US2011033937A1PendingUtilityA1

Down-regulation of gene expression using artificial micrornas

69
Assignee: DU PONTPriority: Dec 18, 2007Filed: Dec 17, 2008Published: Feb 10, 2011
Est. expiryDec 18, 2027(~1.4 yrs left)· nominal 20-yr term from priority
Inventors:Brian Mcgonigle
C12N 15/825C12N 15/8247C12N 15/8218
69
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Claims

Abstract

Isolated nucleic acid fragments comprising precursor miRNA, and artificial miRNAs and their use in down-regulating gene expression are described.

Claims

exact text as granted — not AI-modified
1 . An isolated nucleic acid fragment comprising a precursor miRNA said precursor miRNA corresponding substantially to the deoxyribonucleotide sequence set forth in SEQ ID NO:11 (i) wherein nucleotides 430 to 450 of SEQ ID NO:11 are replaced by a first variable nucleotide subsequence ranging in size from about 19 to about 30 nucleotides depending upon the target sequence whose expression is to be reduced and (ii) further wherein nucleotides 244 to 264 of SEQ ID NO:11 are replaced by a second variable nucleotide subsequence ranging in size from about 19 to about 30 nucleotides, said second variable nucleotide subsequence being capable of hybridizing to the first variable subsequence of the precursor miRNA. 
     
     
         2 . A recombinant construct comprising the isolated nucleic acid fragment of  claim 1  operably linked to at least one regulatory sequence. 
     
     
         3 . A plant cell comprising the recombinant construct of  claim 2 . 
     
     
         4 . The plant cell of  claim 3  wherein the plant cell is a monocot plant cell. 
     
     
         5 . A method for reducing expression of a target sequence in a plant cell, said method comprising:
 (a) transforming at least one plant cell with a nucleic acid construct comprising a comprising a precursor miRNA said precursor miRNA corresponding substantially to the deoxyribonucleotide sequence set forth in SEQ ID NO:11 (i) wherein nucleotides 430 to 450 of SEQ ID NO:11 are replaced by a first variable nucleotide subsequence ranging in size from about 19 to about 30 nucleotides depending upon the target sequence whose expression is to be reduced and (ii) further wherein nucleotides 244 to 264 of SEQ ID NO:11 are replaced by a second variable nucleotide subsequence ranging in size from about 19 to about 30 nucleotides, said second variable nucleotide subsequence being capable of hybridizing to the first variable subsequence of the precursor miRNA; and   (b) selecting those transformed plant cell(s) whose level of expression of the target sequence is reduced when compared to the level of expression of the target gene in a wild type plant cell.   
     
     
         6 . The method of  claim 5  wherein the plant cell is a monocot plant cell. 
     
     
         7 . An isolated nucleic acid fragment comprising a precursor miRNA said precursor miRNA corresponding substantially to the deoxyribonucleotide sequence set forth in SEQ ID NO:12 (i) wherein nucleotides 94 to 114 SEQ ID NO:12 are replaced by a first variable nucleotide subsequence ranging in size from about 19 to about 30 nucleotides depending upon the target sequence whose expression is to be reduced and (ii) further wherein nucleotides 163 to 183 of SEQ ID NO:12 are replaced by a second variable nucleotide subsequence ranging in size from about 19 to about 30 nucleotides, said second variable nucleotide subsequence being capable of hybridizing to the first variable subsequence of the precursor miRNA. 
     
     
         8 . A recombinant construct comprising the isolated nucleic acid fragment of  claim 7  operably linked to at least one regulatory sequence. 
     
     
         9 . A plant cell comprising the recombinant construct of  claim 8 . 
     
     
         10 . The plant cell of  claim 9  wherein the plant cell is a monocot plant cell. 
     
     
         11 . A method for reducing expression of a target sequence in a plant cell, said method comprising:
 (a) transforming at least one plant cell with a nucleic acid construct comprising a comprising a precursor miRNA said precursor miRNA corresponding substantially to the deoxyribonucleotide sequence set forth in SEQ ID NO:12 (i) wherein nucleotides 94 to 114 of SEQ ID NO:12 are replaced by a first variable nucleotide subsequence ranging in size from about 19 to about 30 nucleotides depending upon the target sequence whose expression is to be reduced and (ii) further wherein nucleotides 163 to 183 of SEQ ID NO:12 are replaced by a second variable nucleotide subsequence ranging in size from about 19 to about 30 nucleotides, said second variable nucleotide subsequence being capable of hybridizing to the first variable subsequence of the precursor miRNA; and   (b) selecting those transformed plant cell(s) whose level of expression of the target sequence is reduced when compared to the level of expression of the target gene in a wild type plant cell.   
     
     
         12 . The method of  claim 11  wherein the plant cell is a monocot plant cell. 
     
     
         13 . An isolated nucleic acid fragment comprising a precursor miRNA said precursor miRNA corresponding substantially to the deoxyribonucleotide sequence set forth in SEQ ID NO:13 (i) wherein nucleotides 53 to 73 of SEQ ID NO:13 are replaced by a first variable nucleotide subsequence ranging in size from about 19 to about 30 nucleotides depending upon the target sequence whose expression is to be reduced and (ii) further wherein nucleotides 97 to 117 of SEQ ID NO:13 are replaced by a second variable nucleotide subsequence ranging in size from about 19 to about 30 nucleotides, said second variable nucleotide subsequence being capable of hybridizing to the first variable subsequence of the precursor miRNA. 
     
