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 . A method of inhibiting expression of a target sequence in a cell comprising: 
 (a) introducing into the cell a nucleic acid construct comprising a promoter operably linked to a polynucleotide, the polynucleotide comprising in the following order: 
 (i) at least about 20 contiguous nucleotides in the region from nucleotides 1-38 of SEQ ID NO: 3,  
 (ii) a first oligonucleotide of 10 to about 50 contiguous nucleotides, wherein the first oligonucleotide is substantially complementary to a second oligonucleotide,  
 (iii) at least about 20 contiguous nucleotides in the region from nucleotides 60-106 of SEQ ID NO: 3,  
 (iv) the second oligonucleotide of about 10 to about 50 contiguous nucleotides, wherein the second oligonucleotide encodes a mRNA, and wherein the second oligonucleotide is substantially complementary to the target sequence, and  
 (v) at least about 20 contiguous nucleotides in the region from nucleotides 128-159 of SEQ ID NO:3;  
   wherein the polynucleotide encodes an RNA precursor capable of forming a hairpin, and    (b) expressing the nucleic acid construct for a time sufficient to produce the mRNA, wherein the mRNA inhibits expression of the target sequence.    
     
     
         2 . The method of  claim 1 , wherein the nucleic acid construct further comprises a promoter operably linked to the polynucleotide  
     
     
         3 . The method of  claim 1 , wherein the cell is a plant cell.  
     
     
         4 . A cell comprising the nucleic acid construct of  claim 1 .  
     
     
         5 . The cell of  claim 4 , wherein the cell is a plant cell.  
     
     
         6 . The method of  claim 1 , wherein target sequence expression is inhibited by at least 10%.  
     
     
         7 . The method of  claim 1 , wherein inhibition of the target sequence generates a loss-of-function phenotype.  
     
     
         8 . The method of  claim 2 , wherein the promoter is a pathogen-inducible promoter and inhibition of the target sequence confers resistance to a pathogen.  
     
     
         9 . An isolated polynucleotide comprising in the following order at least 20 contiguous nucleotides in the region from nucleotides 1-38 of SEQ ID NO: 3, a first oligonucleotide of about 10 to about 50 contiguous nucleotides, wherein the first oligonucleotide is substantially complementary to a second oligonucleotide, at least about 20 contiguous nucleotides in the region from nucleotides 60-106 of SEQ ID NO: 3, a second oligonucleotide of about 10 to about 50 contiguous nucleotides, wherein the second oligonucleotide encodes a mRNA, and the second oligonucleotide is substantially complementary to the target sequence, and at least about 20 contiguous nucleotides in the region from nucleotides 128-159 of SEQ ID NO: 3, wherein the polynucleotide encodes an RNA precursor capable of forming a hairpin.  
     
     
         10 . The isolated polynucleotide of  claim 9 , further comprising an operably linked promoter.  
     
     
         11 . A cell comprising the isolated polynucleotide of  claim 9 .  
     
     
         12 . The cell of  claim 11 , wherein the cell is a plant cell.  
     
     
         13 . A transgenic plant comprising the isolated polynucleotide of  claim 9 .  
     
     
         14 . A transgenic seed comprising the isolated polynucleotide of  claim 9 .  
     
     
         15 . The isolated polynucleotide of  claim 10 , wherein the promoter is a pathogen-inducible promoter.  
     
     
         16 . The isolated polynucleotide of  claim 9 , wherein the polynucleotide suppresses expression of a target sequence.  
     
     
         17 . A method of inhibiting expression of a target sequence in a cell comprising: 
 (a) introducing into the cell a nucleic acid construct comprising a promoter operably linked to a polynucleotide, wherein the polynucleotide comprises in the following order: 
 (i) nucleotides 1-38 of SEQ ID NO: 3,  
 (ii) a first oligonucleotide of 21 contiguous nucleotides, wherein the first oligonucleotide is substantially complementary to a second oligonucleotide,  
 (iii) nucleotides 60-106 of SEQ ID NO: 3,  
 (iv) a second oligonucleotide of 21 contiguous nucleotides, wherein the second oligonucleotide encodes a mRNA, and wherein the second oligonucleotide is substantially complementary to the target sequence, and  
 (v) nucleotides 128-159 of SEQ ID NO:3;  
   wherein the polynucleotide encodes an RNA precursor capable of forming a hairpin, and    (b) expressing the nucleic acid construct for a time sufficient to produce the mRNA, wherein the mRNA inhibits expression of the target sequence.    
     
