Plant microRNAs and methods of use thereof
This invention discloses novel microRNAs and their precursors, and recombinant DNA constructs including such novel miRNAs, miRNA precursors, miRNA promoters, and miRNA recognition sites corresponding to the miRNAs. Included are novel miRNA and miRNA precursors that exhibit nutrient-responsive expression. Also disclosed are miRNA decoy sequences. Further provided are non-natural transgenic plant cells, plants, and seeds containing in their genome a recombinant DNA construct of this invention and methods of controlling gene expression using recombinant DNA constructs of this invention.
1. A recombinant DNA construct comprising a heterologous promoter functional in a plant cell and operably linked to DNA encoding an RNA transcript consisting essentially of at least one miRNA decoy sequence that is recognized and bound by an endogenous mature plant miRNA but not cleaved, wherein
said miRNA decoy sequence is an RNA sequence of between about 19 to about 36 contiguous RNA nucleotides, wherein said miRNA decoy sequence is recognized and bound by said endogenous mature plant miRNA, resulting in base-pairing between said miRNA decoy sequence and said endogenous mature plant miRNA, thereby forming a cleavage-resistant RNA duplex comprising at least one mismatch between said miRNA decoy sequence and said endogenous mature plant miRNA at positions 9, 10, or 11 of said endogenous mature plant miRNA, or at least one insertion at a position in said miRNA decoy sequence corresponding to positions 10-11 of said endogenous mature plant miRNA.
2. The recombinant DNA construct of claim 1 , wherein said at least one miRNA decoy sequence comprises (a) a naturally occurring miRNA decoy sequence, or (b) a synthetic miRNA decoy sequence, or (c) both a naturally occurring miRNA decoy sequence and a synthetic miRNA decoy sequence.
3. A non-natural transgenic plant cell comprising the recombinant DNA construct of claim 1 .
4. A non-natural transgenic plant regenerated from the non-natural transgenic plant cell of claim 3 , or a progeny plant of a regenerated plant prepared from the non-natural transgenic plant cell of claim 3 , wherein said non-natural transgenic plant comprises said recombinant DNA construct and has at least one altered trait, relative to a plant lacking said recombinant DNA construct, selected from the group of traits consisting of:
(a) improved abiotic stress tolerance;
(b) improved biotic stress tolerance;
(c) improved resistance to a pest or pathogen of said plant;
(d) modified primary metabolite composition;
(e) modified secondary metabolite composition;
(f) modified trace element, carotenoid, or vitamin composition;
(g) improved yield;
(h) improved ability to use nitrogen or other nutrients;
(i) modified agronomic characteristics;
(j) modified growth or reproductive characteristics; and
(k) improved harvest, storage, or processing quality.
5. A method of providing a non-natural transgenic crop plant having at least one altered trait, comprising expressing said recombinant DNA construct of claim 1 in said non-natural transgenic crop plant, thereby resulting in said non-natural transgenic crop plant exhibiting at least one altered trait, relative to a crop plant in which said recombinant DNA construct is not expressed, selected from the group of traits consisting of:
(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.
6. A recombinant DNA construct of claim 1 , wherein said cleavage-resistant RNA duplex further comprises
(a) 1 or 2 mismatches between said miRNA decoy sequence and said endogenous mature plant miRNA at positions 1, 2, 3, 4, 5, 6, 7, 8, and 9 of said endogenous mature plant miRNA,
(b) 1, 2, or 3 mismatches between said miRNA decoy sequence and said endogenous mature plant miRNA at positions 12 through the last position of said endogenous mature plant miRNA, wherein each of said mismatches at positions 12 through the last position of said endogenous mature plant miRNA is adjacent to at least one complementary base-pair, or
(c) a combination of (a) and (b).