Dissimilar promoters for gene suppression

Abstract

Methods of gene suppression comprise transforming eukaryotic cells with recombinant DNA constructs including promoters with dissimilar expression patterns operably linked to one or more gene suppression elements and, optionally, one or more gene expression elements.

1 . Transgenic seed having in its genome a recombinant DNA construct comprising: 
 (a) a plant endosperm-specific promoter operably linked to at least one first gene suppression element, and    (b) a plant embryo-specific promoter in the opposite orientation to said plant endosperm-specific promoter and located 3′ to said at least one first gene suppression element.    
     
     
         2 . The transgenic seed of  claim 1 , wherein said at least one first gene suppression element comprises a gene suppression element for silencing an amino acid catabolism gene.  
     
     
         3 . The transgenic seed of  claim 2 , wherein said recombinant DNA construct further comprises one or more elements selected from: 
 (a) at least one second gene suppression element operably linked to said plant embryo-specific promoter;    (b) an amino acid biosynthesis gene operably linked to either said plant endosperm-specific promoter or plant embryo-specific promoter; and    (c) a selectable marker gene.    
     
     
         4 . The transgenic seed of  claim 2 , wherein said transgenic seed is transgenic maize seed, and said amino acid catabolism gene is a lysine catabolism gene.  
     
     
         5 . The transgenic seed of  claim 4 , wherein said recombinant DNA construct further comprises one or more elements selected from: 
 (a) at least one second gene suppression element for silencing a lysine catabolism gene operably linked to said plant embryo-specific promoter;    (b) a lysine biosynthesis gene operably linked to said plant endosperm-specific promoter;    (c) an aspartate kinase gene operably linked to either said plant endosperm-specific promoter or plant embryo-specific promoter; and    (d) a selectable marker gene.    
     
     
         6 . The transgenic seed of  claim 2 , wherein: 
 (a) said recombinant DNA construct comprises: 
 (i) a plant endosperm-specific promoter operably linked to at least one first gene suppression element comprising DNA that transcribes to RNA for silencing a lysine catabolism gene by forming double-stranded RNA, and  
 (ii) a plant embryo-specific promoter in the opposite orientation to said first promoter and operably linked to said at least one first gene suppression element; or  
   (b) said recombinant DNA construct comprises: 
 (i) a plant endosperm-specific promoter operably linked to at least one first gene suppression element comprising DNA that transcribes to RNA for silencing a lysine catabolism gene by forming double-stranded RNA,  
 (ii) a plant embryo-specific promoter in the opposite orientation to said first promoter and operably linked to said at least one first gene suppression element, and  
 (iii) at least one terminator operably linked to either said first or second promoters; or  
   (c) said recombinant DNA construct comprises: 
 (i) a plant endosperm-specific promoter operably linked to at least one first intron-embedded gene suppression element for silencing a lysine catabolism gene, at least one lysine biosynthesis gene, and a first terminator,  
 (ii) a plant embryo-specific promoter in the opposite orientation to said first promoter and operably linked to at least one second gene suppression element for silencing a lysine catabolism gene; or  
   (d) said recombinant DNA construct comprises: 
 (i) a first gene suppression cassette comprising a plant endosperm-specific promoter operably linked to at least one first intron-embedded gene suppression element for silencing a lysine catabolism gene, at least one lysine biosynthesis gene, and a first terminator, and  
 (ii) a second gene suppression cassette comprising a plant embryo-specific promoter operably linked to at least one second gene suppression element for silencing a lysine catabolism gene, and a second terminator,  
 wherein said first and second gene suppression cassettes are in opposite orientations; or  
   (e) said recombinant DNA construct comprises: 
 (i) a first gene suppression cassette comprising a plant endosperm-specific promoter operably linked to at least one first intron-embedded gene suppression element for silencing a lysine catabolism gene, at least one lysine biosynthesis gene and a first terminator, and  
 (ii) a second gene suppression cassette comprising a plant embryo-specific promoter operably linked to at least one intron-embedded second gene suppression element for silencing a lysine catabolism gene, at least one lysine biosynthesis gene and a second terminator,  
 wherein said first and second gene suppression cassettes are in opposite orientations; or  
   (f) said recombinant DNA construct comprises: 
 (i) a first gene suppression cassette comprising a plant endosperm-specific promoter operably linked to at least one first gene suppression element for silencing a lysine catabolism gene, and a first terminator, and  
 (ii) a second gene suppression cassette comprising a plant embryo-specific promoter operably linked to at least one second gene suppression element for silencing a lysine catabolism gene, and a second terminator,  
 wherein said first and second gene suppression cassettes are in opposite orientations.  
   
     
     
         7 . Stably transgenic plant cells having in their genome a recombinant DNA construct comprising: 
 (a) a first promoter operably linked to at least one first gene suppression element for silencing at least one first target gene, and (b) a second promoter that is in the opposite orientation to said first promoter and is located 3′ to said at least one first gene suppression element,    wherein said first and said second promoters have dissimilar expression patterns, and    wherein transcription of said recombinant DNA construct in a plant cell results in silencing of said at least one first target gene.    
     
     
         8 . The stably transgenic plant cells of  claim 7 , wherein said first and said second promoters comprise a plant embryo-specific promoter and a plant endosperm-specific promoter and said stably transgenic plant cells comprise seed embryo and endosperm cells of a crop plant.  
     
