Recombinant DNA Constructs and Methods for Modulating Expression of a Target Gene

Abstract

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 - 10 . (canceled) 
     
     
         11 . 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 at an miRNA recognition site for an endogenous mature miRNA, and forms, through complementary base-pairing, a hybridized segment of from 19 to 24 nucleotides in length at said miRNA recognition site in said target RNA transcript,
 wherein said hybridized segment comprises an A, G, or C in said single-stranded cleavage blocker RNA at a position corresponding to the 5′ terminus of said endogenous mature miRNA, and matches through complementary base-pairing between said single-stranded cleavage blocker RNA and said miRNA recognition site at positions corresponding to positions 9, 10, and 11 in 3′ to 5′ direction of said endogenous mature miRNA, and   wherein said single-stranded cleavage blocker RNA interferes with the binding of said endogenous mature miRNA to said target RNA transcript at said miRNA recognition site.   
     
     
         12 . The recombinant DNA construct of  claim 11 , wherein formation of said hybridized segment inhibits cleavage of said target RNA transcript mediated by said endogenous mature miRNA. 
     
     
         13 . A method of modulating expression of at least one target gene, comprising expressing in said plant cell the recombinant DNA construct of  claim 11 , wherein said at least one target gene encodes said target RNA transcript. 
     
     
         14 . The method of  claim 13 , wherein formation of said hybridized segment inhibits suppression of said at least one target gene by said endogenous mature miRNA. 
     
     
         15 . A non-natural plant chromosome or plastid comprising the recombinant DNA construct of  claim 11 . 
     
     
         16 . A non-natural transgenic plant cell having in its genome the recombinant DNA construct of  claim 11 , 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. 
     
     
         17 . A non-natural partially transgenic plant, wherein:
 (a) said non-natural partially transgenic plant comprises the non-natural transgenic plant cell of  claim 16  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 16  and further comprises a non-transgenic scion.   
     
     
         18 . The recombinant DNA construct of  claim 11 , wherein said target RNA transcript is transcribed from at least one target gene comprising:
 (a) coding sequence, non-coding sequence, or both coding and non-coding sequence; or   (b) a single target gene, or multiple target genes; or   (c) one or more of the group consisting of:
 (1) an endogenous gene of a eukaryote, 
 (2) a transgene of a transgenic plant, 
 (3) an endogenous gene of a pest or pathogen of a plant, and 
   (4) an endogenous gene of a symbiont associated with a pest or pathogen of a plant.