US20150247154A1 - Recombinant DNA Constructs and Methods for Modulating Expression of a Target Gene

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Abstract

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US20150247154A1

Publication number: US20150247154A1
Application number: US14/691,281
Authority: US
Inventors: Sergey I. Ivashuta; Barbara E. Wiggins; Yuanji Zhang
Original assignee: Monsanto Technology LLC
Current assignee: Monsanto Technology LLC
Priority date: 2008-07-01
Filing date: 2015-04-20
Publication date: 2015-09-03
Also published as: ES2563485T3; CN102149821A; US9040774B2; EP2924118A1; CN106995819A; CN106995819B; AR114027A2; AU2016203359B2; AU2018203835B2; AU2016203359A1; CA2729713C; EP2304030B1; BRPI0913657A2; UY32066A; AU2009267007A1; WO2010002984A1; AU2009267007B2; US20110296555A1; CA2729713A1; AU2018203835A1

This invention provides recombinant DNA constructs and methods for manipulating expression of a target gene that is regulated by a small RNA, by interfering with the binding of the small RNA to its target gene. More specifically, this invention discloses recombinant DNA constructs encoding cleavage blockers, 5-modified cleavage blockers, and translational inhibitors useful for modulating expression of a target gene and methods for their use. Further disclosed are miRNA targets useful for designing recombinant DNA constructs including miRNA-unresponsive transgenes, miRNA decoys, cleavage blockers, 5-modified cleavage blockers, and translational inhibitors, as well as methods for their use, and transgenic eukaryotic cells and organisms containing such constructs.

CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION OF SEQUENCE LISTINGS

This application is a continuation of U.S. National Stage application Ser. No. 12/999,777 filed on Jan. 5, 2011, which claims the benefit of priority of U.S. Provisional Patent Application No. 61/077,244 filed on Jul. 1, 2008, which is incorporated by reference in its entirety herein. A computer readable form of the sequence listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The sequence listing is contained in the file named “P34155US02_SEQLIST.txt”, which is 2,674,688 bytes (measured in operating system MS windows) and was created on Apr. 20, 2015.

FIELD OF THE INVENTION

Disclosed herein are recombinant DNA constructs with DNA that undergoes processing to an RNA providing RNase III cleavage resistance to a target gene transcript. Such RNAs serve as cleavage blockers and translational inhibitors useful for modulating expression of a target gene. Further disclosed are miRNA recognition site sequences and their use in designing recombinant DNA constructs including miRNA-unresponsive transgenes, miRNA decoys, cleavage blockers, and translational inhibitors. Also disclosed are non-natural transgenic plant cells, plants, and seeds containing in their genome a recombinant DNA construct of this invention. Further disclosed are methods of modulating expression of a target gene using recombinant DNA constructs of this invention.

BACKGROUND OF THE INVENTION

Several cellular pathways involved in RNA-mediated gene suppression have been described, each distinguished by a characteristic pathway and specific components. Generally, RNA-mediated gene suppression involves a double-stranded RNA (dsRNA) intermediate that is formed intramolecularly within a single RNA molecule or intermolecularly between two RNA molecules. This longer dsRNA intermediate is processed by a ribonuclease of the RNase III family (Dicer or Dicer-like ribonuclease) to one or more small double-stranded RNAs, one strand of which is incorporated by the ribonuclease into the RNA-induced silencing complex (“RISC”). Which strand is incorporated into RISC is believed to depend on certain thermodynamic properties of the double-stranded small RNA, such as those described by Schwarz et al. (2003) Cell, 115:199-208, and Khvorova et al. (2003) Cell, 115:209-216.
The siRNA pathway involves the non-phased cleavage of a longer double-stranded RNA intermediate to small interfering RNAs (“siRNAs”). The size of siRNAs is believed to range from about 19 to about 25 base pairs, but common classes of siRNAs include those containing 21 base pairs or 24 base pairs. See, for example, Hamilton et al. (2002) EMBO J., 21:4671-4679.
The microRNA pathway involves microRNAs (“miRNAs”), non-protein coding RNAs generally of between about 19 to about 25 nucleotides (commonly about 20-24 nucleotides in plants) that guide cleavage in trans of target transcripts, negatively regulating the expression of genes involved in various regulation and development pathways; see Ambros et al. (2003) RNA, 9:277-279. Naturally occurring miRNAs are derived from a primary transcript (“pri-miRNA”) that is naturally processed to a shorter transcript (“pre-miRNA”) which itself is further processed to the mature miRNA. For a recent review of miRNA biogenesis in both plants and animals, see Kim (2005) Nature Rev. Mol. Cell Biol., 6:376-385. Gene regulation of biological pathways by miRNAs can occur at multiple levels and in different ways, including regulation of single or multiple genes, regulation of transcriptional regulators, and regulation of alternative splicing; see Makeyev & Maniatis (2008) Science, 319:1789-1790. Various utilities of miRNAs, their precursors, their recognition sites, and their promoters are described in detail in co-assigned U.S. Patent Application Publication 2006/0200878 A1, specifically incorporated by reference herein, which include: (1) the expression of a native miRNA or miRNA precursor sequence to suppress a target gene; (2) the expression of an engineered (non-native) miRNA or miRNA precursor sequence to suppress a target gene; (3) expression of a transgene with a miRNA recognition site, wherein the transgene is suppressed when the corresponding mature miRNA is expressed, either endogenously or transgenically; and (4) expression of a transgene driven by a miRNA promoter.
In the trans-acting siRNA (“ta-siRNA”) pathway, miRNAs serve to guide in-phase processing of siRNA primary transcripts in a process that requires an RNA-dependent RNA polymerase for production of a double-stranded RNA precursor; trans-acting siRNAs are defined by lack of secondary structure, a miRNA target site that initiates production of double-stranded RNA, requirements of DCL4 and an RNA-dependent RNA polymerase (RDR6), and production of multiple perfectly phased ˜21-nt small RNAs with perfectly matched duplexes with 2-nucleotide 3′ overhangs (see Allen et al. (2005) Cell, 121:207-221; Vazquez et al. (2004) Mol. Cell, 16:69-79).
The phased small RNA (“phased sRNA”) pathway (see PCT patent application PCT/US2007/019283, published as WO 2008/027592) is based on an endogenous locus termed a “phased small RNA locus”, which transcribes to an RNA transcript forming a single foldback structure that is cleaved in phase in vivo into multiple small double-stranded RNAs (termed “phased small RNAs”) capable of suppressing a target gene. In contrast to siRNAs, a phased small RNA transcript is cleaved in phase. In contrast to miRNAs, a phased small RNA transcript is cleaved by DCL4 or a DCL4-like orthologous ribonuclease (not DCL1) to multiple abundant small RNAs capable of silencing a target gene. In contrast to the ta-siRNA pathway, the phased small RNA locus transcribes to an RNA transcript that forms hybridized RNA independently of an RNA-dependent RNA polymerase and without a miRNA target site that initiates production of double-stranded RNA.
Gene suppression mediated by small RNAs processed from natural antisense transcripts has been reported in at least two pathways. In the natural antisense transcript small interfering RNA (“nat-siRNA”) pathway (Borsani et al. (2005) Cell, 123:1279-1291), siRNAs are generated by DCL1 cleavage of a double-stranded RNA formed between the antisense transcripts of a pair of genes (cis-antisense gene pairs). A similar natural anti-sense transcript microRNA (“nat-miRNA”) pathway (Lu et al. (2008) Proc. Natl. Acad. Sci. USA, 105: 4951-4956) has also been reported. In metazoan animals, small RNAs termed Piwi-interacting RNAs (“piRNAs”) have been reported to also have gene-silencing activity (Lau et al. (2006) Science, 313:363-367; O'Donnell & Boeke (2007) Cell, 129:37-44).

SUMMARY OF THE INVENTION

In one aspect, this invention provides a recombinant DNA construct including DNA that undergoes processing to an RNA including single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to the transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of the hybridized segment.
Another aspect of this invention provides a recombinant DNA construct encoding a “cleavage blocker” for inhibiting double-stranded RNA-mediated suppression of the at least one target gene, thereby increasing expression of the target gene (relative to expression in the absence of the cleavage blocker). One embodiment is a recombinant DNA construct including DNA that undergoes processing to an RNA including single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to the transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of the hybridized segment, wherein the binding of the single-stranded RNA to the transcript (and the resultant formation of the hybridized segment) inhibits double-stranded RNA-mediated suppression of the at least one target gene.
Another aspect of this invention provides a recombinant DNA construct encoding a a “5′-modified cleavage blocker”. One embodiment includes a recombinant DNA construct including DNA that undergoes processing to an RNA including single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to the transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of the hybridized segment, wherein the binding of the single-stranded RNA to the transcript (and the resultant formation of the hybridized segment) inhibits double-stranded RNA-mediated suppression of the at least one target gene, wherein the cleavage by an RNase III ribonuclease is mediated by binding of a mature miRNA, the binding is at a miRNA recognition site (that is recognized by the mature miRNA) in the transcript, the cleavage of the transcript occurs at the miRNA recognition site, and the hybridized segment is formed at least partially within the miRNA recognition site, and the hybridized segment includes an A, G, or C (but not a U) at a position corresponding to the 5′ terminus of the mature miRNA that natively binds to the recognition site, but does not require mismatches between the single-stranded RNA and the miRNA recognition site at positions of the miRNA recognition site corresponding to positions 9, 10, or 11 (in 3′ to 5′ direction) of the mature miRNA, or insertions at a position in the single-stranded RNA at positions of the miRNA recognition site corresponding to positions 10 or 11 (in 3′ to 5′ direction) of the mature miRNA.
Another aspect of this invention provides a recombinant DNA construct encoding a “translational inhibitor” for inhibiting translation of the transcript, thereby decreasing expression of the target gene (relative to expression in the absence of expression of the construct). One embodiment is a recombinant DNA construct including DNA that undergoes processing to an RNA including single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to the transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of the hybridized segment, wherein the binding of the single-stranded RNA to the transcript (and the formation of the hybridized segment) inhibits translation of the transcript.
Other aspects of this invention provide methods for modulating expression of miRNA target genes from plant species. Embodiments of this invention include methods to increase or improve yield of crop plants by expressing in such plants recombinant DNA constructs of this invention, for example, recombinant DNA constructs encoding a native miRNA precursor sequence or an artificial precursor sequence, or recombinant DNA constructs encoding a cleavage blocker or translational inhibitor or decoy.
Further aspects of this invention provide non-natural transgenic plant cells having in their genome a recombinant DNA construct of this invention. Also provided are a non-natural transgenic plant containing the transgenic plant cell of this invention, a non-natural transgenic plant grown from the transgenic plant cell of this invention, and non-natural transgenic seed produced by the transgenic plants, as well as commodity products produced from a non-natural transgenic plant cell, plant, or seed of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the predicted fold-back structures of the native miRNA miRMON1 precursor (Panel A), the synthetic miRNA miRGL1 precursor (Panel B), the synthetic cleavage blocker miRGL1-CB (Panel C), and the synthetic 5′-modified miRGL1 cleavage blocker (Panel D), as well as an alignment (Panel E) of the miRNA recognition site in the target gene GL1, the mature miRGL1, the mature miRGL1-CB, and the artificial GL1 recognition site in the miRGL1-sensor, as described in Examples 1 and 2.

FIG. 2 depicts a maize transformation base vector (pMON93039, SEQ ID NO: 2065), as described in Example 5.

FIG. 3 depicts a soybean or cotton transformation base vector (pMON82053, SEQ ID NO: 2066), as described in Example 5.

FIG. 3 depicts a cotton transformation base vector (pMON99053, SEQ ID NO: 2067), as described in Example 5.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise stated, nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5′ to 3′ direction. Nucleic acid sequences may be provided as DNA or as RNA, as specified; disclosure of one necessarily defines the other, as is known to one of ordinary skill in the art. The term “miRNA precursor”, as used herein, refers to an RNA transcript that is naturally processed to produce a mature miRNA. Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term.
Recombinant DNA Constructs that are Processed to RNA Providing Rnase III Resistance to a Target Gene Transcript
In one aspect, this invention provides a recombinant DNA construct including DNA that undergoes processing to an RNA including single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to the transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of the hybridized segment. The recombinant DNA construct is made by techniques known in the art, such as those described under the heading “Making and Using Recombinant DNA Constructs” and illustrated in the working Examples. The recombinant DNA construct is particularly useful for making transgenic plant cells, transgenic plants, and transgenic seeds as discussed below under “Making and Using Transgenic Plant Cells and Transgenic Plants”. This invention therefore includes embodiments wherein the recombinant DNA construct is located within a vector for transforming a plant cell (such as within a plasmid or viral vector), or on a biolistic particle for transforming a plant cell, or within a chromosome or plastid of a non-natural transgenic plant cell, or within a non-natural transgenic cell, non-natural transgenic plant tissue, non-natural transgenic plant seed, non-natural transgenic pollen grain, or a non-natural transgenic or partially transgenic plant. Further included are embodiments wherein the recombinant DNA construct is in a commodity product produced from a non-natural transgenic cell, non-natural transgenic plant tissue, non-natural transgenic plant seed, non-natural transgenic pollen grain, or a non-natural transgenic or partially transgenic plant of this invention; such commodity products include, but are not limited to harvested leaves, roots, shoots, tubers, stems, fruits, seeds, or other parts of a plant, meals, oils, extracts, fermentation or digestion products, crushed or whole grains or seeds of a plant, or any food or non-food product including such commodity products produced from a transgenic plant cell, plant, or seed of this invention.
The processing of the DNA includes transcription of the DNA to a primary RNA transcript, which may undergo one or more additional natural processing steps that result in the single-stranded RNA that binds to the transcript of at least one target gene. In one embodiment, the processing of the DNA includes transcription of the DNA to an RNA intermediate including one or more double-stranded RNA stems; the double-stranded RNA stem or stems is further processed to single-stranded RNA. A final product of the DNA processing is the RNA including single-stranded RNA that binds to the transcript of at least one target gene.
For example, the recombinant DNA construct includes DNA that is transcribed to a primary transcript with a sequence derived from a native pri-miRNA or pre-miRNA sequence that forms secondary structure including one or more double-stranded stems, followed by processing of the primary transcript to a shorter, at least partially double-stranded intermediate (similar to a pre-miRNA) which is then cleaved by an RNase III ribonuclease (ribonuclease III, e.g., Drosha or DCL1 or a DCL1-like orthologous ribonuclease) to a pair of single-stranded RNAs (similar to a miRNA and a miRNA*pair). In another example, the recombinant DNA construct includes DNA that is transcribed to a primary transcript that forms secondary structure including one or more double-stranded stems, followed by cleavage of the double-stranded RNA stem(s) by an RNase III ribonuclease to one or more pairs of single-stranded small RNAs (similar to an siRNA duplex). In another example, the recombinant DNA construct includes DNA that is transcribed to a primary transcript that includes one or more spliceable introns that are removed by intronic processing. In yet another example, the recombinant DNA construct includes DNA that is transcribed to a primary transcript including one or more self-cleaving ribozymes (see, e.g., Tang & Breaker (2000) Proc. Natl. Acad. Sci. USA, 97:5784-5789); removal of the ribozyme(s) results in the RNA including single-stranded RNA that binds to the transcript of at least one target gene.
The RNA resulting from processing of the DNA includes at least single-stranded RNA that binds to the transcript of at least one target gene. In one embodiment, the RNA resulting from processing of the DNA consists of one single-stranded RNA molecule that binds to the transcript of one target gene. In another embodiment, the RNA resulting from processing of the DNA consists of one single-stranded RNA molecule that binds to the transcripts of multiple target genes. In another embodiment, the RNA resulting from processing of the DNA consists of multiple molecules of single-stranded RNA that bind to the transcript of at least one target gene; this can result, e.g., from processing of a primary RNA transcript having multiple segments, each including single-stranded RNA that binds to the transcript of at least one target gene, for example, where the multiple segments (which can have the same or different sequence) are separated by self-cleaving ribozymes and cleavage of the ribozymes yields the multiple single-stranded RNAs. In another embodiment, the RNA resulting from processing of the DNA includes single-stranded RNA that binds to the transcript of at least one target gene, as well as additional RNA elements (which may be single-stranded or double-stranded or both), such as, but not limited to, an RNA aptamer, an RNA riboswitch, a ribozyme, site-specific recombinase recognition sites, or an RNA sequence that serves to regulate transcription of the single-stranded RNA that binds to the transcript of at least one target gene.
In various embodiments, the at least one target gene includes: coding sequence, non-coding sequence, or both coding and non-coding sequences; a single target gene or multiple target genes (for example, multiple alleles of a target gene, or multiple different target genes); or one or more of (a) an endogenous gene of a eukaryote, (b) a transgene of a transgenic plant, (c) an endogenous gene of a pest or pathogen of a plant, and (d) an endogenous gene of a prokaryotic or eukaryotic symbiont associated with a pest or pathogen of a plant. Target genes that can be regulated by a recombinant DNA construct of this invention are described in detail below under the heading “Target Genes”.
The single-stranded RNA binds to the transcript of at least one target gene to form a hybridized segment of at least partially (in some cases perfectly) double-stranded RNA. In some embodiments the percent complementarity between the single-stranded RNA and the transcript of at least one target gene is 100%. However, it is clear that Watson-Crick base-pairing need not be complete between the single-stranded RNA and the transcript of at least one target gene, but is at least sufficient so that under physiological conditions a stably hybridized segment of at least partially double-stranded RNA is formed between the two.
The hybridized segment of double-stranded RNA imparts to the transcript resistance to cleavage by an RNase III ribonuclease (for example, Drosha or Dicer or Dicer-like proteins, including, but not limited to, DCL1, DCL2, DCL3, DCL4, DCL1-like, DCL2-like, DCL3-like, or DCL4-like proteins) within or in the vicinity of the hybridized segment. In many instances, the resistance imparted is resistance to cleavage by an RNase III ribonuclease within the hybridized segment. For example, where the single-stranded RNA binds to the transcript of at least one target gene at a miRNA recognition site in the transcript recognized and bound by an endogenous miRNA, such that the hybridized segment encompasses the miRNA recognition site, the hybridized segment of double-stranded RNA imparts to the transcript resistance to cleavage by an RNase III ribonuclease at the miRNA recognition site (i.e., within the hybridized segment). In other instances, the resistance imparted is resistance to cleavage by an RNase III ribonuclease in the vicinity of the hybridized segment. For example, where the single-stranded RNA binds to the transcript of at least one target gene immediately or closely adjacent to a miRNA recognition site in the transcript recognized and bound by an endogenous miRNA, such that the hybridized segment does not encompass the miRNA recognition site but is sufficiently close to prevent binding by the endogenous miRNA to the transcript, the hybridized segment of double-stranded RNA imparts to the transcript resistance to cleavage by an RNase III ribonuclease at the miRNA recognition site (i.e., in the vicinity of, but not within, the hybridized segment).
The length of the single-stranded RNA is not necessarily equal to the length of the hybridized segment, since not all of the single-stranded RNA necessarily binds to the transcript of at least one target gene. In some embodiments, the length of the single-stranded RNA is about equal to, or exactly equal to, the length of the hybridized segment. In other embodiments, the length of the single-stranded RNA is greater than the length of the hybridized segment. Expressed in terms of numbers of contiguous nucleotides, the length of the single-stranded RNA is generally from between about 10 nucleotides to about 500 nucleotides, or from between about 20 nucleotides to about 500 nucleotides, or from between about 20 nucleotides to about 100 nucleotides, for example, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 120, about 140, about 160, about 180, about 200, about 240, about 280, about 320, about 360, about 400, or about 500 nucleotides. Expressed in terms of numbers of contiguous nucleotides (and recognizing that the hybridized segment can include nucleotides that are not base-paired), the length of the hybridized segment is generally from between about 10 nucleotides to about 100 nucleotides, or from between about 10 nucleotides to about 24 nucleotides, or from between about 20 nucleotides to about 100 nucleotides, or from between about 26 nucleotides to about 100 nucleotides, although it can be greater than about 100 nucleotides, and in some preferred embodiments it is preferably smaller than 100 nucleotides (such as in some embodiments of translational inhibitors, described below under the heading “Translational Inhibitors”). In preferred embodiments, the length of the hybridized segment is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, or about 100 nucleotides. In one particularly preferred embodiment, the length of the hybridized segment is between about 10 to about 24 nucleotides, e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides.
In many embodiments, the recombinant DNA construct of this invention includes other DNA elements in addition to the DNA that undergoes processing to an RNA including single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to the transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of the hybridized segment. These additional DNA elements include at least one element selected from the group consisting of:

- (a) a promoter functional in a eukaryotic (plant, animal, fungus, or protist) cell, such as any of the promoters described under the heading “Promoters”;
- (b) a Pol III promoter (see “Promoters”, below) operably linked to the DNA that undergoes processing to an RNA including single-stranded RNA;
- (c) DNA that is processed to an RNA aptamer (as described under the heading “Aptamers”)
- (d) a transgene transcription unit (as described under the heading “Transgene Transcription Units”);
- (e) DNA encoding a spliceable intron (as described under the heading “Introns”);
- (f) DNA encoding a self-splicing ribozyme (as described under the heading “Ribozymes”);
- (g) DNA encoding a site-specific recombinase recognition site (as described under the heading “Recombinases”);
- (h) DNA encoding a gene suppression element (as described under the heading “Gene Suppression Elements”); and
- (i) DNA encoding a transcription regulatory element (as described under the heading “Transcription Regulatory Elements”).

The recombinant DNA construct of this invention is particularly useful for providing an RNA that functions as a “cleavage blocker” or a “translational inhibitor”, according to the RNA's interaction with the transcript of the target gene(s). Cleavage blockers and translational inhibitors are described in more detail below.

Cleavage Blockers

One aspect of this invention is a recombinant DNA construct including DNA that undergoes processing to an RNA including single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to the transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of the hybridized segment, wherein the binding of the single-stranded RNA to the transcript (and the resultant formation of the hybridized segment) inhibits double-stranded RNA-mediated suppression of the at least one target gene. In this context, the term “cleavage blocker” generally refers to the RNA including single-stranded RNA that binds to the transcript of at least one target gene, and more specifically refers to the portion(s) of the single-stranded RNA that forms a hybridized segment of at least partially double-stranded RNA with the transcript. Cleavage blockers inhibit double-stranded RNA-mediated suppression of the at least one target gene, thereby increasing expression of the target gene (relative to expression in the absence of the cleavage blocker).
Generally, the cleavage by an RNase III ribonuclease is mediated by binding of a small RNA (most preferably a small RNA that is associated with a silencing complex) to the transcript. In preferred embodiments, the small RNA is selected from the group consisting of a miRNA, an siRNA, a trans-acting siRNA, a phased small RNA, a natural antisense transcript siRNA, and a natural antisense transcript miRNA; however, the small RNA can be any small RNA associated with a silencing complex such as RISC or an Argonaute or Argonaute-like protein. In some embodiments, the small RNA is an endogenous small RNA (e.g., an endogenous miRNA); in other embodiments, the small RNA is a transgenic small RNA (e.g., a transgenically expressed engineered miRNA).
In various embodiments, the length of the hybridized segment includes between about 10 base pairs to about 100 base pairs, although it can be greater than about 100 base pairs. In preferred embodiments (and recognizing that the hybridized segment can include nucleotides that are not base-paired), the length of the hybridized segment includes between about 10 base pairs to about 100 base pairs, such as from between about 10 to about 20, or between about 10 to about 24, or between about 10 to about 30, or between about 10 to about 40, or between about 10 to about 50, or between about 18 to about 28, or between about 18 to about 25, or between about 18 to about 24, or between about 20 to about 30, or between about 20 to about 40, or between about 20 to about 50 base pairs. In preferred embodiments, the length of the hybridized segment is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, about 30, about 34, about 40, about 45, about 50, about 60, about 70, about 80, about 90, or about 100 base pairs, wherein the hybridized segment optionally includes additional nucleotides that are not base-paired and that are not counted in the length of the hybridized segment when this is expressed in terms of base pairs. In particularly preferred embodiments, the length of the hybridized segment is between about 18 to about 28 base pairs (that is, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 base pairs), or between about 10 to about 24 base pairs (that is, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 base pairs), or between about 18 to about 24 base pairs (that is, 18, 19, 20, 21, 22, 23, or 24 base pairs) wherein the hybridized segment optionally includes additional nucleotides that are not base-paired and that are not counted in the length of the hybridized segment when this is expressed in terms of base pairs. One of skill in the art is able to determine what number of unpaired nucleotides is acceptable for a given hybridized segment, i.e., that will still allow formation hybridized segment that is stable under physiological conditions and is resistant to RNase III ribonuclease cleavage.
In some instances, the hybridized segment is completely base-paired, that is, contains a contiguous ribonucleotide sequence that is the same length as, and is perfectly complementary to, a contiguous ribonucleotide sequence of the target gene transcript. In particularly preferred embodiments, however, the hybridized segment is not completely base-paired, and includes at least one mismatch or at least one insertion in the hybridized segment at a position that results in inhibiting cleavage of the transcript by the RNase III ribonuclease.
One aspect of this invention provides a “miRNA cleavage blocker”. One preferred embodiment is a recombinant DNA construct including DNA that undergoes processing to an RNA including single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to the transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of the hybridized segment, wherein the binding of the single-stranded RNA to the transcript (and the resultant formation of the hybridized segment) inhibits double-stranded RNA-mediated suppression of the at least one target gene, wherein the cleavage by an RNase III ribonuclease is mediated by binding of a mature miRNA, the binding is at a miRNA recognition site (that is recognized by the mature miRNA) in the transcript, the cleavage of the transcript occurs at the miRNA recognition site, and the hybridized segment is formed at least partially within the miRNA recognition site. In this embodiment, the recombinant DNA construct yields a miRNA cleavage blocker RNA that binds to (or in the vicinity of) a miRNA recognition site in a target gene transcript, forming a hybridized segment that is itself resistant to RNase III ribonuclease cleavage (or that prevents RNase III ribonuclease cleavage of the transcript in the vicinity of the hybridized segment), thus preventing the mature miRNA that normally recognizes the miRNA recognition site from binding to the miRNA recognition site and mediating RNase III ribonuclease cleavage of the target gene transcript. In particularly preferred embodiments, the hybridized segment includes: (a) at least one mismatch between the single-stranded RNA and the miRNA recognition site at positions of the miRNA recognition site corresponding to positions 9, 10, or 11 (in 3′ to 5′ direction) of the mature miRNA, or (b) at least one insertion at a position in the single-stranded RNA at positions of the miRNA recognition site corresponding to positions 10 or 11 (in 3′ to 5′ direction) of the mature miRNA. In some preferred embodiments, the single-stranded RNA that binds to the transcript of at least one target gene has a nucleotide sequence to allow a stably hybridized segment to be formed between it and the target gene transcript, but that inhibits binding of an Argonaute or Argonaute-like protein to the hybridized segment, as described by Mi et al. (2008) Cell, 133:1-12; for example, the single-stranded RNA has a nucleotide sequence that includes an A, G, or C (but not a U) at a position corresponding to the 5′ terminus of the mature miRNA that natively binds to the recognition site. Most preferably, the binding of a miRNA cleavage blocker to the target gene transcript results in inhibition of miRNA-mediated suppression of the at least one target gene, thereby increasing expression of the target gene (relative to expression in the absence of the miRNA cleavage blocker).
Another aspect of this invention includes a “5′-modified cleavage blocker”. A preferred embodiment includes a recombinant DNA construct including DNA that undergoes processing to an RNA including single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to the transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of the hybridized segment, wherein the binding of the single-stranded RNA to the transcript (and the resultant formation of the hybridized segment) inhibits double-stranded RNA-mediated suppression of the at least one target gene, wherein the cleavage by an RNase III ribonuclease is mediated by binding of a mature miRNA, the binding is at a miRNA recognition site (that is recognized by the mature miRNA) in the transcript, the cleavage of the transcript occurs at the miRNA recognition site, and the hybridized segment is formed at least partially within the miRNA recognition site, and the hybridized segment includes an A, G, or C (but not a U) at a position corresponding to the 5′ terminus of the mature miRNA that natively binds to the recognition site, but does not include mismatches between the single-stranded RNA and the miRNA recognition site at positions of the miRNA recognition site corresponding to positions 9, 10, or 11 (in 3′ to 5′ direction) of the mature miRNA, or insertions at a position in the single-stranded RNA at positions of the miRNA recognition site corresponding to positions 10 or 11 (in 3′ to 5′ direction) of the mature miRNA. Binding of such a 5′-modified cleavage blocker to the target gene transcript results in inhibition of miRNA-mediated suppression of the at least one target gene, thereby increasing expression of the target gene (relative to expression in the absence of the cleavage blocker).
One of ordinary skill in the art easily recognizes that various aspects of this invention include analogous recombinant DNA constructs that are processed to provide RNA including single-stranded RNA that serve as an “siRNA cleavage blocker”, a “trans-acting siRNA cleavage blocker”, a “phased small RNA cleavage blocker”, a “natural antisense transcript siRNA cleavage blocker”, or a “natural antisense transcript miRNA cleavage blocker” (or, in general terms, a “small RNA cleavage blocker”), according to whether the RNase III ribonuclease cleavage that is inhibited is mediated by, respectively, an siRNA, a trans-acting siRNA, a phased small RNA, a natural antisense transcript siRNA, or a natural antisense transcript miRNA (or, in general terms, any small RNA associated with a silencing complex such as RISC or an Argonaute or Argonaute-like protein). In these cases, the formation of the RNase III ribonuclease cleavage-resistant hybridized segment generally prevents the respective small RNA from binding to the target gene transcript and mediating RNase III ribonuclease cleavage of the transcript. Most preferably, the binding of such a small RNA cleavage blocker to the target gene transcript results in inhibition of double-stranded RNA-mediated suppression of the at least one target gene, thereby increasing expression of the target gene (relative to expression in the absence of the small RNA cleavage blocker). One of ordinary skill in the art is able to devise a nucleotide sequence for such an RNA including single-stranded RNA that, upon binding to the transcript of at least one target gene, forms a hybridized segment that is stable under physiological conditions and is resistant to RNase III ribonuclease cleavage, for example, (1) by selecting a nucleotide sequence that inhibits binding of an Argonaute or Argonaute-like protein to the hybridized segment, as described by Mi et al. (2008) Cell, doi:10.1016/j.cell.2008.02.034; (2) by selecting a nucleotide sequence such that the difference in free energy (“ΔΔG”, see Khvorova et al. (2003) Cell, 115, 209-216) between the portions of the single-stranded RNA and the target gene transcript that form the hybridized segment inhibit association with a silencing complex such as RISC or an Argonaute or Argonaute-like protein; or (3) by selecting a nucleotide sequence such that mismatches or insertions at a potential small RNA-mediated RNase III ribonuclease cleavage site prevents cleavage of the transcript. Knowledge of the target gene itself is not required, merely the sequence of the mature miRNA sequence or of a miRNA precursor that is processed to the mature miRNA—or, alternatively, knowledge of the miRNA recognition site sequence—in combination with the teachings of this application, in order to identify or design a cleavage blocker (or 5′-modified cleavage blocker) for inhibiting the target gene silencing effects of a given miRNA.
One approach to manipulating a miRNA-regulated pathway has been disclosed (see co-assigned U.S. patent application Ser. No. 11/974,469, published as U.S. Patent Application Publication 2009-0070898 A1, which disclosure including rules for predicting or designing a miRNA decoy sequence is specifically incorporated by reference herein) as a novel miRNA “decoy”, a sequence that can be recognized and bound by an endogenous mature miRNA resulting in base-pairing between the miRNA decoy sequence and the endogenous mature miRNA, thereby forming a stable RNA duplex that is not cleaved because of the presence of mismatches between the miRNA decoy sequence and the mature miRNA.
The Examples of this application specifically identify miRNA targets recognized by particular miRNAs. Provided with this information and Applicants' teachings, one of ordinary skill in the art would be able to design and use various additional embodiments of this invention, including a recombinant DNA construct transcribable in a plant cell, including a promoter that is functional in the plant cell and operably linked to at least one polynucleotide selected from: (a) DNA encoding a cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target; (b) DNA encoding a 5′-modified cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target; (c) DNA encoding a translational inhibitor to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target; (d) DNA encoding a decoy to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target; (e) DNA encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of at least one miRNA target, wherein a miRNA recognition site in the native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage; (f) DNA encoding a miRNA precursor which is processed into a miRNA for suppressing expression of at least one miRNA target; (g) DNA encoding double-stranded RNA which is processed into siRNAs for suppressing expression of at least one miRNA target; and (h) DNA encoding a ta-siRNA which is processed into siRNAs for suppressing expression of at least one miRNA target.