     
         9 . A recombinant DNA construct for transformation of a plant cell, comprising: 
 (a) a first promoter operably linked to at least one first gene suppression element for silencing at least one first target gene, and    (b) a second promoter that is in the opposite orientation to said first promoter and is located 3′ to said at least one first gene suppression element,    wherein said first and said second promoters have dissimilar expression patterns, and    wherein transcription of said recombinant DNA construct in a plant cell results in silencing of said at least one first target gene.    
     
     
         10 . The recombinant DNA construct of  claim 9 , wherein first and second promoters have dissimilar spatial expression patterns, and said silencing occurs in at least two distinct spatial locations.  
     
     
         11 . The recombinant DNA construct of  claim 9 , wherein first and second promoters have dissimilar temporal expression patterns, and said silencing occurs in at least two distinct times or developmental stages.  
     
     
         12 . The recombinant DNA construct of  claim 9 , wherein said at least one gene suppression element is under transcriptional control of both said first and said second promoters.  
     
     
         13 . The recombinant DNA construct of  claim 9 , further comprising one or more of: 
 (a) a second gene suppression element operably linked to said second promoter;    (b) at least one gene expression element for expressing at least one exogenous gene,    (c) at least one terminator, and    (d) at least one T-DNA border.    
     
     
         14 . The recombinant DNA construct of  claim 9 , wherein said at least one first gene suppression element comprises at least one element selected from the group consisting of: 
 (a) DNA that comprises at least one anti-sense DNA segment that is anti-sense to at least one segment of said at least one first target gene;    (b) DNA that comprises multiple copies of at least one anti-sense DNA segment that is anti-sense to at least one segment of said at least one first target gene;    (c) DNA that comprises at least one sense DNA segment that is at least one segment of said at least one first target gene;    (d) DNA that comprises multiple copies of at least one sense DNA segment that is at least one segment of said at least one first target gene;    (e) DNA that transcribes to RNA for suppressing said at least one first target gene by forming double-stranded RNA and comprises at least one anti-sense DNA segment that is anti-sense to at least one segment of said at least one target gene and at least one sense DNA segment that is at least one segment of said at least one first target gene;    (f) DNA that transcribes to RNA for suppressing said at least one first target gene by forming a single double-stranded RNA and comprises multiple serial anti-sense DNA segments that are anti-sense to at least one segment of said at least one first target gene and multiple serial sense DNA segments that are at least one segment of said at least one first target gene;    (g) DNA that transcribes to RNA for suppressing said at least one first target gene by forming multiple double strands of RNA and comprises multiple anti-sense DNA segments that are anti-sense to at least one segment of said at least one first target gene and multiple sense DNA segments that are at least one segment of said at least one first target gene, and wherein said multiple anti-sense DNA segments and said multiple sense DNA segments are arranged in a series of inverted repeats;    (h) DNA that comprises nucleotides derived from a plant miRNA;    (i) DNA that comprises nucleotides of a siRNA;    (j) DNA that transcribes to an RNA aptamer capable of binding to a ligand; and    (k) DNA that transcribes to an RNA aptamer capable of binding to a ligand, and DNA that transcribes to regulatory RNA capable of regulating expression of said first target gene, wherein said regulation is dependent on the conformation of said regulatory RNA, and said conformation of said regulatory RNA is allosterically affected by the binding state of said RNA aptamer.    
     
     
         15 . The recombinant DNA construct of  claim 9 , wherein said first gene suppression element is embedded in an intron.  
     
     
         16 . The recombinant DNA construct of  claim 9 , further comprising a second gene suppression element operably linked to said second promoter, wherein said first and second gene suppression elements are embedded in an intron.  
     
     
         17 . The recombinant DNA construct of  claim 9 , wherein said first and said second promoters comprise a plant embryo-specific promoter and a plant endosperm-specific promoter.  
     
     
         18 . A method of gene silencing in a plant, comprising: 
 (a) transforming a plant cell with the recombinant DNA construct of  claim 9 , thereby providing a transgenic plant cell;    (b) preparing a regenerated transgenic plant from said transgenic plant cell, or a transgenic progeny seed or plant of said regenerated transgenic plant;    (c) transcribing said recombinant DNA construct in said regenerated transgenic plant or said transgenic progeny seed or plant    whereby said at least one first target gene is silenced in said regenerated transgenic plant or said transgenic progeny seed or plant.    
     
     
         19 . The method of  claim 18 , wherein said plant is a crop plant.  
     
     
         20 . The method of  claim 18 , wherein said recombinant DNA construct is transcribed in a transgenic progeny seed having substantial endosperm, and said first and said second promoters comprise a plant embryo-specific promoter and a plant endosperm-specific promoter.  
     
     
         21 . The method of  claim 20 , wherein said transgenic progeny seed is transgenic progeny maize seed, said at least one first target gene is at least one lysine catabolism gene, and said at least one lysine catabolism gene is silenced in embryo and endosperm cells of said transgenic progeny seed.  
     
     
         22 . The method of  claim 21 , wherein said recombinant DNA construct further comprises at least one lysine biosynthesis gene operably linked to said endosperm-specific promoter.