Recombinant DNA constructs and methods for controlling gene expression

Abstract

The present invention provides molecular constructs and methods for use thereof, including constructs including heterologous miRNA recognition sites, constructs for gene suppression including a gene suppression element embedded within an intron flanked on one or on both sides by non-protein-coding sequence, constructs containing engineered miRNA or miRNA precursors, and constructs for suppression of production of mature microRNA in a cell. Also provided are transgenic plant cells, plants, and seeds containing such constructs, and methods for their use. The invention further provides transgenic plant cells, plants, and seeds containing recombinant DNA for the ligand-controlled expression of a target sequence, which may be endogenous or exogenous. Also disclosed are novel miRNAs and miRNA precursors from crop plants including maize and soy.

1 . A transgenic plant cell having in its genome recombinant DNA comprising transcribable DNA comprising DINA that transcribes to an RNA aptamer capable of binding to a ligand.  
     
     
         2 . The transgenic plant cell of  claim 1 , wherein said transcribable DNA further comprises DNA that transcribes to regulatory RNA capable of regulating expression of a target sequence, 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.  
     
     
         3 - 4 . (canceled)  
     
     
         5 . The transgenic plant cell of  claim 1 , wherein: 
 (a) said transcribable DNA is flanked by sequence encoding a ribozyme; or    (b) said transcribable DNA is embedded in an intron; or    (c) said transcribable DNA is flanked by DNA that transcribes to RNA capable of forming double-stranded RNA; or    (d) said transcribable DNA is flanked by DNA that transcribes to RNA comprising a MicroRNA recognition site; or    (e) said transcribable DNA further comprises at least one gene expression element.    
     
     
         6 . The transgenic plant cell of  claim 2 , wherein said regulatory RNA is operably linked to said target sequence.  
     
     
         7 . The transgenic plant cell of  claim 2 , wherein said regulatory RNA is not operably linked to said target sequence.  
     
     
         8 . The transgenic plant cell of  claim 2 , wherein: 
 (a) binding of said ligand to said RNA aptamer results in an increase of expression of said target sequence relative to expression in the absence of said binding.    (b) binding of said ligand to said RNA aptamer results in a decrease of expression of said target sequence relative to expression in the absence of said binding.    (c) binding of said ligand to said RNA aptamer results in an increase of expression of said target sequence relative to expression in the absence of said binding, said increase of expression resulting in a level of said ligand sufficient to maintain said increase of expression.    (d) binding of said ligand to said RNA aptamer results in a decrease of expression or said target sequence relative to expression in the absence of said binding, said decrease of expression resulting in a level Of said ligand sufficient to maintain said increase of expression.    
     
     
         9 . A transgenic plant comprising a regenerated plant prepared from said transgenic plant cell of  claim 1 , or a progeny plant of said regenerated plant.  
     
     
         10 - 15 . (canceled)  
     
     
         16 . A method of reducing damage to a plant by a pest or pathogen of said plant, comprising transcribing in a plant cell a recombinant DNA construct comprising transcribable DNA comprising DNA that transcribes to an RNA aptamer capable of binding to a ligand, whereby a plant cell of  claim 1  is produced; wherein said ligand comprises at least part of a molecule endogenous to said pest or pathogen, and whereby binding of said RNA aptamer to said ligand reduces damage to said plant by said pest or pathogen, relative to damage in the absence of transcription of said recombinant DNA construct.  
     
     
         17 - 18 . (canceled)  
     
     
         19 . A recombinant DNA constrict useful for producing a plant cell of  claim 1  comprising: 
 (a) transcribable DNA comprising DNA that transcribes to an RNA aptamer capable of binding to a ligand; and    (b) DNA sequence that transcribes to double-stranded RNA flanking said transcribable DNA.    
     
     
         20 . (canceled)  
     
     
         21 . A recombinant DNA construct for plant transformation comprising a promoter operably linked to a first gene suppression element for suppressing at least one first target gene, wherein said first gene suppression element is embedded in an intron flanked on one or on both sides by non-protein-coding DNA.  
     
     
         22 - 24 . (canceled)  
     
     
         25 . The recombinant DNA construct Of  claim 21 , wherein said intron is 3′ to a terminator.  
     
     
         26 - 27 . (canceled)  
     
     
         28 . The recombinant DNA construct of  claim 21 , wherein said first gone 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.    
     
     
         29 - 32 . (canceled)  
     
     
         33 . A method of effecting gene suppression, comprising: 
 (a) providing a transgenic plant comprising a regenerated plant prepared from a transgenic plant cell containing the recombinant DNA construct of  claim 21 , or a progeny plant of said regenerated plant; and    (b) transcribing said recombinant DNA construct in said transgenic plant;    wherein said transcribing produces RNA that is capable of suppressing said at least one first target gene in said transgenic plant, whereby said at least one first target gene is suppressed relative to its expression in the absence of transcription of said recombinant DNA construct.    
     
     
         34 - 42 . (canceled)  
     
     
         43 . A recombinant DNA construct for suppressing production of mature microRNA in a cell, comprising a promoter element operably linked to a gene suppression element for suppression of at least one target sequence selected from said at least one target microRNA precursor or a promoter of said at least one target microRNA precursor or both.  
     
