Nucleic acid constructs and methods for producing altered seed oil compositions

Active Publication Date: 2005-02-10
MONSANTO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013] Further provided by the present invention is a recombinant nucleic acid molecule comprising a first set of DNA sequences that is capable, when expressed in a host cell, of forming a dsRNA construct and suppressing the endogenous expression of at least one, preferably two, genes selected from the group consisting of FAD2, FAD3, and FA TB genes, where the first set of DNA sequences comprises a first non-coding sequence that expresses a first RNA sequence that exhibits at least 90% identity to a non-coding region of a FAD2 gene, a first antisense sequence that expresses a first antisense RNA sequence capable of forming a double-stranded RNA molecule with the first RNA sequence, a second non-coding sequence that expresses a second RNA sequence that exhibits at least 90% identity to a non-coding region of a FAD3 gene, and a second antisense sequence that expresses a second antisense RNA sequence capable of forming a double-stranded RNA molecule with the second RNA sequence; and a second set of DNA sequences that is capable, when expressed in a host cell, of increasing the endogenous expression of at least one gene selected from the group consisting of a beta-ketoacyl-ACP synthase I gene, a beta-ketoacyl-ACP synthase IV gene, and a delta-9 desaturase gene.
[0014] The present invention provides methods of transforming plants with these recombinant nucleic acid molecules. The methods include a method of producing a transformed plant having seed with an increased oleic acid content, reduced saturated fatty acid content, and reduced polyunsaturated fatty acid content, comprising (A) transforming a plant cell with a recombinant nucleic acid molecule which comprises a first set of DNA sequences that is capable, when expressed in a host cell, of suppressing the endogenous expression of at least one, preferably two, genes selected from the group consisting of FAD2, FAD3, and FATB genes, and a second set of DNA sequences that is capable, when expressed in a host cell, of increasing the endogenous expression of at least one gene selected from the group consisting of a beta-ketoacyl-ACP synthase I gene, a beta-ketoacyl-ACP synthase IV gene, and a delta-9 desaturase gene; and (B) growing the transformed plant, where the transformed plant produces seed with an increased oleic acid content, reduced saturated fatty acid content, and reduced polyunsaturated fatty acid content relative to seed from a plant having a similar genetic background but lacking the recombinant nucleic acid molecule.
[0015] Further provided are methods of transforming plant cells with the recombinant nucleic acid molecules. The methods include a method of altering the oil composition of a plant cell comprising (A) transforming a plant cell with a recombinant nucleic acid molecule which comprises a first set of DNA sequences that is capable, when expressed in a host cell, of suppressing the endogenou

Problems solved by technology

However, a soybean oil that is broadly beneficial to major soybean oil users such as consumers of salad oil, cooking oil and frying oil, and industrial markets such as biodiesel and biolube markets, is not available.
Prior soybean oils were either

Method used

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  • Nucleic acid constructs and methods for producing altered seed oil compositions
  • Nucleic acid constructs and methods for producing altered seed oil compositions
  • Nucleic acid constructs and methods for producing altered seed oil compositions

Examples

Experimental program
Comparison scheme
Effect test

example 1

Suppression Constructs

[0163] 1A. FAD2-1 Constructs

[0164] The FAD2-1A intron (SEQ ID NO: 1) is cloned into the expression cassette, pCGN3892, in sense and antisense orientations. The vector pCGN3892 contains the soybean 7S promoter and a pea rbcS 3′. Both gene fusions are then separately ligated into pCGN9372, a vector that contains the CP4 EPSPS gene regulated by the FMV promoter. The resulting expression constructs (pCGN5469 sense and pCGN5471 antisense) are used for transformation of soybean.

