Compositions and methods for modulating expression of gene products

Abstract

Compositions and methods for modulating expression of gene products are provided. Compositions comprise suppression cassettes that comprise a convergent promoter pair operably linked to a silencing element that, upon expression, is capable of decreasing the expression of one or more target polynucleotides of interest. Compositions of the invention also include transformed plants, plant cells, plant tissues, and plant seeds. Methods of transformation and regeneration of plants comprising the novel constructs are provided. The methods find use in regulating gene expression, particularly genes associated with agronomic traits of interest. Further provided are promoter sequences, cells, plants, and vectors comprising these promoter sequences and methods of their use.

1 . A DNA suppression cassette comprising a silencing element flanked by a first operably linked convergent promoter at one terminus of the silencing element and a second operably linked convergent promoter at the opposing terminus of the silencing element, wherein the first and the second convergent promoters are capable of driving expression of the silencing element, said silencing element is transcribed as a hairpin RNA, and expression of said silencing element decreases the expression level of a target polynucleotide of interest.  
     
     
         2 . The DNA suppression cassette of  claim 1 , wherein the first and the second convergent promoters have a different expression profile.  
     
     
         3 . The DNA suppression cassette of  claim 1 , wherein said silencing element comprises, in the following order, a first segment, a second segment, and a third segment, wherein 
 a) said first segment comprises at least about 20 nucleotides having at least 90% sequence complementary to the target polynucleotide of interest;    b) said second segment comprises a loop of sufficient length to allow the silencing element to be transcribed as a hairpin RNA; and,    c) said third segment comprises at least about 20 nucleotides having at least 85% complementary to the first segment.    
     
     
         4 . The DNA suppression cassette of  claim 1 , wherein said first and said second convergent promoters are active in a plant cell.  
     
     
         5 . The DNA suppression cassette of  claim 4 , wherein said first and said second convergent promoters are tissue-preferred promoters.  
     
     
         6 . The DNA suppression cassette of  claim 5 , wherein said first and said second convergent promoters are selected from the group consisting of a seed-preferred promoter, an embryo-preferred promoter, an aleurone-preferred promoter, a pericarp-preferred promoter, and an endosperm-preferred promoter.  
     
     
         7 . The DNA suppression cassette of  claim 6 , wherein said first convergent promoter is an endosperm-preferred promoter and said second convergent promoter is an embryo-preferred promoter.  
     
     
         8 . The DNA suppression cassette of  claim 7 , wherein said first convergent promoter is a gamma-zein promoter and said second convergent promoter is an oleosin promoter.  
     
     
         9 . The DNA suppression cassette of  claim 8 , wherein said silencing element comprises the nucleotide sequence set forth in SEQ ID NO:2.  
     
     
         10 . A vector comprising the DNA suppression cassette of  claim 1 .  
     
     
         11 . A plant comprising the DNA suppression cassette of  claim 4 .  
     
     
         12 . The plant of  claim 11 , wherein said plant is a monocot.  
     
     
         13 . The plant of  claim 11 , wherein said plant is a dicot.  
     
     
         14 . The plant of  claim 11 , wherein said plant is selected from the group consisting of corn, oat, soybean, wheat, rice, canola,  Brassica  sp., sorghum, sunflower, barley, millet, cotton, peanut, flax, safflower, palm, olive, castor bean, and coconut.  
     
     
         15 . A method for decreasing the expression level of at least one polynucleotide of interest in an organism, said method comprising 
 a) introducing into a cell of said organism a DNA suppression cassette comprising a silencing element flanked by a first operably linked convergent promoter at one terminus of the silencing element and a second operably linked convergent promoter at the opposing terminus of the silencing element, wherein the first and the second convergent promoters are capable of driving expression of the silencing element, said silencing element is transcribed as a hairpin RNA; and,    b) expressing said silencing element from both the first and the second convergent promoters, and thereby decreasing the level of expression of the target polynucleotide of interest.    
     
     
         16 . The method of  claim 15 , wherein the first and the second convergent promoters have a different expression profile.  
     