Translational Inhibitors

Another aspect of this invention is a recombinant DNA construct including DNA that undergoes processing to an RNA including single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to the transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of the hybridized segment, wherein the binding of the single-stranded RNA to the transcript (and the formation of the hybridized segment) inhibits translation of the transcript. In this context, the term “translational inhibitor” generally refers to the RNA including single-stranded RNA that binds to the transcript of at least one target gene, and more specifically refers to the portion(s) of the single-stranded RNA that forms a hybridized segment of at least partially double-stranded RNA with the transcript. Translational inhibitors inhibit translation of the transcript, thereby decreasing expression of the target gene (relative to expression in the absence of expression of the construct).
Binding of the translational inhibitor is to a location of the mRNA that is wholly or at least partially within the coding sequence or in a location such that the formation of the hybridized segment interferes with translation. In one embodiment, the binding of the single-stranded RNA to the transcript (and the formation of the hybridized segment) occurs at least partially within the 5′ untranslated region of the transcript; this embodiment is often preferred where the transcript is of a plant target gene. In another embodiment, the binding of the single-stranded RNA to the transcript (and the formation of the hybridized segment) occurs at least partially within the 3′ untranslated region of the transcript; this embodiment is preferred where the transcript is of an animal target gene. In yet another embodiment, the binding of the single-stranded RNA to the transcript occurs within or in the vicinity of the start codon or of the 5′ cap, preferably preventing translation initiation.
In preferred embodiments, the hybridized segment is resistant to cleavage by the RNase III ribonuclease. In preferred embodiments, the length of the hybridized segment includes between about 10 base pairs to about 50 base pairs, although it can be greater than about 50 base pairs. In preferred embodiments (and recognizing that the hybridized segment can include nucleotides that are not base-paired), the length of the hybridized segment includes between about 10 base pairs to about 50 base pairs, such as from between about 10 to about 20, or between about 10 to about 30, or between about 10 to about 40, or between about 10 to about 50, or between about 18 to about 28, or between about 18 to about 25, or between about 18 to about 23, or between about 20 to about 30, or between about 20 to about 40, or between about 20 to about 50 base pairs. In preferred embodiments, the length of the hybridized segment is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, about 30, about 34, about 40, about 45, or about 50 base pairs, wherein the hybridized segment optionally includes additional nucleotides that are not base-paired and that are not counted in the length of the hybridized segment when this is expressed in terms of base pairs. In particularly preferred embodiments, the length of the hybridized segment is between about 18 to about 28 base pairs, that is, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 base pairs, wherein the hybridized segment optionally includes additional nucleotides that are not base-paired and that are not counted in the length of the hybridized segment when this is expressed in terms of base pairs. One of skill in the art is able to determine what number of unpaired nucleotides is acceptable for a given hybridized segment, i.e., that will still allow formation hybridized segment that is stable under physiological conditions and is resistant to RNase III ribonuclease cleavage.
One of ordinary skill in the art is able to devise a nucleotide sequence for such an RNA including single-stranded RNA that, upon binding to the transcript of at least one target gene, forms a hybridized segment that is stable under physiological conditions and is resistant to RNase III ribonuclease cleavage, for example, (1) by selecting a nucleotide sequence that inhibits binding of an Argonaute or Argonaute-like protein to the hybridized segment, as described by Mi et al. (2008) Cell, doi:10.1016/j.cell.2008.02.034; (2) by selecting a nucleotide sequence such that the difference in free energy (“ΔΔG”, see Khvorova et al. (2003) Cell, 115, 209-216) between the portions of the single-stranded RNA and the target gene transcript that form the hybridized segment inhibit association with a silencing complex such as RISC or an Argonaute or Argonaute-like protein; or (3) by selecting a nucleotide sequence such that mismatches or insertions at a potential small RNA-mediated RNase III ribonuclease cleavage site prevents cleavage of the transcript. In a particularly preferred embodiment, the length of the hybridized segment includes between about 19 to about 50 base pairs, the hybridized segment includes smaller segments of 9 or fewer contiguous, perfectly complementary base pairs, and at least one mismatch or insertion is between each pair of the smaller segments.

Methods of Modulating Expression of a Target Gene

In another aspect, this invention provides a method of modulating expression of a target gene, including expressing in a cell a recombinant DNA construct of this invention, that is, a recombinant DNA construct including DNA that undergoes processing to an RNA including single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to the transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of the hybridized segment. Expressing in vivo in a cell a recombinant DNA construct of this invention provides an RNA that functions as a “cleavage blocker” or a “translational inhibitor”.
By “modulating expression of a target gene” is meant either: (a) increasing expression of the target gene, e.g., where the recombinant DNA construct expressed in the cell provides a cleavage blocker, or (b) decreasing expression of the target gene, e.g., where the recombinant DNA construct expressed in the cell provides a translational inhibitor. By “expressing in a cell” is meant carrying out in vivo the process of transcription, as well as any additional natural processing steps necessary to provide the RNA including single-stranded RNA that binds to the transcript of at least one target gene.
The cell in which the recombinant DNA construct is expressed is in many embodiments a eukaryotic cell (such as a plant, animal, fungus, or protist cell), and in other embodiments is a prokaryotic cell (such as a bacterial cell). The target gene that has its expression modulated by the method of this invention is not necessarily an endogenous gene of the cell in which the recombinant DNA construct is expressed. For example, this invention encompasses a method including expressing in cells of a plant a recombinant DNA construct including DNA that undergoes processing to an RNA including single-stranded RNA that binds to the transcript of at least one target gene of a pest or pathogen of the plant to form a hybridized segment of at least partially double-stranded RNA that imparts to the transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of the hybridized segment, thereby either (a) increasing expression of the target gene of the pest or pathogen, when the recombinant DNA construct provides a cleavage blocker, or (b) decreasing expression of the target gene of the pest or pathogen, when the recombinant DNA construct provides a translational inhibitor. Where the target gene is not an endogenous gene of the cell wherein the recombinant DNA construct is transcribed (such as in cells of a plant), additional processing steps may occur either in the cell where transcription occurred, or in other cells (such as in cells of a pest or pathogen of the plant).
In one embodiment of the method, the recombinant DNA construct is expressed in a cell to provide a cleavage blocker RNA. In this embodiment, the binding of the single-stranded RNA to the transcript (and the formation of the hybridized segment) inhibits double-stranded RNA-mediated suppression of the at least one target gene, thereby increasing expression of the target gene, relative to expression in the absence of expression of the construct.
In one embodiment of the method, the recombinant DNA construct is expressed in a cell to provide a translational blocker RNA. In this embodiment, the binding of the single-stranded RNA to the transcript (and the formation of the hybridized segment) inhibits translation of the transcript, thereby decreasing expression of the target gene, relative to expression in the absence of expression of the construct.
MicroRNAs (miRNAs) are believed to generally regulate gene expression post-transcriptionally in plants by directing sequence-specific cleavage of messenger RNAs (“mRNAs”). One aspect of this invention is a method to control the rate of post-transcriptional suppression of a plant gene that transcribes to a mRNA containing a miRNA recognition site that is normally recognized and bound by a specific miRNA in complex with Argonaute (Ago), followed by cleavage of the resulting miRNA/mRNA hybridized segment by an RNase III ribonuclease such as a Dicer-like ribonuclease. This method uses a “cleavage blocker” construct to transgenically express in planta an RNA including single-stranded RNA that binds to the mRNA transcript of the target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to the transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of the hybridized segment, wherein the binding of the single-stranded RNA to the transcript (and the resultant formation of the hybridized segment) inhibits double-stranded RNA-mediated suppression of the at least one target gene. The “cleavage blocker” RNA generally competes with endogenous mature miRNAs, for binding with an mRNA that is normally regulated by that miRNA; the cleavage blocker protects the mRNA from cleavage by the miRNA-Ago complex by binding to the miRNA target site on the mRNA to form a non-cleavable hybridized segment. Thus, a cleavage blocker protects the target mRNA's cleavage site (miRNA recognition site) from being cleaved by miRNA and prevents down-regulation of that particular target gene. Preferably, a cleavage blocker increases expression of the target gene (relative to its expression in the absence of the cleavage blocker). This method allows for regulation of gene expression in a specific manner and is a useful alternative to upregulating the level of a gene's transcript or its encoded protein by over-expression of the gene.
One aspect of this invention is a method for providing a cleavage blocker by generating the cleavage blocker single-stranded RNA in planta from a “cleavage blocker construct” based on a recombinant miRNA-precursor-like sequence. A miRNA-precursor-like sequence is created by placing the cleavage blocker sequence in the backbone of a miRNA primary transcript, while maintaining the predicted secondary structure in the transcript's fold-back in such a way that resulting transcript is processed by Dicer-like ribonucleases to single-stranded RNA, which is then able to associate with the miRNA recognition site on the target mRNA and prevent the mRNA from being cleaved by a mature miRNA. The cleavage blocker sequence is selected such that, upon hybridization of the cleavage blocker to the target mRNA, a hybridized segment is formed that includes: (a) at least one mismatch between the single-stranded RNA and the miRNA recognition site at positions of the miRNA recognition site corresponding to positions 9, 10, or 11 of the mature miRNA, or (b) at least one insertion at a position in the single-stranded RNA at positions of the miRNA recognition site corresponding to positions 10-11 of the mature miRNA. In especially preferred embodiments, the single-stranded RNA that binds to the transcript of at least one target gene has a nucleotide sequence to allow a stably hybridized segment to be formed between it and the target gene transcript, but that inhibits binding of an Argonaute or Argonaute-like protein to the hybridized segment, as described by Mi et al. (2008) Cell, doi:10.1016/j.cell.2008.02.034; for example, the single-stranded RNA has a nucleotide sequence that includes an A, G, or C (but not a U) at a position corresponding to the 5′ terminus of the mature miRNA that natively binds to the recognition site. For cleavage blockers expressed in transgenic plants, there is in many embodiments preferably also a mismatch between the single-stranded RNA and the miRNA recognition site at the position of the miRNA recognition site corresponding to positions 1 of the mature miRNA to prevent transitivity of the suppression effect.
An alternative method for generating a cleavage blocker in vivo or in planta is to express short single-stranded RNA from a strong promoter such as Pol II or Pol III promoters. This single-stranded RNA preferably includes sequence that is complimentary to the mRNA only at the miRNA recognition site. Because producing a cleavage blocker using this method does not require the association of the RNA with an Argonaute or Ago protein, mismatches at positions 10 and 11 are not required.

Target Genes

The recombinant DNA construct of this invention can be designed to modulate the expression of any target gene or genes. The target gene can be translatable (coding) sequence, or can be non-coding sequence (such as non-coding regulatory sequence), or both, and can include at least one gene selected from the group consisting of a eukaryotic target gene, a non-eukaryotic target gene, a microRNA precursor DNA sequence, and a microRNA promoter. The target gene can be native (endogenous) to the cell (e.g., a cell of a plant or animal) in which the recombinant DNA construct is transcribed, or can be native to a pest or pathogen (or a symbiont of the pest or pathogen) of the plant or animal in which the recombinant DNA construct is transcribed. The target gene can be an exogenous gene, such as a transgene in a plant. A target gene can be a native gene targetted for suppression, with or without concurrent expression of an exogenous transgene, for example, by including a gene expression element in the recombinant DNA construct, or in a separate recombinant DNA construct. For example, it can be desirable to replace a native gene with an exogenous transgene homologue.
The target gene can include a single gene or part of a single gene that is targetted for suppression, or can include, for example, multiple consecutive segments of a target gene, multiple non-consecutive segments of a target gene, multiple alleles of a target gene, or multiple target genes from one or more species. A target gene can include any sequence from any species (including, but not limited to, non-eukaryotes such as bacteria, and viruses; fungi; plants, including monocots and dicots, such as crop plants, ornamental plants, and non-domesticated or wild plants; invertebrates such as arthropods, annelids, nematodes, and molluscs; and vertebrates such as amphibians, fish, birds, domestic or wild mammals, and even humans.
In one embodiment, the target gene is exogenous to the plant in which the recombinant DNA construct is to be transcribed, but endogenous to a pest or pathogen (e.g., viruses, bacteria, fungi, oomycetes, and invertebrates such as insects, nematodes, and molluscs), or to a symbiont of the pest or pathogen, of the plant. The target gene can include multiple target genes, or multiple segments of one or more genes. In one embodiment, the target gene or genes is a gene or genes of an invertebrate pest or pathogen of the plant. Thus, a recombinant DNA construct of this invention can be transcribed in a plant and used to modulate the expression of a gene of a pathogen or pest that may infest the plant. These embodiments are particularly useful in providing non-natural transgenic plants having resistance to one or more plant pests or pathogens, for example, resistance to a nematode such as soybean cyst nematode or root knot nematode or to a pest insect.
Where the target gene is that of an invertebrate pest, the invertebrate pest is at least one or more invertebrate selected from the group consisting of insects, arachnids (e.g., mites), nematodes, molluscs (e.g., slugs and snails), and annelids, and can include an invertebrate associated with an invertebrate pest in a symbiotic relationship (e.g., the mutualistic relationship between some ant and aphid species). The term “symbiotic” relationship as used herein encompasses both facultative (non-obligate) and obligate symbioses wherein at least one of the two or more associated species benefits, and further includes mutualistic, commensal, and parasitic relationships. Symbionts also include non-invertebrate symbionts, such as prokaryotes and eukaryotic protists. An invertebrate pest can be controlled indirectly by targetting a symbiont that is associated, internally or externally, with the invertebrate pest. For example, prokaryotic symbionts are known to occur in the gut or other tissues of many invertebrates, including invertebrate pests of interest. examples of a targetted symbiont associated with an invertebrate pest include the aphid endosymbiotic bacteria Buchnera; Wolbachia bacteria that infect many insects; Baumannia cicadellinicola and Sulcia muelleri, the co-symbiotic bacteria of the glassy-winged sharpshooter (Homalodisca coagulata), which transmits the Pierce's disease pathogen Xylella fastidiosa; and eukaryotic protist (flagellate) endosymbionts in termites. In an alternative approach, expression of an endogenous target gene of the invertebrate pest can be modified in such a way as to control a symbiont of the invertebrate, in turn affecting the host invertebrate.
The target gene can be translatable (coding) sequence, or can be non-coding sequence (such as non-coding regulatory sequence), or both. examples of a target gene include non-translatable (non-coding) sequence, such as, but not limited to, 5′ untranslated regions, promoters, enhancers, or other non-coding transcriptional regions, 3′ untranslated regions, terminators, and introns. Target genes include genes encoding microRNAs, small interfering RNAs, and other small RNAs associated with a silencing complex (RISC) or an Argonaute protein; RNA components of ribosomes or ribozymes; small nucleolar RNAs; and other non-coding RNAs. Target genes can also include genes encoding transcription factors and genes encoding enzymes involved in the biosynthesis or catabolism of molecules of interest (such as, but not limited to, amino acids, fatty acids and other lipids, sugars and other carbohydrates, biological polymers, and secondary metabolites including alkaloids, terpenoids, polyketides, non-ribosomal peptides, and secondary metabolites of mixed biosynthetic origin).
In many embodiments, the target gene is an essential gene of a plant pest or pathogen (or of a symbiont of the pest or pathogen). Essential genes include genes that are required for development of the pest or pathogen to a fertile reproductive adult. Essential genes include genes that, when silenced or suppressed, result in the death of the organism (as an adult or at any developmental stage, including gametes) or in the organism's inability to successfully reproduce (e.g., sterility in a male or female parent or lethality to the zygote, embryo, or larva). A description of nematode essential genes is found, e.g., in Kemphues, K. “Essential Genes” (Dec. 24, 2005), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.57.1, available on line at www.wormbook.org. A description of insect genes is publicly available at the Drosophila genome database (available on line at flybase.bio.indiana.edu/), and 438 essential genes have been identified for Drosophila as a representative insect; see Boutros et al. (2004) Science, 303:832-835, and supporting material available on line at www.sciencemag.org/cgi/content/full/303/5659/832/DC1. A description of bacterial and fungal essential genes is provided in the Database of Essential Genes (“DEG”, available on line at tubic.tju.edu.cn/deg/). Essential genes include those that influence other genes, where the overall effect is the death of the invertebrate pest or loss of the invertebrate pest's inability to successfully reproduce. In an example, suppression of the Drosophila homeobox gene Caudal leads eventually to host mortality caused by disequilibrium of the insect's commensal gut bacterial population (Ryu et al. (2008) Science, 319:777-782) and thus Caudal as well as the antimicrobial peptide genes directly controlled by Caudal are both considered essential genes.
Plant pest invertebrates include, but are not limited to, pest nematodes, pest molluscs (slugs and snails), pest annelids, and pest insects. Plant pathogens of interest include fungi, oomycetes, bacteria (e.g., the bacteria that cause leaf spotting, fireblight, crown gall, and bacterial wilt), mollicutes, and viruses (e.g., the viruses that cause mosaics, vein banding, flecking, spotting, or abnormal growth). See also G. N. Agrios, “Plant Pathology” (Fourth Edition), Academic Press, San Diego, 1997, 635 pp., for descriptions of fungi, bacteria, mollicutes (including mycoplasmas and spiroplasmas), viruses, nematodes, parasitic higher plants, and flagellate protozoans, all of which are plant pests or pathogens of interest. See also the updated compilation of plant pests and pathogens and the diseases caused by such on the American Phytopathological Society's “Common Names of Plant Diseases”, available online at www.apsnet.org/online/common/top.asp.
Examples of fungal plant pathogens of particular interest include, e.g., the fungi that cause powdery mildew, rust, leaf spot and blight, damping-off, root rot, crown rot, cotton boll rot, stem canker, twig canker, vascular wilt, smut, or mold, including, but not limited to, Fusarium spp., Phakospora spp., Rhizoctonia spp., Aspergillus spp., Gibberella spp., Pyricularia spp., and Alternaria spp., and the numerous fungal species provided in Tables 4 and 5 of U.S. Pat. No. 6,194,636, which is specifically incorporated in its entirety by reference herein. examples of plant pathogens include pathogens previously classified as fungi but more recently classified as oomycetes. Specific examples of oomycete plant pathogens of particular interest include members of the genus Pythium (e.g., Pythium aphanidermatum) and Phytophthora (e.g., Phytophthora infestans, Phytophthora sojae,) and organisms that cause downy mildew (e.g., Peronospora farinosa).
Examples of invertebrate pests include cyst nematodes Heterodera spp. especially soybean cyst nematode Heterodera glycines, root knot nematodes Meloidogyne spp., corn rootworms (Diabrotica spp.), Lygus spp., aphids and similar sap-sucking insects such as phylloxera (Daktulosphaira vitifoliae), corn borers, cutworms, armyworms, leafhoppers, Japanese beetles, grasshoppers, and other pest coleopterans, dipterans, and lepidopterans.
Specific examples of suitable target genes also include genes involved in amino acid or fatty acid synthesis, storage, or catabolism, genes involved in multi-step biosynthesis pathways, where it may be of interest to regulate the level of one or more intermediate; and genes encoding cell-cycle control proteins. Target genes can include genes encoding undesirable proteins (e.g., allergens or toxins) or the enzymes for the biosynthesis of undesirable compounds (e.g., undesirable flavor or odor components).
The recombinant DNA construct can be designed to be more specifically modulate the expression of the target gene, for example, by designing the recombinant DNA construct to include DNA that undergoes processing to an RNA including single-stranded RNA that binds to the target gene transcript, wherein the single-stranded RNA includes a nucleotide sequence substantially non-identical (or non-complementary) to a non-target gene sequence (and is thus less likely to bind to a non-target gene transcript). In one example, the recombinant DNA construct is designed to suppress a target gene that is a gene endogenous to a single species (e.g., Western corn rootworm, Diabrotica virgifera virgifera LeConte) but to not suppress a non-target gene such as genes from related, even closely related, species (e.g., Northern corn rootworm, Diabrotica barberi Smith and Lawrence, or Southern corn rootworm, Diabrotica undecimpunctata). In other embodiments, the recombinant DNA construct is designed to modulate the expression of a target gene sequence common to multiple species in which the target gene is to be silenced. For example, a recombinant DNA construct for modulating a target gene in corn rootworm can be selected to be specific to all members of the genus Diabrotica. In a further example of this embodiment, such a Diabrotica-targetted recombinant DNA construct can be selected so as to not target any gene sequence from beneficial insect species.

Promoters

Generally, the recombinant DNA construct of this invention includes a promoter, functional in the cell in which the construct is intended to be transcribed, and operably linked to the DNA that undergoes processing to an RNA including single-stranded RNA that binds to the transcript of at least one target gene. In various embodiments, the promoter is selected from the group consisting of a constitutive promoter, a spatially specific promoter, a temporally specific promoter, a developmentally specific promoter, and an inducible promoter.
Non-constitutive promoters suitable for use with the recombinant DNA constructs of the invention include spatially specific promoters, temporally specific promoters, and inducible promoters. Spatially specific promoters can include organelle-, cell-, tissue-, or organ-specific promoters (e.g., a plastid-specific, a root-specific, a pollen-specific, or a seed-specific promoter for suppressing expression of the first target RNA in plastids, roots, pollen, or seeds, respectively). In many cases a seed-specific, embryo-specific, aleurone-specific, or endosperm-specific promoter is especially useful. Temporally specific promoters can include promoters that tend to promote expression during certain developmental stages in a plant's growth cycle, or during different times of day or night, or at different seasons in a year. Inducible promoters include promoters induced by chemicals or by environmental conditions such as, but not limited to, biotic or abiotic stress (e.g., water deficit or drought, heat, cold, high or low nutrient or salt levels, high or low light levels, or pest or pathogen infection). Of particular interest are microRNA promoters, especially those having a temporally specific, spatially specific, or inducible expression pattern; examples of miRNA promoters, as well as methods for identifying miRNA promoters having specific expression patterns, are provided in U.S. Patent Application Publications 2006/0200878, 2007/0199095, and 2007/0300329, which are specifically incorporated herein by reference. An expression-specific promoter can also include promoters that are generally constitutively expressed but at differing degrees or “strengths” of expression, including promoters commonly regarded as “strong promoters” or as “weak promoters”.
Promoters of particular interest include the following examples: an opaline synthase promoter isolated from T-DNA of Agrobacterium; a cauliflower mosaic virus 35S promoter; enhanced promoter elements or chimeric promoter elements such as an enhanced cauliflower mosaic virus (CaMV) 35S promoter linked to an enhancer element (an intron from heat shock protein 70 of Zea mays); root specific promoters such as those disclosed in U.S. Pat. Nos. 5,837,848; 6,437,217 and 6,426,446; a maize L3 oleosin promoter disclosed in U.S. Pat. No. 6,433,252; a promoter for a plant nuclear gene encoding a plastid-localized aldolase disclosed in U.S. Patent Application Publication 2004/0216189; cold-inducible promoters disclosed in U.S. Pat. No. 6,084,089; salt-inducible promoters disclosed in U.S. Pat. No. 6,140,078; light-inducible promoters disclosed in U.S. Pat. No. 6,294,714; pathogen-inducible promoters disclosed in U.S. Pat. No. 6,252,138; and water deficit-inducible promoters disclosed in U.S. Patent Application Publication 2004/0123347 A1. All of the above-described patents and patent publications disclosing promoters and their use, especially in recombinant DNA constructs functional in plants are incorporated herein by reference.
Plant vascular- or phloem-specific promoters of interest include a rolC or rolA promoter of Agrobacterium rhizogenes, a promoter of a Agrobacterium tumefaciens T-DNA gene 5, the rice sucrose synthase RSs1 gene promoter, a Commelina yellow mottle badnavirus promoter, a coconut foliar decay virus promoter, a rice tungro bacilliform virus promoter, the promoter of a pea glutamine synthase GS3A gene, a invCD111 and invCD141 promoters of a potato invertase genes, a promoter isolated from Arabidopsis shown to have phloem-specific expression in tobacco by Kertbundit et al. (1991) Proc. Natl. Acad. Sci. USA., 88:5212-5216, a VAHOX1 promoter region, a pea cell wall invertase gene promoter, an acid invertase gene promoter from carrot, a promoter of a sulfate transporter gene Sultrl; 3, a promoter of a plant sucrose synthase gene, and a promoter of a plant sucrose transporter gene.
Promoters suitable for use with a recombinant DNA construct of this invention include polymerase II (“pol II”) promoters and polymerase III (“pol III”) promoters. RNA polymerase II transcribes structural or catalytic RNAs that are usually shorter than 400 nucleotides in length, and recognizes a simple run of T residues as a termination signal; it has been used to transcribe siRNA duplexes (see, e.g., Lu et al. (2004) Nucleic Acids Res., 32:e171). Pol II promoters are therefore preferred in certain embodiments where a short RNA transcript is to be produced from a recombinant DNA construct of this invention. In one embodiment, the recombinant DNA construct includes a pol II promoter to express an RNA transcript flanked by self-cleaving ribozyme sequences (e.g., self-cleaving hammerhead ribozymes), resulting in a processed RNA, including single-stranded RNA that binds to the transcript of at least one target gene, with defined 5′ and 3′ ends, free of potentially interfering flanking sequences. An alternative approach uses pol III promoters to generate transcripts with relatively defined 5′ and 3′ ends, i.e., to transcribe an RNA with minimal 5′ and 3′ flanking sequences. In some embodiments, Pol III promoters (e.g., U6 or H1 promoters) are preferred for adding a short AT-rich transcription termination site that results in 2 base-pair overhangs (UU) in the transcribed RNA; this is useful, e.g., for expression of siRNA-type constructs. Use of pol III promoters for driving expression of siRNA constructs has been reported; see van de Wetering et al. (2003) EMBO Rep., 4: 609-615, and Tuschl (2002) Nature Biotechnol., 20: 446-448.
The promoter element can include nucleic acid sequences that are not naturally occurring promoters or promoter elements or homologues thereof but that can regulate expression of a gene. Examples of such “gene independent” regulatory sequences include naturally occurring or artificially designed RNA sequences that include a ligand-binding region or aptamer (see “Aptamers”, below) and a regulatory region (which can be cis-acting). See, for example, Isaacs et al. (2004) Nat. Biotechnol., 22:841-847, Bayer and Smolke (2005) Nature Biotechnol., 23:337-343, Mandal and Breaker (2004) Nature Rev. Mol. Cell Biol., 5:451-463, Davidson and Ellington (2005) Trends Biotechnol., 23:109-112, Winkler et al. (2002) Nature, 419:952-956, Sudarsan et al. (2003) RNA, 9:644-647, and Mandal and Breaker (2004) Nature Struct. Mol. Biol., 11:29-35. Such “riboregulators” could be selected or designed for specific spatial or temporal specificity, for example, to regulate translation of the DNA that undergoes processing to an RNA including single-stranded RNA that binds to the transcript of at least one target gene only in the presence (or absence) of a given concentration of the appropriate ligand. One example is a riboregulator that is responsive to an endogenous ligand (e.g., jasmonic acid or salicylic acid) produced by the plant when under stress (e.g., abiotic stress such as water, temperature, or nutrient stress, or biotic stress such as attach by pests or pathogens); under stress, the level of endogenous ligand increases to a level sufficient for the riboregulator to begin transcription of the DNA that undergoes processing to an RNA including single-stranded RNA that binds to the transcript of at least one target gene.

