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Polyunsaturated fatty acids in plants

a technology plants, applied in the field of polyunsaturated fatty acids in plants, can solve the problems of uncontrollable fluctuations in availability of natural sources, depletion of fish stocks, and natural variation or depletion of fish stocks, and achieve the effect of modifying the fatty acid profil

Inactive Publication Date: 2010-02-16
CALGENE LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009]Novel compositions and methods are provided for preparation of poly-unsaturated long chain fatty acids and desaturases in plants and plant cells. The methods involve growing a host plant cell of interest transformed with an expression cassette functional in a host plant cell, the expression cassette comprising a transcriptional and translational initiation regulatory region, joined in reading frame 5′ to a DNA sequence encoding a desaturase polypeptide capable of modulating the production of PUFAs. Expression of the desaturase polypeptide provides for an alteration in the PUFA profile of host plant cells as a result of altered concentrations of enzymes involved in PUFA biosynthesis. Of particular interest is the selective control of PUFA production in plant tissues and / or plant parts such as leaves, roots, fruits and seeds. The invention finds use for example in the large scale production of DHA, Mead Acid, EPA, ARA, DGLA, stearidonic acid GLA and other fatty acids and for modification of the fatty acid profile of edible plant tissues and / or plant parts.

Problems solved by technology

However, there are several disadvantages associated with commercial production of PUFAs from natural sources.
Natural sources also are subject to uncontrollable fluctuations in availability.
Fish stocks may undergo natural variation or may be depleted by overfishing.
Fish oils have unpleasant tastes and odors, which may be impossible to economically separate from the desired product, and can render such products unacceptable as food supplements.
Animal oils, and particularly fish oils, can accumulate environmental pollutants.
Weather and disease can cause fluctuation in yields from both fish and plant sources.
Crops which do produce PUFAs, such as borage, have not been adapted to commercial growth and may not perform well in monoculture.
Growth of such crops is thus not economically competitive where more profitable and better established crops can be grown.
Large scale fermentation of organisms such as Mortierella is also expensive.
Natural animal tissues contain low amounts of ARA and are difficult to process.
Microorganisms such as Porphyridium and Mortierella are difficult to cultivate on a commercial scale.
Supplements such as fish oil capsules can contain low levels of the particular desired component and thus require large dosages.
High dosages result in ingestion of high levels of undesired components, including contaminants.
Care must be taken in providing fatty acid supplements, as overaddition may result in suppression of endogenous biosynthetic pathways and lead to competition with other necessary fatty acids in various lipid fractions in vivo, leading to undesirable results.
Unpleasant tastes and odors of the supplements can make such regimens undesirable, and may inhibit compliance by the patient.
An expansive supply of poly-unsaturated fatty acids from natural sources and from chemical synthesis are not sufficient for commercial needs.

Method used

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Examples

Experimental program
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example 1

Expression of ω-3 Desaturase from C. Elegans in Transgenic Plants

[0047]The Δ15 / ω-3 activity of Brassica napus can be increased by the expression of an ω-3 desaturase from C. elegans. The fat-1 cDNA clone (Genbank accession L41807; Spychalla, J. P., Kinney, A. J., and Browse, J. 1997 P.A.A.S. 94, 1142-1147, SEQ ID NO:1 and SEQ ID NO:2) was obtained from john Browse at Washington State University. The fat-1 cDNA was modified by PCR to introduce cloning sites using the following primers:[0048]Fat-1forward (SEQ ID NO:3):[0049]5′-CUACUACUACUACTGCAGACAATGGTCGCTCATTCCTCAGA-3′[0050]Fat-1reverse (SEQ ID NO:4):[0051]5′-CAUCAUCAUCAUGCGGCCGCTTACTTGGCCTTTGCCTT-3′

[0052]These primers allowed the amplification of the entire coding region and added PstI and NotI sites to the 5′- and 3′-ends, respectively. The PCR product was subcloned into pAMP1 (GIBCOBRL) using the CloneAmp system (GIBCOBRL) to create pCGN5562. The sequence was verified by sequencing of both strands to be sure no changes were intro...

example 2

Over-Expression of Δ15-desaturase Activity in Transgenic Canola

[0060]The Δ15-desaturase activity of Brassica napus can be increased by over-expression of the Δ15-desaturase cDNA clone.