     
         17 . The method of  claim 15 , wherein said silencing element comprises, in the following order, a first segment, a second segment, and a third segment, wherein 
 a) said first segment comprises about 20 nucleotides having at least 90% sequence complementary to the target polynucleotide of interest;    b) said second segment comprises a loop of sufficient length to allow the silencing element to be transcribed as a hairpin RNA; and,    c) said third segment comprises about 20 nucleotides having at least 85% complementary to the first segment.    
     
     
         18 . The method of  claim 15 , wherein said organism is a plant or part thereof.  
     
     
         19 . The method of  claim 18 , wherein said first and said second convergent promoters are tissue-preferred promoters.  
     
     
         20 . The method of  claim 19 , wherein said first and said second convergent promoters are selected from the group consisting of a seed-preferred promoter, an embryo-preferred promoter, an aleurone-preferred promoter, a pericarp-preferred promoter, and an endosperm-preferred promoter.  
     
     
         21 . The method of  claim 20 , wherein said first convergent promoter is the endosperm-preferred promoter and said second convergent promoter is the embryo-preferred promoter.  
     
     
         22 . The method of  claim 21 , wherein said first convergent promoter is a gamma-zein promoter and said second convergent promoter is an oleosin promoter.  
     
     
         23 . The method of  claim 22 , wherein said silencing element comprises the nucleotide sequence as set forth in SEQ ID NO:1.  
     
     
         24 . The method of  claim 18 , wherein said plant is a monocot.  
     
     
         25 . The method of  claim 18 , wherein said plant is a dicot.  
     
     
         26 . The method of  claim 18 , wherein said plant is selected from the group consisting of corn, oat, soybean, wheat, rice, canola, Brassica sp., sorghum, sunflower, barley, millet, cotton, peanut, flax, safflower, palm, olive, castor bean, and coconut.  
     
     
         27 . The method of  claim 18 , wherein the level of expression of the target polynucleotide of interest is decreased in a part of the plant.  
     
     
         28 . The method of  claim 27 , wherein said plant part is a seed or a fruit.  
     
     
         29 . The method of  claim 28 , wherein said plant part is an embryo of a seed.  
     
     
         30 . An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: 
 a) a nucleotide sequence comprising the sequence set forth in SEQ ID NO:3 or a complement thereof;    b) a nucleotide sequence comprising at least 20 contiguous nucleotides of the sequence set forth in SEQ ID NO:3, wherein said sequence initiates transcription in a plant cell; and,    c) a nucleotide sequence comprising a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:3, wherein said sequence initiates transcription in the plant cell.    
     
     
         31 . An expression cassette comprising the polynucleotide of  claim 30  operably linked to a heterologous nucleotide sequence of interest.  
     
     
         32 . A vector comprising the expression cassette of  claim 31 .  
     
     
         33 . A plant cell comprising the expression cassette of  claim 31 .  
     
     
         34 . The plant cell of  claim 33 , wherein said expression cassette is stably incorporated into the genome of the plant cell.  
     
     
         35 . The plant cell of  claim 33 , wherein said plant cell is from a monocot.  
     
     
         36 . The plant cell of  claim 35 , wherein said monocot is maize.  
     
     
         37 . The plant cell of  claim 33 , wherein said plant cell is from a dicot.  
     
     
         38 . A plant comprising the expression cassette of  claim 31 .  
     
     
         39 . The plant of  claim 38 , wherein said plant is a monocot.  
     
     
         40 . The plant of  claim 39 , wherein said monocot is maize.  
     
     
         41 . The plant of  claim 38 , wherein said plant is a dicot.  
     
     
         42 . The plant of  claim 38 , wherein said expression cassette is stably incorporated into the genome of the plant.  
     
     
         43 . A transgenic seed of the plant of  claim 38 , wherein the seed comprises the expression cassette.  
     
     
         44 . A method for expressing a polynucleotide of interest in a plant or a plant cell, said method comprising introducing into the plant or the plant cell an expression cassette comprising a promoter operably linked to a heterologous nucleotide sequence of interest, wherein said promoter comprises a polynucleotide of  claim 30 .  
     
     
         45 . A method for expressing a polynucleotide in an embryo-preferred manner in a plant, said method comprising introducing into a plant cell an expression cassette and regenerating a plant from said plant cell, said plant having stably incorporated into its genome the expression cassette, said expression cassette comprising a promoter operably linked to a heterologous polynucleotide of interest, wherein said promoter comprises a polynucleotide of  claim 30.