Aptamers

In some embodiments, the recombinant DNA construct of this invention includes DNA that is processed to an RNA aptamer, that is, an RNA that binds to a ligand through binding mechanism that is not primarily based on Watson-Crick base-pairing (in contrast, for example, to the base-pairing that occurs between complementary, anti-parallel nucleic acid strands to form a double-stranded nucleic acid structure). See, for example, Ellington and Szostak (1990) Nature, 346:818-822. Examples of aptamers can be found, for example, in the public Aptamer Database, available on line at aptamer.icmb.utexas.edu (Lee et al. (2004) Nucleic Acids Res., 32(1):D95-100). Aptamers useful in the invention can, however, be monovalent (binding a single ligand) or multivalent (binding more than one individual ligand, e.g., binding one unit of two or more different ligands).
Ligands useful in the invention include any molecule (or part of a molecule) that can be recognized and be bound by a nucleic acid secondary structure by a mechanism not primarily based on Watson-Crick base pairing. In this way, the recognition and binding of ligand and aptamer is analogous to that of antigen and antibody, or of biological effector and receptor. Ligands can include single molecules (or part of a molecule), or a combination of two or more molecules (or parts of a molecule), and can include one or more macromolecular complexes (e.g., polymers, lipid bilayers, liposomes, cellular membranes or other cellular structures, or cell surfaces). Examples of specific ligands include vitamins such as coenzyme B₁₂and thiamine pyrophosphate, flavin mononucleotide, guanine, adenosine, S-adenosylmethionine, S-adenosylhomocysteine, coenzyme A, lysine, tyrosine, dopamine, glucosamine-6-phosphate, caffeine, theophylline, antibiotics such as chloramphenicol and neomycin, herbicides such as glyphosate and dicamba, proteins including viral or phage coat proteins and invertebrate epidermal or digestive tract surface proteins, and RNAs including viral RNA, transfer-RNAs (t-RNAs), ribosomal RNA (rRNA), and RNA polymerases such as RNA-dependent RNA polymerase (RdRP). One class of RNA aptamers useful in the invention are “thermoswitches” that do not bind a ligand but are thermally responsive, that is to say, the aptamer's conformation is determined by temperature; see, for example, Box 3 in Mandal and Breaker (2004) Nature Rev. Mol. Cell Biol., 5:451-463.

Transgene Transcription Units

In some embodiments, the recombinant DNA construct of this invention includes a transgene transcription unit. A transgene transcription unit includes DNA sequence encoding a gene of interest, e.g., a natural protein or a heterologous protein. A gene of interest can be any coding or non-coding sequence from any species (including, but not limited to, non-eukaryotes such as bacteria, and viruses; fungi, protists, plants, invertebrates, and vertebrates. Genes of interest include those genes also described above as target genes, under the heading “Target Genes”. The transgene transcription unit can further include 5′ or 3′ sequence or both as required for transcription of the transgene.

Introns

In some embodiments, the recombinant DNA construct of this invention includes DNA encoding a spliceable intron. By “intron” is generally meant a segment of DNA (or the RNA transcribed from such a segment) that is located between exons (protein-encoding segments of the DNA or corresponding transcribed RNA), wherein, during maturation of the messenger RNA, the intron present is enzymatically “spliced out” or removed from the RNA strand by a cleavage/ligation process that occurs in the nucleus in eukaryotes. The term “intron” is also applied to non-coding DNA sequences that are transcribed to RNA segments that can be spliced out of a maturing RNA transcript, but are not introns found between protein-coding exons. One example of these are spliceable sequences that that have the ability to enhance expression in plants (in some cases, especially in monocots) of a downstream coding sequence; these spliceable sequences are naturally located in the 5′ untranslated region of some plant genes, as well as in some viral genes (e.g., the tobacco mosaic virus 5′ leader sequence or “omega” leader described as enhancing expression in plant genes by Gallie and Walbot (1992) Nucleic Acids Res., 20:4631-4638). These spliceable sequences or “expression-enhancing introns” can be artificially inserted in the 5′ untranslated region of a plant gene between the promoter but before any protein-coding exons. Examples of such expression-enhancing introns include, but are not limited to, a maize alcohol dehydrogenase (Zm-Adhl), a maize Bronze-1 expression-enhancing intron, a rice actin 1 (Os-Actl) intron, a Shrunken-1 (Sh-1) intron, a maize sucrose synthase intron, a heat shock protein 18 (hsp18) intron, and an 82 kilodalton heat shock protein (hsp82) intron. U.S. Pat. Nos. 5,593,874 and 5,859,347, specifically incorporated by reference herein, describe methods of improving recombinant DNA constructs for use in plants by inclusion of an expression-enhancing intron derived from the 70 kilodalton maize heat shock protein (hsp70) in the non-translated leader positioned 3′ from the gene promoter and 5′ from the first protein-coding exon.

Ribozymes

In some embodiments, the recombinant DNA construct of this invention includes DNA encoding one or more ribozymes. Ribozymes of particular interest include a self-cleaving ribozyme, a hammerhead ribozyme, or a hairpin ribozyme. In one embodiment, the recombinant DNA construct includes DNA encoding one or more ribozymes that serve to cleave the transcribed RNA to provide defined segments of RNA, such as the single-stranded RNA that binds to the target gene transcript.

Recombinases

In some embodiments, the recombinant DNA construct of this invention includes DNA encoding one or more site-specific recombinase recognition sites. In one embodiment, the recombinant DNA construct includes at least a pair of loxP sites, wherein site-specific recombination of DNA between the loxP sites is mediated by a Cre recombinase. The position and relative orientation of the loxP sites is selected to achieve the desired recombination; for example, when the loxP sites are in the same orientation, the DNA between the loxP sites is excised in circular form. In another embodiment, the recombinant DNA construct includes DNA encoding one loxP site; in the presence of Cre recombinase and another DNA with a loxP site, the two DNAs are recombined.

Gene Suppression Elements

In some embodiments, the recombinant DNA construct of this invention further includes DNA encoding a gene suppression element. Gene suppression elements include any DNA sequence (or RNA sequence encoded therein) designed to specifically suppress a gene or genes of interest, which can be a gene endogenous to the cell in which the recombinant DNA construct is transcribed, or a gene exogenous to that cell. The gene to be suppressed can be any of those disclosed as target genes under the heading “Target Genes”.
Suitable gene suppression elements are described in detail in U.S. Patent Application Publication 2006/0200878, which disclosure is specifically incorporated herein by reference, and include one or more of:

- (a) DNA that includes at least one anti-sense DNA segment that is anti-sense to at least one segment of the gene to be suppressed;
- (b) DNA that includes multiple copies of at least one anti-sense DNA segment that is anti-sense to at least one segment of the gene to be suppressed e;
- (c) DNA that includes at least one sense DNA segment that is at least one segment of the gene to be suppressed;
- (d) DNA that includes multiple copies of at least one sense DNA segment that is at least one segment of the gene to be suppressed;
- (e) DNA that transcribes to RNA for suppressing the gene to be suppressed by forming double-stranded RNA and includes at least one anti-sense DNA segment that is anti-sense to at least one segment of the gene to be suppressed and at least one sense DNA segment that is at least one segment of the gene to be suppressed;
- (f) DNA that transcribes to RNA for suppressing the gene to be suppressed by forming a single double-stranded RNA and includes multiple serial anti-sense DNA segments that are anti-sense to at least one segment of the gene to be suppressed and multiple serial sense DNA segments that are at least one segment of the gene to be suppressed;
- (g) DNA that transcribes to RNA for suppressing the gene to be suppressed by forming multiple double strands of RNA and includes multiple anti-sense DNA segments that are anti-sense to at least one segment of the gene to be suppressed and multiple sense DNA segments that are at least one segment of the gene to be suppressed, and wherein the multiple anti-sense DNA segments and the multiple sense DNA segments are arranged in a series of inverted repeats;
- (h) DNA that includes nucleotides derived from a plant miRNA;
- (i) DNA that includes nucleotides of a siRNA;
- (j) DNA that transcribes to an RNA aptamer capable of binding to a ligand; and
- (k) DNA that transcribes to an RNA aptamer capable of binding to a ligand, and DNA that transcribes to regulatory RNA capable of regulating expression of the gene to be suppressed, wherein the regulation is dependent on the conformation of the regulatory RNA, and the conformation of the regulatory RNA is allosterically affected by the binding state of the RNA aptamer.

In some embodiments, an intron is used to deliver a gene suppression element in the absence of any protein-coding exons (coding sequence). In one example, an intron, such as an expression-enhancing intron (preferred in certain embodiments), is interrupted by embedding within the intron a gene suppression element, wherein, upon transcription, the gene suppression element is excised from the intron. Thus, protein-coding exons are not required to provide the gene suppressing function of the recombinant DNA constructs disclosed herein.

Transcription Regulatory Elements

In some embodiments, the recombinant DNA construct of this invention includes DNA encoding a transcription regulatory element. Transcription regulatory elements include elements that regulate the expression level of the recombinant DNA construct of this invention (relative to its expression in the absence of such regulatory elements). Examples of suitable transcription regulatory elements include riboswitches (cis- or trans-acting), transcript stabilizing sequences, and miRNA recognition sites, as described in detail in U.S. Patent Application Publication 2006/0200878, specifically incorporated herein by reference.

Making and Using Recombinant DNA Constructs

The recombinant DNA constructs of this invention are made by any method suitable to the intended application, taking into account, for example, the type of expression desired and convenience of use in the plant in which the construct is to be transcribed. General methods for making and using DNA constructs and vectors are well known in the art and described in detail in, for example, handbooks and laboratory manuals including Sambrook and Russell, “Molecular Cloning: A Laboratory Manual” (third edition), Cold Spring Harbor Laboratory Press, NY, 2001. An example of useful technology for building DNA constructs and vectors for transformation is disclosed in U.S. Patent Application Publication 2004/0115642 A1, specifically incorporated herein by reference. DNA constructs can also be built using the GATEWAY™ cloning technology (available from Invitrogen Life Technologies, Carlsbad, Calif.), which uses the site-specific recombinase LR cloning reaction of the Integrase/att system from bacteriophage lambda vector construction, instead of restriction endonucleases and ligases. The LR cloning reaction is disclosed in U.S. Pat. Nos. 5,888,732 and 6,277,608, and in U.S. Patent Application Publications 2001/283529, 2001/282319 and 2002/0007051, all of which are specifically incorporated herein by reference. Another alternative vector fabrication method employs ligation-independent cloning as disclosed by Aslandis et al. (1990) Nucleic Acids Res., 18:6069-6074 and Rashtchian et al. (1992) Biochem., 206:91-97, where a DNA fragment with single-stranded 5′ and 3′ ends is ligated into a desired vector which can then be amplified in vivo.
In certain embodiments, the DNA sequence of the recombinant DNA construct includes sequence that has been codon-optimized for the plant in which the recombinant DNA construct is to be expressed. For example, a recombinant DNA construct to be expressed in a plant can have all or parts of its sequence (e.g., the first gene suppression element or the gene expression element) codon-optimized for expression in a plant by methods known in the art. See, e.g., U.S. Pat. No. 5,500,365, incorporated by reference, for a description of codon-optimization methodology for plants; see also De Amicis and Marchetti (2000) Nucleic Acid Res., 28:3339-3346.

Non-Natural Transgenic Plant Cells, Plants, and Seeds

In another aspect, this invention provides a non-natural transgenic plant cell having in its genome a recombinant DNA construct of this invention including DNA that undergoes processing to an RNA including single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to the transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of the hybridized segment. This invention further provides a non-natural transgenic plant including the non-natural transgenic plant cell. In one embodiment, the non-natural transgenic plant is wholly composed of transgenic tissue. In another embodiment, the non-natural plant is a partially transgenic plant and includes non-transgenic tissue; in one example, the non-natural partially transgenic plant includes a non-transgenic scion and a transgenic rootstock including the non-natural transgenic plant cell. Further provided by this invention is a non-natural transgenic seed including the non-natural transgenic plant cell.
A non-natural transgenic plant of this invention includes plants of any developmental stage, and includes a non-natural regenerated plant prepared from the non-natural transgenic plant cells disclosed herein, or a non-natural progeny plant (which can be an inbred or hybrid progeny plant) of the regenerated plant, or seed of such a non-natural transgenic plant. Also provided is a non-natural transgenic seed having in its genome a recombinant DNA construct of this invention. The non-natural transgenic plant cells, transgenic plants, and transgenic seeds of this invention are made by methods well-known in the art, as described below under the heading “Making and Using Transgenic Plant Cells and Transgenic Plants”.
The non-natural transgenic plant cell can include an isolated plant cell (e.g., individual plant cells or cells grown in or on an artificial culture medium), or can include a plant cell in undifferentiated tissue (e.g., callus or any aggregation of plant cells). The non-natural transgenic plant cell can include a plant cell in at least one differentiated tissue selected from the group consisting of leaf (e.g., petiole and blade), root, stem (e.g., tuber, rhizome, stolon, bulb, and corm) stalk (e.g., xylem, phloem), wood, seed, fruit, and flower (e.g., stamen, filament, anther, pollen, microspore, carpel, pistil, ovary, ovules). The non-natural transgenic plant cell or non-natural transgenic plant of the invention can be stably transformed, e.g., fertile transgenic plants and their non-natural transgenic seed also containing the recombinant construct of this invention.
In some embodiments of this invention, the non-natural plant is a non-natural transgenic plant. In such embodiments, all cells (with the possible exception of haploid cells) and tissues of the non-natural plant contain the recombinant DNA construct of this invention. In other embodiments, the non-natural plant is partially transgenic, and includes natural non-transgenic tissue (for example, non-natural transgenic tissue grafted onto natural non-transgenic tissue). In one embodiment, the non-natural plant includes a natural non-transgenic scion and a non-natural transgenic rootstock including the transgenic plant cell, wherein the non-transgenic scion and transgenic rootstock are grafted together. Such embodiments are particularly useful where the plant is one that is commonly vegetatively grown as a scion grafted onto a rootstock (wherein scion and rootstock can be of the same species or variety or of different species or variety); examples include grapes, apples, pears, quince, avocados, citrus, stone fruits, kiwifruit, roses, and other plants of agricultural or ornamental importance. Specifically claimed embodiments include embodiments where (a) the non-natural partially transgenic plant includes a natural non-transgenic grape scion and a non-natural transgenic grape rootstock; and (b) the non-natural partially transgenic plant includes a natural non-transgenic fruit tree (e.g., pear) scion and a non-natural transgenic fruit tree (e.g., quince) rootstock.

Making and Using Transgenic Plant Cells and Transgenic Plants

Where a recombinant DNA construct of this invention is used to produce a non-natural transgenic plant cell, plant, or seed of this invention, ransformation can include any of the well-known and demonstrated methods and compositions. Suitable methods for plant transformation include virtually any method by which DNA can be introduced into a cell. One method of plant transformation is microprojectile bombardment, for example, as illustrated in U.S. Pat. No. 5,015,580 (soybean), U.S. Pat. No. 5,538,880 (maize), U.S. Pat. No. 5,550,318 (maize), U.S. Pat. No. 5,914,451 (soybean), U.S. Pat. No. 6,153,812 (wheat), U.S. Pat. No. 6,160,208 (maize), U.S. Pat. No. 6,288,312 (rice), U.S. Pat. No. 6,365,807 (rice), and U.S. Pat. No. 6,399,861 (maize), and U.S. Pat. No. 6,403,865 (maize), all of which are incorporated by reference for enabling the production of transgenic plants.
Another useful method of plant transformation is Agrobacterium-mediated transformation by means of Agrobacterium containing a binary Ti plasmid system, wherein the Agrobacterium carries a first Ti plasmid and a second, chimeric plasmid containing at least one T-DNA border of a wild-type Ti plasmid, a promoter functional in the transformed plant cell and operably linked to a gene suppression construct of the invention. See, for example, the binary system described in U.S. Pat. No. 5,159,135, incorporated by reference. Also see De Framond (1983) Biotechnology, 1:262-269; and Hoekema et al., (1983) Nature, 303:179. In such a binary system, the smaller plasmid, containing the T-DNA border or borders, can be conveniently constructed and manipulated in a suitable alternative host, such as E. coli, and then transferred into Agrobacterium.
Detailed procedures for Agrobacterium-mediated transformation of plants, especially crop plants, include procedures disclosed in U.S. Pat. Nos. 5,004,863, 5,159,135, and 5,518,908 (cotton); U.S. Pat. Nos. 5,416,011, 5,569,834, 5,824,877 and 6,384,301 (soybean); U.S. Pat. Nos. 5,591,616 and 5,981,840 (maize); U.S. Pat. No. 5,463,174 (brassicas including canola), U.S. Pat. No. 7,026,528 (wheat), and U.S. Pat. No. 6,329,571 (rice), and in U.S. Patent Application Publications 2004/0244075 (maize) and 2001/0042257 A1 (sugar beet), all of which are specifically incorporated by reference for enabling the production of transgenic plants. Similar methods have been reported for many plant species, both dicots and monocots, including, among others, peanut (Cheng et al. (1996) Plant Cell Rep., 15: 653); asparagus (Bytebier et al. (1987) Proc. Natl. Acad. Sci. U.S.A., 84:5345); barley (Wan and Lemaux (1994) Plant Physiol., 104:37); rice (Toriyama et al. (1988) Bio/Technology, 6:10; Zhang et al. (1988) Plant Cell Rep., 7:379; wheat (Vasil et al. (1992) Bio/Technology, 10:667; Becker et al. (1994) Plant J., 5:299), alfalfa (Masoud et al. (1996) Transgen. Res., 5:313); and tomato (Sun et al. (2006) Plant Cell Physiol., 47:426-431). See also a description of vectors, transformation methods, and production of transformed Arabidopsis thaliana plants where transcription factors are constitutively expressed by a CaMV35S promoter, in U. S. Patent Application Publication 2003/0167537 A1, incorporated by reference. Various methods of transformation of other plant species are well known in the art, see, for example, the encyclopedic reference, “Compendium of Transgenic Crop Plants”, edited by Chittaranjan Kole and Timothy C. Hall, Blackwell Publishing Ltd., 2008; ISBN 978-1-405-16924-0 (available electronically at mrw.interscience.wiley.com/emrw/9781405181099/hpt/toc), which describes transformation procedures for cereals and forage grasses (rice, maize, wheat, barley, oat, sorghum, pearl millet, finger millet, cool-season forage grasses, and bahiagrass), oilsee crops (soybean, oilseed brassicas, sunflower, peanut, flax, sesame, and safflower), legume grains and forages (common bean, cowpea, pea, faba bean, lentil, tepary bean, Asiatic beans, pigeonpea, vetch, chickpea, lupin, alfalfa, and clovers), temperate fruits and nuts (apple, pear, peach, plums, berry crops, cherries, grapes, olive, almond, and Persian walnut), tropical and subtropical fruits and nuts (citrus, grapefruit, banana and plantain, pineapple, papaya, mango, avocado, kiwifruit, passionfruit, and persimmon), vegetable crops (tomato, eggplant, peppers, vegetable brassicas, radish, carrot, cucurbits, alliums, asparagus, and leafy vegetables), sugar, tuber, and fiber crops (sugarcane, sugar beet, stvia, potato, sweet potato, cassava, and cotton), plantation crops, ornamentals, and turf grasses (tobacco, coffee, cocoa, tea, rubber tree, medicinal plants, ornamentals, amd turf grasses), and forest tree species One of ordinary skill in the art has various transformation methodologies for production of stable transgenic plants.
Transformation methods to provide transgenic plant cells and transgenic plants containing stably integrated recombinant DNA are preferably practiced in tissue culture on media and in a controlled environment. “Media” refers to the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism. Recipient cell targets include, but are not limited to, meristem cells, callus, immature embryos or parts of embryos, and gametic cells such as microspores, pollen, sperm, and egg cells. Any cell from which a fertile plant can be regenerated is contemplated as a useful recipient cell for practice of the invention. Callus can be initiated from various tissue sources, including, but not limited to, immature embryos or parts of embryos, seedling apical meristems, microspores, and the like. Those cells which are capable of proliferating as callus can serve as recipient cells for genetic transformation. Practical transformation methods and materials for making transgenic plants of this invention (e.g., various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) are disclosed, for example, in U.S. Pat. Nos. 6,194,636 and 6,232,526 and U. S. Patent Application Publication 2004/0216189, which are specifically incorporated by reference.
In general transformation practice, DNA is introduced into only a small percentage of target cells in any one transformation experiment. Marker genes are generally used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a transgenic DNA construct into their genomes. Preferred marker genes provide selective markers which confer resistance to a selective agent, such as an antibiotic or herbicide. Any of the antibiotics or herbicides to which a plant cell may be resistant can be a useful agent for selection. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene is integrated and expressed at sufficient levels to permit cell survival. Cells can be tested further to confirm stable integration of the recombinant DNA. Commonly used selective marker genes include those conferring resistance to antibiotics such as kanamycin or paromomycin (nptll), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (EPSPS). Examples of useful selective marker genes and selection agents are illustrated in U.S. Pat. Nos. 5,550,318, 5,633,435, 5,780,708, and 6,118,047, all of which are specifically incorporated by reference. Screenable markers or reporters, such as markers that provide an ability to visually identify transformants can also be employed. Examples of useful screenable markers include, for example, a gene expressing a protein that produces a detectable color by acting on a chromogenic substrate (e.g., beta glucuronidase (GUS) (uidA) or luciferase (luc)) or that itself is detectable, such as green fluorescent protein (GFP) (gfp) or an immunogenic molecule. Those of skill in the art will recognize that many other useful markers or reporters are available for use.
Detecting or measuring transcription of the recombinant DNA construct in the transgenic plant cell of the invention can be achieved by any suitable method, including protein detection methods (e.g., western blots, ELISAs, and other immunochemical methods), measurements of enzymatic activity, or nucleic acid detection methods (e.g., Southern blots, northern blots, PCR, RT-PCR, fluorescent in situ hybridization).
Other suitable methods for detecting or measuring transcription of the recombinant DNA construct in the transgenic plant cell of the invention include measurement of any other trait that is a direct or proxy indication of the level of expression of the target gene in the transgenic plant cell in which the recombinant DNA construct is transcribed, relative to the level of expression in one in which the recombinant DNA is not transcribed, e.g., gross or microscopic morphological traits, growth rates, yield, reproductive or recruitment rates, resistance to pests or pathogens, or resistance to biotic or abiotic stress (e.g., water deficit stress, salt stress, nutrient stress, heat or cold stress). Such methods can use direct measurements of a phenotypic trait or proxy assays (e.g., in plants, these assays include plant part assays such as leaf or root assays to determine tolerance of abiotic stress). Such methods include direct measurements of resistance to an invertebrate pest or pathogen (e.g., damage to plant tissues) or proxy assays (e.g., plant yield assays, or bioassays such as the Western corn rootworm (Diabrotica virgifera virgifera LeConte) larval bioassay described in International Patent Application Publication WO2005/110068 A2 and U. S. Patent Application Publication US 2006/0021087 A1, specifically incorporated by reference, or the soybean cyst nematode bioassay described by Steeves et al. (2006) Funct. Plant Biol., 33:991-999, wherein cysts per plant, cysts per gram root, eggs per plant, eggs per gram root, and eggs per cyst are measured.
The recombinant DNA constructs of the invention can be stacked with other recombinant DNA for imparting additional traits (e.g., in the case of transformed plants, traits including herbicide resistance, pest resistance, cold germination tolerance, water deficit tolerance, and the like) for example, by expressing or suppressing other genes. Constructs for coordinated decrease and increase of gene expression are disclosed in U.S. Patent Application Publication 2004/0126845 A1, specifically incorporated by reference.
Seeds of fertile transgenic plants can be harvested and used to grow progeny generations, including hybrid generations, of transgenic plants of this invention that include the recombinant DNA construct in their genome. Thus, in addition to direct transformation of a plant with a recombinant DNA construct of this invention, transgenic plants of the invention can be prepared by crossing a first plant having the recombinant DNA with a second plant lacking the construct. For example, the recombinant DNA can be introduced into a plant line that is amenable to transformation to produce a transgenic plant, which can be crossed with a second plant line to introgress the recombinant DNA into the resulting progeny. A transgenic plant of the invention can be crossed with a plant line having other recombinant DNA that confers one or more additional trait(s) (such as, but not limited to, herbicide resistance, pest or disease resistance, environmental stress resistance, modified nutrient content, and yield improvement) to produce progeny plants having recombinant DNA that confers both the desired target sequence expression behavior and the additional trait(s).
In such breeding for combining traits the transgenic plant donating the additional trait can be a male line (pollinator) and the transgenic plant carrying the base traits can be the female line. The progeny of this cross segregate such that some of the plant will carry the DNA for both parental traits and some will carry DNA for one parental trait; such plants can be identified by markers associated with parental recombinant DNA Progeny plants carrying DNA for both parental traits can be crossed back into the female parent line multiple times, e.g., usually 6 to 8 generations, to produce a homozygous progeny plant with substantially the same genotype as one original transgenic parental line as well as the recombinant DNA of the other transgenic parental line.
Yet another aspect of the invention is a transgenic plant grown from the transgenic seed of the invention. This invention contemplates transgenic plants grown directly from transgenic seed containing the recombinant DNA as well as progeny generations of plants, including inbred or hybrid plant lines, made by crossing a transgenic plant grown directly from transgenic seed to a second plant not grown from the same transgenic seed. Crossing can include, for example, the following steps:

- (a) plant seeds of the first parent plant (e.g., non-transgenic or a transgenic) and a second parent plant that is transgenic according to the invention;
- (b) grow the seeds of the first and second parent plants into plants that bear flowers;
- (c) pollinate a flower from the first parent with pollen from the second parent; and
- (d) harvest seeds produced on the parent plant bearing the fertilized flower.

It is often desirable to introgress recombinant DNA into elite varieties, e.g., by backcrossing, to transfer a specific desirable trait from one source to an inbred or other plant that lacks that trait. This can be accomplished, for example, by first crossing a superior inbred (“A”) (recurrent parent) to a donor inbred (“B”) (non-recurrent parent), which carries the appropriate gene(s) for the trait in question, for example, a construct prepared in accordance with the current invention. The progeny of this cross first are selected in the resultant progeny for the desired trait to be transferred from the non-recurrent parent “B”, and then the selected progeny are mated back to the superior recurrent parent “A”. After five or more backcross generations with selection for the desired trait, the progeny can be essentially hemizygous for loci controlling the characteristic being transferred, but are like the superior parent for most or almost all other genes. The last backcross generation would be selfed to give progeny which are pure breeding for the gene(s) being transferred, i.e., one or more transformation events.
Through a series of breeding manipulations, a selected DNA construct can be moved from one line into an entirely different line without the need for further recombinant manipulation. One can thus produce inbred plants which are true breeding for one or more DNA constructs. By crossing different inbred plants, one can produce a large number of different hybrids with different combinations of DNA constructs. In this way, plants can be produced which have the desirable agronomic properties frequently associated with hybrids (“hybrid vigor”), as well as the desirable characteristics imparted by one or more DNA constructs.
In certain transgenic plant cells and transgenic plants of the invention, it may be desirable to concurrently express a gene of interest while also modulating expression of a target gene. Thus, in some embodiments, the transgenic plant contains recombinant DNA further including a gene expression element for expressing at least one gene of interest, and transcription of the recombinant DNA construct of this invention is preferably effected with concurrent transcription of the gene expression element.
The recombinant DNA constructs of this invention can be transcribed in any plant cell or tissue or in a whole plant of any developmental stage. Transgenic plants can be derived from any monocot or dicot plant, such as, but not limited to, plants of commercial or agricultural interest, such as crop plants (especially crop plants used for human food or animal feed), wood- or pulp-producing trees, vegetable plants, fruit plants, and ornamental plants. Examples of plants of interest include grain crop plants (such as wheat, oat, barley, maize, rye, triticale, rice, millet, sorghum, quinoa, amaranth, and buckwheat); forage crop plants (such as forage grasses and forage dicots including alfalfa, vetch, clover, and the like); oilseed crop plants (such as cotton, safflower, sunflower, soybean, canola, rapeseed, flax, peanuts, and oil palm); tree nuts (such as walnut, cashew, hazelnut, pecan, almond, and the like); sugarcane, coconut, date palm, olive, sugarbeet, tea, and coffee; wood- or pulp-producing trees; vegetable crop plants such as legumes (for example, beans, peas, lentils, alfalfa, peanut), lettuce, asparagus, artichoke, celery, carrot, radish, the brassicas (for example, cabbages, kales, mustards, and other leafy brassicas, broccoli, cauliflower, Brussels sprouts, turnip, kohlrabi), edible cucurbits (for example, cucumbers, melons, summer squashes, winter squashes), edible alliums (for example, onions, garlic, leeks, shallots, chives), edible members of the Solanaceae (for example, tomatoes, eggplants, potatoes, peppers, groundcherries), and edible members of the Chenopodiaceae (for example, beet, chard, spinach, quinoa, amaranth); fruit crop plants such as apple, pear, citrus fruits (for example, orange, lime, lemon, grapefruit, and others), stone fruits (for example, apricot, peach, plum, nectarine), banana, pineapple, grape, kiwifruit, papaya, avocado, and berries; plants grown for biomass or biofuel (for example, Miscanthus grasses, switchgrass, jatropha, oil palm, eukaryotic microalgae such as Botryococcus braunii, Chlorella spp., and Dunaliella spp., and eukaryotic macroalgae such as Gracilaria spp., and Sargassum spp.); and ornamental plants including ornamental flowering plants, ornamental trees and shrubs, ornamental groundcovers, and ornamental grasses.
This invention also provides commodity products produced from a non-natural transgenic plant cell, plant, or seed of this invention, including, but not limited to, harvested leaves, roots, shoots, tubers, stems, fruits, seeds, or other parts of a plant, meals, oils, extracts, fermentation or digestion products, crushed or whole grains or seeds of a plant, or any food or non-food product including such commodity products produced from a transgenic plant cell, plant, or seed of this invention. The detection of one or more of nucleic acid sequences of the recombinant DNA constructs of this invention in one or more commodity or commodity products contemplated herein is de facto evidence that the commodity or commodity product contains or is derived from a non-natural transgenic plant cell, plant, or seed of this invention.
In various embodiments, the non-natural transgenic plant having in its genome a recombinant DNA construct of this invention has at least one additional altered trait, relative to a plant lacking the recombinant DNA construct, selected from the group of traits consisting of:

- (a) improved abiotic stress tolerance;
- (b) improved biotic stress tolerance;
- (c) modified primary metabolite composition;
- (d) modified secondary metabolite composition;
- (e) modified trace element, carotenoid, or vitamin composition;
- (f) improved yield;
- (g) improved ability to use nitrogen, phosphate, or other nutrients;
- (h) modified agronomic characteristics;
- (i) modified growth or reproductive characteristics; and
- (j) improved harvest, storage, or processing quality.

