Recombinant DNA Constructs and Methods for Controlling Gene Expression

A method of directing in-phase production of siRNAs in a trans-acting siRNA suppression system in a plant cell, comprising providing to a plant cell a 22-nucleotide engineered mature miRNA,

• wherein said 22-nucleotide engineered mature miRNA is produced from an engineered miRNA precursor comprising a nucleotide sequence derived from the sequence of a native miR167 precursor selected from the group consisting of SEQ ID NOs:6, 7, 8, and 9 by:
• replacing one or more nucleotides at the location of said mature miRNA in said native miR167 precursor with nucleotides complementary to a portion of a target sequence, and
• maintaining the position and number of mismatches in the stem portion of the fold-back structure of said native miR167 precursor by altering, as needed, one or more nucleotides at the location of the miRNA*, and
• leaving unchanged the nucleotides other than those at positions at the location of the mature miRNA and miRNA*;
• whereby said 22-nucleotide engineered mature miRNA is provided to said plant cell and in-phase production of siRNAs in a trans-acting siRNA suppression system is initiated.

A method of designing a recombinant miRNA precursor to produce a 22-nucleotide RNA molecule in a plant cell,

• wherein the nucleotide sequence of said recombinant miRNA precursor is obtained by:
• replacing one or more nucleotides at the location of the mature miRNA in the sequence of a native miR167 precursor having a sequence selected from the group consisting of SEQ ID NOs:6, 7, 8, and 9 with nucleotides complementary to a portion of a target sequence,
• maintaining the position and number of mismatches in the stem portion of the fold-back structure of said native miR167 precursor by altering, as needed, one or more nucleotides at the location of the miRNA*, and
• leaving unchanged the nucleotides other than those at positions at the location of the mature miRNA and miRNA*;
• and
• wherein said recombinant miRNA precursor produces a 22-nucleotide RNA molecule when expressed in a plant cell.