Gene silencing

Patent metadata

Field	Value
Publication number	US-9706724-B2
Application number	US-201514617488-A
Country	US
Kind code	B2
Filing date	Feb 9, 2015
Priority date	Sep 24, 2004
Publication date	Jul 18, 2017
Grant date	Jul 18, 2017

Abstract

The present invention relates to unique strategies and constructs for producing a nucleic acid product that downregulates or prevents expression of a desired target polynucleotide. The present invention contemplates the suppression of gene expression in plants by constructs that trigger transcriptional or post-transcriptional gene silencing. The methods of gene silencing may suppress gene expression through a variety of mechanisms.

First claim

What is claimed is:

1. A plant comprising an expression cassette for reducing gene expression of a plant target gene sequence, comprising:
(i) a polynucleotide that shares sequence identity with at least a part of the plant target gene sequence; and
(ii) an inverted repeat of the polynucleotide,
wherein the polynucleotide and its inverted repeat are positioned in between, and operably linked to, two promoters that are each functional in plants,
wherein (a) the polynucleotide and its inverted repeat are fully identical in sequence over at least 23 nucleotides, (b) the direction of transcription is from one promoter toward the other promoter, and (c) neither the polynucleotide nor its inverted repeat is operably linked to a terminator.

2. The plant of claim 1 , wherein the polynucleotide of (i) shares sequence identity with at least a part of: a regulatory element, an exon, an intron, a 5′-untranslated region, or a 3′-untranslated region, of an endogenous plant target gene sequence.

3. The plant of claim 2 , wherein the endogenous plant target gene sequence is a COMT gene involved in lignin biosynthesis, a CCOMT gene involved in lignin biosynthesis, any other gene involved in lignin biosynthesis, an R1 gene involved in starch phosphorylation, a phosphorylase gene involved in starch phosphorylation, a PPO gene involved in oxidation of polyphenols, a polygalacturonase gene involved in pectin degradation, a gene involved in the production of allergens, or a gene involved in fatty acid biosynthesis.

4. The plant of claim 1 , wherein the expression cassette is located between transfer-DNA border sequences of a plasmid that is suitable for bacterium-mediated plant transformation, wherein the bacterium is a strain of Agrobacterium, Rhizobium , or Phyllobacterium.

5. The plant of claim 1 , wherein the expression cassette further comprises:
a spacer polynucleotide, positioned between the polynucleotide and the inverted repeat of the polynucleotide,
wherein the spacer polynucleotide is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, or more than 500 nucleotides long.

6. The plant of claim 1 , wherein each promoter is selected from the group consisting of: a constitutive promoter, a tissue-specific promoter, or an inducible promoter, and
wherein the second promoter is a constitutive promoter, a near-constitutive promoter, a tissue-specific promoter, or an inducible promoter.

7. The plant of claim 1 , wherein the expression cassette is stably transformed into the plant genome.

8. The plant of claim 1 , wherein at least one of the promoters is specific to a plant tissue selected from the group consisting of: tuber, seed, leaf, root, vascular system, flower, pollen, and ovule.

9. The plant of claim 1 , wherein the plant is selected from the group consisting of:
potato, tomato, tobacco, avocado, alfalfa, lettuce, carrot, strawberry, sugarbeet, cassava, sweet potato, soybean, pea, bean, cucumber, grape, brassica , maize, turf grass, wheat, rice, barley, sorghum, oat, oak, eucalyptus , walnut, palm, orchid, lily, onion, banana, sugarcane, pepper, broccoli, cotton, poinsettia, legumes, rose, mint, squash, daisy, and cactus.

10. The plant of claim 1 , wherein the plant is a potato plant.

11. A potato grown from the plant of claim 10 .

12. A food product made from the plant of claim 1 , wherein the food product contains the expression cassette.

13. A food product made from the plant of claim 1 , wherein the plant is a potato plant, and wherein the food product contains the expression cassette.

14. A food product made from the plant of claim 1 , wherein the plant is a potato plant and the food product is made from the tuber of said plant, and wherein the food product contains the expression cassette.

15. A food product made from the plant of claim 1 , wherein the plant is a potato plant and the food product is a cut potato, and wherein the food product contains the expression cassette.

16. A food product made from the plant of claim 1 , wherein the plant is a potato plant and the food product is a French fry or chip, and wherein the food product contains the expression cassette.

17. A potato comprising an expression cassette for reducing gene expression of a plant target gene sequence, comprising:
(i) a polynucleotide that shares sequence identity with at least a part of the plant target gene sequence; and
(ii) an inverted repeat of the polynucleotide,
wherein the polynucleotide and its inverted repeat are positioned in between, and operably linked to, two promoters that are each functional in plants,
wherein (a) the polynucleotide and its inverted repeat are fully identical in sequence over at least 23 nucleotides, (b) the direction of transcription is from one promoter toward the other promoter, and (c) neither the polynucleotide nor its inverted repeat is operably linked to a terminator.

18. A food product made from the potato of claim 17 , wherein the food product contains the expression cassette.

19. The food product of claim 18 , wherein the food product is a cut potato, and wherein the food product contains the expression cassette.

20. The food product of claim 18 , wherein the food product is a French fry or chip, and wherein the food product contains the expression cassette.

21. A method of making a food product, the method comprising:
processing a potato comprising an expression cassette,
wherein the expression cassette comprises
(i) a polynucleotide that shares sequence identity with at least a part of the plant target gene sequence; and
(ii) an inverted repeat of the polynucleotide,

wherein the polynucleotide and its inverted repeat are positioned in between, and operably linked to, two promoters that are each functional in plants,
wherein (a) the polynucleotide and its inverted repeat are fully identical in sequence over at least 23 nucleotides, (b) the direction of transcription is from one promoter toward the other promoter, and (c) neither the polynucleotide nor its inverted repeat is operably linked to a terminator.

22. The method of claim 21 , wherein the food product is a French fry or chip.

23. The method of claim 21 , wherein processing the potato comprises slicing or cutting the potato.

24. The method of claim 23 , wherein processing the potato comprises cutting the potato into wedges.

Assignees

• Simplot Co J R

Inventors

• Rommens Caius
• Yan Hua
• Bougri Oleg
• Swords Kathleen M M

Classifications

• C12N15/8242

  with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits · CPC title

• C12N15/8243

  involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine · CPC title

• C12N15/8255

  involving lignin biosynthesis · CPC title

• A23V2002/00

  Food compositions, function of food ingredients or processes for food or foodstuffs · CPC title

• C12N15/8247

  involving modified lipid metabolism, e.g. seed oil composition · CPC title