In some embodiments, the non-natural transgenic plant is characterized by: improved tolerance of abiotic stress (e.g., tolerance of water deficit or drought, heat, cold, non-optimal nutrient or salt levels, non-optimal light levels) or of biotic stress (e.g., crowding, allelopathy, or wounding); by a modified primary metabolite (e.g., fatty acid, oil, amino acid, protein, sugar, or carbohydrate) composition; a modified secondary metabolite (e.g., alkaloids, terpenoids, polyketides, non-ribosomal peptides, and secondary metabolites of mixed biosynthetic origin) composition; a modified trace element (e.g., iron, zinc), carotenoid (e.g., beta-carotene, lycopene, lutein, zeaxanthin, or other carotenoids and xanthophylls), or vitamin (e.g., tocopherols) composition; improved yield (e.g., improved yield under non-stress conditions or improved yield under biotic or abiotic stress); improved ability to use nitrogen, phosphate, or other nutrients; modified agronomic characteristics (e.g., delayed ripening; delayed senescence; earlier or later maturity; improved shade tolerance; improved resistance to root or stalk lodging; improved resistance to “green snap” of stems; modified photoperiod response); modified growth or reproductive characteristics (e.g., intentional dwarfing; intentional male sterility, useful, e.g., in improved hybridization procedures; improved vegetative growth rate; improved germination; improved male or female fertility); improved harvest, storage, or processing quality (e.g., improved resistance to pests during storage, improved resistance to breakage, improved appeal to consumers); or any combination of these traits.
In another embodiment, non-natural transgenic seed, or seed produced by the non-natural transgenic plant, has modified primary metabolite (e.g., fatty acid, oil, amino acid, protein, sugar, or carbohydrate) composition, a modified secondary metabolite composition, a modified trace element, carotenoid, or vitamin composition, an improved harvest, storage, or processing quality, or a combination of these. In another embodiment, it can be desirable to change levels of native components of the transgenic plant or seed of a transgenic plant, for example, to decrease levels of an allergenic protein or glycoprotein or of a toxic metabolite.
Generally, screening a population of transgenic plants each regenerated from a transgenic plant cell is performed to identify transgenic plant cells that develop into transgenic plants having the desired trait. The transgenic plants are assayed to detect an enhanced trait, e.g., enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein, and enhanced seed oil. Screening methods include direct screening for the trait in a greenhouse or field trial or screening for a surrogate trait. Such analyses are directed to detecting changes in the chemical composition, biomass, physiological properties, or morphology of the plant. Changes in chemical compositions such as nutritional composition of grain are detected by analysis of the seed composition and content of protein, free amino acids, oil, free fatty acids, starch, tocopherols, or other nutrients. Changes in growth or biomass characteristics are detected by measuring plant height, stem diameter, internode length, root and shoot dry weights, and (for grain-producing plants such as maize, rice, or wheat) ear or seed head length and diameter. Changes in physiological properties are identified by evaluating responses to stress conditions, e.g., assays under imposed stress conditions such as water deficit, nitrogen or phosphate deficiency, cold or hot growing conditions, pathogen or insect attack, light deficiency, or increased plant density. Other selection properties include days to pollen shed, days to silking in maize, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, staying green, stalk lodging, root lodging, plant health, fertility, green snap, and pest resistance. In addition, phenotypic characteristics of harvested seed may be evaluated; for example, in maize this can include the number of kernels per row on the ear, number of rows of kernels on the ear, kernel abortion, kernel weight, kernel size, kernel density and physical grain quality. The following illustrates examples of screening assays useful for identifying desired traits in maize plants. These can be readily adapted for screening other plants such as canola, cotton, and soybean either as hybrids or inbreds.
Transgenic maize plants having nitrogen use efficiency are identified by screening in fields with three levels of nitrogen fertilizer being applied, e.g. low level (0 pounds/acre), medium level (80 pounds/acre) and high level (180 pounds/acre). Plants with enhanced nitrogen use efficiency provide higher yield as compared to control plants.
Transgenic maize plants having enhanced yield are identified by screening the transgenic plants over multiple locations with plants grown under optimal production management practices and maximum weed and pest control. A useful target for improved yield is a 5% to 10% increase in yield as compared to yield produced by plants grown from seed for a control plant. Selection methods may be applied in multiple and diverse geographic locations and over one or more planting seasons to statistically distinguish yield improvement from natural environmental effects.
Transgenic maize plants having enhanced water use efficiency are identified by screening plants in an assay where water is withheld for period to induce stress followed by watering to revive the plants. For example, a useful selection process imposes 3 drought/re-water cycles on plants over a total period of 15 days after an initial stress free growth period of 11 days. Each cycle consists of 5 days, with no water being applied for the first four days and a water quenching on the 5th day of the cycle. The primary phenotypes analyzed by the selection method are the changes in plant growth rate as determined by height and biomass during a vegetative drought treatment.
Transgenic maize plants having enhanced cold tolerance are identified by screening plants in a cold germination assay and/or a cold tolerance field trial. In a cold germination assay trays of transgenic and control seeds are placed in a dark growth chamber at 9.7 degrees Celsius for 24 days. Seeds having higher germination rates as compared to the control are identified as having enhanced cold tolerance. In a cold tolerance field trial plants with enhanced cold tolerance are identified from field planting at an earlier date than conventional spring planting for the field location. For example, seeds are planted into the ground around two weeks before local farmers begin to plant maize so that a significant cold stress is exerted onto the crop. As a control, seeds also are planted under local optimal planting conditions such that the crop has little or no exposure to cold condition. At each location, seeds are planted under both cold and normal conditions preferably with multiple repetitions per treatment.
The foregoing description and the examples presented in this disclosure describe the subject matter of this invention, which includes the following: (I) a recombinant DNA construct comprising DNA that undergoes processing to an RNA comprising single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to said transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of said hybridized segment; (II) a recombinant DNA construct comprising DNA that undergoes processing to an RNA comprising single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to said transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of said hybridized segment, wherein said processing of DNA to an RNA comprising single-stranded RNA comprises transcription of said DNA to an RNA intermediate comprising one or more double-stranded RNA stems; (III) a recombinant DNA construct comprising DNA that undergoes processing to an RNA comprising single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to said transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of said hybridized segment, wherein length of said single-stranded RNA comprises between about 10 to about 100 nucleotides; (IV) a recombinant DNA construct comprising DNA that undergoes processing to an RNA comprising single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to said transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of said hybridized segment, further comprising at least one element selected from the group consisting of: (A) promoter functional in a eukaryotic cell; (B) a Pol III promoter operably linked to said DNA that undergoes processing to an RNA comprising single-stranded RNA; (C) DNA that is processed to an RNA aptamer; (D) a transgene transcription unit; (E) DNA encoding a spliceable intron; (F) DNA encoding a self-splicing ribozyme; (G) DNA encoding a site-specific recombinase recognition site; (H) DNA encoding a gene suppression element; and (I) DNA encoding a transcription regulatory element; (V) a recombinant DNA construct comprising DNA that undergoes processing to an RNA comprising single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to said transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of said hybridized segment, wherein said at least one target gene comprises: (A) coding sequence, non-coding sequence, or both coding and non-coding sequence; or (B) a single target gene, or multiple target genes; or (C) one or more of the group consisting of: (1) an endogenous gene of a eukaryote, (2) a transgene of a transgenic plant, (3) an endogenous gene of a pest or pathogen of a plant, and (4) an endogenous gene of a symbiont associated with a pest or pathogen of a plant; (VI) a recombinant DNA construct comprising DNA that undergoes processing to an RNA comprising single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to said transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of said hybridized segment, wherein said binding of said single-stranded RNA to said transcript: (A) inhibits double-stranded RNA-mediated suppression of said at least one target gene; or (B) inhibits translation of said transcript; (VII) a recombinant DNA construct comprising DNA that undergoes processing to an RNA comprising single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to said transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of said hybridized segment, wherein said binding of said single-stranded RNA to said transcript: (A) inhibits double-stranded RNA-mediated suppression of said at least one target gene; or (B) inhibits translation of said transcript; and wherein: (1) said binding of said single-stranded RNA to said transcript inhibits double-stranded RNA-mediated suppression of said at least one target gene and the length of said hybridized segment comprises between about 10 to about 100 base pairs; (2) said binding of said single-stranded RNA to said transcript inhibits translation of said transcript and the length of said hybridized segment comprises between about 10 to about 50 base pairs; or (3) said binding of said single-stranded RNA to said transcript inhibits translation of said transcript and the length of said hybridized segment comprises between about 19 to about 50 base pairs, said hybridized segment comprises smaller segments of 9 or fewer contiguous, perfectly complementary base pairs, and at least one mismatch or insertion is between each pair of said smaller segments; (VIII) a recombinant DNA construct comprising DNA that undergoes processing to an RNA comprising single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to said transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of said hybridized segment, wherein said binding of said single-stranded RNA to said transcript: (A) inhibits double-stranded RNA-mediated suppression of said at least one target gene; or (B) inhibits translation of said transcript; and wherein said binding of said single-stranded RNA to said transcript inhibits double-stranded RNA-mediated suppression of said at least one target gene and the length of said hybridized segment comprises between about 10 to about 100 base pairs, and said double-stranded RNA-mediated suppression comprises cleavage of said transcript by said RNase III ribonuclease, and said cleavage is mediated by binding of a small RNA to said transcript; (IX) a recombinant DNA construct comprising DNA that undergoes processing to an RNA comprising single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to said transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of said hybridized segment, wherein said binding of said single-stranded RNA to said transcript: (A) inhibits double-stranded RNA-mediated suppression of said at least one target gene; or (B) inhibits translation of said transcript; and wherein said small RNA is: (1) an endogenous small RNA or a transgenic small RNA; or (2) selected from the group consisting of a miRNA, an siRNA, a trans-acting siRNA, a phased small RNA, a natural antisense transcript siRNA, and a natural antisense transcript miRNA; (X) a recombinant DNA construct comprising DNA that undergoes processing to an RNA comprising single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to said transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of said hybridized segment, wherein said binding of said single-stranded RNA to said transcript: (A) inhibits double-stranded RNA-mediated suppression of said at least one target gene; or (B) inhibits translation of said transcript; and wherein said binding of said single-stranded RNA to said transcript inhibits double-stranded RNA-mediated suppression of said at least one target gene and the length of said hybridized segment comprises between about 10 to about 100 base pairs, and said double-stranded RNA-mediated suppression comprises cleavage of said transcript by said RNase III ribonuclease, and said cleavage is mediated by binding of a small RNA to said transcript; and wherein said hybridized segment comprises at least one mismatch or at least one insertion in said hybridized segment at a position that results in inhibiting cleavage of said transcript by said RNase III ribonuclease; (XI) a recombinant DNA construct comprising DNA that undergoes processing to an RNA comprising single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to said transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of said hybridized segment, wherein said binding of said single-stranded RNA to said transcript: (A) inhibits double-stranded RNA-mediated suppression of said at least one target gene; or (B) inhibits translation of said transcript; and wherein said binding of said single-stranded RNA to said transcript inhibits double-stranded RNA-mediated suppression of said at least one target gene and the length of said hybridized segment comprises between about 10 to about 100 base pairs, and said double-stranded RNA-mediated suppression comprises cleavage of said transcript by said RNase III ribonuclease, and said cleavage is mediated by binding of a small RNA to said transcript; and wherein said small RNA is a mature miRNA, said binding is at a miRNA recognition site in said transcript, said cleavage of said transcript occurs at said miRNA recognition site, and said hybridized segment is formed at least partially within said miRNA recognition site; (XII) a recombinant DNA construct comprising DNA that undergoes processing to an RNA comprising single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to said transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of said hybridized segment, wherein said binding of said single-stranded RNA to said transcript: (A) inhibits double-stranded RNA-mediated suppression of said at least one target gene; or (B) inhibits translation of said transcript; and wherein said binding of said single-stranded RNA to said transcript inhibits double-stranded RNA-mediated suppression of said at least one target gene and the length of said hybridized segment comprises between about 10 to about 100 base pairs, and said double-stranded RNA-mediated suppression comprises cleavage of said transcript by said RNase III ribonuclease, and said cleavage is mediated by binding of a small RNA to said transcript; and wherein said small RNA is a mature miRNA, said binding is at a miRNA recognition site in said transcript, said cleavage of said transcript occurs at said miRNA recognition site, and said hybridized segment is formed at least partially within said miRNA recognition site; and wherein said hybridized segment comprises: (1) at least one mismatch between said single-stranded RNA and said miRNA recognition site at positions corresponding to positions 9, 10, or 11 of said mature miRNA, or (2) at least one insertion at a position in said single-stranded RNA at positions corresponding to positions 10-11 of said mature miRNA, or (3) an A, G, or C (but not a U) at a position corresponding to the 5′ terminus of said mature miRNA, but does not include (a) mismatches between said single-stranded RNA and said miRNA recognition site at positions of said miRNA recognition site corresponding to positions 9, 10, or 11 (in 3′ to 5′ direction) of said mature miRNA, or (b) insertions at a position in said single-stranded RNA at positions of said miRNA recognition site corresponding to positions 10 or 11 (in 3′ to 5′ direction) of said mature miRNA; (XIII) a recombinant DNA construct comprising DNA that undergoes processing to an RNA comprising single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to said transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of said hybridized segment, wherein said binding of said single-stranded RNA to said transcript: (A) inhibits double-stranded RNA-mediated suppression of said at least one target gene; or (B) inhibits translation of said transcript; and wherein said binding of said single-stranded RNA to said transcript inhibits translation of said transcript, and said binding of said single-stranded RNA to said transcript occurs: (i) at least partially within the 5′ untranslated region or 3′ untranslated region of said transcript; or (ii) within or in the vicinity of the start codon or of the 5′ cap; (XIV) a recombinant DNA construct comprising DNA that undergoes processing to an RNA comprising single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to said transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of said hybridized segment, wherein said binding of said single-stranded RNA to said transcript: (A) inhibits double-stranded RNA-mediated suppression of said at least one target gene; or (B) inhibits translation of said transcript; and wherein said binding of said single-stranded RNA to said transcript inhibits translation of said transcript, and said hybridized segment is resistant to cleavage by said RNase III ribonuclease; (XV) a method of modulating expression of a target gene, comprising expressing in a cell a recombinant DNA construct comprising DNA that undergoes processing to an RNA comprising single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to said transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of said hybridized segment; (XVI) a method of modulating expression of a target gene, comprising expressing in a cell a recombinant DNA construct comprising DNA that undergoes processing to an RNA comprising single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to said transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of said hybridized segment; and wherein said binding of said single-stranded RNA to said transcript: (A) inhibits double-stranded RNA-mediated suppression of said at least one target gene, thereby increasing expression of said target gene; or (B) inhibits translation of said transcript, thereby decreasing expression of said target gene; (XVII) a non-natural plant chromosome or plastid comprising a recombinant DNA construct comprising DNA that undergoes processing to an RNA comprising single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to said transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of said hybridized segment; (XVIII) a non-natural transgenic plant cell having in its genome a recombinant DNA construct comprising DNA that undergoes processing to an RNA comprising single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to said transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of said hybridized segment, or a non-natural transgenic plant or a non-natural transgenic plant seed or a non-natural transgenic pollen grain comprising said non-natural transgenic plant cell; (XIX) a non-natural partially transgenic plant comprising: (A) a non-natural transgenic plant cell having in its genome a recombinant DNA construct comprising DNA that undergoes processing to an RNA comprising single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to said transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of said hybridized segment and further comprising non-transgenic tissue: or (B) a transgenic rootstock comprising a non-natural transgenic plant cell having in its genome a recombinant DNA construct comprising DNA that undergoes processing to an RNA comprising single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to said transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of said hybridized segment and further comprising a non-transgenic scion; (XX) a recombinant DNA construct transcribable in a plant cell, comprising a promoter that is functional in said plant cell and operably linked to at least one polynucleotide selected from: (A) DNA encoding a cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (B) DNA encoding a 5′-modified cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (C) DNA encoding a translational inhibitor to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (D) DNA encoding a decoy to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (E) DNA encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of at least one miRNA target identified in Tables 2 or 3, wherein a miRNA recognition site in said native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage; (F) DNA encoding a miRNA precursor which is processed into a miRNA for suppressing expression of at least one miRNA target identified in Tables 2 or 3; (G) DNA encoding double-stranded RNA which is processed into siRNAs for suppressing expression of at least one miRNA target identified in Tables 2 or 3; and (H) DNA encoding a ta-siRNA which is processed into siRNAs for suppressing expression of at least one miRNA target identified in Tables 2 or 3; (XXI) a recombinant DNA construct transcribable in a plant cell, comprising a promoter that is functional in said plant cell and operably linked to at least one polynucleotide selected from: (A) DNA encoding a cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (B) DNA encoding a 5′-modified cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (C) DNA encoding a translational inhibitor to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (D) DNA encoding a decoy to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (E) DNA encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of at least one miRNA target identified in Tables 2 or 3, wherein a miRNA recognition site in said native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage; (F) DNA encoding a miRNA precursor which is processed into a miRNA for suppressing expression of at least one miRNA target identified in Tables 2 or 3; (G) DNA encoding double-stranded RNA which is processed into siRNAs for suppressing expression of at least one miRNA target identified in Tables 2 or 3; and (H) DNA encoding a ta-siRNA which is processed into siRNAs for suppressing expression of at least one miRNA target identified in Tables 2 or 3; and wherein said at least one miRNA target identified in Tables 2 or 3 is at least one selected from the group consisting of a miR156 target, a miR160 target, a miR164 target, a miR166 target, a miR167 target, a miR169 target, a miR171 target, a miR172 target, a miR319 target, miR395 target, a miR396 target, a miR398 target, a miR399 target, a miR408 target, a miR444 target, a miR528 target, a miR167g target, a miR169g target, COP1 (constitutive photomorphogenesis1), GA2ox (gibberellic acid 2 oxidase), GA20ox (gibberellic acid 20 oxidase), HB2 (homeobox 2), HB2-4 (homeobox 2 and homeobox 4), HB4 (homeobox 4), LG1 (liguleless1), SPX (SYG1, PHO81 and XPR1 domain; PFAM entry PF03105 at www.sanger.ac.uk), VIMla (variant in methlylation 1a), DHS1 (deoxyhypusine synthase), DHS2 (deoxyhypusine synthase), DHS3 (deoxyhypusine synthase), DHS4 (deoxyhypusine synthase), DHS5 (deoxyhypusine synthase), DHS6 (deoxyhypusine synthase), DHS7 (deoxyhypusine synthase), DHS8 (deoxyhypusine synthase), CRF (corn RING finger; RNF169), G1543a (maize orthologue of Arabidopsis thaliana homeobox 17), G1543b (maize orthologue of Arabidopsis thaliana homeobox 17), GS3 (grain size 3), and GW2 (grain weight 2); (XXII) a recombinant DNA construct transcribable in a plant cell, comprising a promoter that is functional in said plant cell and operably linked to at least one polynucleotide selected from: (A) DNA encoding a cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (B) DNA encoding a 5′-modified cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (C) DNA encoding a translational inhibitor to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (D) DNA encoding a decoy to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (E) DNA encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of at least one miRNA target identified in Tables 2 or 3, wherein a miRNA recognition site in said native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage; (F) DNA encoding a miRNA precursor which is processed into a miRNA for suppressing expression of at least one miRNA target identified in Tables 2 or 3; (G) DNA encoding double-stranded RNA which is processed into siRNAs for suppressing expression of at least one miRNA target identified in Tables 2 or 3; and (H) DNA encoding a ta-siRNA which is processed into siRNAs for suppressing expression of at least one miRNA target identified in Tables 2 or 3; and wherein said at least one miRNA target identified in Tables 2 or 3 is at least one selected from the group consisting of a miR156 target, a miR160 target, a miR164 target, a miR166 target, a miR167 target, a miR169 target, a miR171 target, a miR172 target, a miR319 target, miR395 target, a miR396 target, a a miR398 target, a miR399 target, a miR408 target, a miR444 target, a miR528 target, a miR167g target, a miR169g target, COP1 (constitutive photomorphogenesis1), GA2ox (gibberellic acid 2 oxidase), GA20ox (gibberellic acid 20 oxidase), HB2 (homeobox 2), HB2-4 (homeobox 2 and homeobox 4), HB4 (homeobox 4), LG1 (liguleless1), SPX (SYG1, PHO81 and XPR1 domain; PFAM entry PF03105 at www.sanger.ac.uk), VIMla (variant in methlylation 1a), DHS1 (deoxyhypusine synthase), DHS2 (deoxyhypusine synthase), DHS3 (deoxyhypusine synthase), DHS4 (deoxyhypusine synthase), DHS5 (deoxyhypusine synthase), DHS6 (deoxyhypusine synthase), DHS7 (deoxyhypusine synthase), DHS8 (deoxyhypusine synthase), CRF (corn RING finger; RNF169), G1543a (maize orthologue of Arabidopsis thaliana homeobox 17), G1543b (maize orthologue of Arabidopsis thaliana homeobox 17), GS3 (grain size 3), and GW2 (grain weight 2); and wherein said at least one polynucleotide is at least one selected from the group consisting of DNA encoding a nucleotide sequence selected from SEQ ID NOs: 1120, 1121, 1122, 1248, 1257, 1313, 1314, 1364, 1387, 1478, 1489, 1490, 1491, 1492, 1493, 1585, 1597, 1598, 1599, 1713, 1752, 1753, 1801, 1802, 1820, 1927, 1929, 1931, 1971, 2006, 2007, 2008, 2010, 2012, 2014, 2016, 2018, 2022, 2023, 2025, 2027, 2029, 2031, 2033, 2035, 2037, 2039, 2041, 2043, 2045, 2047, 2049, 2051, 2053, 2055, 2056, 2057, 2059, 2060, 2061, and 2063; and (XXIII) a recombinant DNA construct transcribable in a plant cell, comprising a promoter that is functional in said plant cell and operably linked to at least one polynucleotide selected from: (A) DNA encoding a cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (B) DNA encoding a 5′-modified cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (C) DNA encoding a translational inhibitor to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (D) DNA encoding a decoy to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (E) DNA encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of at least one miRNA target identified in Tables 2 or 3, wherein a miRNA recognition site in said native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage; (F) DNA encoding a miRNA precursor which is processed into a miRNA for suppressing expression of at least one miRNA target identified in Tables 2 or 3; (G) DNA encoding double-stranded RNA which is processed into siRNAs for suppressing expression of at least one miRNA target identified in Tables 2 or 3; and (H) DNA encoding a ta-siRNA which is processed into siRNAs for suppressing expression of at least one miRNA target identified in Tables 2 or 3; amd wherein said recombinant DNA construct is stably integrated into a plastid or a chromosome of said plant cell.

EXAMPLES

Example 1

This example illustrates the making and using of a “cleavage blocker” recombinant DNA construct including DNA that undergoes processing to an RNA including single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA that imparts to the transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of the hybridized segment, wherein the binding of the single-stranded RNA to the transcript (and the resultant formation of the hybridized segment) inhibits double-stranded RNA-mediated suppression of a target gene. More specifically, this example describes constructs for producing in planta an artificial or engineered miRNA or a cleavage blocker and use of the cleavage blocker to inhibit miRNA-mediated suppression of an Arabidopsis GL1 gene in transformed plant cells.
Target Gene:
The Arabidopsis GLABROUS1 (GL1) gene is required for trichome synthesis; GL1 mutants lack leaf trichomes. GL1 is encoded by the DNA sequence

(SEQ ID NO: 1)

ATGAGAATAAGGAGAAGAGATGAAAAAGAGAATCAAGAATACAAGAAAGG

TTTATGGACAGTTGAAGAAGACAACATCCTTATGGACTATGTTCTTAATC

ATGGCACTGGCCAATGGAACCGCATCGTCAGAAAAACTGGGCTAAAGAGA

TGTGGGAAAAGTTGTAGACTGAGATGGATGAATTATTTGAGCCCTAATGT

GAACAAAGGCAATTTCACTGAACAAGAAGAAGACCTCATTATTCGTCTCC

ACAAGCTCCTCGGCAATAGATGGTCTTTGATAGCTAAAAGAGTACCGGGA

AGAACAGATAACCAAGTCAAGAACTACTGGAACACTCATCTCAGCAAAAA

ACTCGTCGGAGATTACTCCTCCGCCGTCAAAACCACCGGAGAAGACGACG

ACTCTCCACCGTCATTGTTCATCACTGCCGCCACACCTTCTTCTTGTCAT

CATCAACAAGAAAATATCTACGAGAATATAGCCAAGAGCTTTAACGGCGT

CGTATCAGCTTCGTACGAGGATAAACCAAAACAAGAACTGGCTCAAAAAG

ATGTCCTAATGGCAACTACTAATGATCCAAGTCACTATTATGGCAATAAC

GCTTTATGGGTTCATGACGACGATTTTGAGCTTAGTTCACTCGTAATGAT

GAATTTTGCTTCTGGTGATGTTGAGTACTGCCTTTAG,

includes a miRNA recognition site, which has the sequence CTCCACCGTCATTGTTCATCA (SEQ ID NO: 2) and which is also indicated by the underlined text at nucleotide positions 404 to 424 of SEQ ID NO: 1.

MicroRNA:
Selected as a scaffold or initial sequence for designing an artificial miRNA was DNA derived from a soybean“miRMON1” precursor having the sequence

(SEQ ID NO: 3)

AATTCATTACATTGATAAAACACAATTCAAAAGATCAATGTTCCACTTCA

TGCAAAGACATTTCCAAAATATGTGTAGGTAGAGGGGTTTTACAGGATCG

TCCTGAGACCAAATGAGCAGCTGACCACATGATGCAGCTATGTTTGCTAT

TCAGCTGCTCATCTGTTCTCAGGTCGCCCTTGTTGGACTGTCCAACTCCT

ACTGATTGCGGATGCACTTGCCACAAATGAAAATCAAAGCGAGGGGAAAA

GAATGTAGAGTGTGACTACGATTGCATGCATGTGATTTAGGTAATTAAGT

TACATGATTGTCTAATTGTGTTTATGGAATTGTATA,

where nucleotides of the mature miRNA (“miRMON1”) are indicated by underlined text at nucleotide positions 104 to 124 of SEQ ID NO: 3. The encoded transcript was predicted to have the fold-back structure depicted in FIG. 1, Panel A, and is a segment of a longer miRMON1 precursor having the sequence

(SEQ ID NO: 4)

AAAATTCATTACATTGATAAAACACAATTCAAAAGATCAATGTTCCACTT

CATGCAAAGACATTTCCAAAATATGTGTAGGTAGAGGGGTTTTACAGGAT

CGTCCTGAGACCAAATGAGCAGCTGACCACATGATGCAGCTATGTTTGCT

ATTCAGCTGCTCATCTGTTCTCAGGTCGCCCTTGTTGGACTGTCCAACTC

CTACTGATTGCGGATGCACTTGCCACAAATGAAAATCAAAGCGAGGGGAA

AAGAATGTAGAGTGTGACTACGATTGCATGCATGTGATTTAGGTAATTAA

GTTACATGATTGTCTAATTGTGTTTATGGAATTGTATATTTTCAGACCAG

GCACCTGTAACTAATTATAGGTACCATACCTTAAAATAAGTCCAACTAAG

TCCATGTCTGTGATTTTTTAGTGTCACAAATCACAATCCATTGCCATTGG

TTTTTTAATTTTTCATTGTCTGTTGTTTAACTAACTCTAGCTTTTTAGCT

GCTTCAAGTACAGATTCCTCAAAGTGGAAAATGTTCTTTGAAGTCAATAA

AAAGAGCTTTGATGATCATCTGCATTGTCTAAGTTGGATAAACTAATTAG

AGAGAACTTTTGAACTTTGTCTACCAAATATCTGTCAGTGTCATCTGTCA

GTTCTGCAAGCTGAAGTGTTGAATCCACGAGGTGCTTGTTGCAAAGTTGT

GATATTAAAAGACATCTACGAAGAAGTTCAAGCAAAACTCTTTTTGGC,

where nucleotides of the mature miRMON1 are indicated by underlined text at nucleotide positions 106 to 126 of SEQ ID NO: 4; this longer miRMON1 precursor was previously disclosed as SEQ ID NO: 38 in U.S. patent application Ser. No. 11/303,745, published as U. S. Patent Application Publication 2006/200878, and is specifically incorporated herein by reference). The longer precursor (SEQ ID NO: 4) is also suitable as a scaffold.