     
         18 . An isolated polynucleotide comprising in the following order nucleotides 1-38 of SEQ ID NO: 3, a first oligonucleotide of 21 contiguous nucleotides, wherein the first oligonucleotide is substantially complementary to a second oligonucleotide, nucleotides 60-106 of SEQ ID NO: 3, a second oligonucleotide of 21 contiguous nucleotides, wherein the second oligonucleotide encodes a mRNA, and the second oligonucleotide is substantially complementary to the target sequence, and nucleotides 128-159 of SEQ ID NO:3, wherein the polynucleotide encodes an RNA precursor capable of forming a hairpin.  
     
     
         19 . A method of inhibiting expression of a target sequence in a cell comprising: 
 (a) introducing into the cell a nucleic acid construct comprising a promoter operably linked to a polynucleotide encoding a modified plant mRNA precursor comprising a first and a second oligonucleotide, wherein at least one of the first or the second oligonucleotides is heterologous to the precursor, wherein the first oligonucleotide is substantially complementary to the second oligonucleotide, and the second oligonucleotide encodes a mRNA substantially complementary to the target sequence, wherein the precursor is capable of forming a hairpin; and    (b) expressing the nucleic acid construct for a time sufficient to produce the mRNA, wherein the mRNA inhibits expression of the target sequence.    
     
     
         20 . A method of inhibiting expression of a target sequence in a cell comprising: 
 (a) introducing into the cell a nucleic acid construct comprising a promoter operably linked to a polynucleotide encoding a modified plant miR172 mRNA precursor comprising a first and a second oligonucleotide, wherein at least one of the first or the second oligonucleotides is heterologous to the precursor, wherein the first oligonucleotide is substantially complementary to the second oligonucleotide, and the second oligonucleotide encodes a mRNA substantially complementary to the target sequence, wherein the precursor is capable of forming a hairpin; and    (b) expressing the nucleic acid construct for a time sufficient to produce the mRNA, wherein the mRNA inhibits expression of the target sequence.    
     
     
         21 . The method of  claim 20 , wherein the plant miR172 mRNA precursor is from a dicot or a monocot.  
     
     
         22 . The method of  claim 20 , wherein the plant miR172 mRNA precursor is from a plant selected from the group consisting of  Arabidopsis , tomato, soybean, rice, and corn.  
     
     
         23 . The method of  claim 20 , wherein the plant miR172 mRNA precursor comprises SEQ ID NO: 3.  
     
     
         24 . A method of inhibiting expression of a target sequence in a cell comprising: 
 (a) introducing into the cell a nucleic acid construct comprising a promoter operably linked to a polynucleotide encoding a modified  Arabidopsis  miR172 mRNA precursor comprising a first and a second oligonucleotide, wherein at least one of the first or the second oligonucleotides is heterologous to the precursor, wherein the first oligonucleotide is substantially complementary to the second oligonucleotide, and the second oligonucleotide encodes a mRNA substantially complementary to the target sequence, wherein the precursor is capable of forming a hairpin; and    (b) expressing the nucleic acid construct for a time sufficient to produce the mRNA, wherein the mRNA inhibits expression of the target sequence.    
     
     
         25 . The method of  claim 24 , wherein the  Arabidopsis  miR172 mRNA precursor comprises SEQ ID NO: 3.  
     
     
         26 . A cell produced by the method of  claim 1 .  
     
     
         27 . The cell of  claim 26 , wherein the cell is a plant cell.  
     
     
         28 . The cell of  claim 27 , wherein the plant cell is from a monocot or a dicot.  
     
     
         29 . The cell of  claim 28 , wherein the cell is selected from the group consisting of corn, wheat, rice, barley, oats, sorghum, millet, sunflower, safflower, cotton, soy, canola, alfalfa,  Arabidopsis , and tobacco.  
     
     
         30 . The cell of  claim 29 , wherein the cell is from  Arabidopsis.    
     
     
         31 . The cell of  claim 29 , wherein the cell is from corn.  
     
     
         32 . A cell produced by the method of  claim 19 .  
     
     
         33 . The cell of  claim 32 , wherein the cell is a plant cell.  
     
     
         34 . The cell of  claim 33 , wherein the plant cell is from a monocot or a dicot.  
     
     
         35 . The cell of  claim 34 , wherein the 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 cell of  claim 35 , wherein the cell is from  Arabidopsis.    
     
     
         37 . The cell of  claim 35 , wherein the cell is from corn.  
     
     
         38 . A cell produced by the method of  claim 20 .  
     
     
         39 . The cell of  claim 38 , wherein the cell is a plant cell.  
     
     
         40 . The cell of  claim 39 , wherein the plant cell is from a monocot or a dicot.  
     
     
         41 . The cell of  claim 40 , wherein the cell is selected from the group consisting of corn, wheat, rice, barley, oats, sorghum, millet, sunflower, safflower, cotton, soy, canola, alfalfa,  Arabidopsis , and tobacco.  
     
     
         42 . The cell of  claim 41 , wherein the cell is from  Arabidopsis.    
     
     
         43 . The cell of  claim 41 , wherein the cell is from corn.  
     
