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Fatty acid desaturase genes from plants

a technology of fatty acid desaturase and genes, applied in the field of nucleic acid fragments encoding fatty acid desaturase enzymes, can solve the problems of unstable oil, reduced polyunsaturated fatty acid levels, and development of disagreeable odors and flavors, and achieve the effect of controlling the nature and level of unsaturated fatty acids

Inactive Publication Date: 2009-08-13
EI DU PONT DE NEMOURS & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011]Applicants have discovered a means to control the nature and levels of unsaturated fatty acids in plants. Nucleic acid fragments from glycerolipid desaturase cDNAs or genes are used to create chimeric genes. The chimeric genes may be used to transform various plants to modify the fatty acid composition of the plant or the oil produced by the plant. More specifically, one embodiment of the invention is an isolated nucleic ac

Problems solved by technology

However, in use, the high level of poly-unsaturated fatty acids in canola oil renders the oil unstable, easily oxidized, and susceptible to development of disagreeable odors and flavors (Gailliard, 1980, Vol. 4, pp.
The levels of poly-unsaturates may be reduced by hydrogenation, but the expense of this process and the concomitant production of nutritionally questionable trans isomers of the remaining unsaturated fatty acids reduces the overall desirability of the hydrogenated oil (Mensink et al., New England J. Medicine (1990) N323: 439-445).
Similar problems exist with soybean and corn oils.
In the USA flax is highly susceptible to rust infection.
Similar commercial progress with the other plants shown in Table 1 has been largely elusive due to the difficult nature of the procedure and the pleiotropic effects of the mutational regime on plant hardiness and yield potential.
However, biochemical characterization of the desaturase reactions has been meager.
The instability of the enzymes and the intractability of their proper assay has largely limited researchers to investigations of enzyme activities in crude membrane preparations.
However, these genes have not proven useful for isolating plant fatty acid desaturases other than stearoyl-ACP desaturase via sequence-dependent protocols, and the present art does not indicate how to obtain plant fatty acid desaturases other than stearoyl-ACP desaturases or how to obtain fatty acid desaturase-related enzymes.
Thus, the present art does not teach how to obtain glycerolipid desaturases from plants.
Furthermore, there is no evidence that a method to control the nature and levels of unsaturated fatty acids in plants using nucleic acids encoding fatty acid desaturases other than stearoyl-ACP desaturase is known in the art.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Isolation of Genomic DNA Flanking the T-DNA Site of Insertion in Arabidopsis thaliana Mutant Line 3707 Identification of an Arabidopsis thaliana T-DNA Mutant with Low Linolenic Acid Content

[0133]A population of Arabidopsis thaliana (geographic race Wassilewskija) transformants containing the T-DNA of Agrobacterium tumefaciens was generated by seed transformation as described by Feldmann et al., (Mol. Gen. Genetics (1987) 208:1-9). In this population the transformants contain DNA sequences encoding the pBR322 bacterial vector, nopaline synthase, neomycin phosphotransferase (NPTII, confers kanamycin resistance), and b-lactamase (confers ampicillin resistance) within the T-DNA border sequences. The integration of the T-DNA into different areas of the chromosomes of individual transformants may cause a disruption of plant gene function at or near the site of insertion, and phenotypes associated with this loss of gene function can be analyzed by screening the population for the phenotype...

example 2

Cloning of Arabidopsis thaliana Delta-15 Desaturase cDNA using Genomic DNA Flanking The T-DNA Site of Insertion in Arabidopsis thaliana Mutant Line 3707 as a Hybridization Probe

[0151]The 5.2 kb Hind III fragment from plasmid pF1 was purified by electrophoresis in agarose after digestion of the plasmid with Hind III and radiolabeled with 32P as described above. For the preparation of an Arabidopsis cDNA library, polyadenylated mRNA was prepared from 3 day-old, etiolated Arabidopsis (ecotype Columbia) seedling hypocotyls using standard protocols (Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. (1989) Cold Spring Harbor Laboratory Press). Five micrograms of this mRNA were used as template with an oligo d(T) primer, and Moloney Murine Leukemia Virus reverse transcriptase (Pharmacia) was used to catalyze first strand cDNA synthesis. Second-strand cDNA was made according to Gubler et al., (Gene (1983) 25:263-272) except that DNA ligase was omitted. After the second stran...

example 3

Cloning of an Arabidopsis cDNA Encoding a Plastid Delta-15 Fatty Acid Desaturase

[0153]A related fatty acid desaturase was cloned in a similar fashion, except that the probe used was not derived from a PCR reaction on pCF3, but rather was the actual 1.4 kb Not I fragment isolated from pCF3 which was purified and radiolabeled as described above.

[0154]Approximately 80,000 phage from the Arabidopsis etiolated hypocotyl cDNA library described above were plated out and screened essentially as before, except as indicated below. The filters were soaked in 1M NaCl, 50 mM Tris-HCl, pH 7.5, 1% SDS, 5% dextran sulfate, 0.1 mg / mL denatured salmon sperm DNA during the pre-hybridization step (8 hr at 50° C.). Then probe was added and the hybridization proceeded over 16 hr at the same temperature. Filters were washed sequentially with 2×SSPE, 0.1% SDS at room temperature for 5 min and then again with fresh solution for 10 min, and finally with 0.5×SSPE, 0.1% SDS at 50° C. for 5 min. Approximately 1...

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Abstract

The preparation and use of nucleic acid fragments encoding fatty acid desaturase enzymes are described. The invention permits alteration of plant lipid composition. Chimeric genes incorporating such nucleic acid fragments with suitable regulatory sequences may be used to create transgenic plants with altered levels of unsaturated fatty acids.

Description

FIELD OF THE INVENTION[0001]The invention relates to the preparation and use of nucleic acid fragments encoding fatty acid desaturase enzymes to modify plant lipid composition.BACKGROUND OF THE INVENTION[0002]Plant lipids have a variety of industrial and nutritional uses and are central to plant membrane function and climatic adaptation. These lipids represent a vast array of chemical structures, and these structures determine the physiological and industrial properties of the lipid. Many of these structures result either directly or indirectly from metabolic processes that alter the degree of unsaturation of the lipid. Different metabolic regimes in different plants produce these altered lipids, and either domestication of exotic plant species or modification of agronomically adapted species is usually required to economically produce large amounts of the desired lipid.[0003]Plant lipids find their major use as edible oils in the form of triacylglycerols. The specific performance a...

Claims

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Application Information

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IPC IPC(8): A01H1/00C07H21/04A01H5/00C11B1/00C12Q1/68A01H5/10C11C3/00C12N5/10C12N9/00C12N9/02C12N15/09C12N15/53C12N15/82
CPCA01H5/10C12Y114/19006C12N15/8247C12N9/0083
Inventor BROWSE, JOHNGRAU, LUIS PEREZKINNEY, ANTHONY J.PIERCE, JR., JOHN W.WIERZBICKI, ANNA M.YADAV, NARENDRA S.
Owner EI DU PONT DE NEMOURS & CO