DNA encoding an engineered “miRGL1” miRNA precursor derived from SEQ ID NO: 3 was designed to produce an engineered miRGL1 precursor transcript that is processed to an artificial “miRGL1” mature miRNA for suppressing the Arabidopsis endogenous gene, GL1. The miRGL1 precursor had the sequence

(SEQ ID NO: 5)

AATTCATTACATTGATAAAACACAATTCAAAAGATCAATGTTCCACTTCA

TGCAAAGACATTTCCAAAATATGTGTAGGTAGAGGGGTTTTACAGGATCG

TCCTGATGAACAATGACGGTGGAGCCACATGATGCAGCTATGTTTGCTAT

CTCCACCGTCATCGTCCATCAGGTCGCCCTTGTTGGACTGTCCAACTCCT

ACTGATTGCGGATGCACTTGCCACAAATGAAAATCAAAGCGAGGGGAAAA

GAATGTAGAGTGTGACTACGATTGCATGCATGTGATTTAGGTAATTAAGT

TACATGATTGTCTAATTGTGTTTATGGAATTGTATA,

where nucleotides of the mature miRNA (“miRGL1”) are indicated by underlined text at nucleotide positions 104 to 124 of SEQ ID NO: 5 and nucleotides of the corresponding opposite strand designated miRNA* (“miRGL1*”) are indicated by italicized text at nucleotide positions 151 to 171 of SEQ ID NO: 5. This miRGL1 precursor was predicted to have the fold-back structure depicted in FIG. 1, Panel B and is processed in planta to the mature miRGL1, which has the sequence (in 5′ to 3′ direction) TGATGAACAATGACGGTGGAG (SEQ ID NO: 6, alternatively written in 3′ to 5′ direction as GAGGTGGCAGTAACAAGTAGT).

Cleavage Blocker:
DNA encoding a cleavage blocker (“miRGL1-CB”) precursor derived from SEQ ID NO: 3 was designed to transcribe to an engineered “cleavage blocker”-type miRNA precursor that is processed to an RNA including single-stranded RNA that binds to the transcript of the target gene GL1 to form a hybridized segment of at least partially double-stranded RNA that imparts to the GL1 transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of the hybridized segment, wherein the binding of the single-stranded RNA to the transcript (and the resultant formation of the hybridized segment) inhibits double-stranded RNA-mediated suppression of the at least one target gene, wherein the suppression is mediated by miRGL1. The miRGL1-CB precursor had the sequence

(SEQ ID NO: 7)

AATTCATTACATTGATAAAACACAATTCAAAAGATCAATGTTCCACTTCA

TGCAAAGACATTTCCAAAATATGTGTAGGTAGAGGGGTTTTACAGGATCG

TCCTGATGAACATAGACGGTGGAGCCACATGATGCAGCTATGTTTGCTAT

CTCCACCGTCTACGTCCATCAGGTCGCCCTTGTTGGACTGTCCAACTCCT

ACTGATTGCGGATGCACTTGCCACAAATGAAAATCAAAGCGAGGGGAAAA

GAATGTAGAGTGTGACTACGATTGCATGCATGTGATTTAGGTAATTAAGT

TACATGATTGTCTAATTGTGTTTATGGAATTGTATA,

where nucleotides of the mature cleavage blocker (“miRGL1-CB”) are indicated by underlined text at nucleotide positions 104 to 124 of SEQ ID NO: 7 and nucleotides of the corresponding opposite strand miRNA* (“miRGL1-CB*”) are indicated by italicized text at nucleotide positions 151 to 171 of SEQ ID NO: 7. Nucleotides at positions 113 and 114 of SEQ ID NO: 7 are indicated by bold underlined text and correspond to positions 10 and 11 (in 3′ to 5′ direction) of the mature miRGL1-CB1; these two nucleotides were selected to be intentionally mismatched to nucleotides of the miRNA recognition site (SEQ ID NO: 2) of GL1 (SEQ ID NO: 1) to prevent cleavage by an RNase III ribonuclease. The encoded miRGL1-CB RNA precursor was predicted to have the fold-back structure depicted in FIG. 1, Panel C and is processed in planta to the mature miRGL1-CB, which has the sequence (in 5′ to 3′ direction) TGATGAACATAGACGGTGGAG (SEQ ID NO: 8, alternatively written in 3′ to 5′ direction as GAGGTGGCAGATACAAGTAGT). FIG. 1, Panel E depicts an alignment of the GL1 miRNA recognition site (SEQ ID NO: 2), the mature miRGL1 in 3′ to 5′ direction (SEQ ID NO: 6), and the mature miRGL1-CB in 3′ to 5′ direction (SEQ ID NO: 8).

miRGL1 Sensor:
DNA encoding a “miRGL1-sensor” having the sequence
(SEQ ID NO: 9)

TccagctgctcatttggtctcaTGATCACTGCGGCCGCAATACAgccata

gatcacttgatgtcaC GAccaccgtcattgttcatcagatttctctctgc

aagcg

was designed to include an artificial miRGL1 recognition site having the sequence GACCACCGTCATTGTTCATCA (SEQ ID NO: 10), which is also indicated by underlined text at nucleotide positions 67 and 87 of SEQ ID NO: 9. Nucleotides at positions 67 and 68 of SEQ ID NO: 9 (or nucleotides at positions 1 and 2 of SEQ ID NO: 10) are indicated by bold underlined text and correspond to positions 1 and 2 (in 3′ to 5′ direction) of the mature miRGL1; these two nucleotides were selected to be intentionally mismatched to the last two nucleotides on the 3′ end of the mature miRGL1 (SEQ ID NO: 6) to prevent transitivity.
Three plasmids for Agrobacterium-mediated transformation were constructed:

- (1) “35S/miRGL1/Term”—this plasmid included a construct containing, in 5′ to 3′ direction, (a) a 35S promoter driving expression of (b) a miRGL1 precursor (SEQ ID NO: 5), and (c) a nos terminator;
- (2) “35S/GFP/miRGL1-sensor/Term”—this plasmid included a construct containing, in 5′ to 3′ direction, (a) a 35S promoter operably linked to (b) a green fluorescent protein (GFP) coding sequence, (c) a miRGL1-sensor sequence (SEQ ID NO: 9), and (d) a nos terminator;
- (3) “35S/miRGL1-CB”—this plasmid included a construct containing, in 5′ to 3′ direction, (a) a 35S promoter driving expression of (b) a miRGL1-CB precursor (SEQ ID NO: 7).

An aspect of this invention was demonstrated using protocols described in Kościańska et al. (2005) Plant Mol. Biol., 59:647-661). Nicotiana benthamiana plants were transiently transformed using Agrobacterium with various combinations of these plasmids and, where necessary, “filler” (null plasmid) Agrobacterium to ensure infiltration of equal amounts of Agrobacterium.
Nicotiana benthamiana plants transformed with plasmid (2) exhibited GFP (green) fluorescence when visualized under UV light. In plants transformed with plasmids (1) and (2), GFP fluorescence was abolished with only chlorophyll (red) fluorescence observed under UV light, indicating that the mature miRGL1 microRNA suppressed expression of GFP. In plants transformed with plasmids (1), (2) and (3), GFP fluorescence was restored, indicating that the miRGL1-CB cleavage blocker inhibited double-stranded RNA-mediated (i.e., mRGL1-mediated) suppression of the target gene GFP by protecting the miRGL1 recognition site from being cleaved by the mature miRGL1, resulting in increased expression (fluorescence) of the target gene GFP relative to its expression in the absence of the cleavage blocker.
In another demonstration of this invention, stably transformed Arabidopsis thaliana plants were produced by Agrobacterium-mediated transformation with a plasmid expressing a miRGL1 precursor (SEQ ID NO: 5), which is processed in planta to a “miRGL1” mature miRNA for suppressing the Arabidopsis endogenous gene, GL1. The resulting transformed Arabidopsis plants exhibited leaves without trichomes, indicating suppression of the target gene GLABROUS1. Arabidopsis plants homozygous for miRGL1 DNA are further transformed with a plasmid expressing a miRGL1-CB precursor (SEQ ID NO: 7) and selected using kanamycin resistance. In these double transformant plants, in planta expression of the mature cleavage blocker miRGL1-CB (in 3′ to 5′ direction, SEQ ID NO: 8) inhibits double-stranded RNA-mediated (i.e., mRGL1-mediated) suppression of the target gene GLABROUS1 (GL1) by protecting the miRGL1 recognition site from being cleaved by the mature miRGL1, resulting in restoration of trichome production (indicating increased expression of the target gene GL1 relative to its expression in the absence of the cleavage blocker).

Example 2

This example illustrates an alternative “cleavage blocker” recombinant DNA construct having modification at a position corresponding to the 5′ terminus of the mature miRNA that natively binds to the recognition site of the target gene, i.e., a “5′-modified cleavage blocker” that is transgenically produced in planta and a method of use of this cleavage blocker to inhibit miRNA-mediated suppression of a target gene in transformed plant cells.
In one example, DNA encoding an artificial miRNA (miRGL1) precursor (SEQ ID NO: 6) was modified by a single nucleotide change (changing the 5′ terminus of the mature miRGL1 from a U to a C) to yield the 5′-modified cleavage blocker precursor sequence

(SEQ ID NO: 11)

AATTCATTACATTGATAAAACACAATTCAAAAGATCAATGTTCCACTTCA

TGCAAAGACATTTCCAAAATATGTGTAGGTAGAGGGGTTTTACAGGATCG

TCCCGATGAACAATGACGGTGGAGCCACATGATGCAGCTATGTTTGCTAT

CTCCACCGTCATCGTCCATCGGGTCGCCCTTGTTGGACTGTCCAACTCCT

ACTGATTGCGGATGCACTTGCCACAAATGAAAATCAAAGCGAGGGGAAAA

GAATGTAGAGTGTGACTACGATTGCATGCATGTGATTTAGGTAATTAAGT

TACATGATTGTCTAATTGTGTTTATGGAATTGTATA,

where nucleotides of the mature 5′-modified cleavage blocker are indicated by underlined text at nucleotide positions 104 to 124 of SEQ ID NO: 11 (for comparison, nucleotides of SEQ ID NO: 11 that correspond to miRGL1* nucleotides in SEQ ID NO: 6 are indicated by italicized text at nucleotide positions 151 to 171 of SEQ ID NO: 11). This 5′-modified cleavage blocker RNA precursor was predicted to have the fold-back structure depicted in FIG. 1, Panel D and is processed in planta to the mature 5′-modified cleavage blocker, which has the sequence (in 5′ to 3′ direction) CGATGAACAATGACGGTGGAG (SEQ ID NO: 12, alternatively written in 3′ to 5′ direction as GAGGTGGCAGTAACAAGTAGC). Nicotiana benthaminiana was transiently transfected using procedures similar to those described in Example 2. The resulting mature small RNA processed from this 5′-modified cleavage blocker RNA precursor was unexpectedly observed to function as a cleavage blocker, inhibiting miRGL1-mediated suppression of the target gene GFP.

Two 5′-modified variants of the miRGL1-CB precursor (SEQ ID NO: 7) were made, wherein the position corresponding to the 5′ terminus of the mature miRGL1-CB was changed from a T to an A or from a T to a C, respectively, but wherein the mismatches corresponding to positions 10 or 11 (in 3′ to 5′ direction) of the mature miRGL1 were preserved. Both variants were predicted to have a fold-back structure (not shown) similar to those shown in FIG. 1, Panels A through D. The “5′-A variant” had the nucleotide sequence

(SEQ ID NO: 13)

AATTCATTACATTGATAAAACACAATTCAAAAGATCAATGTTCCACTTCA

TGCAAAGACATTTCCAAAATATGTGTAGGTAGAGGGGTTTTACAGGATCG

TCCAGATGAACATAGACGGTGGAGCCACATGATGCAGCTATGTTTGCTAT

CTCCACCGTCTACGTCCA T CTGGTCGCCCTTGTTGGACTGTCCAACTCCT

ACTGATTGCGGATGCACTTGCCACAAATGAAAATCAAAGCGAGGGGAAAA

GAATGTAGAGTGTGACTACGATTGCATGCATGTGATTTAGGTAATTAAGT

TACATGATTGTCTAATTGTGTTTATGGAATTGTATA

and the “5′-C variant” had the nucleotide sequence

(SEQ ID NO: 14)

AATTCATTACATTGATAAAACACAATTCAAAAGATCAATGTTCCACTTCA

TGCAAAGACATTTCCAAAATATGTGTAGGTAGAGGGGTTTTACAGGATCG

TCCCGATGAACATAGACGGTGGAGCCACATGATGCAGCTATGTTTGCTAT

CTCCACCGTCTACGTCCATC T GGTCGCCCTTGTTGGACTGTCCAACTCCT

ACTGATTGCGGATGCACTTGCCACAAATGAAAATCAAAGCGAGGGGAAAA

GAATGTAGAGTGTGACTACGATTGCATGCATGTGATTTAGGTAATTAAGT

TACATGATTGTCTAATTGTGTTTATGGAATTGTATA,

where nucleotides of the mature cleavage blocker are indicated by underlined text at nucleotide positions 104 to 124 of SEQ ID NO: 13 or of SEQ ID NO: 14 (for comparison, nucleotides of SEQ ID NO: 13 or of SEQ ID NO: 14 that correspond to miRGL1* nucleotides in SEQ ID NO: 6 are indicated by italicized text at nucleotide positions 151 to 171 of SEQ ID NO: 13 or of SEQ ID NO: 14).

The “5′-C variant” (SEQ ID NO: 14) was transiently transfected into Nicotiana benthaminiana (using procedures similar to those of Example 2); co-inoculation of the “5′-C” variant and 35S/miRGL1-sensor/Term (without miRGL1) resulted in GFP fluorescence, indicating that the “5′-C variant” was unable to cleave the miRGL1 recognition site and did not have miRNA-like activity.
Both the “5′-A variant” (SEQ ID NO: 13) (plasmid pMON115363) and the “5′-C variant” (SEQ ID NO: 14) (plasmid pMON115349) were tested using transient transfection of Nicotiana benthaminiana (similar to the experiment described in Example 2), and found to also inhibit miRGL1-mediated suppression of the target gene GFP, although not to as great an extent as the original cleavage blocker miRGL1-CB (SEQ ID NO: 7).
The above example serves as guidance in making and using a cleavage blocker (or 5′-modified cleavage blocker) useful for inhibiting miRNA-mediated suppression of a target gene. It is clear to one of ordinary skill in the art that knowledge of the target gene itself is not required, merely the sequence of the mature miRNA sequence or of a miRNA precursor that is processed to the mature miRNA—or, alternatively, knowledge of the miRNA recognition site sequence—in combination with the teachings of this application, in order to devise a cleavage blocker (or 5′-modified cleavage blocker) to inhibit the target gene silencing effects of a given miRNA.
Thus, this application further provides and claims novel cleavage blockers and 5′-modified cleavage blockers for all miRNA sequences that have been publicly disclosed, including, but not limited to, the miRNAs available at miRBase (microrna.sanger.ac.uk), and the mature miRNAs and miRNA precursors disclosed in U.S. patent application Ser. No. 11/303,745 (published as U. S. Patent Application Publication 2006/0200878), Ser. No. 11/974,469 (published as U. S. Patent Application Publication 2009-0070898 A1), Ser. No. 11/868,081 (published as U. S. Patent Application Publication 2008/0115240), Ser. No. 10/884,374 (published as U. S. Patent Application Publication 2005/0144669), and Ser. No. 10/490,955 (now U.S. Pat. No. 7,232,806), which patent application disclosures including the respective sequence listings are specifically incorporated by reference herein.

Example 3

This example provides embodiments of target genes identified as “validated miRNA targets” (i.e., containing a validated miRNA recognition site). Recombinant DNA constructs of this invention are useful for modulating expression of such target genes and for making non-natural transgenic plant cells, plant tissues, and plants (especially non-natural transgenic crop plants) having improved yield or other desirable traits.
Prediction of a recognition site is achieved using methods known in the art, such as sequence complementarity rules as described by Zhang (2005) Nucleic Acids Res., 33:W701-704 and by Rhoades et al. (2002) Cell, 110:513-520. One method to experimentally validate predicted miRNA recognition sites is the technique known as RNA ligase-mediated rapid amplification of cDNA 5′ ends (“5′ RLM-RACE” or “5′ RACE”), which identifies miRNA cleavage patterns; see, for example, Kasschau et al. (2003) Dev. Cell, 4:205-217, and Llave et al. (2002) Science, 297:2053-2056. This approach relies on ligation of an RNA adapter molecule to the 5′ end of the cleavage site and is dependent on the 5′ phosphate left by RNase III enzymes including Ago1. The resulting PCR products are sequenced and the relative number of clones which align to the predicted miRNA cleavage site between nucleotides 10 and 11 relative to the miRNA 5′ end provide an estimate of miRNA activity.
While the standard for validation of a predicted miRNA target is experimental verification of the predicted cleavage, computational validation is also extremely useful for providing a set of potential target genes that is of manageable or practical size. At least two computational validation approaches based on homology of miRNAs and predicted miRNA targets can be used. One approach compares the predicted targets with experimentally verified targets; the predicted target is computationally validated if it is homologous to an experimentally validated target. This approach is expected to identify miRNA targets with high confidence and to become increasingly important as more experimentally validated targets become available. The second approach compares sequences from two species when no known miRNA target information is available. If both miRNAs and predicted miRNA targets are conserved in both species, then predicted targets in both species are deemed validated.
In this example, the first approach was used, wherein computational validation of predicted miRNA targets was based on homology of predicted targets and known targets. A list of experimentally verified plant miRNA target genes was created through mining the literature on miRNA targets from rice (Sunkar et al. (2005) Plant Cell, 17:1397-1411; Luo et al. (2006) FEBS Lett., 580:5111-5116), moss (Physcomitrella patens) (Axtell et al. (2007) Plant Cell, 19:1750-1769; Fattash et al. (2007) BMC Plant Biol., 7:13), poplar (Lu et al. (2005) Plant Cell, 17:2186-2203), green algae (Molnar et al. (2007) Nature, 447:1126-1130), and maize (Lauter et al. (2005) Proc. Natl. Acad. Sci. USA, 102:9412-9417). To this list were added 203 Arabidopsis thaliana loci from the publicly accessible Arabidopsis Small RNA Project (available on line at asrp.cgrb.oregonstate.edu/db/microRNAfamily.html). From this list, a gene function keyword “dictionary” from the available functional annotation was compiled, including known keyword variants (Table 1).
Any functional annotation of a given predicted miRNA target was searched for a match to the dictionary's keywords. A computational algorithm was developed to match the longest keyword first, second longest keyword second, and so on, to reduce false positives in keyword match. Where a match was found, the predicted target was deemed validated. This approach was applied to miRNA targets that had been predicted from proprietary sequence databases from various plant species; the computationally validated miRNA targets thus identified are given in Table 2.
Identification of validated miRNA targets allows the manipulation of the interaction between a given miRNA and its target gene (whether a native gene or a transgene that contains a validated miRNA recognition site). For example, over-expression of a target gene containing a validated miRNA target (validated miRNA recognition site) is expected to reduce the effect of that particular miRNA in the biochemical network or networks involving the miRNA.
Alternatively, an artificial transcript that includes the same miRNA target sequence (or one modified to prevent cleavage by an RNase II ribonuclease) can be used as a miRNA “decoy” (as described in co-assigned U.S. patent application Ser. No. 11/974,469, published as U. S. Patent Application Publication 2009-0070898 A1, which disclosure is specifically incorporated by reference herein), competing with the endogenous target gene to bind to that particular miRNA and thereby reducing the effect of the miRNA (e.g., suppression of the target gene and reduction of the effect of the miRNA on other genes downstream of the target gene) in the biochemical network or networks involving the miRNA. Knowledge of the validated miRNA targets disclosed herein allows one of ordinary skill in the art to use the miRNA target sequences as scaffolds for designing artificial sequences useful as transgenic miRNA decoys to reduce the effect of the miRNA on its target gene(s), or to identify endogenous sequences that are similarly useful as miRNA decoys. Thus, this application further provides and claims miRNA decoys for the validated miRNA targets disclosed herein, as well as miRNA decoys for all miRNA sequences that have been publicly disclosed, including, but not limited to, the miRNAs available at miRBase (microrna.sanger.ac.uk), and the mature miRNAs and miRNA precursors disclosed in U.S. patent application Ser. No. 11/303,745 (published as U.S. Patent Application Publication 2006/0200878), Ser. No. 11/974,469 (published as U. S. Patent Application Publication 2009-0070898 A1), Ser. No. 11/868,081 (published as U. S. Patent Application Publication 2008/0115240), Ser. No. 10/884,374 (published as U. S. Patent Application Publication 2005/0144669), and Ser. No. 10/490,955 (now U.S. Pat. No. 7,232,806), which specifications are specifically incorporated by reference in their entirety herein.
In yet another embodiment, this invention further provides a miRNA-unresponsive transgene by modifying the sequence of a validated miRNA recognition site in the transgene to prevent binding and/or cleavage by that particular miRNA. In one example, increased expression of a gene that is normally modulated by an endogenous miRNA may be achieved by expressing a recombinant DNA construct encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of the gene but wherein a miRNA recognition site in the native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage. In still another embodiment, this invention provides a transgene sequence that is modified by the addition of a validated miRNA recognition site in order to place that transgene under the control of that particular miRNA; in a variation on this, a transgenic plant is made by introducing into its genome both the transgene as well as an exogenous precursor of the particular miRNA that is to regulate the transgene.

TABLE 1

miRNA target keyword dictionary

miR156	Squamosa Promoter Binding Protein, Squamosa Promoter Binding, Squamosa
	Promoter-Binding, SBP-like, SPL, SPL2, SPL15, SPL9, SPL13, SPL4, SPL10, SPL6,
	SPL11, SBP domain containing protein, SBP domain, SBP-domain, teosinte glume
	architecture, tga1
miR157	Squamosa Promoter Binding Protein, Squamosa Promoter Binding, Squamosa
	Promoter-Binding, SBP-like, SPL, SPL2, SPL15, SPL9, SPL13, SPL4, SPL10, SPL6,
	SPL11, SBP domain containing protein, SBP domain, SBP-domain, teosinte glume
	architecture, tga1
miR158	Pentatricopeptide repeat, pentatricopeptide (PPR), PPR, PPR-repeat, pentatricopeptide
miR159	MYB, AtMYB65, AtMYB101, AtMYB104, GAMyB, myb domain protein, myb
	domain, myb protein, DUO1, MYB120, MYB97, MYB65, MYB33, myb-like DNA-
	binding domain, myb-like, myb-like DNA-binding
miR160	Auxin Response Factor, ARF, ARF10, ARF16, ARF17, B3 DNA binding domain
	containing protein, B3 domain, B3 DNA-binding domain, B3 Domain-Containing
miR161	Pentatricopeptide repeat, pentatricopeptide (PPR), PPR, PPR-repeat, EMB2654,
	EMBRYO DEFECTIVE 2654, pentatricopeptide
miR162	Dicer-like 1, Dicer-like1, Dicer like 1, DCL, DCL1, CAF, SUS1, SIN1, ASU1,
	EMB76, EMB60, Dicer
miR163	S-adenosylmethionine-dependent methyltransferase, SAMT, S-adenosyl-L-
	methionine:carboxyl methyltransferase, methyltransferase
miR164	Cup-shaped cotyledon, Cup shaped cotyledon, CUC, NAM, NAC, CUC2, CUC1,
	NAM-like, NAC1, No Apical Meristem, ATAF, ANAC079/ANAC080, ANAC100,
	ANAC092, NAC domain protein, NAC domain, NAC domain-containing protein, NAC
	domain-containing
miR165	Phavoluta, Phabulosa, Revoluta, Corona, PHB, PFV, CNA, HD-ZIPIII, HD-ZIP, HD
	ZIP, REV, PHV, AtHB8, AtHB15, ICU4, ATHB-15, INCURVATA 4, IFL, IFL1, HD-
	ZIP class III HD-Zip protein, HD-ZIP class III, HD-Zip protein, class III HD-Zip
	protein, class III HD-Zip, homeodomain/leucine zipper, rolled leaf1 (rld1), rolled leaf 1
	rolled leaf, rld1, HB1 gene, HB1, HD-ZIP III
miR166	Phavoluta, Phabulosa, Revoluta, Corona, PHB, PFV, CNA, HD-ZIPIII, HD-ZIP, HD
	ZIP, REV, PHV, AtHB8, AtHB15, ICU4, ATHB-15, INCURVATA 4, IFL, IFL1, HD-
	ZIP class III HD-Zip protein, HD-ZIP class III, HD-Zip protein, class III HD-Zip
	protein, class III HD-Zip, homeodomain/leucine zipper, rolled leaf1 (rld1), rolled leaf 1
	rolled leaf, rld1, HB1 gene, HB1, HD-ZIP III
miR167	Auxin Response Factor, ARF, ARF6, ARF8
miR168	Argonaute, AGO, AGO1, PINHEAD, ZWILLE, ZLL, AGO2, AGO3, AGO4, AGO5,
	AGO6, AGO7, AGO8, AGO9, AGO10, PNH/ZLL
miR169	nuclear transcription factor Y, HAP2, CCAAT, CCAAT-binding, NFYa, HAP2b,
	HAP2b-like, HAP2ab-like, HAP2c-like, HAP2c, HAP2a, HAP2a-like
miR170
	Scarecrow/GRAS transcription factors, GRAS, Scarecrow/GRAS, nodulation signaling
	pathway 2 protein, nodulation signaling pathway 2, Nodulation-Signaling Pathway 2,
	NSP2, nodulation signaling pathway, nodulation-Signaling Pathway, NSP1
miR171
	Scarecrow/GRAS transcription factors, GRAS, Scarecrow/GRAS, nodulation signaling
	pathway 2 protein, nodulation signaling pathway 2, Nodulation-Signaling Pathway 2,
	NSP2, nodulation signaling pathway, nodulation-Signaling Pathway, NSP1
miR172	Apetala, AP2, TOE1, TOE2, TOE3, SMZ, SNZ, Target of EAT, TOE, Schnarchzapfen,
	SCHLAFMUTZE, Glossy15, Glossy-15, Glossy 15, AP2 domain containing protein,
	AP2 domain protein, AP2 domain, Apetala floral homeotic protein APETALA2,
	Apetala floral homeotic protein, Apetala protein, APETALA2
miR173	TAS
miR319	Teosinte Branched, Cycloidea, PCF, TCP, TCP2, TCP3, TCP4, TCP 10, TCP24, TCP
	family transcription factor, TCP family, TCP domain protein, TCP-domain protein,
	maternal effect embryo arrest, Cyclin, CyCA, CyCB, CyCC, CyCD, CyCH, CyCT,
	CyCU
miR390	TAS3, TAS, Ser/Thr/Tyr protein kinase, Ser/Thr/Tyr
miR393	Transport inhibitor response, TIR, TIR1, F-box, F box, F-box family protein, F box
	family protein, F-box family, F box family, IPS1, GRH1, GRR1-LIKE, ubiquitin-
	protein ligase, ubiquitin protein ligase, basic helix-loop-helix (bHLH) family protein,
	bHLH, basic helix-loop-helix, F-box domain containing protein, F-box domain protein,
	F-box domain
miR394	F-box, F box, F-box family protein, F box family protein, F-box family, F box family,
	F-box domain containing protein, F-box domain protein, F-box domain
miR395	APS, AST, ATP-sulfurylase, sulfate transporter, sulphate transporter, AST68, APS1,
	APS3, APS4, ATP sulfurylase, sulfate adenylyltransferase, Sulfate transporter
miR396	Growth regulation factor, GRL, GRF, GROWTH-REGULATING FACTOR,
	GROWTH REGULATING FACTOR, AtGRF3, AtGRF4, AtGRF8, AtGRF7, AtGRF1
	AtGRF2, AtGRF
miR397	Laccase, LAC, PCL, plantacyanin, plastacyanin, blue copper binding protein, IRX12,
	copper ion binding
miR398	Copper superoxide dismutase, superoxide dismutase 2, CSD, CSD2, COPPER/ZINC
	SUPEROXIDE DISMUTASE, COPPER ZINC SUPEROXIDE DISMUTASE,
	COPPER-ZINC SUPEROXIDE DISMUTASE, cytochrome c oxidase, cytochromec
	oxidase, cytochrome-c oxidase
miR399	E2 ubiquitin conjugating enzyme, PHO2, ubiquitin-protein ligase, ubiquitin protein
	ligase, UBC24, ubiquitin conjugating enzyme, ubiquitin conjugating
miR400	Pentatricopeptide repeat, pentatricopeptide (PPR), PPR, EMB2745, EMBRYO
	DEFECTIVE 2745, pentatricopeptide
miR402	DML3, DEMETER-LIKE PROTEIN 3, DEMETER-LIKE PROTEIN, DEMETER
	LIKE PROTEIN
miR403	AGO, Argonaute, AGO2
miR408	Laccase, LAC, LAC3, PCL, plantacyanin, plastacyanin, blue copper binding protein,
	blue copper binding, ARPN, copper ion binding, blue copper protein
miR444	MADS box, MADS-box, MADS
miR447	2-phosphoglycerate kinase-related, 2-phosphoglycerate kinase, phosphoglycerate kinas
miR472	RFL1, RPS5, RPS5-LIKE 1, ATP binding, RPS5, RESISTANT TO P. SYRINGAE 5,
	disease resistance protein (CC-NBS-LRR class), disease resistance protein, CC-NBS-
	LRR, NBS-LRR disease resistance protein, NBS-LRR type disease resistance protein
miR473	GRAS domain-containing protein, AtGAI, AtLAS, AtPAT1, AtRGA, AtRGL1,
	AtRGL2, AtRGL3, AtSCL1, AtSCL11, AtSCL13, AtSCL14, AtSCL15, AtSCL16,
	AtSCL18, AtSCL21, AtSCL22, AtSCL23, AtSCL26, AtSCL27, AtSCL28, AtSCL29,
	AtSCL3, AtSCL30, AtSCL31, AtSCL32, AtSCL33, AtSCL4, AtSCL5, AtSCL6,
	AtSCL7, AtSCL8, AtSCL9, AtSCR, AtSHR, REPRESSOR, RGA2, RGA-LIKE 1,
	RGL, RGL1, SGR7, VHS4, VHS5
miR474	Pentatricopeptide repeat, pentatricopeptide (PPR), PPR, PPR-
	repeat, EMB2654, EMBRYO DEFECTIVE 2654, pentatricopeptide
miR475	Pentatricopeptide repeat, pentatricopeptide (PPR), PPR, PPR-
	repeat, EMB2654, EMBRYO DEFECTIVE 2654, pentatricopeptide
miR476	Pentatricopeptide repeat, pentatricopeptide (PPR), PPR, PPR-
	repeat, EMB2654, EMBRYO DEFECTIVE 2654, pentatricopeptide
miR477	Basic helix-loop helix (bHLH) transcription factor, transcription factor/zinc ion binding
	CONSTANS-like, GRAS domain-containing protein, bHLH, GRAS, CONSTANS-
	like, CONSTANS
miR478	Organic anion transporter-like protein, Organic anion transporter
miR480	Proton-dependent oligopeptide transport family protein, Proton-dependent oligopeptide
	transport, Proton dependent oligopeptide transport
miR482	Putative disease resistance protein, disease resistance protein, disease resistance
miR529	Ethylene-response factor/AP2 domain transcription factor, erf/ap2, Ethylene-response
	factor/AP2
miR534	Ankyrin-repeat proteins, Ankyrin repeat proteins, Ankyrin-repeat protein, Ankyrin-
	repeat, Ankyrin repeat
miR536	F-box, F box, F-box family protein, F box family protein, F-box family, F box family,
	F-box protein
miR538	MADS-box, MADS
miR771	eukaryotic translation initiation factor 2 family protein, eIF-2 family protein, eIF-2,
	eIF2
miR773	DMT02, DMT2, MET02, MET2, DNA methyltransferase 2, DNA (cytosine-5-)-
	methyltransferase
miR774	F-box family, F-box, F box, F-box domain containing protein, F-box domain protein, F
	box domain
miR775	galactosyltransferase family protein, galactosyltransferase family, galactosyltransferase
miR776	IRE, INCOMPLETE ROOT HAIR ELONGATION
miR777	COP 1-interacting protein-related, COP1-interacting protein, COP1-interacting, COP1
	interacting
miR778	SET-domain, SET, SUVH6, SUVH5, SU(VAR)3-9 homolog
miR779	leucine-rich repeat transmembrane protein kinase, leucine-rich repeat, leucine rich
	repeat, transmembrane protein kinase, transmembrane
miR780	CHX18, ATCHX18, cation/hydrogen exchanger 18, monovalent cation:proton
	antiporter, proton antiporter
miR781	InterPro:IPR003169, SWIB complex BAF60b domain-containing protein, SWIB
	complex BAF60b domain, SWIB, BAF60b, plus-3 domain-containing protein, plus-3
	domain, plus-3, GYF domain-containing protein, GYF domain
miR809	Mlo disease resistant protein gene, Mlo-like, Mlo
miR818	ENT domain protein gene, ENT domain, ENT-domain
miR820	DNA cytosine methyltransferase, cytosine methyltransferase
miR823	CMT3, CHROMOMETHYLASE 3, CHROMOMETHYLASE
miR824	MADS-box, MADS, AGL16, AGAMOUS-LIKE, AGAMOUS
miR827	SPX, NLA, SYG/Pho81/XPR1, zinc finger, zinc-finger, C3HC4-type RING finger,
	C3HC4
miR828	MYB, myb domain protein, myb protein, AtMYB113, MYB113, MYB-like protein,
	myb-like, myb-like DNA-binding
miR842	JR/MBP, jacalin lectin family protein, jacalin lectin family, jacalin lectin, jacalin, lectin
miR844	protein kinase family protein, protein kinase family, protein kinase
miR846	JR/MBP, InterPro:IPR001229, jasmonate inducible protein, jacalin lectin family
	protein, jacalin lectin family, jacalin lectin, jacalin, lectin
miR856	Zinc transporter, Zinc-transporter, ACHX18, ATCHX18 \| ATCHX18, cation/hydrogen
	exchanger 18, cation/hydrogen exchanger, monovalent cation:proton antiporter, proton
	antiporter, antiporter
miR857	LAC, LAC7, laccase 7, copper ion binding, copper-ion binding
miR858	MYB, myb domain protein, myb protein, MYB12, AtMYB12, AtMYB83, MYB83,
	MYB-like protein, myb-like, myb-like DNA-binding
miR859	F-box, F box, F-box family protein, F box family protein, F-box family, F box family,
	F-box protein, InterPro:IPR006527, UDP-3-O-acyl N-acetylglycosamine deacetylase
	family protein, UDP-3-O-acyl N-acetylglycosamine deacetylase family, UDP-3-O-acyl
	N-acetylglycosamine deacetylase, UDP-3-O-acyl N-acetylglycosamine, F-box domain
	containing protein, F-box domain protein, F-box domain
miR902	Basic helix-loop helix (bHLH) transcription factor, bHLH
miR904	AGO, Argonaute
miR1029	Ethylene-response factor/AP2 domain transcription factor, Ethylene-response factor,
	Ethylene response factor, erf/AP2
miR1219c	Auxin Response Factors, Auxin Response Factor, arf

