Source: UNIVERSITY OF NORTH TEXAS submitted to NRP
MODIFICATION OF COTTONSEED FATTY ACID COMPOSITION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0190101
Grant No.
2001-35503-10991
Cumulative Award Amt.
(N/A)
Proposal No.
2001-01491
Multistate No.
(N/A)
Project Start Date
Sep 15, 2001
Project End Date
Sep 30, 2004
Grant Year
2001
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF NORTH TEXAS
1155 UNION CIR #305250
DENTON,TX 76203-5017
Performing Department
BIOLOGICAL SCIENCES
Non Technical Summary
Recent research indicates that it is possible to metabolically engineer changes in seed oil composition without adversely affecting crop performance. These capabilities suggest that selective modification of enzyme activities involved in fatty acid metabolism in cottonseeds will lead to newly designed cottonseed oils. Although fiber will always be the primary seed-derived product from cotton, increased value in secondary, processed seed products will increase the overall value of the cotton crop and could stimulate a greatly improved utilization of this renewable domestic agricultural resource. With support from our prior USDA-NRI Value-Added Products Award (Number 98-35503-6339), we have focused on cottonseed oil as our primary target for increasing the value of the cotton crop, and have adopted the long-term goal of developing novel cottonseed fatty acid compositions as value-added resources for the cotton industry. Our objectives for this award are: (1) to continue the development of new edible oils in cottonseed, substantially different from that of normal cottonseed oil, for
Animal Health Component
70%
Research Effort Categories
Basic
30%
Applied
70%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
50218101040100%
Knowledge Area
502 - New and Improved Food Products;

Subject Of Investigation
1810 - Cottonseed;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
The overall goal of this research project is to develop novel cottonseed fatty acid composition in seed oils of cotton plants as value-added resources for the cotton industry. Specifically, we will continue the development of new edible oils in cottonseed, for "mid-oleic", "low saturate", and "high saturate" oils, in transgenic cotton plants (cultivar Coker 312) by transformation with plasmid vectors harboring fatty acid-modifying gene constructs. These genes encode two key enzymes regulating fatty acid compositions in cottonseeds. The first is designated fatB and encodes an acyl-ACP (acyl carrier protein) thioesterase that regulates the palmitic acid content in cottonseed oil, and the second is fad2 and encodes a fatty acid desaturase that regulates the proportion of monounsaturated fatty acid (mainly oleic acid, C18:1) to diunsaturated fatty acid (C18:2). At present, we are characterizing the transgenic plants with altered oleic acid content by biochemical and molecular genetic approaches, in conjunction with breeders in west Texas and the Mississippi Delta region. Also, we plan to begin the development of transgenic cotton plants expressing heterologous plant genes for the production of conjugated polyunsaturated and epoxy fatty acids in cottonseed oil. These genes are closely related to the fatty acid desaturase gene (fad2 gene), and their expression in cottonseeds should lead to the production of novel industrial fatty acids in cottonseed oils.
Project Methods
Sense and antisense plasmid constructs with various gene regulatory elements and permutations of cotton fatB1 and fad2 DNA sequences have been developed in the binary "parent" vector pBI121 for Agrobacterium-mediated transformation of cotton plants. Also, expression vectors (developed at Dupont Agricultural Products Experimental Station, Wilmington, DE) harboring heterologous fatty acid desaturase-like sequences, such as a fad2-epoxygenase and fadX-conjugase, will be introduced into cotton plants by Agrobacterium-mediated transformation. This will provide a novel family of cottonseed oils with enriched levels of epoxy and conjugated fatty acids for potential industrial applications. The transgenic To cotton plants are grown to maturity, selfed, and progeny screened for expression of the transgenes by gas chromatography analysis of total fatty acids. Transgenic T1 plants are evaluated for integration of the plasmid construct DNA sequences by genomic blotting. Experiments with the strong constitutively active 35S CaMV and seed-specific phaseolin promoters are compared to determine the optimum combination of plant performance and seed oil composition. These new cotton lines will be developed in consultation with our advisors in the cottonseed products industry and cotton breeders in west Texas and the Mississippi Delta.