     
         44 - 53 . (canceled)  
     
     
         54 . A method of effecting suppression of mature microRNA production in a eukaryotic cell, comprising transcribing in a eukaryotic cell the recombinant DNA construct of  claim 43 , whereby mature microRNA production is suppressed relative to its production in the absence of transcription of said recombinant DNA construct.  
     
     
         55 - 59 . (canceled)  
     
     
         60 . The method of claim  58 , wherein said target microRNA precursor molecule is derived from the fold-back structure of a maize or soybean MIR sequence selected from tile MIR sequences identified in Tables 3, 4, 5, 6, 9, and 10, and their complements.  
     
     
         61 . (canceled)  
     
     
         62 . A recombinant DNA construct comprising a transcribable engineered miRNA precursor designed to suppress a target sequence, wherein said transcribable engineered miRNA precursor is derived from the fold-back structure of a maize or soybean MIR sequence selected from the group consisting of the MIR sequences identified in Tables 3, 4, 5, 6, 9, and 10 and their complements.  
     
     
         63 . A method to suppress expression of a target sequence in a plant cell, comprising transcribing in a plant cell the recombinant DNA construct of  claim 62 , whereby expression of said target sequence is suppressed relative to its expression in the absence of transcription of said recombinant DNA construct.  
     
     
         64 . A recombinant DNA construct comprising a promoter, a terminator, transcribable sequence between said promoter and said terminator, and at least one gene suppression element that is 3′ to said terminator.  
     
     
         65 . A recombinant DNA construct comprising a promoter operably linked to DNA that transcribes to RNA comprising: 
 (a) at least one exogenous miRNA recognition site recognizable by a mature miRNA expressed in a specific cell of a multicellular eukaryote, and    (b) target RNA to be suppressed in said specific cell,    wherein said target RNA is to be expressed in cells of said multicellular eukaryote other than said specific cell.    
     
     
         66 - 69 . (canceled)  
     
     
         70 . A transgenic plant comprising a regenerated plant prepared from a transgenic plant cell having in its genome the recombinant DNA construct of  claim 65 , or a progeny plant of said regenerated plant, wherein said mature miRNA is a maize or soybean mature miRNA.  
     
     
         71 . (canceled)  
     
     
         72 . A method for suppressing expression of a target RNA in a specific cell of a multicellular eukaryote, comprising transcribing in said multicellular eukaryote the recombinant DNA construct of  claim 65 , wherein said mature miRNA guides cleavage of target RNA in said specific cell, whereby expression of said target RNA is suppressed in said specific cell relative to its expression in cells lacking expression of said mature miRNA.  
     
     
         73 - 75 . (canceled)  
     
     
         78 . A transgenic plant cell having in its genome a recombinant DNA construct selected from the recombinant DNA constructs of claims  19 ,  21 ,  43 ,  62 ,  64 , and  65 .  
     
     
         79 . (canceled)  
     
     
         80 . A transgenic plant comprising a regenerated plant prepared From said transgenic plant cell of  claim 78 , or a progeny plant of said regenerated plant.  
     
     
         81 . A method of providing at least one altered plant tissue, comprising: 
 (a) providing a transgenic plant comprising a regenerated plant prepared from a transgenic plant cell of  claim 78 , or a progeny plant of said regenerated plant; and    (b) transcribing said recombinant DNA construct in at least one tissue of said transgenic plant, whereby an altered trait in said at least one tissue results, relative to tissue wherein said recombinant DNA construct is not transcribed, said altered trait being selected from: 
 (i) improved abiotic stress tolerance;  
 (ii) improved biotic stress tolerance;  
 (iii) improved resistance to a pest or pathogen of said plant;  
 (iv) modified primary metabolite composition;  
 (v) modified secondary metabolite composition;  
 (vi) modified trace element, carotenoid, or vitamin composition;  
 (vii) improved yield;  
 (viii) improved ability to use nitrogen or other nutrients;  
 (ix) modified agronomic characteristics;  
 (x) modified growth or reproductive characteristics; and  
 (xi)improved harvest, storage, or processing quality.  
   
     
     
         82 - 87 . (canceled)  
     
     
         88 . A transgenic plant cell having in its genome the recombinant DNA construct of  claim 21  or a progeny transgenic plant regenerated from said transgenic plant cell or progeny plant or seed of said progeny transgenic plant.  
     
     
         89 . A transgenic plant cell having in its genome the recombinant DNA construct of  claim 43  or a progeny transgenic plant regenerated from said transgenic plant cell or progeny plant or seed of said progeny transgenic plant.  
     
     
         90 . A transgenic plant cell having in its genome the recombinant DNA construct of  claim 62  or a progeny transgenic plant regenerated from said transgenic plant cell or progeny plant or seed of said progeny transgenic plant.  
     
     
         91 . A transgenic plant cell having in its genome the recombinant DNA construct of  claim 65  or a progeny transgenic plant regenerated front said transgenic plant cell or progeny plant or seed of said progeny transgenic plant.