Nucleic acid constructs and methods for producing altered seed oil compositions

• US 20060206963A1
• Filed: 03/16/2006
• Published: 09/14/2006
• Est. Priority Date: 03/21/2002
• Status: Active Grant

• Current Assignee: Monsanto Technology LLC (Bayer AG)
• Original Assignee: Monsanto Technology LLC (Bayer AG)
• Inventors: Ulmasov, Tim, Voelker, Toni, Bringe, Neal A., Fillatti, JoAnne J.
• Granted Patent: US 7,566,813 B2
• Time in Patent Office: Days
• US Class Current: 800/281
• CPC Class Codes: A23D 9/00 Other edible oils or fats, e.g. shortenings, cooking oils
• CPC Class Codes: A23K 10/30 from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms obtained by microbiological or biochemical processes, e.g. using yeasts or enzymes, A23K10/10
• CPC Class Codes: A23K 20/147 Polymeric derivatives, e.g. peptides or proteins
• CPC Class Codes: A23K 20/158 Fatty acids; Fats; Products containing oils or fats
• CPC Class Codes: C10L 1/026 for compression ignition
• CPC Class Codes: C11B 1/00 Production of fats or fatty oils from raw materials
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 15/8237 Externally regulated expression systems
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 9/0083 Miscellaneous (1.14.99)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

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.

Claims

1. A soybean seed exhibiting a seed oil fatty acid composition comprising an oleic acid content of about 42% to about 85% by weight of the total fatty acids and a saturated fatty acid content of less than 8% by weight of the total fatty acids.

2. The soybean seed of claim 1, wherein said seed further comprises a genome with a nucleic acid sequence that suppresses the expression of endogenous soybean FAD2-1 and FATB, wherein said nucleic acid sequence comprises a fragment of soybean FAD2-1 intron that is between about 20 and about 420 nucleotides in length and a fragment of a soybean FATB gene that is between about 40 to about 450 nucleotides in length;

and said genome further comprises a nucleic acid sequence that increases the expression of one or both of beta-ketoacyl-ACP synthase IV and delta-9 desaturase.

3. The soybean seed of claim 2, wherein said fragment of soybean FAD2-1 intron and said fragment of soybean FATB are each transcribed in sense- and antisense-orientations, resulting in RNA which is at least in part double-stranded.

4. The soybean seed of claim 2, wherein said nucleic acid sequence that suppresses the expression of endogenous soybean FAD2-1 and FATB is assembled as a functional transcription unit after insertion into a plant chromosome.

5. The soybean seed of claim 1, wherein said seed further comprises a genome with a nucleic acid sequence comprising a fragment of a soybean FAD2-1 intron that is between about 20 and about 420 contiguous nucleotides in length and a fragment of a soybean FATB gene that is between about 40 to about 450 contiguous nucleotides in length.

6. The soybean seed of claim 5, wherein said fragment of soybean FAD2-1 intron and said fragment of soybean FATB are each transcribed in sense- and antisense-orientations, resulting in RNA which is at least in part double-stranded.

7. The soybean seed of claim 5, wherein said nucleic acid sequence is assembled as a functional transcription unit after insertion into a plant chromosome.

8. The soybean seed of claim 1, wherein said seed further comprises a genome with a nucleic acid sequence that suppresses the expression of endogenous soybean FAD2-1, comprising a fragment of soybean FAD2-1 intron that is between about 20 and about 420 nucleotides in length;

and said genome further comprises a nucleic acid sequence that increases the expression of one or both of beta-ketoacyl-ACP synthase IV and delta-9 desaturase.

9. A method of reducing the amount of FAD2 gene suppression relative to the amount of FAD2 gene suppression obtained by expressing a dsRNAi construct comprising a heterologous FAD2 sequence consisting of an entire FAD2 intron or an entire FAD2 UTR, said method comprising:

i) expressing a heterologous FAD2 sequence in a plant cell, wherein said heterologous FAD2 sequence is derived from an endogenous FAD2 gene in a plant cell and consists of a FAD2 intron fragment or a FAD2 UTR fragment; and

ii) suppressing an endogenous FAD2 gene with said heterologous FAD2 sequence, wherein the amount of FAD2 gene suppression is less than the amount of gene expression obtained by expressing a heterologous FAD2 sequence consisting of the entire length of a FAD2 intron or the entire length of a FAD2 UTR.

10. The method of claim 9, wherein said FAD2 intron fragment is between about 40 and about 320 contiguous nucleotides of an entire FAD2 intron 1 and said a FAD2 UTR fragment is about 100 to about 140 contiguous nucleotides of an entire FAD2 3′

UTR.

11. A method of altering the oil composition of a plant cell comprising:

transforming a plant cell with a heterologous FAD2 sequence derived from part of an endogenous FAD2 gene, wherein said heterologous FAD2 sequence consists of a FAD2 intron fragment or a FAD2 UTR fragment; and

growing said plant cell under conditions wherein transcription of said heterologous FAD2 sequence is initiated, whereby said oil composition is altered relative to a plant cell with a similar genetic background but lacking said heterologous FAD2 sequence.

