Duplicated cassava vein mosaic virus enhancers and uses thereof

Abstract

The invention features duplicated enhancer domains and enhancer cassettes. The invention also features expression constructs having one or more duplicated enhancer domains and a promoter under the regulation of the duplicated enhancer domains. The expression construct is useful for increasing the expression of any nucleic acid molecule that is operably linked to the expression construct. Accordingly, the invention also features a method for expressing a nucleic acid molecule through the use of an expression construct of the present invention.

1 . An enhancer cassette comprising a duplicated enhancer derived from a cassaya vein mosaic virus.  
     
     
         2 . The enhancer cassette of  claim 1 , said enhancer cassette comprising a component having the formula (X—Y) n , wherein X corresponds to said enhancer domain derived from a cassaya vein mosaic virus, Y is an intervening spacer domain comprising a sequence of between zero and thirty nucleotides inclusive, and n is an integer between 2 and 8 inclusive.  
     
     
         3 . The enhancer cassette of  claim 2 , wherein X comprises nucleotides 1 to 261 of SEQ ID NO: 1, nucleotides 1 to 332 of SEQ ID NO: 1, or nucleotides 262 to 332 of SEQ ID NO: 1.  
     
     
         4 . The enhancer cassette of  claim 2 , wherein n is 2.  
     
     
         5 . An expression construct comprising, as a first component, an enhancer cassette comprising a duplicated enhancer derived from a cassaya vein mosaic virus; and, as a second component, a promoter having an RNA polymerase binding site and an mRNA initiation site.  
     
     
         6 . The expression construct of  claim 5 , wherein said promoter is a cassaya vein mosaic virus promoter.  
     
     
         7 . The expression construct of  claim 6 , wherein said cassaya vein mosaic virus promoter comprises nucleotides 333-444 of SEQ ID NO: 1.  
     
     
         8 . The expression construct of  claim 5 , wherein said promoter is a heterologous promoter.  
     
     
         9 . The expression construct  claim 5 , further comprising, as a third component, a nucleic acid molecule of interest, wherein said first, second, and third components are operably linked so that the nucleic acid molecule is transcribed.  
     
     
         10 . The expression construct of  claim 9 , wherein said third component encodes a protein providing disease or insect resistance.  
     
     
         11 . The expression construct of  claim 9 , wherein said third component encodes an antisense RNA.  
     
     
         12 . The expression construct of  claim 9 , wherein said third component encodes a selectable marker  
     
     
         13 . The expression construct of  claim 9 , wherein transcription of said nucleic acid molecule is increased relative to transcription of said nucleic acid molecule operably linked to an expression construct not comprising a duplicated CsVMV enhancer domain.  
     
     
         14 . An expression vector comprising an enhancer construct comprising, as a first component, an enhancer cassette comprising a duplicated enhancer derived from a cassaya vein mosaic virus; as a second component, a promoter having an RNA polymerase binding site and an mRNA initiation site; and, as a third component, a nucleic acid molecule of interest, wherein said first, second, and third components are operably linked so that the nucleic acid molecule is transcribed.  
     
     
         15 . A cell comprising an enhancer construct comprising, as a first component, an enhancer cassette comprising a duplicated enhancer derived from a cassaya vein mosaic virus; as a second component, a promoter having an RNA polymerase binding site and an mRNA initiation site; and, as a third component, a nucleic acid molecule of interest, wherein said first, second, and third components are operably linked so that the nucleic acid molecule is transcribed.  
     
     
         16 . The cell of  claim 15 , wherein said cell is a eukaryotic cell.  
     
     
         17 . The cell of  claim 16 , wherein said cell is a plant cell.  
     
     
         18 . A transgenic plant comprising an enhancer cassette comprising a duplicated enhancer derived from a cassaya vein mosaic virus.  
     
     
         19 . A method for expressing a nucleic acid molecule in a cell, said method comprising: 
 (i) transforming said cell with an expression construct comprising    (a) a first component having the formula (X—Y) n , wherein X corresponds to an enhancer derived from a cassaya vein mosaic virus, Y is an intervening spacer domain comprising a sequence between zero and thirty nucleotides inclusive, and n is an integer between 2 and 8 inclusive;    (b) a second component consisting of a promoter comprising an RNA polymerase binding site and an mRNA initiation site; and    (c) a third component consisting of the nucleic acid molecule to be expressed, wherein the first, second, and third components are operably linked so that the nucleic acid molecule is transcribed, and    (ii) providing conditions that allow for expression of said nucleic acid in said cell.