Progress 09/15/01 to 09/30/04

Outputs
The overall goal of this project was to develop transgenic cotton lines with novel fatty acid compositions in their seed oil. Several new oil combinations were developed, including modified oleic acid- and modified palmitic acid- lines. These transgenic plant lines are in various stages of testing and will be available for further research and/or commercialization where warranted and/or feasible. Other fatty acid modifications were made at the cellular level, such as production of conjugated fatty acids in cotton callus, but in these cases regeneration of transgenic plants was not achieved. The objectives of this project (initially funded in 1998) essentially have been accomplished and now related efforts are being supported by funds from a private seed company. No additional requests for support from the NRI for this work are anticipated at this time.

Impacts
Results from this research indicate that modification of seed oil composition in cotton plants through genetic engineering approaches is technically feasible and can produce new lines with desired seed fatty acid profiles. This raises the possibility that novel, added-value oleochemicals could be produced in combination with fiber increasing the value of the overall crop for producers.

Publications

  • Pirtle, R.M., Huynh, T.T., Yoder, D., Nampaisansuk, M., Pirtle, I., Chapman, K.D. (1999) Characterization of a palmitoyl-acyl carrier protein thioesterase (FatB1) in Cotton (Gossypium hirsutum L.). Plant Cell Physiology 40: 155-163
  • Yoder, D.W., Nampaisansuk, M., Pirtle, I. Chapman, K.D., Pirtle R.M. (1999) Molecular cloning and nucleotide sequence of a gene encoding a cotton palmitoyl-acyl carrier protein thioesterase. Biochim Biophys Acta, 1446: 403-413
  • Chapman, K.D., Austin-Brown, S. Sparace, S.A., Kinney, A.J., Ripp, K.G., Pirtle, I.L, Pirtle, R.M. (2001) Transgenic cotton plants with increased seed oleic acid content. Journal of the American Oil Chemists' Society (JAOCS) 78(9): 941-947
  • Pirtle, I.L., W. Kongcharoensuntorn, M. Nampaisansuk, K.D. Chapman, J. Knesek, and R.M Pirtle (2001) Molecular cloning and analysis of a gene for a cotton fatty acid desaturase 2 (FAD2). Proceedings of the Beltwide Cotton Conference. Volume 1: 551-554.
  • Chapman, K.D., Austin-Brown, S., Wessler, H., Huynh, T., Hoang, C., Sparace, S.A., Ricchiuti, T. Kinney, A.J., Ripp, K.G., Pirtle, I.L, Pirtle, R.M., Nampaisansuk, M. (2001) Metabolic enginneering for increased cottonseed oleic acid content. Proceedings of the Beltwide Cotton Conference. Volume 2: 1434-1436.
  • Pirtle, I., Kongcharoensuntorn, W., Nampaisansuk, M., Knesek, J. Chapman K.D., Pirtle R.M. (2001) Isolation and nucleotide sequence of functional delta-12 desaturase (fad2) gene from cotton (Gossypium hirsutum, L). Biochim Biophys Acta, 1522(2): 122-129.
  • Huynh, T., Pirtle, R.M., Chapman, K.D. (2002) Expression of a cotton (Gossypium hirsutum, L) FatB palmitoyl-acyl carrier protein thioesterase in Eschericia coli. Plant Physiology and Biochemistry,40: 1-9.
  • Huynh, T. (2001) Palmitoyl-Acyl Carrier Protein Thioesterase in Cotton (Gossypium hirsutum L.): Biochemical and Molecular Characterization of a Major Mechanism for the Regulation of Palmitic Acid Content. Ph.D. Dissertation. University of North Texas, 148 pp (K.D. Chapman, advisor).
  • Kongcharoensuntorn, W. (2001) Isolation and analysis of cotton genomic clones encompassing a fatty acid desaturase (FAD2) gene. Ph. D. Dissertation. University of North Texas, 129 pp (R. Pirtle, advisor).
  • Nampaisansuk, M. (2002) Molecular Cloning and Analysis of the Genes for Cotton Palmitoyl-Acyl Carrier Protein Thioesterase (PATE) and Delta-12 Fatty Acid Desaturase (Fad2-3) and Construction of Sense and Antisense Plasmid Vectors for Altering Oil Composition of Transgenic Cotton Plants. Ph.D. Dissertation. University of North Texas, 233 pp (R.Pirtle, advisor).
  • Yoder, D.W. (2002) Characterization of cDNA and Genomic Clones for a Palmitoyl-Acyl Carrier Protein Thioesterase (FatB1). Ph. D. Dissertation. University of North Texas, 113 pp (R. Pirtle, advisor).