12. A method to enhance oleic acid content and reduce saturated fatty acid content in a plant seed comprising:

i) shortening the length of a first heterologous FAD2 sequence until the amount of FAD2 gene suppression from a plant transformed with said first heterologous FAD2 sequence is at least partially reduced relative to the amount of FAD2 gene suppression in a plant cell comprising a similar genetic background and a second heterologous FAD2 sequence, wherein said second heterologous FAD2 sequence consists of more endogenous FAD2 sequence than said first heterologous FAD2 sequence;

ii) expressing a heterologous FATB sequence capable of at least partially reducing FATB gene expression in a plant cell relative to the suppression of FATB in a plant cell with a similar genetic background but without said heterologous FATB sequence;

iii) growing a plant comprising a genome with said first heterologous FAD2 sequence and said heterologous FATB sequence; and

iv) cultivating a plant that produces seed with a reduced saturated fatty acid content relative to seed from a plant having a similar genetic background but lacking said first heterologous FAD2 sequence and said heterologous FATB sequence.

13. A method of modulating the fatty acid composition of oil from a seed of a temperate oilseed crop comprising, isolating a fragment of a genetic element of at least 40 nucleotides in length that is capable of suppressing the expression of an endogenous gene in the fatty acid synthesis pathway;

introducing said genetic element into a plant cell of said temperate oilseed crop;

producing a transgenic plant; and

selecting a transgenic plant seed comprising said genetic element that modulates the fatty acid composition of oil from said seed.

14. The method of claim 13, wherein said an endogenous gene is two or more endogenous genes in the fatty acid synthesis pathway.

15. The method of claim 13, wherein said genetic element is selected from the group consisting of an intron, an exon, a transit peptide coding DNA sequence, a 3′

-UTR, a 5′

-UTR, an open reading frame or a transcript of said endogenous gene in the fatty acid synthesis pathway.

16. The method of claim 13, wherein said endogenous gene is selected from the group consisting of FAD2-1, FAD3, FATB, and combinations thereof.

17. The method of claim 13, wherein said temperate oilseed crop is selected from the group consisting of soybean, canola, sunflower, peanut, maize, and cotton.

18. A cell of a soybean seed exhibiting a seed oil fatty acid composition comprising an oleic acid content of about 42% to about 85% by weight of the total fatty acids and a saturated fatty acid content of less than 8% by weight of the total fatty acids.

19. A recombinant nucleic acid molecule comprising a fragment of soybean FAD2-1 intron that is between about 20 and about 420 contiguous nucleotides in length and a fragment of soybean FATB gene that is between about 40 and about 450 contiguous nucleotides in length.

20. A recombinant nucleic acid molecule comprising a nucleic acid sequence comprising a fragment of soybean FAD2-1 intron that is between about 20 and about 420 nucleotides in length, a fragment of a soybean FATB gene that is between about 40 to about 450 nucleotides in length, and a nucleic acid sequence that increases the expression of one or both of beta-ketoacyl-ACP synthase IV and delta-9 desaturase.

21. A non-blended soybean oil having a fatty acid composition comprising an oleic acid content of from about 42% to about 85% by weight of the total fatty acids and a saturated fatty acid content of about 8% or less by weight of the total fatty acids.

22. A soybean meal derived from a soybean seed exhibiting a seed oil fatty acid composition comprising an oleic acid content of about 42% to about 85% by weight of the total fatty acids and a saturated fatty acid content of less than 8% by weight of the total fatty acids.

23. A method for decreasing linolenic acid content of a soybean seed comprising:

i) introducing into a soybean cell a heterologous nucleic acid molecule comprising nucleic acid sequence from at least two members of a FAD3 gene family;

ii) expressing a nucleic acid sequence from a FAD3 gene capable of at least partially reducing endogenous FAD3 gene expression in a plant cell;

iii) growing a plant cell comprising a genome with said nucleic acid sequence from at least two members of said FAD3 gene family; and

iv) cultivating said plant cell with a reduced linolenic acid content relative to a plant cell having a similar genetic background but lacking said at least two members of said FAD3 gene family.

24. The method of claim 23, wherein said FAD3 gene family is selected from the group consisting of FAD3-1A, FAD3-1B, and FAD3-1C.

25. The method of claim 23, wherein said DNA fragments are from the 5′

- or 3′

-UTR of a transcribed region of FAD3.

26. A recombinant DNA construct comprising DNA fragments from at least two members of FAD3 gene family.

27. The recombinant DNA construct of claim 26, further comprising a fragment of soybean FAD2-1 intron that is between about 20 and about 420 contiguous nucleotides in length and a fragment of soybean FATB gene that is between about 40 and about 450 contiguous nucleotides in length.

29. A soybean seed comprising the recombinant DNA construct of claim 26.

30. A soybean meal derived from the soybean seed of claim 29.

28. A non-blended soybean oil having a fatty acid composition comprising an oleic acid content of from about 42% to about 85% by weight of the total fatty acids, a saturated fatty acid content of about 8% or less by weight of the total fatty acids, and a linolenic acid content of about 1.5% or less by weight of the total fatty acids.