indicates data missing or illegible when filed

TABLE 2

Computationally validated miRNA targets

		SEQ ID
miRNA	Gene Function	NO:	Gene ID	Species of origin*

miR156/157	SPL	15	PHE0014564	Arabidopsis
				thaliana
miR156/157	SPL	16	PHE0014996	A. thaliana
miR156/157	Squamosa Promoter Binding Protein	17	PHE0004508	A. thaliana
miR156/157	Squamosa Promoter Binding Protein	18	PHE0004925	A. thaliana
miR160	ARF	19	PHE0003525	A. thaliana
miR164	ANAC092	20	PHE0013733	A. thaliana
miR164	NAC domain protein	21	PHE0001074	A. thaliana
miR165/166	Revoluta	22	PHE0008129	A. thaliana
miR165/166	Revoluta	23	PHE0010493	A. thaliana
miR165/166	Revoluta	24	PHE0012654	A. thaliana
miR165/166	Revoluta	25	PHE0007271	A. thaliana
miR165/166	Revoluta	26	PHE0007467	A. thaliana
miR165/166	Revoluta	27	PHE0007720	A. thaliana
miR165/166	Revoluta	28	PHE0010355	A. thaliana
miR165/166	Revoluta	29	PHE0010473	A. thaliana
miR165/166	Revoluta	30	PHE0010494	A. thaliana
miR165/166	Revoluta	31	PHE0010495	A. thaliana
miR165/166	Revoluta	32	PHE0010537	A. thaliana
miR166	Revoluta	33	PHE0010496	A. thaliana
miR166	Revoluta	34	PHE0010497	A. thaliana
miR166	Revoluta	35	PHE0010500	A. thaliana
miR167	ARF	36	PHE0003428	A. thaliana
miR172	AP2	37	PHE0003881	A. thaliana
miR172	AP2 domain	38	PHE0006606	A. thaliana
miR393	F-box	39	PHE0007151	A. thaliana
miR393	F-box	40	PHE0007164	A. thaliana
miR393	F-box	41	PHE0007167	A. thaliana
miR393	Transport inhibitor response	42	PHE0004988	A. thaliana
miR396	GRL	43	PHE0004617	A. thaliana
miR778	SET-domain	44	PHE0006443	A. thaliana
miR779	leucine-rich repeat transmembrane	45	PHE0002993	A. thaliana
	protein kinase
miR858	MYB	46	PHE0001073	A. thaliana
miR858	MYB	47	PHE0001093	A. thaliana
miR858	MYB	48	PHE0002073	A. thaliana
miR858	MYB	49	PHE0010073	A. thaliana
miR858	MyB	50	PHE0011722	A. thaliana
miR858	MyB	51	PHE0015935	A. thaliana
miR859	F-box	52	PHE0003311	A. thaliana
miR859	F-box	53	PHE0006468	A. thaliana
miR902	bHLH	54	PHE0000658	A. thaliana
miR902	bHLH	55	PHE0006524	A. thaliana
miR156	Squamosa Promoter Binding Protein	56	MRT3708_37334C.1	Canola (Brassica
				napus or Brassica
				rapa)
miR156/157	Squamosa Promoter Binding Protein	57	MRT3708_10628C.4	Canola
miR156/157	Squamosa Promoter Binding Protein	58	MRT3708_22559C.1	Canola
miR156/157	Squamosa Promoter Binding Protein	59	MRT3708_30289C.3	Canola
miR156/157	Squamosa Promoter Binding Protein	60	MRT3708_39670C.2	Canola
miR156/157	Squamosa Promoter Binding Protein	61	MRT3708_53675C.1	Canola
miR156/157	Squamosa Promoter Binding Protein	62	MRT3708_58630C.1	Canola
miR159	MYB	63	MRT3708_33278C.1	Canola
miR159	MYB	64	MRT3708_33279C.1	Canola
miR163	methyltransferase	65	MRT3708_16440C.1	Canola
miR163	methyltransferase	66	MRT3708_28174C.1	Canola
miR163	methyltransferase	67	MRT3708_52155C.2	Canola
miR164	NAM	68	MRT3708_39966C.1	Canola
miR164	No Apical Meristem	69	MRT3708_51022C.1	Canola
miR164	No Apical Meristem	70	MRT3708_7877C.4	Canola
miR165/166	class III HD-Zip protein	71	MRT3708_45624C.1	Canola
miR165/166	HD-Zip protein	72	MRT3708_5493C.1	Canola
miR167	Auxin Response Factor	73	MRT3708_37499C.2	Canola
miR167	Auxin Response Factor	74	MRT3708_50323C.1	Canola
miR169	CCAAT-binding	75	MRT3708_45516C.2	Canola
miR169	CCAAT-binding	76	MRT3708_46224C.1	Canola
miR169	CCAAT-binding	77	MRT3708_56325C.1	Canola
miR169	nuclear transcription factor Y	78	MRT3708_42756C.1	Canola
miR170/171	SCARECROW gene regulator	79	MRT3708_34048C.2	Canola
miR172	AP2	80	MRT3708_39387C.1	Canola
miR172	AP2 domain	81	MRT3708_36942C.2	Canola
miR393	Transport inhibitor response	82	MRT3708_31301C.1	Canola
miR393	Transport inhibitor response	83	MRT3708_52518C.1	Canola
miR393	Transport inhibitor response	84	MRT3708_55951C.1	Canola
miR394	F-box	85	MRT3708_61891C.1	Canola
miR395	ATP sulfurylase	86	MRT3708_35187C.3	Canola
miR395	sulfate adenylyltransferase	87	MRT3708_36129C.1	Canola
miR395	sulfate adenylyltransferase	88	MRT3708_55043C.1	Canola
miR396	Growth-regulating factor	89	MRT3708_29578C.1	Canola
miR396	Growth-regulating factor	90	MRT3708_51563C.1	Canola
miR398	cytochrome c oxidase	91	MRT3708_47361C.2	Canola
miR400	PPR	92	MRT3708_57455C.1	Canola
miR408	blue copper protein	93	MRT3708_29149C.3	Canola
miR472	ATP binding	94	MRT3708_45273C.1	Canola
miR472	ATP binding	95	MRT3708_55890C.1	Canola
miR472	ATP binding	96	MRT3708_55902C.2	Canola
miR824	MADS-box	97	MRT3708_59018C.1	Canola
miR827	zinc finger	98	MRT3708_29390C.1	Canola
miR828	myb-like DNA-binding	99	MRT3708_31708C.1	Canola
miR856	antiporter	100	MRT3708_61144C.1	Canola
miR857	LAC	101	MRT3708_24461C.1	Canola
miR858	MYB	102	MRT3708_31372C.1	Canola
miR858	myb-like DNA-binding	103	MRT3708_16589C.4	Canola
miR858	myb-like DNA-binding	104	MRT3708_29291C.3	Canola
miR858	myb-like DNA-binding	105	MRT3708_54665C.1	Canola
miR858	myb-like DNA-binding	106	MRT3708_61897C.1	Canola
miR859	F-box domain	107	MRT3708_51653C.1	Canola
miR167	Auxin Response Factor	108	MRT3711_1592C.1	Field mustard
				(Brassica rapa or
				Brassica
				campestris)
miR168	Argonaute	109	MRT3711_4500C.2	Field mustard
miR169	nuclear transcription factor Y	110	MRT3711_4547C.1	Field mustard
miR172	AP2	111	MRT3711_6838C.1	Field mustard
miR319	PCF	112	MRT3711_7220C.1	Field mustard
miR393	Transport inhibitor response	113	MRT3711_1771C.1	Field mustard
miR395	sulfate adenylyltransferase	114	MRT3711_3394C.1	Field mustard
miR395	sulfate adenylyltransferase	115	MRT3711_4165C.1	Field mustard
miR395	sulfate adenylyltransferase	116	MRT3711_4313C.1	Field mustard
miR472	ATP binding	117	MRT3711_7972C.1	Field mustard
miR827	zinc finger	118	MRT3711_10064C.1	Field mustard
miR858	myb-like DNA-binding	119	MRT3711_7980C.1	Field mustard
miR156/157	SBP domain	120	MRT3847_197471C.3	Glycine max
miR156/157	SBP domain	121	MRT3847_202791C.3	G. max
miR156/157	SBP domain	122	MRT3847_28990C.5	G. max
miR156/157	SBP domain	123	MRT3847_39715C.7	G. max
miR156/157	Squamosa Promoter Binding Protein	124	MRT3847_207934C.2	G. max
miR156/157	Squamosa Promoter Binding Protein	125	MRT3847_257545C.4	G. max
miR156/157	Squamosa Promoter Binding Protein	126	MRT3847_217782C.3	G. max
miR156/157	Squamosa Promoter Binding Protein	127	MRT3847_235081C.4	G. max
miR156/157	Squamosa Promoter Binding Protein	128	MRT3847_235082C.6	G. max
miR156/157	Squamosa Promoter Binding Protein	129	MRT3847_289291C.3	G. max
miR156/157	Squamosa Promoter Binding Protein	130	MRT3847_335568C.1	G. max
miR156/157	Squamosa Promoter Binding Protein	131	MRT3847_350831C.1	G. max
miR156/157	Squamosa Promoter Binding Protein	132	MRT3847_14683C.5	G. max
miR156/157	Squamosa Promoter Binding Protein	133	MRT3847_237444C.4	G. max
miR156/157	Squamosa Promoter Binding Protein	134	MRT3847_329752C.1	G. max
miR156/157	Squamosa Promoter Binding Protein	135	MRT3847_334134C.1	G. max
miR156/157	teosinte glume architecture	136	MRT3847_338602C.1	G. max
miR159	myb-like DNA-binding domain	137	MRT3847_345009C.1	G. max
miR159	myb-like DNA-binding domain	138	MRT3847_346338C.1	G. max
miR160	ARF	139	PHE0003526	G. max
miR160	Auxin Response Factor	140	MRT3847_139013C.5	G. max
miR160	Auxin Response Factor	141	MRT3847_197785C.3	G. max
miR160	Auxin Response Factor	142	MRT3847_239685C.2	G. max
miR160	Auxin Response Factor	143	MRT3847_269589C.4	G. max
miR160	Auxin Response Factor	144	MRT3847_28328C.3	G. max
miR160	Auxin Response Factor	145	MRT3847_289982C.2	G. max
miR160	Auxin Response Factor	146	MRT3847_37862C.4	G. max
miR160	Auxin Response Factor	147	MRT3847_41982C.5	G. max
miR160	Auxin Response Factor	148	MRT3847_52071C.7	G. max
miR161	pentatricopeptide	149	MRT3847_4014C.4	G. max
miR161	PPR	150	MRT3847_20482C.2	G. max
miR161	PPR	151	MRT3847_227121C.4	G. max
miR164	NAC domain protein	152	MRT3847_46332C.2	G. max
miR164	NAC domain protein	153	MRT3847_46333C.6	G. max
miR164	NAC1	154	PHE0001363	G. max
miR164	NAM	155	MRT3847_244824C.2	G. max
miR164	No Apical Meristem	156	MRT3847_259513C.2	G. max
miR164	No Apical Meristem	157	MRT3847_270117C.3	G. max
miR164	No Apical Meristem	158	MRT3847_48464C.4	G. max
miR164	No Apical Meristem	159	MRT3847_48465C.6	G. max
miR165/166	class III HD-Zip protein	160	MRT3847_209034C.4	G. max
miR165/166	class III HD-Zip protein	161	MRT3847_233286C.5	G. max
miR165/166	class III HD-Zip protein	162	MRT3847_248020C.5	G. max
miR165/166	class III HD-Zip protein	163	MRT3847_288367C.4	G. max
miR165/166	class III HD-Zip protein	164	MRT3847_296736C.1	G. max
miR165/166	class III HD-Zip protein	165	MRT3847_326691C.1	G. max
miR165/166	class III HD-Zip protein	166	MRT3847_345104C.1	G. max
miR165/166	class III HD-Zip protein	167	MRT3847_348410C.1	G. max
miR166	Homeobox	168	PHE0003454	G. max
miR167	ARF	169	PHE0003655	G. max
miR167	Auxin Response Factor	170	MRT3847_195447C.5	G. max
miR167	Auxin Response Factor	171	MRT3847_263906C.5	G. max
miR167	Auxin Response Factor	172	MRT3847_305421C.4	G. max
miR167	Auxin Response Factor	173	MRT3847_340154C.1	G. max
miR167	Auxin Response Factor	174	MRT3847_41926C.6	G. max
miR167	Auxin Response Factor	175	MRT3847_55334C.5	G. max
miR169	CCAAT-binding	176	MRT3847_251095C.3	G. max
miR169	CCAAT-binding	177	MRT3847_259875C.4	G. max
miR169	CCAAT-binding	178	MRT3847_293871C.3	G. max
miR169	CCAAT-binding	179	MRT3847_305217C.3	G. max
miR169	CCAAT-binding	180	MRT3847_347487C.1	G. max
miR169	CCAAT-binding	181	MRT3847_40604C.6	G. max
miR169	CCAAT-binding	182	MRT3847_53466C.6	G. max
miR169	CCAAT-binding	183	MRT3847_53467C.5	G. max
miR169	CCAAT-binding	184	MRT3847_54675C.6	G. max
miR169	NFYa	185	PHE0011547	G. max
miR169	nuclear transcription factor Y	186	MRT3847_25786C.5	G. max
miR169	nuclear transcription factor Y	187	MRT3847_289667C.3	G. max
miR169	nuclear transcription factor Y	188	MRT3847_312701C.1	G. max
miR169	nuclear transcription factor Y	189	MRT3847_335193C.1	G. max
miR169	nuclear transcription factor Y	190	MRT3847_51286C.6	G. max
miR169	nuclear transcription factor Y	191	MRT3847_54010C.4	G. max
miR170/171	Scarecrow-like	192	MRT3847_41579C.4	G. max
miR171	GRAS	193	MRT3847_267119C.3	G. max
miR171	GRAS	194	MRT3847_270988C.3	G. max
miR171	GRAS	195	MRT3847_275596C.2	G. max
miR171	GRAS	196	MRT3847_294457C.2	G. max
miR171	GRAS	197	MRT3847_344862C.1	G. max
miR172	AP2 domain	198	PHE0000638	G. max
miR172	AP2 domain	199	MRT3847_202930C.3	G. max
miR172	AP2 domain	200	MRT3847_21933C.5	G. max
miR172	AP2 domain	201	MRT3847_235857C.3	G. max
miR172	AP2 domain	202	MRT3847_257655C.4	G. max
miR172	AP2 domain	203	MRT3847_289890C.3	G. max
miR172	AP2 domain	204	MRT3847_289891C.3	G. max
miR172	AP2 domain	205	MRT3847_295726C.1	G. max
miR172	AP2 domain	206	MRT3847_326790C.1	G. max
miR172	AP2 domain	207	MRT3847_329301C.1	G. max
miR172	AP2 domain	208	MRT3847_43925C.7	G. max
miR172	AP2 domain	209	MRT3847_46007C.5	G. max
miR172	AP2 domain	210	MRT3847_51633C.3	G. max
miR172	AP2 domain	211	MRT3847_59804C.6	G. max
miR172	APETALA2	212	MRT3847_196945C.3	G. max
miR319	Cyclin	213	MRT3847_238163C.3	G. max
miR319	PCF	214	MRT3847_262919C.1	G. max
miR319	TCP family transcription factor	215	MRT3847_230131C.1	G. max
miR319	TCP family transcription factor	216	MRT3847_304168C.2	G. max
miR319	TCP family transcription factor	217	MRT3847_336868C.1	G. max
miR319	TCP family transcription factor	218	MRT3847_343365C.1	G. max
miR319	TCP family transcription factor	219	MRT3847_38312C.5	G. max
miR319	TCP family transcription factor	220	MRT3847_103008C.6	G. max
miR319	TCP family transcription factor	221	MRT3847_12165C.5	G. max
miR319	TCP family transcription factor	222	MRT3847_247420C.4	G. max
miR319	TCP family transcription factor	223	MRT3847_294519C.4	G. max
miR319	TCP family transcription factor	224	MRT3847_334277C.1	G. max
miR390	TAS	225	MRT3847_133706C.5	G. max
miR390	TAS	226	MRT3847_298568C.2	G. max
miR390	TAS	227	MRT3847_60306C.8	G. max
miR393	TIR1	228	MRT3847_238705C.4	G. max
miR393	TIR1	229	MRT3847_27973C.7	G. max
miR393	TIR1	230	MRT3847_313402C.3	G. max
miR393	Transport inhibitor response	231	MRT3847_329954C.2	G. max
miR393	Transport inhibitor response	232	MRT3847_335477C.1	G. max
miR393	Transport inhibitor response	233	MRT3847_44371C.6	G. max
miR394	F-box domain	234	MRT3847_249313C.3	G. max
miR394	F-box domain	235	MRT3847_260044C.4	G. max
miR395	AST	236	MRT3847_118061C.7	G. max
miR395	AST	237	MRT3847_120571C.4	G. max
miR395	AST	238	MRT3847_161863C.4	G. max
miR395	AST	239	MRT3847_233832C.4	G. max
miR395	AST	240	MRT3847_294717C.3	G. max
miR395	AST	241	MRT3847_303988C.3	G. max
miR395	AST	242	MRT3847_336528C.1	G. max
miR395	AST	243	MRT3847_55707C.5	G. max
miR395	ATP sulfurylase	244	MRT3847_14792C.7	G. max
miR395	sulfate adenylyltransferase	245	MRT3847_331787C.1	G. max
miR395	sulfate transporter	246	MRT3847_10451C.5	G. max
miR395	sulfate transporter	247	MRT3847_245035C.3	G. max
miR396	GRF	248	PHE0001215	G. max
miR396	Growth-regulating factor	249	MRT3847_183050C.6	G. max
miR396	Growth-regulating factor	250	MRT3847_200704C.5	G. max
miR396	Growth-regulating factor	251	MRT3847_21877C.7	G. max
miR396	Growth-regulating factor	252	MRT3847_275465C.2	G. max
miR396	Growth-regulating factor	253	MRT3847_285089C.5	G. max
miR396	Growth-regulating factor	254	MRT3847_307974C.3	G. max
miR396	Growth-regulating factor	255	MRT3847_34351C.6	G. max
miR396	Growth-regulating factor	256	MRT3847_39577C.5	G. max
miR397	Laccase	257	MRT3847_148737C.1	G. max
miR397	Laccase	258	MRT3847_196074C.1	G. max
miR397	Laccase	259	MRT3847_240006C.2	G. max
miR397	Laccase	260	MRT3847_256982C.1	G. max
miR397	Laccase	261	MRT3847_25859C.5	G. max
miR397	Laccase	262	MRT3847_29767C.4	G. max
miR397	Laccase	263	MRT3847_297900C.1	G. max
miR397	Laccase	264	MRT3847_309594C.2	G. max
miR397	Laccase	265	MRT3847_33656C.5	G. max
miR397	Laccase	266	MRT3847_347553C.1	G. max
miR397	Laccase	267	MRT3847_36695C.5	G. max
miR397	Laccase	268	MRT3847_49069C.6	G. max
miR397	Laccase	269	MRT3847_7864C.1	G. max
miR397	Laccase	270	MRT3847_99867C.5	G. max
miR398	COPPER/ZINC SUPEROXIDE	271	MRT3847_235546C.3	G. max
	DISMUTASE
miR400	pentatricopeptide	272	MRT3847_12750C.4	G. max
miR400	pentatricopeptide	273	MRT3847_17367C.3	G. max
miR400	PPR	274	MRT3847_10096C.3	G. max
miR400	PPR	275	MRT3847_139832C.5	G. max
miR400	PPR	276	MRT3847_141759C.5	G. max
miR400	PPR	277	MRT3847_218904C.2	G. max
miR400	PPR	278	MRT3847_267668C.2	G. max
miR400	PPR	279	MRT3847_57083C.4	G. max
miR408	blue copper protein	280	PHE0000330	G. max
miR408	blue copper protein	281	MRT3847_273288C.3	G. max
miR408	blue copper protein	282	MRT3847_329905C.2	G. max
miR408	blue copper protein	283	MRT3847_336704C.1	G. max
miR408	blue copper protein	284	MRT3847_343250C.1	G. max
miR408	blue copper protein	285	MRT3847_346770C.1	G. max
miR408	blue copper protein	286	MRT3847_349900C.1	G. max
miR408	blue copper protein	287	MRT3847_350132C.1	G. max
miR408	blue copper protein	288	MRT3847_60064C.6	G. max
miR408	blue copper protein	289	MRT3847_66506C.8	G. max
miR408	Laccase	290	MRT3847_296270C.2	G. max
miR408	Laccase	291	MRT3847_31127C.7	G. max
miR444	MADS box	292	PHE0002647	G. max
miR444	MADS box	293	PHE0002648	G. max
miR444	MADS box	294	PHE0015540	G. max
miR444	MADS-box	295	MRT3847_247970C.2	G. max
miR444	MADS-box	296	MRT3847_259952C.3	G. max
miR472	ATP binding	297	MRT3847_324977C.1	G. max
miR472	ATP binding	298	MRT3847_335756C.1	G. max
miR472	disease resistance protein	299	MRT3847_348618C.1	G. max
miR472	NBS-LRR type disease resistance	300	MRT3847_292513C.3	G. max
	protein
miR472	NBS-LRR type disease resistance	301	MRT3847_34971C.6	G. max
	protein
miR472/482	disease resistance protein	302	MRT3847_159134C.1	G. max
miR472/482	disease resistance protein	303	MRT3847_208382C.4	G. max
miR472/482	disease resistance protein	304	MRT3847_229943C.2	G. max
miR472/482	disease resistance protein	305	MRT3847_262606C.4	G. max
miR472/482	NBS-LRR type disease resistance	306	MRT3847_223192C.5	G. max
	protein
miR472/482	NBS-LRR type disease resistance	307	MRT3847_264890C.3	G. max
	protein
miR475	Pentatricopeptide repeat	308	MRT3847_204627C.1	G. max
miR475	Pentatricopeptide repeat	309	MRT3847_234253C.2	G. max
miR475	Pentatricopeptide repeat	310	MRT3847_289449C.2	G. max
miR475	Pentatricopeptide repeat	311	MRT3847_342062C.1	G. max
miR475	PPR	312	MRT3847_137370C.4	G. max
miR475	PPR	313	MRT3847_196480C.3	G. max
miR475	PPR	314	MRT3847_241148C.2	G. max
miR475	PPR	315	MRT3847_30662C.4	G. max
miR475	PPR	316	MRT3847_44502C.5	G. max
miR475	PPR-repeat	317	MRT3847_235882C.3	G. max
miR477	bHLH	318	MRT3847_117808C.5	G. max
miR477	bHLH	319	MRT3847_330789C.2	G. max
miR477	GRAS	320	MRT3847_161254C.2	G. max
miR477	GRAS	321	MRT3847_250541C.3	G. max
miR482	disease resistance protein	322	MRT3847_216742C.1	G. max
miR482	disease resistance protein	323	MRT3847_221164C.1	G. max
miR482	disease resistance protein	324	MRT3847_28447C.6	G. max
miR482	disease resistance protein	325	MRT3847_302802C.3	G. max
miR482	disease resistance protein	326	MRT3847_146432C.5	G. max
miR482	disease resistance protein	327	MRT3847_184524C.6	G. max
miR482	disease resistance protein	328	MRT3847_268743C.4	G. max
miR482	disease resistance protein	329	MRT3847_272693C.2	G. max
miR482	disease resistance protein	330	MRT3847_297146C.2	G. max
miR482	disease resistance protein	331	MRT3847_314629C.2	G. max
miR482	disease resistance protein	332	MRT3847_335514C.1	G. max
miR482	disease resistance protein	333	MRT3847_335735C.1	G. max
miR482	disease resistance protein	334	MRT3847_337518C.1	G. max
miR482	disease resistance protein	335	MRT3847_340947C.1	G. max
miR482	disease resistance protein	336	MRT3847_352235C.1	G. max
miR482	disease resistance protein	337	MRT3847_63055C.5	G. max
miR482	disease resistance protein	338	MRT3847_66636C.5	G. max
miR482	Putative disease resistance protein	339	MRT3847_184595C.4	G. max
miR824	MADS box	340	PHE0001395	G. max
miR824	MADS box	341	PHE0003427	G. max
miR824	MADS box	342	PHE0013854	G. max
miR824	MADS-box	343	MRT3847_14550C.4	G. max
miR824	MADS-box	344	MRT3847_39202C.7	G. max
miR828	MyB	345	PHE0001477	G. max
miR828	MYB	346	MRT3847_346366C.1	G. max
miR828	myb-like DNA-binding	347	MRT3847_215219C.3	G. max
miR828	myb-like DNA-binding	348	MRT3847_215220C.2	G. max
miR828/858	myb-like DNA-binding	349	MRT3847_22767C.2	G. max
miR857	LAC	350	MRT3847_13225C.3	G. max
miR858	MyB	351	PHE0000380	G. max
miR858	MYB	352	PHE0001408	G. max
miR858	MyB	353	PHE0004448	G. max
miR858	MyB	354	PHE0012029	G. max
miR858	MyB	355	PHE0015929	G. max
miR858	MYB	356	MRT3847_212141C.3	G. max
miR858	MYB	357	MRT3847_347736C.1	G. max
miR858	MYB	358	MRT3847_38379C.5	G. max
miR858	MYB	359	MRT3847_40737C.7	G. max
miR858	MYB	360	MRT3847_41334C.3	G. max
miR858	MYB12	361	MRT3847_51246C.6	G. max
miR858	myb-like DNA-binding	362	MRT3847_131164C.6	G. max
miR858	myb-like DNA-binding	363	MRT3847_137726C.5	G. max
miR858	myb-like DNA-binding	364	MRT3847_228792C.3	G. max
miR858	myb-like DNA-binding	365	MRT3847_255360C.1	G. max
miR858	myb-like DNA-binding	366	MRT3847_255362C.6	G. max
miR858	myb-like DNA-binding	367	MRT3847_260391C.1	G. max
miR858	myb-like DNA-binding	368	MRT3847_261508C.2	G. max
miR858	myb-like DNA-binding	369	MRT3847_270136C.3	G. max
miR858	myb-like DNA-binding	370	MRT3847_290332C.2	G. max
miR858	myb-like DNA-binding	371	MRT3847_294239C.3	G. max
miR858	myb-like DNA-binding	372	MRT3847_322770C.2	G. max
miR858	myb-like DNA-binding	373	MRT3847_32417C.5	G. max
miR858	myb-like DNA-binding	374	MRT3847_332192C.1	G. max
miR858	myb-like DNA-binding	375	MRT3847_335664C.1	G. max
miR858	myb-like DNA-binding	376	MRT3847_34082C.5	G. max
miR858	myb-like DNA-binding	377	MRT3847_39825C.5	G. max
miR858	myb-like DNA-binding	378	MRT3847_40203C.4	G. max
miR858	myb-like DNA-binding	379	MRT3847_41332C.5	G. max