[0165] The FAD2-1B intron (SEQ ID NO: 2) is fused to the 3′ end of the FAD2-1A intron in plasmid pCGN5468 (contains the soybean 7S promoter fused to the FAD2-1A intron (sense) and a pea rbcS 3′) or pCGN5470 (contains the soybean 7S promoter fused to the FAD2-1A intron (antisense) and a pea rbcS 3′) in sense or antisense orientation respectively. The resulting intron combination fusions are then ligated separately into pCGN9372, a vector that contains the CP4 EPSPS gene regulated by the FMV ...

example 2

Combination Constructs

[0177] In FIGS. 7-15, promoters are indicated by arrows, encoding sequences (both coding and non-coding) are indicated by pentagons which point in the direction of transcription, sense sequences are labeled in normal text, and antisense sequences are labeled in upside-down text. The abbreviations used in these Figures include: 7Sa=7Sα promoter; 7Sa′=7Sα′ promoter; Br napin=Brassica napin promoter; FMV=an FMV promoter; ARC=arcelin promoter; RBC E9 3′=Rubisco E9 termination signal; Nos 3′=nos termination signal; TML 3′=tml termination signal; napin 3′=napin termination signal; ′3 (in the same box as FAD or FAT)=3′ UTR; 5′ (in the same box as FAD or FAT)=5′UTR; Cr=Cuphea pulcherrima; Gm=Glycine max; Rc=Ricinus communis; FAB2=a FAB2 allele of a stearoyl-desaturase gene; and Intr or Int=intron.

[0178] 2A. dsRNA Constructs

[0179]FIGS. 7-9 depict nucleic acid molecules of the present invention in which the first sets of DNA sequences are capable of expressing dsRNA c...

example 3

Plant Transformation and Analysis

[0219] The constructs of Examples 1 and 2 are stably introduced into soybean (for example, Asgrow variety A4922 or Asgrow variety A3244 or Asgrow variety A3525) by the methods described earlier, including the methods of McCabe et al., Bio / Technology, 6:923-926 (1988), or Agrobacterium-mediated transformation. Transformed soybean plants are identified by selection on media containing glyphosate. Fatty acid compositions are analyzed from seed of soybean lines transformed with the constructs using gas chromatography. In addition, any of the constructs may contain other sequences of interest, as well as different combinations of promoters.

[0220] For some applications, modified fatty acid compositions are detected in developing seeds, whereas in other instances, such as for analysis of oil profile, detection of fatty acid modifications occurring later in the FAS pathway, or for detection of minor modifications to the fatty acid composition, analysis of ...

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Abstract

The present invention is in the field of plant genetics and provides recombinant nucleic acid molecules, constructs, and other agents associated with the coordinate manipulation of multiple genes in the fatty acid synthesis pathway. In particular, the agents of the present invention are associated with the simultaneous enhanced expression of certain genes in the fatty acid synthesis pathway and suppressed expression of certain other genes in the same pathway. Also provided are plants incorporating such agents, and in particular plants incorporating such constructs where the plants exhibit altered seed oil compositions.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60 / 365,794 filed Mar. 21, 2002, and No. 60 / 390,185 filed Jun. 21, 2002, each of which is herein incorporated by reference in its entirety.INCORPORATION OF SEQUENCE LISTING [0002] A paper copy of the Sequence Listing and a computer readable form of the sequence listing on diskette, containing the file named “OmniSeq for US.txt”, which is 45,810 bytes in size (measured in MS-DOS), and which was recorded on Mar. 21, 2003, are herein incorporated by reference. FIELD OF THE INVENTION [0003] The present invention is directed to recombinant nucleic acid molecules, constructs, and other agents associated with the coordinate manipulation of multiple genes in the fatty acid synthesis pathway. In particular, the agents of the present invention are associated with the simultaneous enhanced expression of certain genes in the fatty acid synthesis pathway a...

Claims

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Application Information

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IPC IPC(8): A01H1/00A01H5/00C12N5/04C12N9/10C12N15/82C12P21/02
CPCA23D9/00A23K1/14A23K1/1631A23K1/164Y02E50/13C11B1/00C12N9/0083C12N15/8237C12N15/8247C10L1/026A23K10/30A23K20/147A23K20/158Y02E50/10
Inventor FILLATTI, JOANNEBRINGE, NEALDEHESH, KATAYOON
Owner MONSANTO TECH LLC
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