     
         14 . A recombinant construct comprising the isolated nucleic acid fragment of  claim 13  operably linked to at least one regulatory sequence. 
     
     
         15 . A plant cell comprising the recombinant construct of  claim 14 . 
     
     
         16 . The plant cell of  claim 14  wherein the plant cell is a monocot plant cell. 
     
     
         17 . A method for reducing expression of a target sequence in a plant cell, said method comprising:
 (a) transforming at least one plant cell with a nucleic acid construct comprising a comprising a precursor miRNA said precursor miRNA corresponding substantially to the deoxyribonucleotide sequence set forth in SEQ ID NO:13 (i) wherein nucleotides 53 to 73 of SEQ ID NO:13 are replaced by a first variable nucleotide subsequence ranging in size from about 19 to about 30 nucleotides depending upon the target sequence whose expression is to be reduced and (ii) further wherein nucleotides 97 to 117 of SEQ ID NO:13 are replaced by a second variable nucleotide subsequence ranging in size from about 19 to about 30 nucleotides, said second variable nucleotide subsequence being capable of hybridizing to the first variable subsequence of the precursor miRNA; and   (b) selecting those transformed plant cell(s) whose level of expression of the target sequence is reduced when compared to the level of expression of the target gene in a wild type plant cell.   
     
     
         18 . The method of  claim 17  wherein the plant cell is a monocot plant cell. 
     
     
         19 . An isolated nucleic acid fragment comprising a precursor miRNA said precursor miRNA corresponding substantially to the deoxyribonucleotide sequence set forth in SEQ ID NO:14 (i) wherein nucleotides 110 to 130 of SEQ ID NO:14 are replaced by a first variable nucleotide subsequence ranging in size from about 19 to about 30 nucleotides depending upon the target sequence whose expression is to be reduced and (ii) further wherein nucleotides 184 to 203 of SEQ ID NO:14 are replaced by a second variable nucleotide subsequence ranging in size from about 19 to about 30 nucleotides, said second variable nucleotide subsequence being capable of hybridizing to the first variable subsequence of the precursor miRNA. 
     
     
         20 . A recombinant construct comprising the isolated nucleic acid fragment of  claim 19  operably linked to at least one regulatory sequence. 
     
     
         21 . A plant cell comprising the recombinant construct of  claim 20 . 
     
     
         22 . The plant cell of  claim 21  wherein the plant cell is a monocot plant cell. 
     
     
         23 . A method for reducing expression of a target sequence in a plant cell, said method comprising:
 (a) transforming at least one plant cell with a nucleic acid construct comprising a comprising a precursor miRNA said precursor miRNA corresponding substantially to the deoxyribonucleotide sequence set forth in SEQ ID NO:14 (i) wherein nucleotides 110 to 130 of SEQ ID NO:14 are replaced by a first variable nucleotide subsequence ranging in size from about 19 to about 30 nucleotides depending upon the target sequence whose expression is to be reduced and (ii) further wherein nucleotides 184 to 203 of SEQ ID NO:14 are replaced by a second variable nucleotide subsequence ranging in size from about 19 to about 30 nucleotides, said second variable nucleotide subsequence being capable of hybridizing to the first variable subsequence of the precursor miRNA; and   (b) selecting those transformed plant cell(s) whose level of expression of the target sequence is reduced when compared to the level of expression of the target sequence in a wild type plant cell.   
     
     
         24 . The method of  claim 23  wherein the plant cell is a monocot plant cell. 
     
     
         25 . An isolated nucleic acid fragment comprising a precursor miRNA said precursor miRNA corresponding substantially to the deoxyribonucleotide sequence set forth in SEQ ID NO:15 (i) wherein nucleotides 83 to 103 of SEQ ID NO:15 are replaced by a first variable nucleotide subsequence ranging in size from about 19 to about 30 nucleotides depending upon the target sequence whose expression is to be reduced and (ii) further wherein nucleotides 172 to 192 of SEQ ID NO:15 are replaced by a second variable nucleotide subsequence ranging in size from about 19 to about 30 nucleotides, said second variable nucleotide subsequence being capable of hybridizing to the first variable subsequence of the precursor miRNA. 
     
     
         26 . A recombinant construct comprising the isolated nucleic acid fragment of  claim 25  operably linked to at least one regulatory sequence. 
     
     
         27 . A plant cell comprising the recombinant construct of  claim 26 . 
     
     
         28 . The plant cell of  claim 27  wherein the plant cell is a monocot plant cell. 
     
     
         29 . A method for reducing expression of a target sequence in a plant cell, said method comprising:
 (a) transforming at least one plant cell with a nucleic acid construct comprising a comprising a precursor miRNA said precursor miRNA corresponding substantially to the deoxyribonucleotide sequence set forth in SEQ ID NO:15 (i) wherein nucleotides 83 to 103 of SEQ ID NO:15 are replaced by a first variable nucleotide subsequence ranging in size from about 19 to about 30 nucleotides depending upon the target sequence whose expression is to be reduced and (ii) further wherein nucleotides 172 to 192 of SEQ ID NO:15 are replaced by a second variable nucleotide subsequence ranging in size from about 19 to about 30 nucleotides, said second variable nucleotide subsequence being capable of hybridizing to the first variable subsequence of the precursor miRNA; and   (b) selecting those transformed plant cell(s) whose level of expression of the target sequence is reduced when compared to the level of expression of the target sequence in a wild type plant cell.   
     
     
         30 . The method of  claim 29  wherein the plant cell is a monocot plant cell.

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