Recombinant DNA constructs encoding ribonuclease cleavage blockers and methods for modulating expression of a target gene
This invention provides recombinant DNA constructs and methods for manipulating expression of a target gene that is regulated by a small RNA, by interfering with the binding of the small RNA to its target gene. More specifically, this invention discloses recombinant DNA constructs encoding cleavage blockers, 5-modified cleavage blockers, and translational inhibitors useful for modulating expression of a target gene and methods for their use. Further disclosed are miRNA targets useful for designing recombinant DNA constructs including miRNA-unresponsive transgenes, miRNA decoys, cleavage blockers, 5-modified cleavage blockers, and translational inhibitors, as well as methods for their use, and transgenic eukaryotic cells and organisms containing such constructs.
1. A recombinant DNA construct comprising a promoter operable in a plant cell, operably linked to DNA encoding a single-stranded cleavage blocker RNA that binds in vivo to a target RNA transcript in said plant cell, at an miRNA recognition site for an endogenous mature miRNA, and forms, through complementary base-pairing, a hybridized segment of between 19 to 24 nucleotides in length at said miRNA recognition site in said target RNA transcript, wherein said hybridized segment comprises:
at least one nucleotide in said single-stranded cleavage blocker RNA that does not match through complementary base-pairing with said miRNA recognition site at positions corresponding to positions 10 or 11 of said endogenous mature miRNA;
and wherein the single-stranded cleavage blocker RNA interferes with the binding of said endogenous mature miRNA to said target RNA transcript at said miRNA recognition site.
2. The recombinant DNA construct of claim 1 , wherein formation of said hybridized segment inhibits cleavage of said target RNA transcript mediated by said endogenous mature miRNA.
3. A method of modulating expression of a target gene in a plant cell, comprising expressing in said plant cell the recombinant DNA construct of claim 1 , wherein said target gene encodes said, target RNA transcript.
4. The method of claim 3 , wherein formation of said hybridized segment inhibits suppression of said at least one target gene by said endogenous mature miRNA.
5. A non-natural plant chromosome or plastid comprising the recombinant DNA construct of claim 1 .
6. A non-natural transgenic plant cell having in its genome the recombinant DNA construct of claim 1 , or a non-natural transgenic plant or a non-natural transgenic plant seed or a non-natural transgenic pollen grain each comprising said non-natural transgenic plant cell.
7. A non-natural partially transgenic plant, wherein:
a. said non-natural partially transgenic plant comprises the non-natural transgenic plant cell of claim 6 and further comprises non-transgenic tissue; or
b. said non-natural partially transgenic plant comprises a transgenic rootstock comprising the non-natural transgenic plant cell of claim 6 and further comprises a non-transgenic scion.