MicroRNAs

Abstract

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

What is claimed: 
     
         1 . A nucleic acid construct comprising a promoter functional in a plant cell operatively linked to a polynucleotide comprising two or more modified miRNA precursors operatively linked together, wherein each modified miRNA precursor comprises a fragment of a plant miRNA primary transcript that is modified in the miRNA sequence and the miRNA complementary sequence, further wherein each modified miRNA precursor is capable of forming a double-stranded RNA or an RNA hairpin, wherein each modified miRNA is an miRNA modified to be (i) fully complementary to a target sequence, (ii) fully complementary to a target sequence except for GU base pairing or (iii) fully complementary to a target sequence in the first ten nucleotides counting from the 5′ end of the miRNA, and wherein the target sequence of each modified miRNA is different. 
     
     
         2 . The nucleic acid contruct of  claim 1 , wherein the plant miRNA transcripts are selected from the group consisting of  Arabidopsis , tomato, soybean, rice and corn primary miRNA transcripts. 
     
     
         3 . The nucleic acid contruct of  claim 1 , wherein at least two of the plant miRNA primary transcripts are the same. 
     
     
         4 . The nucleic acid contruct of  claim 1 , wherein at least two of the plant miRNA primary transcripts are different. 
     
     
         5 . The nucleic acid contruct of  claim 1 , wherein a target sequence is in a non-coding region of RNA. 
     
     
         6 . The nucleic acid contruct of  claim 1 , wherein a target sequence is in a coding region of RNA. 
     
     
         7 . The nucleic acid contruct of  claim 1 , wherein a target sequence contains a splice site of RNA. 
     
     
         8 . The nucleic acid contruct of  claim 1 , wherein at least one of the plant miRNA primary transcripts is a plant miR159 primary transcript. 
     
     
         9 . The nucleic acid contruct of  claim 1 , wherein at least one of the plant miRNA primary transcripts is a plant miR169 primary transcript. 
     
     
         10 . A plant cell comprising the nucleic acid construct of  claim 1 . 
     
     
         11 . The plant cell of  claim 10 , 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. 
     
     
         12 . The plant cell of  claim 10 , wherein the nucleic acid construct is inserted in an intron of a gene or transgene of the plant cell. 
     
     
         13 . A transgenic plant comprising the nucleic acid construct of  claim 1 . 
     
     
         14 . The transgenic plant of  claim 13 , 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. 
     
     
         15 . The transgenic plant of  claim 13 , wherein the nucleic acid construct is inserted into an intron of a gene or transgene of the transgenic plant. 
     
     
         16 . A seed of the transgenic plant of of  claim 13 . 
     
     
         17 . A seed of the transgenic plant of of  claim 15 . 
     
     
         18 . A method for down regulating two or more different target sequences in a plant cell comprising:
 (a) transforming a plant cell with a nucleic acid construct comprising a promoter functional in a plant cell operatively linked to a polynucleotide comprising two or more modified miRNA precursors operatively linked together, wherein each modified miRNA precursor comprises a fragment of a plant miRNA primary transcript that is modified in the miRNA sequence and the miRNA complementary sequence, further wherein each modified miRNA precursor is capable of forming a double-stranded RNA or an RNA hairpin, wherein each modified miRNA is an miRNA modified to be (i) fully complementary to a target sequence, (ii) fully complementary to a target sequence except for GU base pairing or (iii) fully complementary to a target sequence in the first ten nucleotides counting from the 5′ end of the miRNA, and wherein the target sequence of each modified miRNA is different; and   (b) expressing the nucleic acid construct for a time sufficient to produce each modified miRNA, wherein each modified miRNA down regulates a different target sequence.   
     
     
         19 . The method of  claim 18 , wherein each modified miRNA binds to its target sequence and the double-stranded RNA is cleaved. 
     
     
         20 . The method of  claim 18 , wherein the plant miRNA transcripts are selected from the group consisting of  Arabidopsis , tomato, soybean, rice and corn primary miRNA transcripts. 
     
     
         21 . The method of  claim 18 , wherein at least two of the plant miRNA primary transcripts are the same. 
     
     
         22 . The method of  claim 18 , wherein at least two of the plant miRNA primary transcripts are different. 
     
     
         23 . The method of  claim 18 , wherein a target sequence is in a non-coding region of RNA. 
     
     
         24 . The method of  claim 18 , wherein a target sequence is in a coding region of RNA. 
     
     
         25 . The method of  claim 18 , wherein a target sequence contains a splice site of RNA. 
     
     
         26 . The method of  claim 18 , wherein at least one of the plant miRNA primary transcripts is a plant miR159 primary transcript. 
     
     
         27 . The method of  claim 18 , wherein at least one of the plant miRNA primary transcripts is a plant miR169 primary transcript. 
     
     
         28 . The method of  claim 18 , wherein the nucleic acid construct is inserted in an intron of a gene or transgene of the plant cell. 
     
     
         29 . A method for down regulating two or more different target sequences in a plant, the method comprises obtaining a transgenic plant from the transformed plant cell of step (a) of  claim 18  and expressing the nucleic acid construct of step (b) in the transgenic plant. 
     
     
         30 . The method of  claim 29 , wherein each modified miRNA binds to its target sequence and the double-stranded RNA is cleaved. 
     
     
         31 . The method of  claim 29 , wherein the plant miRNA transcripts are selected from the group consisting of  Arabidopsis , tomato, soybean, rice and corn primary miRNA transcripts. 
     
     
         32 . The method of  claim 29 , wherein at least two of the plant miRNA primary transcripts are the same. 
     
     
         33 . The method of  claim 29 , wherein at least two of the plant miRNA primary transcripts are different. 
     
     
         34 . The method of  claim 29 , wherein a target sequence is in a non-coding region of RNA. 
     
     
         35 . The method of  claim 29 , wherein a target sequence is in a coding region of RNA. 
     
     
         36 . The method of  claim 29 , wherein a target sequence contains a splice site of RNA. 
     
     
         37 . The method of  claim 29 , wherein at least one of the plant miRNA primary transcripts is a plant miR159 primary transcript. 
     
     
         38 . The method of  claim 29 , wherein at least one of the plant miRNA primary transcripts is a plant miR169 primary transcript. 
     
     
         39 . The method of  claim 29 , wherein the nucleic acid construct is inserted in an intron of a gene or transgene of the plant cell. 
     
     
         40 . The method of  claim 29 , further comprising harvesting seed from the transgenic plant. 
     
     
         41 . The method of  claim 40 , further comprising growing a plant from the seed and expressing the nucleic acid construct in the growing plant. 
     
     
         42 . The method of  claim 40 , further comprising growing a plant from the seed and crossing the growing plant with a different plant to produce progeny seed comprising the nucleic acid construct.