miR858	myb-like DNA-binding	380	MRT3847_42168C.6	G. max
miR858	myb-like DNA-binding	381	MRT3847_51247C.3	G. max
miR858	myb-like DNA-binding	382	MRT3847_52127C.4	G. max
miR858	myb-like DNA-binding	383	MRT3847_54395C.5	G. max
miR858	myb-like DNA-binding	384	MRT3847_55676C.6	G. max
miR156	SBP domain	385	MRT3635_30868C.2	Gossypium
				hirsutum
miR156/157	SBP domain	386	MRT3635_36657C.2	G. hirsutum
miR156/157	SBP domain	387	MRT3635_65765C.1	G. hirsutum
miR156/157	Squamosa Promoter Binding Protein	388	MRT3635_15791C.2	G. hirsutum
miR156/157	Squamosa Promoter Binding Protein	389	MRT3635_48230C.2	G. hirsutum
miR156/157	Squamosa Promoter Binding Protein	390	MRT3635_69088C.1	G. hirsutum
miR156/157	Squamosa Promoter Binding Protein	391	MRT3635_69159C.1	G. hirsutum
miR156/157	Squamosa Promoter Binding Protein	392	MRT3635_30369C.2	G. hirsutum
miR156/157	Squamosa Promoter Binding Protein	393	MRT3635_56290C.1	G. hirsutum
miR156/157	teosinte glume architecture	394	MRT3635_15393C.1	G. hirsutum
miR159	MYB65	395	MRT3635_249C.2	G. hirsutum
miR159	myb-like DNA-binding	396	MRT3635_54684C.2	G. hirsutum
miR160	Auxin Response Factor	397	MRT3635_36222C.2	G. hirsutum
miR162	CAF	398	MRT3635_16630C.2	G. hirsutum
miR164	NAC domain protein	399	MRT3635_24172C.2	G. hirsutum
miR164	No Apical Meristem	400	MRT3635_48601C.2	G. hirsutum
miR164	No Apical Meristem	401	MRT3635_64345C.1	G. hirsutum
miR165/166	class III HD-Zip protein	402	MRT3635_4809C.2	G. hirsutum
miR165/166	class III HD-Zip protein	403	MRT3635_50942C.2	G. hirsutum
miR165/166	class III HD-Zip protein	404	MRT3635_72188C.1	G. hirsutum
miR166	class III HD-Zip protein	405	MRT3635_12880C.2	G. hirsutum
miR167	Auxin Response Factor	406	MRT3635_13510C.2	G. hirsutum
miR167	Auxin Response Factor	407	MRT3635_14893C.2	G. hirsutum
miR167	Auxin Response Factor	408	MRT3635_24556C.2	G. hirsutum
miR167	Auxin Response Factor	409	MRT3635_59443C.1	G. hirsutum
miR168	AGO1	410	MRT3635_43628C.2	G. hirsutum
miR168	Argonaute	411	MRT3635_68755C.1	G. hirsutum
miR169	CCAAT-binding	412	MRT3635_18720C.2	G. hirsutum
miR169	CCAAT-binding	413	MRT3635_60547C.1	G. hirsutum
miR169	CCAAT-binding	414	MRT3635_63602C.1	G. hirsutum
miR169	CCAAT-binding	415	MRT3635_751C.2	G. hirsutum
miR169	nuclear transcription factor Y	416	MRT3635_57584C.1	G. hirsutum
miR169	nuclear transcription factor Y	417	MRT3635_63203C.1	G. hirsutum
miR169	nuclear transcription factor Y	418	MRT3635_67492C.1	G. hirsutum
miR171	GRAS	419	MRT3635_41132C.2	G. hirsutum
miR172	AP2	420	MRT3635_50596C.2	G. hirsutum
miR172	AP2 domain	421	MRT3635_21738C.2	G. hirsutum
miR172	AP2 domain	422	MRT3635_5937C.2	G. hirsutum
miR172	AP2 domain	423	MRT3635_64989C.1	G. hirsutum
miR172	AP2 domain	424	MRT3635_8244C.2	G. hirsutum
miR319	TCP	425	MRT3635_31917C.2	G. hirsutum
miR319	TCP family transcription factor	426	MRT3635_40862C.2	G. hirsutum
miR319	TCP family transcription factor	427	MRT3635_55735C.1	G. hirsutum
miR393	TIR1	428	MRT3635_18850C.2	G. hirsutum
miR393	TIR1	429	MRT3635_35639C.2	G. hirsutum
miR393	TIR1	430	MRT3635_68504C.1	G. hirsutum
miR393	Transport inhibitor response	431	MRT3635_18188C.2	G. hirsutum
miR393	Transport inhibitor response	432	MRT3635_49076C.2	G. hirsutum
miR395	AST	433	MRT3635_73824C.1	G. hirsutum
miR395	sulfate adenylyltransferase	434	MRT3635_15903C.2	G. hirsutum
miR395	sulfate adenylyltransferase	435	MRT3635_48567C.2	G. hirsutum
miR395	sulfate transporter	436	MRT3635_64866C.1	G. hirsutum
miR396	Growth-regulating factor	437	MRT3635_10089C.2	G. hirsutum
miR396	Growth-regulating factor	438	MRT3635_18322C.2	G. hirsutum
miR396	Growth-regulating factor	439	MRT3635_43733C.2	G. hirsutum
miR396	Growth-regulating factor	440	MRT3635_44225C.2	G. hirsutum
miR396	Growth-regulating factor	441	MRT3635_67643C.1	G. hirsutum
miR396	Growth-regulating factor	442	MRT3635_71085C.1	G. hirsutum
miR396	Growth-regulating factor	443	MRT3635_7854C.2	G. hirsutum
miR397	Laccase	444	MRT3635_2612C.2	G. hirsutum
miR397	Laccase	445	MRT3635_59330C.1	G. hirsutum
miR397	Laccase	446	MRT3635_62379C.1	G. hirsutum
miR400	PPR	447	MRT3635_14024C.2	G. hirsutum
miR400	PPR	448	MRT3635_24425C.2	G. hirsutum
miR400	PPR	449	MRT3635_62540C.1	G. hirsutum
miR400	PPR	450	MRT3635_71976C.1	G. hirsutum
miR408	blue copper protein	451	MRT3635_25321C.2	G. hirsutum
miR408	blue copper protein	452	MRT3635_36078C.2	G. hirsutum
miR408	blue copper protein	453	MRT3635_36080C.2	G. hirsutum
miR408	blue copper protein	454	MRT3635_54561C.2	G. hirsutum
miR408	blue copper protein	455	MRT3635_54936C.2	G. hirsutum
miR444	MADS-box	456	MRT3635_52393C.1	G. hirsutum
miR472	ATP binding	457	MRT3635_16581C.2	G. hirsutum
miR472/482	NBS-LRR type disease resistance	458	MRT3635_77272C.1	G. hirsutum
	protein
miR475	pentatricopeptide	459	MRT3635_73944C.1	G. hirsutum
miR475	Pentatricopeptide repeat	460	MRT3635_35992C.1	G. hirsutum
miR475	Pentatricopeptide repeat	461	MRT3635_51055C.1	G. hirsutum
miR475	PPR	462	MRT3635_36232C.2	G. hirsutum
miR475	PPR	463	MRT3635_65837C.1	G. hirsutum
miR475	PPR	464	MRT3635_6832C.2	G. hirsutum
miR827	SPX	465	MRT3635_71336C.1	G. hirsutum
miR827	zinc finger	466	MRT3635_61225C.1	G. hirsutum
miR828	MYB	467	MRT3635_63902C.1	G. hirsutum
miR828	myb-like DNA-binding	468	MRT3635_11678C.2	G. hirsutum
miR828	myb-like DNA-binding	469	MRT3635_23974C.2	G. hirsutum
miR828	myb-like DNA-binding	470	MRT3635_37632C.1	G. hirsutum
miR828	myb-like DNA-binding	471	MRT3635_46849C.2	G. hirsutum
miR828	myb-like DNA-binding	472	MRT3635_75185C.1	G. hirsutum
miR828/858	MYB	473	MRT3635_12320C.2	G. hirsutum
miR828/858	myb-like DNA-binding	474	MRT3635_25669C.1	G. hirsutum
miR858	MYB	475	MRT3635_11888C.1	G. hirsutum
miR858	MYB	476	MRT3635_17735C.1	G. hirsutum
miR858	MYB	477	MRT3635_3345C.1	G. hirsutum
miR858	MYB	478	MRT3635_46789C.1	G. hirsutum
miR858	myb-like DNA-binding	479	MRT3635_48257C.1	G. hirsutum
miR858	myb-like DNA-binding	480	MRT3635_53024C.2	G. hirsutum
miR858	myb-like DNA-binding	481	MRT3635_55977C.1	G. hirsutum
miR858	myb-like DNA-binding	482	MRT3635_57077C.1	G. hirsutum
miR858	myb-like DNA-binding	483	MRT3635_66730C.1	G. hirsutum
miR858	myb-like DNA-binding	484	MRT3635_67640C.1	G. hirsutum
miR858	myb-like DNA-binding	485	MRT3635_69682C.1	G. hirsutum
miR858	myb-like DNA-binding	486	MRT3635_74072C.1	G. hirsutum
miR156	SBP domain	487	MRT4513_33353C.1	Hordeum vulgare
miR156/157	SBP domain	488	MRT4513_19757C.1	H. vulgare
miR156/157	SBP domain, miR157	489	MRT4513_52153C.1	H. vulgare
miR156/157	SBP-domain, miR157	490	MRT4513_41849C.1	H. vulgare
miR156/157	Squamosa Promoter Binding Protein	491	MRT4513_4449C.1	H. vulgare
miR159	myb-like DNA-binding domain	492	MRT4513_1572C.3	H. vulgare
miR159	myb-like DNA-binding domain	493	MRT4513_55409C.1	H. vulgare
miR160	Auxin Response Factor	494	MRT4513_43004C.1	H. vulgare
miR160	Auxin Response Factor	495	MRT4513_48930C.1	H. vulgare
miR160	Auxin Response Factor	496	MRT4513_51165C.1	H. vulgare
miR160	Auxin Response Factor	497	MRT4513_9322C.2	H. vulgare
miR164	NAC domain protein	498	MRT4513_51143C.2	H. vulgare
miR164	NAC domain protein	499	MRT4513_7890C.1	H. vulgare
miR164	No Apical Meristem	500	MRT4513_26199C.1	H. vulgare
miR167	Auxin Response Factor	501	MRT4513_29483C.2	H. vulgare
miR167	Auxin Response Factor	502	MRT4513_29827C.2	H. vulgare
miR167	Auxin Response Factor	503	MRT4513_31779C.1	H. vulgare
miR167	Auxin Response Factor	504	MRT4513_47791C.1	H. vulgare
miR168	Argonaute	505	MRT4513_31835C.1	H. vulgare
miR168	Argonaute	506	MRT4513_43289C.1	H. vulgare
miR168	PINHEAD	507	MRT4513_28709C.1	H. vulgare
miR169	CCAAT-binding	508	MRT4513_27452C.1	H. vulgare
miR169	CCAAT-binding	509	MRT4513_38912C.1	H. vulgare
miR169	CCAAT-binding	510	MRT4513_51394C.1	H. vulgare
miR170/171	SCL	511	MRT4513_44124C.1	H. vulgare
miR172	AP2	512	MRT4513_6417C.1	H. vulgare
miR172	AP2 domain	513	MRT4513_42015C.1	H. vulgare
miR319	PCF	514	MRT4513_31590C.1	H. vulgare
miR319	PCF	515	MRT4513_52459C.1	H. vulgare
miR393	Transport inhibitor response	516	MRT4513_12741C.1	H. vulgare
miR393	Transport inhibitor response	517	MRT4513_38675C.1	H. vulgare
miR394	F-box	518	MRT4513_23211C.1	H. vulgare
miR396	Growth-regulating factor	519	MRT4513_20166C.2	H. vulgare
miR396	Growth-regulating factor	520	MRT4513_26009C.2	H. vulgare
miR396	Growth-regulating factor	521	MRT4513_33203C.1	H. vulgare
miR396	Growth-regulating factor	522	MRT4513_4600C.1	H. vulgare
miR396	Growth-regulating factor	523	MRT4513_50332C.1	H. vulgare
miR397	Laccase	524	MRT4513_35926C.1	H. vulgare
miR397	Laccase	525	MRT4513_40609C.1	H. vulgare
miR398	Copper/zinc superoxide dismutase	526	MRT4513_43414C.2	H. vulgare
miR398	Copper/zinc superoxide dismutase	527	MRT4513_8559C.2	H. vulgare
miR408	blue copper protein	528	MRT4513_31098C.2	H. vulgare
miR472	NBS-LRR disease resistance protein	529	MRT4513_5784C.1	H. vulgare
miR475	pentatricopeptide	530	MRT4513_47541C.1	H. vulgare
miR475	PPR	531	MRT4513_7525C.2	H. vulgare
miR482	disease resistance	532	MRT4513_11673C.1	H. vulgare
miR858	myb-like DNA-binding	533	MRT4513_11055C.1	H. vulgare
miR858	myb-like DNA-binding	534	MRT4513_42246C.1	H. vulgare
miR858	myb-like DNA-binding	535	MRT4513_4767C.1	H. vulgare
miR858	myb-like DNA-binding	536	MRT4513_5642C.1	H. vulgare
miR156/157	SBP domain	537	MRT3880_19943C.1	Medicago sativa
miR156/157	SBP domain	538	MRT3880_34839C.1	M. sativa
miR156/157	SBP domain	539	MRT3880_54023C.1	M. sativa
miR156/157	Squamosa Promoter Binding Protein	540	MRT3880_59834C.1	M. sativa
miR156/157	Squamosa Promoter Binding Protein	541	MRT3880_62151C.1	M. sativa
miR159	myb-like DNA-binding domain	542	MRT3880_51095C.1	M. sativa
miR160	Auxin Response Factor	543	MRT3880_22965C.1	M. sativa
miR160	Auxin Response Factor	544	MRT3880_28718C.1	M. sativa
miR160	Auxin Response Factor	545	MRT3880_38543C.1	M. sativa
miR160	Auxin Response Factor	546	MRT3880_44036C.1	M. sativa
miR161	PPR	547	MRT3880_11000C.1	M. sativa
miR161/475	Pentatricopeptide repeat	548	MRT3880_37878C.1	M. sativa
miR162	Dicer	549	MRT3880_26893C.1	M. sativa
miR164	NAC domain protein	550	MRT3880_18003C.2	M. sativa
miR164	No Apical Meristem	551	MRT3880_44619C.1	M. sativa
miR165/166	class III HD-Zip protein	552	MRT3880_37546C.1	M. sativa
miR165/166	class III HD-Zip protein	553	MRT3880_39764C.1	M. sativa
miR167	Auxin Response Factor	554	MRT3880_12926C.1	M. sativa
miR167	Auxin Response Factor	555	MRT3880_17672C.1	M. sativa
miR167	Auxin Response Factor	556	MRT3880_25270C.1	M. sativa
miR167	Auxin Response Factor	557	MRT3880_30476C.1	M. sativa
miR167	Auxin Response Factor	558	MRT3880_36150C.1	M. sativa
miR167	Auxin Response Factor	559	MRT3880_470C.1	M. sativa
miR169	nuclear transcription factor Y	560	MRT3880_16272C.2	M. sativa
miR169	nuclear transcription factor Y	561	MRT3880_21811C.2	M. sativa
miR169	nuclear transcription factor Y	562	MRT3880_59679C.1	M. sativa
miR170/171	GRAS	563	MRT3880_12452C.1	M. sativa
miR170/171	GRAS	564	MRT3880_29125C.1	M. sativa
miR170/171	GRAS	565	MRT3880_31130C.1	M. sativa
miR170/171	GRAS	566	MRT3880_40896C.1	M. sativa
miR170/171	GRAS	567	MRT3880_63440C.1	M. sativa
miR172	AP2 domain	568	MRT3880_36568C.1	M. sativa
miR172	AP2 domain	569	MRT3880_39959C.1	M. sativa
miR172	AP2 domain	570	MRT3880_55789C.1	M. sativa
miR319	TCP	571	MRT3880_2628C.1	M. sativa
miR319	TCP family transcription factor	572	MRT3880_44480C.1	M. sativa
miR393	Transport inhibitor response	573	MRT3880_18564C.2	M. sativa
miR393	Transport inhibitor response	574	MRT3880_38847C.1	M. sativa
miR393	Transport inhibitor response	575	MRT3880_67369C.1	M. sativa
miR396	Growth-regulating factor	576	MRT3880_18861C.1	M. sativa
miR396	Growth-regulating factor	577	MRT3880_22460C.1	M. sativa
miR396	Growth-regulating factor	578	MRT3880_41297C.1	M. sativa
miR397	Laccase	579	MRT3880_43121C.1	M. sativa
miR397	Laccase	580	MRT3880_56114C.2	M. sativa
miR400	pentatricopeptide	581	MRT3880_53970C.1	M. sativa
miR400	PPR	582	MRT3880_14263C.1	M. sativa
miR400	PPR	583	MRT3880_65540C.1	M. sativa
miR400/475	Pentatricopeptide repeat	584	MRT3880_27459C.1	M. sativa
miR400/475	Pentatricopeptide repeat	585	MRT3880_49876C.1	M. sativa
miR400/475	PPR	586	MRT3880_44329C.1	M. sativa
miR408	blue copper protein	587	MRT3880_46744C.2	M. sativa
miR408	blue copper protein	588	MRT3880_53025C.1	M. sativa
miR408	blue copper protein	589	MRT3880_5838C.1	M. sativa
miR472	ATP binding	590	MRT3880_29560C.1	M. sativa
miR472	ATP binding	591	MRT3880_30961C.1	M. sativa
miR472	ATP binding	592	MRT3880_48315C.1	M. sativa
miR472	ATP binding	593	MRT3880_53199C.1	M. sativa
miR472	ATP binding	594	MRT3880_54030C.2	M. sativa
miR472	ATP binding	595	MRT3880_57442C.1	M. sativa
miR472	disease resistance protein	596	MRT3880_10080C.1	M. sativa
miR472	disease resistance protein	597	MRT3880_12559C.2	M. sativa
miR472	disease resistance protein	598	MRT3880_17698C.1	M. sativa
miR472	disease resistance protein	599	MRT3880_21650C.1	M. sativa
miR472	disease resistance protein	600	MRT3880_22933C.1	M. sativa
miR472	disease resistance protein	601	MRT3880_26007C.1	M. sativa
miR472	disease resistance protein	602	MRT3880_28379C.1	M. sativa
miR472	disease resistance protein	603	MRT3880_3002C.1	M. sativa
miR472	disease resistance protein	604	MRT3880_38354C.1	M. sativa
miR472	disease resistance protein	605	MRT3880_41496C.1	M. sativa
miR472	disease resistance protein	606	MRT3880_51100C.1	M. sativa
miR472	disease resistance protein	607	MRT3880_5498C.1	M. sativa
miR472	disease resistance protein	608	MRT3880_59891C.1	M. sativa
miR472	NBS-LRR type disease resistance	609	MRT3880_45204C.1	M. sativa
	protein
miR472	NBS-LRR type disease resistance	610	MRT3880_52654C.1	M. sativa
	protein
miR472	NBS-LRR type disease resistance	611	MRT3880_66600C.1	M. sativa
	protein
miR472	NBS-LRR type disease resistance	612	MRT3880_7642C.1	M. sativa
	protein
miR472/482	disease resistance protein	613	MRT3880_19707C.1	M. sativa
miR472/482	disease resistance protein	614	MRT3880_19814C.1	M. sativa
miR472/482	disease resistance protein	615	MRT3880_26877C.1	M. sativa
miR472/482	disease resistance protein	616	MRT3880_2935C.1	M. sativa
miR472/482	disease resistance protein	617	MRT3880_36417C.1	M. sativa
miR472/482	disease resistance protein	618	MRT3880_44875C.1	M. sativa
miR472/482	disease resistance protein	619	MRT3880_5004C.1	M. sativa
miR472/482	disease resistance protein	620	MRT3880_52723C.1	M. sativa
miR472/482	disease resistance protein	621	MRT3880_57846C.1	M. sativa
miR472/482	disease resistance protein	622	MRT3880_63259C.1	M. sativa
miR472/482	disease resistance protein	623	MRT3880_6363C.1	M. sativa
miR472/482	disease resistance protein	624	MRT3880_65083C.1	M. sativa
miR472/482,	disease resistance protein, leucine rich	625	MRT3880_55187C.1	M. sativa
miR779	repeat
miR475	Pentatricopeptide repeat	626	MRT3880_13183C.1	M. sativa
miR475	Pentatricopeptide repeat	627	MRT3880_42014C.1	M. sativa
miR475	Pentatricopeptide repeat	628	MRT3880_46171C.1	M. sativa
miR475	PPR	629	MRT3880_12164C.1	M. sativa
miR475	PPR	630	MRT3880_12471C.1	M. sativa
miR475	PPR	631	MRT3880_16503C.1	M. sativa
miR475	PPR	632	MRT3880_22609C.1	M. sativa
miR475	PPR	633	MRT3880_35917C.1	M. sativa
miR475	PPR	634	MRT3880_39210C.1	M. sativa
miR475	PPR	635	MRT3880_55838C.1	M. sativa
miR475	PPR	636	MRT3880_56789C.1	M. sativa
miR475	PPR	637	MRT3880_65802C.1	M. sativa
miR475	PPR	638	MRT3880_870C.1	M. sativa
miR475	PPR	639	MRT3880_9632C.1	M. sativa
miR476	Pentatricopeptide repeat	640	MRT3880_13782C.1	M. sativa
miR477	GRAS	641	MRT3880_1038C.1	M. sativa
miR477	GRAS	642	MRT3880_14765C.1	M. sativa
miR477	GRAS	643	MRT3880_28393C.1	M. sativa
miR477	GRAS	644	MRT3880_31231C.1	M. sativa
miR477	GRAS	645	MRT3880_42028C.1	M. sativa
miR477	GRAS	646	MRT3880_51782C.1	M. sativa
miR482	disease resistance protein	647	MRT3880_12508C.1	M. sativa
miR482	disease resistance protein	648	MRT3880_16156C.1	M. sativa
miR482	disease resistance protein	649	MRT3880_22305C.1	M. sativa
miR482	disease resistance protein	650	MRT3880_30579C.1	M. sativa
miR482	disease resistance protein	651	MRT3880_38019C.1	M. sativa
miR482	disease resistance protein	652	MRT3880_4159C.1	M. sativa
miR482	disease resistance protein	653	MRT3880_49695C.1	M. sativa
miR482	disease resistance protein	654	MRT3880_54965C.1	M. sativa
miR482	disease resistance protein	655	MRT3880_56400C.1	M. sativa
miR482	disease resistance protein	656	MRT3880_56673C.1	M. sativa
miR482	disease resistance protein	657	MRT3880_58830C.1	M. sativa
miR482	disease resistance protein	658	MRT3880_58849C.1	M. sativa
miR482	disease resistance protein	659	MRT3880_59857C.1	M. sativa
miR482	disease resistance protein	660	MRT3880_60136C.1	M. sativa
miR482	disease resistance protein	661	MRT3880_65552C.2	M. sativa
miR482	disease resistance protein	662	MRT3880_8722C.1	M. sativa
miR482	disease resistance protein	663	MRT3880_9618C.1	M. sativa
miR828	myb-like DNA-binding	664	MRT3880_19611C.1	M. sativa
miR858	myb-like DNA-binding	665	MRT3880_10365C.1	M. sativa
miR858	myb-like DNA-binding	666	MRT3880_12267C.1	M. sativa
miR858	myb-like DNA-binding	667	MRT3880_19438C.1	M. sativa
miR858	myb-like DNA-binding	668	MRT3880_23642C.1	M. sativa
miR858	myb-like DNA-binding	669	MRT3880_33147C.1	M. sativa
miR858	myb-like DNA-binding	670	MRT3880_34889C.1	M. sativa
miR858	myb-like DNA-binding	671	MRT3880_39946C.1	M. sativa
miR858	myb-like DNA-binding	672	MRT3880_55009C.1	M. sativa
miR858	myb-like DNA-binding	673	MRT3880_56414C.1	M. sativa
miR858	myb-like DNA-binding	674	MRT3880_62538C.1	M. sativa
miR858	myb-like DNA-binding	675	MRT3880_801C.1	M. sativa
miR858	myb-like DNA-binding	676	MRT3880_8393C.1	M. sativa
miR859	F-box protein	677	MRT3880_46176C.1	M. sativa
miR859	F-box protein	678	MRT3880_47002C.1	M. sativa
miRMON13	PPR	679	MRT3880_52640C.1	M. sativa
miRMON13	PPR	680	MRT3880_60915C.1	M. sativa
miR156	SBP domain	681	MRT4530_118092C.3	Oryza sativa
miR156	SBP domain	682	MRT4530_135991C.4	O. sativa
miR156	SBP domain	683	MRT4530_257640C.1	O. sativa
miR156	SBP-domain	684	MRT4530_142142C.4	O. sativa
miR156	Squamosa Promoter Binding Protein	685	MRT4530_195506C.2	O. sativa
miR156	Squamosa Promoter Binding Protein	686	MRT4530_220364C.2	O. sativa
miR156	Squamosa Promoter Binding Protein	687	MRT4530_236277C.1	O. sativa
miR156	Squamosa Promoter Binding Protein	688	MRT4530_53217C.5	O. sativa
miR156	Squamosa Promoter Binding Protein	689	MRT4530_6964C.4	O. sativa
miR159	MYB	690	MRT4530_103606C.2	O. sativa
miR159	myb-like	691	MRT4530_82994C.2	O. sativa
miR159	myb-like DNA-binding domain	692	MRT4530_103605C.3	O. sativa
miR159	myb-like DNA-binding domain	693	MRT4530_156102C.3	O. sativa
miR159	myb-like DNA-binding domain	694	MRT4530_181046C.3	O. sativa
miR159	myb-like DNA-binding domain	695	MRT4530_42135C.5	O. sativa
miR160	ARF	696	PHE0003527	O. sativa
miR160	ARF	697	PHE0003528	O. sativa
miR160	Auxin Response Factor	698	MRT4530_228913C.1	O. sativa
miR160	Auxin Response Factor	699	MRT4530_69952C.4	O. sativa
miR160	Auxin Response Factor	700	MRT4530_71017C.4	O. sativa
miR160	Auxin Response Factor	701	MRT4530_75962C.5	O. sativa
miR162	CAF	702	MRT4530_212066C.2	O. sativa
miR164	NAC	703	MRT4530_224181C.2	O. sativa
miR164	NAC domain protein	704	MRT4530_178256C.3	O. sativa
miR164	NAC domain protein	705	MRT4530_221769C.1	O. sativa
miR164	NAC1	706	MRT4530_141528C.5	O. sativa
miR164	No Apical Meristem	707	MRT4530_147737C.4	O. sativa
miR164	No Apical Meristem	708	MRT4530_157393C.3	O. sativa
miR166	HD-ZIP	709	MRT4530_253068C.2	O. sativa
miR167	ARF	710	PHE0003657	O. sativa
miR167	Auxin Response Factor	711	MRT4530_86291C.3	O. sativa
miR168	Argonaute	712	MRT4530_147864C.3	O. sativa
