterize novel cotton lipid metabolic enzymes and regulatory proteins, with particular focus on those that physically interact with the CPS enzymes that catalyze the first step in cotton CPFA biosynthesis; 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 objectives 1 and 2. Promising new lines with enhanced seed CPFA profiles (and other potentially beneficial changes to seed fatty acid composition) will be shared with co-investigator Paton for metabolic studies in a mammalian system.
Project Methods
-Generation of CPS gene-edited plants via issue culture: two different 'broad-spectrum CPS' CRISPR-Cas9 constructs are integrated into the chromosomes of cultured Coker-312 cotton plants via Agrobacterium-mediated transformation. Cotton explants are cultured on selective media containing appropriate hormones and supplements. Rooted plantlets producing shoots will be tested for the presence of the Cas9 gene by PCR, positive plants will be transferred to soil and eventually placed in a greenhouse or growth chamber. Representatives of at least 4-5 independent fertile lines will be subjected to small-scale genomic DNA isolation, followed by PCR amplification of the targeted CPS genes, restriction endonuclease treatment, and amplicon sequencing to assess levels of CPS gene editing in the initial T0 lines. Those showing highest levels of editing activity will be grown to maturity for production of T1 seeds. T1 plants and successive generations will be generated by self-pollination and continued monitoring of both CPS gene editing levels and segregation of the Cas9 nuclease gene. Finished lines containing homozygous CPS edits will be chosen for further analysis of lipid composition of seeds, roots, and other relevant tissues by gas chromatography.- Multiple rapid and high-throughput yeast two-hybrid experimental approaches will be used to identify novel structural, regulatory, and metabolic proteins and enzymes and regulatory proteins that physically interact with cotton CPSs. At least some of the enzymes and other accessory proteins that participate in conversion of DHSA to SA and MA, and help dictate the partitioning of all CPFAs to their intended organellar destinations, will do so by forming protein:protein complexes with CPS, involving direct physical contact. The identity of each interacting protein and the authenticity of its interaction with CPS will be confirmed by co-expression studies in at least one other experimental model system. Each candidate gene will also be tested for its ability to effect altered CPFA production when transiently coexpressed with cotton CPS in Nicotiana benthamiana leaves.- Candidate genes that pass the first and second protein:protein interaction tests will be incorporated into new gene editing plasmid constructs by standard DNA cloning methods, followed by Agrobacterium transformation and transformed into cotton. Early generations of these newly-edited lines with unique genetic alterations will be propagated by self-pollination until reaching homozygosity of the edited loci, with accompanying genomic DNA PCR analyses along the way. Individual and pooled seed samples from these plants will be used for oil content and FA profile. Fiber quality and content analysis, testing for normal morphology and vegetative growth and reproduction, and testing of oil trait consistency across multiple environments will be initiated and carried out by collaboration with ARS and University of North Texas colleagues.-Production of HOa1 seeds, which contain ~34-36% monounsaturated oleic acid (compared to 18% oleic acid in standard upland cottonseed oil) will be scaled up to produce at least 35 lbs of whole seed, the amount necessary to produce approx. 3.5 L of processed oil to be used in a full-scale diet formulation for mammalian feeding studies. USDA-ARS facilities, and resources shared by stakeholders, will be used to help achieve this objective. GC and MS analyses will be conducted on whole seed and extracted oil samples to confirm oil yield and fatty acid composition prior to initiation of feeding studies.