Gene silencing

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.

1 - 13 . (canceled) 
     
     
         14 . A construct, comprising an expression cassette which comprises in the 5′ to 3′ orientation (i) a promoter, (ii) a first polynucleotide that comprises a sequence that shares sequence identity with at least a part of a promoter sequence associated with a target gene, (iii) a second polynucleotide comprising a sequence that is a perfect or imperfect inverse complement of the first polynucleotide, and (iv) a terminator, wherein the first promoter is operably linked to the 5′-end of the first polynucleotide and the second polynucleotide is operably linked to the terminator. 
     
     
         15 - 32 . (canceled) 
     
     
         33 . The construct of  claim 14 , wherein the first polynucleotide and the promoter sequence associated with the target gene are fully identical in sequence over at least 23 nucleotides. 
     
     
         34 . The construct of  claim 14 , wherein the second polynucleotide and the first polynucleotide do not comprise any sequence located downstream of the transcription start of the target gene. 
     
     
         35 . The construct of  claim 14 , wherein (a) at least part of the first desired polynucleotide is in the antisense orientation; or (b) at least part of the first desired polynucleotide is in the sense orientation. 
     
     
         36 . The construct of  claim 14 , further comprising a spacer polynucleotide positioned between the first and second polynucleotides, 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, or more than 500 nucleotides long. 
     
     
         37 . The construct of  claim 14 , wherein the promoters and the terminator are functional in a plant and 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.    
     
     
         38 . The construct of  claim 14 , wherein the promoter is a constitutive promoter, a near-constitutive promoter, a tissue-specific promoter, or an inducible promoter. 
     
     
         39 . The construct of  claim 14 , wherein the promoter is a functional plant promoter specific to a plant tissue selected from the group consisting of in tubers, seeds, leaves, roots, vascular system, flowers, pollen, and ovules. 
     
     
         40 . The construct of  claim 14 , wherein the target gene 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. 
     
     
         41 . The construct of  claim 14 , wherein the promoter sequence associated with the target gene is selected from the group consisting of (1) a starch-associated R1 gene promoter, (2) a polyphenol oxidase gene promoter, (3) a fatty acid desaturase 12 gene promoter, (4) a microsomal omega-6 fatty acid desaturase gene promoter, (5) a cotton stearoyl-acyl-carrier protein delta 9-desaturase gene promoter, (6) an oleoyl-phosphatidylcholine omega 6-desaturase gene promoter, (7) a  Medicago truncatula  caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) gene promoter, (8) a  Medicago sativa  (alfalfa) caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) gene promoter, (9) a  Medicago truncatula  caffeoyl CoA 3-O-methyltransferase (CCOMT) gene promoter, (10) a  Medicago sativa  (alfalfa) caffeoyl CoA 3-O-methyltransferase (CCOMT) gene promoter, (11) a  Zea mays  (maize) COMT gene, (12) a major apple allergen Mal d 1 gene promoter, (13) a major peanut allergen Ara h 2 gene promoter, (14) a major soybean allergen Gly m Bd 30 K gene promoter, (15) a polygalacturonase gene promoter, (16) any other endogenous promoter. 
     
     
         42 . A method for downregulating the expression of a target gene in a plant cell, comprising expressing the construct of  claim 14  in a plant cell, wherein the expression level of the target gene is downregulated compared to the expression level of the target gene in an untransformed plant cell. 
     
     
         43 . A method for enhancing tolerance to black spot bruising in a tuber, comprising expressing the construct of  claim 14  in a cell of a tuber, wherein (a) the first polynucleotide comprises the sequence that shares sequence identity with at least a part of a tuber-expressed polyphenol oxidase gene promoter, (b) the promoter is GBSS or AGP, and (c) expression of the construct in the cell reduces transcription and/or translation of a polyphenol oxidase gene in the tuber cell genome, thereby enhancing the tolerance of the tuber to black spot bruising. 
     
     
         44 . A method for reducing cold-induced sweetening in a tuber, comprising expressing the construct of  claim 14  in a cell of a tuber, wherein (a) the first polynucleotide comprises the sequence that shares sequence identity with at least a part of an R1 gene promoter, (b) the promoters is GBSS or AGP, and (c) expression of the construct in the cell reduces transcription and/or translation of an R1 gene in the tuber cell genome, thereby reducing cold-induced sweetening in the tuber. 
     
     
         45 . A method for increasing oleic acid levels in an oil-bearing plant, comprising expressing the construct of  claim 14  in a cell of a seed of an oil-bearing plant, wherein (a) the first polynucleotide comprises a sequence that shares sequence identity with at least a part of a Fad2 gene promoter, (b) the promoter is a napin gene promoter, a Fad2 gene promoter, or a stearoyl-ACP desaturase gene promoter, and (c) expression of the construct in the cell reduces transcription and/or translation of a Fad2 gene in the cell of the seed of the oil-bearing plant, thereby increasing the oil content of the seed. 
     
     
         46 . A method for reducing lignin content in a plant, comprising expressing the construct of  claim 14  in a cell of the plant, wherein (a) the first polynucleotide comprises a sequence that shares sequence identity with at least part of the sequence of the promoter associated with a gene selected from the group consisting of caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) gene and caffeoyl CoA 3-O-methyltransferase (CCOMT) gene, (b) the promoter is a petE or Pal gene promoter, and (c) expression of the construct in the cell reduces transcription and/or translation of a COMT or CCOMT gene in the cell of the plant, thereby reducing lignin content in a plant. 
     
     
         47 . A method for reducing the degradation of pectin in a fruit of a plant, comprising expressing the construct of  claim 14  in a fruit cell of the plant, wherein (a) the first polynucleotide comprises a sequence that shares sequence identity with at least a part of a polygalacturonase gene promoter, (b) the promoter is a fruit-specific promoter, and (c) expression of the construct in the fruit cell reduces transcription and/or translation of a polygalacturonase gene in the cell of the plant, thereby reducing the degradation of pectin in the fruit. 
     
     
         48 . A method for reducing the allergenicity of a food produced by an apple plant, comprising expressing the construct of  claim 14  in a cell of the apple plant, wherein (a) the first polynucleotide comprises a sequence from the Mal d I gene promoter, and (b) expression of the construct in the apple plant reduces transcription and/or translation of Mal d I in the apple. 
     
     
         49 . A method for reducing the allergenicity of a food produced by a peanut plant, comprising expressing the construct of  claim 14  in a cell of the peanut plant, wherein (a) the first polynucleotide comprises a sequence from the Ara h 2 gene promoter, and (b) expression of the construct in the peanut plant reduces transcription and/or translation of Ara h 2 in the peanut. 
     
     
         50 . A method for reducing the allergenicity of a food produced by a soybean plant, comprising expressing the construct of  claim 14  in a cell of the soybean plant, wherein (a) the first polynucleotide comprises a sequence from the Gly m Bd gene promoter, and (b) expression of the construct in the soybean plant reduces transcription and/or translation of Gly m Bd in the soybean. 
     
     
         51 . A plant cell obtained according to the method of  claim 42 . 
     
     
         52 . A plant comprising the plant cell of  claim 51 . 
     
     
         53 . A food product made from the plant of  claim 52 .