[0061]A. B. napus Δ15-desaturase cDNA clone was obtained by PCR amplification of first-strand cDNA derived from B. napus cv. 212 / 86. The primers were based on published sequence: Genbank #L01418 Arondel et al, 1992 Science 258:1353-1355 (SEQ ID NO:7 and SEQ ID NO:8).

The following primers were used:

[0062]Bnd15-FORWARD (SEQ ID NO:9)[0063]5′-CUACUACUACUAGAGCTCAGCGATGGTTGTTGCTATGGAC-3′[0064]Bnd15-REVERSE (SEQ ID NO:10)[0065]5′-CAUCAUCAUCAUGAATTCTTAATTGATTTTAGATTTG-3′

[0066]These primers allowed the amplification of the entire coding region and added SacI and EcoRI sites to the 5′- and 3′-ends, respectively The PCR product was subcloned into pAMP1 (GIBCOBRL) using the CloneAmp system (GIBCOBRL) to create pCGN5520. The sequence was verified by sequencing of both strands to be sure that the open reading frame ...

example 3

Expression of Δ5 Desaturase in Plants Expression in Leaves

[0072]Ma29 is a putative M. alpina Δ5 desaturase as determined by sequence homology (SEQ ID NO:11 and SEQ ID NO:12). This experiment was designed to determine whether leaves expressing Ma29 (as determined by Northern) were able to convert exogenously applied DGLA (20:3) to ARA (20:4).

[0073]The Ma29 desaturase cDNA was modified by PCR to introduce convenient restriction sites for cloning. The desaturase coding region has been inserted into a d35 cassette under the control of the double 35S promoter for expression in Brassica leaves (pCGN5525) following standard protocols (see U.S. Pat. No. 5,424,200 and U.S. Pat. No. 5,106,739). Transgenic Brassica plants containing pCGN5525 were generated following standard protocols (see U.S. Pat. No. 5,188,958 and U.S. Pat. No. 5,463,174).

[0074]In the first experiment, three plants were used: a control, LPO04-1, and two transgenics, 5525-23 and 5525-29. LP004 is a low-linolenic Brassica var...

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Abstract

The present invention relates to compositions and methods for preparing polyunsaturated long chain fatty acids in plants, plant parts and plant cells, such as leaves, roots, fruits and seeds. Nucleic acid sequences and constructs encoding fatty acid desaturases, including Δ5-desaturases, Δ6-desaturases and Δ12-desaturases, are used to generate transgenic plants, plant parts and cells which contain and express one or more transgenes encoding one or more desaturases. Expression of the desaturases with different substrate specificities in the plant system permit the large scale production of polyunsaturated long chain fatty acids such as docosahexaenoic acid, eicosapentaenoic acid, α-linolenic acid, gamma-linolenic acid, arachidonic acid and the like for modification of the fatty acid profile of plants, plant parts and tissues. Manipulation of the fatty acid profiles allows for the production of commercial quantities of novel plant oils and products.

Description

[0001]This application claims the benefit of U.S. Provisional Application No. 60 / 089,043 filed Jun. 12, 1998.TENHNICAL FIELD[0002]This invention relates to modulating levels of enzymes and / or enzyme components capable of altering the production of long chain polyunsaturated fatty acids (PUFAS) in a host plant. The invention is exemplified by the production of PUFAS in plants.BACKGROUND[0003]Three main families of polyunsaturated fatty acids (PUFAs) are the 3 fatty acids, exemplified by arachidonic acid, the ω9 fatty acids exemplified by Mead acid, and the ω3 fatty acids, exemplified by eicosapentaenoic acid. PUFAs are important components of the plasma membrane of the cell, where they may be found in such forms as phospholipids. PUFAs also serve as precursors to other molecules of importance in human beings and animals, including the prostacyclins, leukotrienes and prostaglandins. PUFAs are necessary for proper development, particularly in the developing infant brain, and for tissue...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): A01H5/00C12N15/82C12N9/02C12P7/64C12N15/09C12P7/6427C12P7/6432C12P7/6434C12P7/6472
CPCC12N9/0083C12P7/6472C12P7/6427C12N15/8247C12P7/6432C12P7/6434
Inventor KNUTZON, DEBBIE
Owner CALGENE LLC
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