US2011091975A1PendingUtilityA1

Down-regulation of gene expression using artificial micrornas

66
Assignee: DU PONTPriority: Dec 18, 2007Filed: Dec 17, 2008Published: Apr 21, 2011
Est. expiryDec 18, 2027(~1.4 yrs left)· nominal 20-yr term from priority
Inventors:Brian Mcgonigle
C12N 15/8242C12N 15/8216C12N 15/8218C12N 15/8261Y02A40/146
66
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Claims

Abstract

Isolated nucleic acid fragments comprising precursor miRNAs, 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:13 (i) wherein nucleotides 513 to 533 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 384 to 407 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. 
     
     
         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: 13 (i) wherein nucleotides 513 to 533 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 384 to 407 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.   
     
     
         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:14 (i) wherein nucleotides 275 to 295 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 121 to 141 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. 
     
     
         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:14 (i) wherein nucleotides 275 to 295 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 121 to 141 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 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:15 (i) wherein nucleotides 262 to 282 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 155 to 175 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. 
     
     
         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 deoxynucleotide sequence set forth in SEQ ID NO:15 (i) wherein nucleotides 262 to 282 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 155 to 175 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 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:16 (i) wherein nucleotides 249 to 269 of SEQ ID NO:16 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 316 to 336 of SEQ ID NO:16 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 deoxynucleotide sequence set forth in SEQ ID NO:16 (i) wherein nucleotides 249 to 269 of SEQ ID NO:16 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 316 to 336 of SEQ ID NO:16 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:17 (i) wherein nucleotides 196 to 216 of SEQ ID NO:17 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 262 to 282 of SEQ ID NO:17 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:17 (i) wherein nucleotides 196 to 216 of SEQ ID NO:17 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 262 to 282 of SEQ ID NO:17 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. 
     
     
         31 . An isolated nucleic acid fragment comprising a precursor miRNA said precursor miRNA corresponding substantially to the deoxyribonucleotide sequence set forth in SEQ ID NO:18 (i) wherein nucleotides 127 to 147 of SEQ ID NO:18 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 53 to 73 of SEQ ID NO:18 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. 
     
     
         32 . A recombinant construct comprising the isolated nucleic acid fragment of  claim 31  operably linked to at least one regulatory sequence. 
     
     
         33 . A plant cell comprising the recombinant construct of  claim 32 . 
     
     
         34 . The plant cell of  claim 33  wherein the plant cell is a monocot plant cell. 
     
     
         35 . 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.

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