     
         44 . A cell produced by the method of  claim 24 .  
     
     
         45 . The cell of  claim 44 , wherein the cell is a plant cell.  
     
     
         46 . The cell of  claim 45 , wherein the plant cell is from a monocot or a dicot.  
     
     
         47 . The cell of  claim 46 , wherein the cell is selected from the group consisting of corn, wheat, rice, barley, oats, sorghum, millet, sunflower, safflower, cotton, soy, canola, alfalfa,  Arabidopsis , and tobacco.  
     
     
         48 . The cell of  claim 47 , wherein the cell is from  Arabidopsis.    
     
     
         49 . The cell of  claim 47 , wherein the cell is from corn.  
     
     
         50 . The method of  claim 3 , the method further comprising producing a transformed plant, wherein the plant comprises the nucleic acid construct which encodes the mRNA.  
     
     
         51 . A plant produced by the method of  claim 50 .  
     
     
         52 . The method of  claim 19 , wherein the cell is a plant cell, the method further comprising producing a transformed plant, wherein the plant comprises the nucleic acid construct which encodes the mRNA.  
     
     
         53 . A plant produced by the method of  claim 52 .  
     
     
         54 . The method of  claim 20 , wherein the cell is a plant cell, the method further comprising producing a transformed plant, wherein the plant comprises the nucleic acid construct which encodes the mRNA.  
     
     
         55 . A plant produced by the method of  claim 50 .  
     
     
         56 . The method of  claim 24 , wherein the cell is a plant cell, the method further comprising producing a transformed plant, wherein the plant comprises the nucleic acid construct which encodes the mRNA.  
     
     
         57 . A plant produced by the method of  claim 56 .  
     
     
         58 . The plant of  claim 57 , wherein the plant is a monocot or a dicot.  
     
     
         59 . The plant of  claim 58 , wherein the plant is selected from the group consisting of corn, wheat, rice, barley, oats, sorghum, millet, sunflower, safflower, cotton, soy, canola, alfalfa,  Arabidopsis , and tobacco.  
     
     
         60 . The plant of  claim 59 , wherein the plant is  Arabidopsis.    
     
     
         61 . The plant of  claim 59 , wherein the plant is corn.  
     
     
         62 . An isolated polynucleotide comprising a polynucleotide which encodes a modified plant mRNA precursor, the modified precursor comprising a first and a second oligonucleotide, wherein at least one of the first or the second oligonucleotides is heterologous to the precursor, wherein the first oligonucleotide is substantially complementary to the second oligonucleotide, and the second oligonucleotide comprises a mRNA substantially complementary to the target sequence, wherein the precursor is capable of forming a hairpin.  
     
     
         63 . A cell comprising the isolated polynucleotide of  claim 62 .  
     
     
         64 . The cell of  claim 63 , wherein the cell is a plant cell.  
     
     
         65 . An isolated polynucleotide comprising a polynucleotide encoding a modified plant miR172 mRNA precursor, the modified precursor comprising a first and a second oligonucleotide, wherein at least one of the first or the second oligonucleotides is heterologous to the precursor, wherein the first oligonucleotide is substantially complementary to the second oligonucleotide, and the second oligonucleotide comprises a mRNA substantially complementary to the target sequence, wherein the precursor is capable of forming a hairpin.  
     
     
         66 . A cell comprising the isolated polynucleotide of  claim 65 .  
     
     
         67 . The cell of  claim 66 , wherein the cell is a plant cell.  
     
     
         68 . An isolated polynucleotide comprising polynucleotide encoding a modified  Arabidopsis  miR172 mRNA precursor, the modified precursor comprising a first and a second oligonucleotide, wherein at least one of the first or the second oligonucleotides is heterologous to the precursor, wherein the first oligonucleotide is substantially complementary to the second oligonucleotide, and the second oligonucleotide comprises a mRNA which is substantially complementary to the target sequence, wherein the precursor is capable of forming a hairpin.  
     
     
         69 . A cell comprising the isolated polynucleotide of  claim 68 .  
     
     
         70 . The cell of  claim 69 , wherein the cell is a plant cell.  
     
     
         71 . The cell of  claim 70 , wherein the plant cell is from a dicot or a monocot.  
     
     
         72 . The cell of  claim 71 , wherein the cell is from  Arabidopsis.    
     
     
         73 . The cell of  claim 71 , wherein the cell is from corn.  
     
     
         74 . The isolated polynucleotide of  claim 62 , wherein the first and the second oligonucleotides are heterologous to the precursor.  
     
     
         75 . The isolated polynucleotide of  claim 65 , wherein the first and the second oligonucleotides are heterologous to the precursor.  
     
     
         76 . The isolated polynucleotide of  claim 68 , wherein the first and the second oligonucleotides are heterologous to the precursor.