Gene silencing

Abstract

The invention provides methods and compositions useful in target sequence suppression and target sequence validation. The invention provides polynucleotide constructs useful for gene silencing, as well as cells, plants and seeds comprising the polynucleotides. The invention also provides a method for using microRNA to silence a target sequence.

1 - 18 . (canceled) 
     
     
         19 . A method of inhibiting expression of a target sequence in a plant or plant cell comprising:
 (a) introducing into the plant or plant cell a nucleic acid construct comprising a promoter operably linked to a polynucleotide encoding a modified full-length plant miRNA precursor, wherein the full-length plant miRNA precursor has been modified in a backside region or a miRNA template region, or both, as follows:
 (1) the backside region consists of a first oligonucleotide of 21 contiguous nucleotides, wherein the first oligonucleotide is either fully complementary to a second oligonucleotide of 21 contiguous nucleotides or has 1, 2, 3, 4, or 5 mismatches, and 
 (2) the miRNA template region consists of the second oligonucleotide of 21 contiguous nucleotides, wherein the second oligonucleotide encodes a miRNA, and wherein the second oligonucleotide is either fully complementary to the target sequence or has 1, 2, 3, 4, or 5 mismatches, and 
 wherein at least one of the first or the second oligonucleotides is heterologous to the precursor, wherein the precursor is capable of forming a hairpin, wherein the precursor has only been modified in the backside region, the miRNA template region, or both, and optionally by addition of a restriction site recognition sequence or a recombination sequence flanking the hairpin; and 
   (b) expressing the nucleic acid construct for a time sufficient to produce the miRNA, wherein the miRNA inhibits expression of the target sequence.   
     
     
         20 . (canceled) 
     
     
         21 . The method of  claim 19 , wherein the full-length plant miRNA precursor is from a dicot or a monocot. 
     
     
         22 . The method of  claim 21 , wherein the full-length plant miRNA precursor is from a plant selected from the group consisting of  Arabidopsis , tomato, soybean, rice, and corn. 
     
     
         23 - 31 . (canceled) 
     
     
         32 . A plant or plant cell produced by the method of  claim 19 . 
     
     
         33 . (canceled) 
     
     
         34 . The plant or plant cell of  claim 32 , wherein the plant or plant cell is a monocot or a dicot plant or plant cell. 
     
     
         35 . The plant or plant cell of  claim 34 , wherein the plant or plant cell is selected from the group consisting of corn, wheat, rice, barley, oats, sorghum, millet, sunflower, safflower, cotton, soy, canola, alfalfa,  Arabidopsis , and tobacco. 
     
     
         36 . The plant or plant cell of  claim 35 , wherein the plant or plant cell is an  Arabidopsis  plant or plant cell. 
     
     
         37 . The plant or plant cell of  claim 35 , wherein the plant or plant cell is a corn plant or plant cell. 
     
     
         38 - 76 . (canceled)