miR169	CCAAT-binding	713	MRT4530_156068C.3	O. sativa
miR169	CCAAT-binding	714	MRT4530_52650C.3	O. sativa
miR169	CCAAT-binding	715	MRT4530_98042C.6	O. sativa
miR171	GRAS	716	MRT4530_157676C.3	O. sativa
miR171	GRAS	717	MRT4530_159257C.2	O. sativa
miR171	GRAS	718	MRT4530_177712C.1	O. sativa
miR171	GRAS	719	MRT4530_64038C.2	O. sativa
miR171	Scarecrow-like	720	MRT4530_146050C.4	O. sativa
miR171	SCL	721	MRT4530_111185C.3	O. sativa
miR171	SCL	722	MRT4530_12928C.2	O. sativa
miR171	SCL	723	MRT4530_88963C.6	O. sativa
miR172	AP2	724	PHE0003882	O. sativa
miR172	AP2 domain	725	MRT4530_160275C.3	O. sativa
miR172	AP2 domain	726	MRT4530_56773C.3	O. sativa
miR319	TCP family transcription factor	727	MRT4530_154891C.2	O. sativa
miR319	TCP family transcription factor	728	MRT4530_9431C.5	O. sativa
miR319	TCP3	729	MRT4530_151800C.2	O. sativa
miR393	Transport inhibitor response	730	MRT4530_241313C.2	O. sativa
miR395	ATP sulfurylase	731	MRT4530_16384C.4	O. sativa
miR395	sulfate transporter	732	MRT4530_33633C.6	O. sativa
miR396	Growth-regulating factor	733	PHE0000026	O. sativa
miR396	Growth-regulating factor	734	MRT4530_140789C.3	O. sativa
miR396	Growth-regulating factor	735	MRT4530_145151C.4	O. sativa
miR396	Growth-regulating factor	736	MRT4530_147352C.3	O. sativa
miR396	Growth-regulating factor	737	MRT4530_180707C.1	O. sativa
miR396	Growth-regulating factor	738	MRT4530_221461C.1	O. sativa
miR396	Growth-regulating factor	739	MRT4530_63308C.3	O. sativa
miR396	Growth-regulating factor	740	MRT4530_73195C.3	O. sativa
miR396	Growth-regulating factor	741	MRT4530_83576C.4	O. sativa
miR397	Laccase	742	MRT4530_148379C.4	O. sativa
miR397	Laccase	743	MRT4530_181828C.1	O. sativa
miR397	Laccase	744	MRT4530_237569C.1	O. sativa
miR397	Laccase	745	MRT4530_60143C.3	O. sativa
miR408	blue copper protein	746	MRT4530_137979C.3	O. sativa
miR408	blue copper protein	747	MRT4530_260849C.1	O. sativa
miR408	blue copper protein	748	MRT4530_40477C.6	O. sativa
miR408	Laccase	749	MRT4530_160612C.2	O. sativa
miR408	Laccase	750	MRT4530_169405C.1	O. sativa
miR444	MADS	751	MRT4530_27947C.3	O. sativa
miR444	MADS	752	MRT4530_78475C.3	O. sativa
miR444	MADS box	753	PHE0001381	O. sativa
miR444	MADS box	754	PHE0015548	O. sativa
miR444	MADS box	755	PHE0015549	O. sativa
miR444	MADS-box	756	PHE0003829	O. sativa
miR444	MADS-box	757	MRT4530_196636C.3	O. sativa
miR809	Mlo	758	MRT4530_59197C.5	O. sativa
miR538	MADS-box	759	PHE0014613	Physcomitrella
				patens
miR156/157	SBP domain	760	MRT4558_6587C.1	Sorghum bicolor
miR156/157	SBP-domain	761	MRT4558_12680C.1	S. bicolor
miR156/157	Squamosa Promoter Binding Protein	762	MRT4558_8644C.2	S. bicolor
miR159	GAMYB	763	MRT4558_37619C.1	S. bicolor
miR160	Auxin Response Factor	764	MRT4558_27799C.1	S. bicolor
miR164	NAC domain protein	765	MRT4558_43436C.1	S. bicolor
miR164	NAC domain protein	766	MRT4558_4564C.2	S. bicolor
miR164	NAC1	767	MRT4558_43081C.1	S. bicolor
miR164	No Apical Meristem	768	MRT4558_41467C.1	S. bicolor
miR165/166	class III HD-Zip protein	769	MRT4558_27560C.1	S. bicolor
miR167	Auxin Response Factor	770	MRT4558_10718C.3	S. bicolor
miR167	Auxin Response Factor	771	MRT4558_1659C.2	S. bicolor
miR167	Auxin Response Factor	772	MRT4558_37108C.1	S. bicolor
miR169	CCAAT-binding	773	MRT4558_11671C.2	S. bicolor
miR169	CCAAT-binding	774	MRT4558_13240C.2	S. bicolor
miR169	CCAAT-binding	775	MRT4558_19368C.2	S. bicolor
miR169	CCAAT-binding	776	MRT4558_8287C.2	S. bicolor
miR170/171	SCL	777	MRT4558_7655C.1	S. bicolor
miR172	AP2 domain	778	MRT4558_25704C.2	S. bicolor
miR393	Transport inhibitor response	779	MRT4558_1226C.2	S. bicolor
miR393	Transport inhibitor response	780	MRT4558_20000C.2	S. bicolor
miR394	F-box domain	781	MRT4558_11973C.2	S. bicolor
miR395	sulfate adenylyltransferase	782	MRT4558_11861C.1	S. bicolor
miR395	Sulfate transporter	783	MRT4558_24400C.2	S. bicolor
miR396	Growth-regulating factor	784	MRT4558_13321C.2	S. bicolor
miR400	Pentatricopeptide repeat	785	MRT4558_43831C.1	S. bicolor
miR408	blue copper protein	786	MRT4558_16166C.2	S. bicolor
miR408	blue copper protein	787	MRT4558_8981C.2	S. bicolor
miR408	Laccase	788	MRT4558_40844C.1	S. bicolor
miR444	MADS-box	789	MRT4558_11440C.2	S. bicolor
miR472	ATP binding	790	MRT4558_33723C.1	S. bicolor
miR475	PPR	791	MRT4558_5261C.2	S. bicolor
miR536	F-box protein	792	MRT4558_34710C.1	S. bicolor
miR858	myb-like DNA-binding	793	MRT4558_5881C.2	S. bicolor
miR858	myb-like DNA-binding	794	MRT4558_642C.1	S. bicolor
miR159	myb protein	795	MRT4565_281735C.1	Triticum aestivum
miR169	CCAAT	796	MRT4565_240119C.2	T. aestivum
miR169	CCAAT	797	MRT4565_270644C.2	T. aestivum
miR172	AP2	798	MRT4565_247090C.1	T. aestivum
miR394	F-box	799	MRT4565_259298C.2	T. aestivum
miR444	MADS box	800	PHE0002649	T. aestivum
miR444	MADS-box	801	MRT4565_247066C.1	T. aestivum
miR444	MADS-box	802	MRT4565_258649C.1	T. aestivum
miR529	AP2	803	MRT4565_278632C.2	T. aestivum
miR858	MYB	804	MRT4565_223049C.1	T. aestivum
miR165/166	REV	805	PHE0012638	unidentified
miR824	MADS box	806	PHE0015528	unidentified
miR824	MADS box	807	PHE0015545	unidentified
miR1029	erf	808	MRT4577_148956C.8	Zea mays
miR1029	erf	809	MRT4577_267494C.5	Z. mays
miR1029	erf	810	MRT4577_389477C.2	Z. mays
miR1029	erf	811	MRT4577_48700C.7	Z. mays
miR1029	erf	812	MRT4577_565542C.1	Z. mays
miR1029	erf	813	MRT4577_600239C.1	Z. mays
miR156	Squamosa Promoter Binding	814	MRT4577_396357C.4	Z. mays
miR156/157	SBP domain	815	MRT4577_122478C.6	Z. mays
miR156/157	SBP domain	816	MRT4577_270892C.4	Z. mays
miR156/157	SBP domain	817	MRT4577_334372C.5	Z. mays
miR156/157	SBP domain	818	MRT4577_532824C.3	Z. mays
miR156/157	SBP domain	819	MRT4577_535297C.2	Z. mays
miR156/157	SBP domain	820	MRT4577_537670C.2	Z. mays
miR156/157	SBP domain	821	MRT4577_565057C.1	Z. mays
miR156/157	SBP domain	822	MRT4577_568647C.1	Z. mays
miR156/157	SBP domain	823	MRT4577_571545C.1	Z. mays
miR156/157	SBP domain	824	MRT4577_644419C.1	Z. mays
miR156/157	SBP-domain	825	MRT4577_23629C.7	Z. mays
miR156/157	SBP-domain	826	MRT4577_295538C.7	Z. mays
miR156/157	SBP-domain	827	MRT4577_31704C.9	Z. mays
miR156/157	Squamosa Promoter Binding	828	MRT4577_427964C.4	Z. mays
miR156/157	Squamosa Promoter Binding	829	MRT4577_461098C.3	Z. mays
miR156/157	Squamosa Promoter Binding Protein	830	MRT4577_137984C.6	Z. mays
miR156/157	Squamosa Promoter Binding Protein	831	MRT4577_188360C.6	Z. mays
miR156/157	Squamosa Promoter Binding Protein	832	MRT4577_205098C.7	Z. mays
miR156/157	Squamosa Promoter Binding Protein	833	MRT4577_26483C.7	Z. mays
miR156/157	Squamosa Promoter Binding Protein	834	MRT4577_341149C.6	Z. mays
miR156/157	Squamosa Promoter Binding Protein	835	MRT4577_383301C.4	Z. mays
miR156/157	Squamosa Promoter Binding Protein	836	MRT4577_42534C.9	Z. mays
miR156/157	Squamosa Promoter Binding Protein	837	MRT4577_564644C.1	Z. mays
miR156/157	Squamosa Promoter Binding Protein	838	MRT4577_619443C.1	Z. mays
miR156/157	Squamosa Promoter-Binding	839	MRT4577_333683C.4	Z. mays
miR156/157	Squamosa Promoter-Binding	840	MRT4577_38044C.8	Z. mays
miR156/157	teosinte glume architecture	841	MRT4577_181019C.5	Z. mays
miR156/157	teosinte glume architecture	842	MRT4577_78773C.8	Z. mays
miR159	GAMYB	843	MRT4577_481577C.2	Z. mays
miR159	MYB	844	MRT4577_210747C.5	Z. mays
miR159	MYB	845	MRT4577_542744C.2	Z. mays
miR159	myb-like	846	MRT4577_298452C.5	Z. mays
miR159	myb-like DNA-binding	847	MRT4577_565447C.1	Z. mays
miR159	myb-like DNA-binding	848	MRT4577_565456C.1	Z. mays
miR159	myb-like DNA-binding domain	849	MRT4577_30813C.8	Z. mays
miR159	myb-like DNA-binding domain	850	MRT4577_390477C.4	Z. mays
miR159	myb-like DNA-binding domain	851	MRT4577_391124C.5	Z. mays
miR159	myb-like DNA-binding domain	852	MRT4577_416957C.3	Z. mays
miR159	myb-like DNA-binding domain	853	MRT4577_545477C.2	Z. mays
miR159	myb-like DNA-binding domain	854	MRT4577_582653C.1	Z. mays
miR159	myb-like DNA-binding domain	855	MRT4577_598088C.1	Z. mays
miR159	myb-like DNA-binding domain	856	MRT4577_605039C.1	Z. mays
miR159	myb-like DNA-binding domain	857	MRT4577_613992C.1	Z. mays
miR159	myb-like DNA-binding domain	858	MRT4577_622542C.1	Z. mays
miR159	myb-like DNA-binding domain	859	MRT4577_709777C.1	Z. mays
miR159	myb-like DNA-binding domain	860	MRT4577_77765C.6	Z. mays
miR160	Auxin Response Factor	861	MRT4577_256734C.4	Z. mays
miR160	Auxin Response Factor	862	MRT4577_258637C.3	Z. mays
miR160	Auxin Response Factor	863	MRT4577_385317C.4	Z. mays
miR160	Auxin Response Factor	864	MRT4577_400043C.5	Z. mays
miR160	Auxin Response Factor	865	MRT4577_41620C.7	Z. mays
miR160	Auxin Response Factor	866	MRT4577_429671C.4	Z. mays
miR160	Auxin Response Factor	867	MRT4577_430512C.4	Z. mays
miR160	Auxin Response Factor	868	MRT4577_448022C.1	Z. mays
miR160	Auxin Response Factor	869	MRT4577_503622C.2	Z. mays
miR160	Auxin Response Factor	870	MRT4577_569655C.1	Z. mays
miR160	Auxin Response Factor	871	MRT4577_605037C.1	Z. mays
miR161	PPR	872	MRT4577_219343C.5	Z. mays
miR161	PPR	873	MRT4577_338127C.1	Z. mays
miR161	PPR	874	MRT4577_381918C.5	Z. mays
miR161	PPR	875	MRT4577_549370C.2	Z. mays
miR161	PPR	876	MRT4577_653452C.1	Z. mays
miR162	Dicer	877	MRT4577_226226C.4	Z. mays
miR162	Dicer	878	MRT4577_50615C.6	Z. mays
miR162	Dicer	879	MRT4577_592675C.1	Z. mays
miR164	NAC domain protein	880	MRT4577_686098C.1	Z. mays
miR164	NAC domain protein	881	MRT4577_98755C.5	Z. mays
miR164	NAC1	882	PHE0003788	Z. mays
miR164	No Apical Meristem	883	MRT4577_105083C.9	Z. mays
miR164	No Apical Meristem	884	MRT4577_16045C.7	Z. mays
miR164	No Apical Meristem	885	MRT4577_256695C.4	Z. mays
miR164	No Apical Meristem	886	MRT4577_29326C.8	Z. mays
miR164	No Apical Meristem	887	MRT4577_317955C.5	Z. mays
miR164	No Apical Meristem	888	MRT4577_370828C.5	Z. mays
miR164	No Apical Meristem	889	MRT4577_394716C.4	Z. mays
miR164	No Apical Meristem	890	MRT4577_586054C.1	Z. mays
miR164	No Apical Meristem	891	MRT4577_625707C.1	Z. mays
miR164	No Apical Meristem	892	MRT4577_629408C.1	Z. mays
miR164	No Apical Meristem	893	MRT4577_705865C.1	Z. mays
miR164	No Apical Meristem	894	MRT4577_9951C.8	Z. mays
miR165/166	class III HD-Zip protein	895	MRT4577_197925C.4	Z. mays
miR165/166	class III HD-Zip protein	896	MRT4577_200605C.3	Z. mays
miR165/166	class III HD-Zip protein	897	MRT4577_320718C.6	Z. mays
miR165/166	class III HD-Zip protein	898	MRT4577_43102C.9	Z. mays
miR165/166	class III HD-Zip protein	899	MRT4577_535928C.2	Z. mays
miR165/166	class III HD-Zip protein	900	MRT4577_568616C.1	Z. mays
miR165/166	class III HD-Zip protein	901	MRT4577_613062C.1	Z. mays
miR165/166	class III HD-Zip protein	902	MRT4577_659410C.1	Z. mays
miR165/166	class III HD-Zip protein	903	MRT4577_673351C.1	Z. mays
miR165/166	HD-ZIP	904	PHE0008043	Z. mays
miR165/166	Rev	905	PHE0007773	Z. mays
miR165/166	Rev	906	PHE0012657	Z. mays
miR165/166	rolled leaf	907	MRT4577_229497C.6	Z. mays
miR165/166	rolled leaf	908	MRT4577_312384C.3	Z. mays
miR165/166	rolled leaf	909	MRT4577_342259C.4	Z. mays
miR165/166	rolled leaf	910	MRT4577_442838C.4	Z. mays
miR165/166	rolled leaf	911	MRT4577_535676C.2	Z. mays
miR165/166	rolled leaf	912	MRT4577_566770C.1	Z. mays
miR165/166	rolled leaf	913	MRT4577_586718C.1	Z. mays
miR167	ARF	914	PHE0003656	Z. mays
miR167	Auxin Response Factor	915	MRT4577_267543C.4	Z. mays
miR167	Auxin Response Factor	916	MRT4577_267545C.6	Z. mays
miR167	Auxin Response Factor	917	MRT4577_306050C.5	Z. mays
miR167	Auxin Response Factor	918	MRT4577_310720C.4	Z. mays
miR167	Auxin Response Factor	919	MRT4577_339989C.4	Z. mays
miR167	Auxin Response Factor	920	MRT4577_35746C.4	Z. mays
miR167	Auxin Response Factor	921	MRT4577_360403C.2	Z. mays
miR167	Auxin Response Factor	922	MRT4577_377896C.4	Z. mays
miR167	Auxin Response Factor	923	MRT4577_45522C.9	Z. mays
miR167	Auxin Response Factor	924	MRT4577_509023C.3	Z. mays
miR167	Auxin Response Factor	925	MRT4577_521851C.2	Z. mays
miR167	Auxin Response Factor	926	MRT4577_536912C.2	Z. mays
miR167	Auxin Response Factor	927	MRT4577_569979C.1	Z. mays
miR167	Auxin Response Factor	928	MRT4577_650810C.1	Z. mays
miR167	Auxin Response Factor	929	MRT4577_676039C.1	Z. mays
miR167	Auxin Response Factor	930	MRT4577_680014C.1	Z. mays
miR167	Auxin Response Factor	931	MRT4577_681088C.1	Z. mays
miR167	Auxin Response Factor	932	MRT4577_681995C.1	Z. mays
miR167	Auxin Response Factor	933	MRT4577_683953C.1	Z. mays
miR167	Auxin Response Factor	934	MRT4577_684325C.1	Z. mays
miR167	Auxin Response Factor	935	MRT4577_8821C.7	Z. mays
miR168	Argonaute	936	MRT4577_247045C.8	Z. mays
miR168	Argonaute	937	MRT4577_29086C.7	Z. mays
miR168	Argonaute	938	MRT4577_418712C.5	Z. mays
miR168	Argonaute	939	MRT4577_57570C.9	Z. mays
miR168	Argonaute	940	MRT4577_577443C.1	Z. mays
miR169	CCAAT-binding	941	MRT4577_40749C.8	Z. mays
miR169	CCAAT-binding	942	MRT4577_428392C.4	Z. mays
miR169	CCAAT-binding	943	MRT4577_434247C.4	Z. mays
miR169	CCAAT-binding	944	MRT4577_536961C.2	Z. mays
miR169	CCAAT-binding	945	MRT4577_536962C.2	Z. mays
miR169	CCAAT-binding	946	MRT4577_540147C.2	Z. mays
miR169	CCAAT-binding	947	MRT4577_556372C.2	Z. mays
miR169	CCAAT-binding	948	MRT4577_570254C.1	Z. mays
miR169	CCAAT-binding	949	MRT4577_668660C.1	Z. mays
miR169	CCAAT-binding	950	MRT4577_693949C.1	Z. mays
miR169	CCAAT-binding	951	MRT4577_701125C.1	Z. mays
miR170/171	SCL	952	PHE0006551	Z. mays
miR170/171	SCL	953	MRT4577_140896C.6	Z. mays
miR170/171	SCL	954	MRT4577_234039C.6	Z. mays
miR170/171	SCL	955	MRT4577_269667C.5	Z. mays
miR170/171	SCL	956	MRT4577_520619C.2	Z. mays
miR170/171	SCL	957	MRT4577_617401C.1	Z. mays
miR170/171	SCL	958	MRT4577_75777C.8	Z. mays
miR171	GRAS	959	MRT4577_26778C.8	Z. mays
miR171	GRAS	960	MRT4577_30852C.6	Z. mays
miR171	GRAS	961	MRT4577_683754C.1	Z. mays
miR171	GRAS	962	MRT4577_687943C.1	Z. mays
miR171	Scarecrow	963	MRT4577_569322C.1	Z. mays
miR172	AP2	964	PHE0006602	Z. mays
miR172	AP2 domain	965	MRT4577_12523C.7	Z. mays
miR172	AP2 domain	966	MRT4577_27478C.9	Z. mays
miR172	AP2 domain	967	MRT4577_304712C.4	Z. mays
miR172	AP2 domain	968	MRT4577_307553C.7	Z. mays
miR172	AP2 domain	969	MRT4577_431122C.3	Z. mays
miR172	AP2 domain	970	MRT4577_455774C.3	Z. mays
miR172	AP2 domain	971	MRT4577_468762C.3	Z. mays
miR172	AP2 domain	972	MRT4577_548310C.2	Z. mays
miR172	AP2 domain	973	MRT4577_556612C.2	Z. mays
miR172	AP2 domain	974	MRT4577_597136C.1	Z. mays
miR172	AP2 domain	975	MRT4577_669210C.1	Z. mays
miR172	AP2 domain	976	MRT4577_676464C.1	Z. mays
miR172	AP2 domain	977	MRT4577_708079C.1	Z. mays
miR172	APETALA2	978	MRT4577_49517C.8	Z. mays
miR172	APETALA2	979	MRT4577_700043C.1	Z. mays
miR172	Glossy15	980	PHE0000011	Z. mays
miR319	Cyclin	981	PHE0001434	Z. mays
miR319	PCF	982	MRT4577_427906C.4	Z. mays
miR319	PCF	983	MRT4577_480991C.1	Z. mays
miR319	PCF	984	MRT4577_568064C.1	Z. mays
miR319	PCF	985	MRT4577_590917C.1	Z. mays
miR319	PCF	986	MRT4577_679533C.1	Z. mays
miR319	PCF	987	MRT4577_680167C.1	Z. mays
miR319	TCP family transcription factor	988	MRT4577_147719C.7	Z. mays
miR319	TCP family transcription factor	989	MRT4577_221733C.7	Z. mays
miR319	TCP family transcription factor	990	MRT4577_275063C.6	Z. mays
miR319	TCP family transcription factor	991	MRT4577_30525C.6	Z. mays
miR319	TCP family transcription factor	992	MRT4577_340633C.4	Z. mays
miR319	TCP family transcription factor	993	MRT4577_557860C.2	Z. mays
miR319	TCP family transcription factor	994	MRT4577_558102C.2	Z. mays
miR319	TCP family transcription factor	995	MRT4577_568063C.1	Z. mays
miR319	TCP family transcription factor	996	MRT4577_571095C.1	Z. mays
miR319	TCP family transcription factor	997	MRT4577_590269C.1	Z. mays
miR319	TCP family transcription factor	998	MRT4577_686625C.1	Z. mays
miR390	TAS	999	MRT4577_306288C.5	Z. mays
miR390	TAS	1000	MRT4577_325578C.3	Z. mays
miR390	TAS	1001	MRT4577_687438C.1	Z. mays
miR390	TAS	1002	MRT4577_72903C.4	Z. mays
miR393	F-box	1003	PHE0000546	Z. mays
miR393	F-box	1004	PHE0000912	Z. mays
miR393	Transport inhibitor response	1005	MRT4577_39097C.9	Z. mays
miR393	Transport inhibitor response	1006	MRT4577_546333C.2	Z. mays
miR393	Transport inhibitor response	1007	MRT4577_560980C.2	Z. mays
miR393	Transport inhibitor response	1008	MRT4577_656737C.1	Z. mays
miR393	Transport inhibitor response	1009	MRT4577_688815C.1	Z. mays
miR394	F-box domain	1010	MRT4577_56429C.8	Z. mays
miR394	F-box domain	1011	MRT4577_613832C.1	Z. mays
miR395	AST	1012	MRT4577_293072C.7	Z. mays
miR395	AST	1013	MRT4577_57393C.8	Z. mays
miR395	AST	1014	MRT4577_594643C.1	Z. mays
miR395	AST	1015	MRT4577_655078C.1	Z. mays
miR395	AST	1016	MRT4577_681126C.1	Z. mays
miR395	ATP sulfurylase	1017	MRT4577_118322C.5	Z. mays
miR395	ATP sulfurylase	1018	MRT4577_453989C.4	Z. mays
miR395	sulfate adenylyltransferase	1019	MRT4577_386324C.4	Z. mays
miR395	sulfate adenylyltransferase	1020	MRT4577_57434C.9	Z. mays
miR395	sulfate adenylyltransferase	1021	MRT4577_694623C.1	Z. mays
miR395	sulfate adenylyltransferase	1022	MRT4577_709359C.1	Z. mays
miR395	sulfate transporter	1023	MRT4577_644561C.1	Z. mays
miR396	Growth-regulating factor	1024	PHE0000025	Z. mays
miR396	Growth-regulating factor	1025	PHE0000289	Z. mays
miR396	Growth-regulating factor	1026	PHE0001216	Z. mays
miR396	Growth-regulating factor	1027	MRT4577_215581C.4	Z. mays
miR396	Growth-regulating factor	1028	MRT4577_215583C.5	Z. mays
miR396	Growth-regulating factor	1029	MRT4577_232004C.7	Z. mays
miR396	Growth-regulating factor	1030	MRT4577_24924C.7	Z. mays
miR396	Growth-regulating factor	1031	MRT4577_266456C.6	Z. mays
miR396	Growth-regulating factor	1032	MRT4577_278593C.3	Z. mays
miR396	Growth-regulating factor	1033	MRT4577_29961C.8	Z. mays
miR396	Growth-regulating factor	1034	MRT4577_356670C.6	Z. mays
miR396	Growth-regulating factor	1035	MRT4577_359461C.1	Z. mays
miR396	Growth-regulating factor	1036	MRT4577_372672C.5	Z. mays
miR396	Growth-regulating factor	1037	MRT4577_410501C.4	Z. mays
miR396	Growth-regulating factor	1038	MRT4577_432229C.3	Z. mays
miR396	Growth-regulating factor	1039	MRT4577_534804C.2	Z. mays
miR396	Growth-regulating factor	1040	MRT4577_551090C.1	Z. mays
miR396	Growth-regulating factor	1041	MRT4577_563407C.1	Z. mays
miR396	Growth-regulating factor	1042	MRT4577_569284C.1	Z. mays
miR396	Growth-regulating factor	1043	MRT4577_597418C.1	Z. mays
miR396	Growth-regulating factor	1044	MRT4577_618948C.1	Z. mays
miR396	Growth-regulating factor	1045	MRT4577_635741C.1	Z. mays
miR397	Laccase	1046	MRT4577_233334C.7	Z. mays
miR397	Laccase	1047	MRT4577_26704C.2	Z. mays
miR397	Laccase	1048	MRT4577_293572C.3	Z. mays
miR397	Laccase	1049	MRT4577_602028C.1	Z. mays
miR398	cytochrome c oxidase	1050	MRT4577_434356C.4	Z. mays
miR398	cytochrome c oxidase	1051	MRT4577_547404C.2	Z. mays
miR399	Cyclin	1052	PHE0002694	Z. mays
miR400	PPR	1053	MRT4577_480700C.2	Z. mays
miR400	PPR	1054	MRT4577_593504C.1	Z. mays
miR408	blue copper protein	1055	MRT4577_325458C.1	Z. mays
miR408	blue copper protein	1056	MRT4577_37590C.9	Z. mays
miR408	blue copper protein	1057	MRT4577_47069C.8	Z. mays
miR408	blue copper protein	1058	MRT4577_528699C.2	Z. mays
miR408	blue copper protein	1059	MRT4577_550892C.1	Z. mays
miR408	Laccase	1060	PHE0003380	Z. mays
miR408	Laccase	1061	MRT4577_245033C.8	Z. mays
miR408	Laccase	1062	MRT4577_380413C.6	Z. mays
miR408	Laccase	1063	MRT4577_388860C.4	Z. mays
miR408	Laccase	1064	MRT4577_461451C.3	Z. mays
miR408	Laccase	1065	MRT4577_625157C.1	Z. mays
miR408	Laccase	1066	MRT4577_629379C.1	Z. mays
miR408	plantacyanin	1067	PHE0000329	Z. mays
miR444	MADS	1068	PHE0013719	Z. mays
miR444	MADS box	1069	PHE0002650	Z. mays
miR444	MADS box	1070	MRT4577_321664C.4	Z. mays
miR444	MADS-box	1071	MRT4577_204116C.4	Z. mays
miR444	MADS-box	1072	MRT4577_537511C.2	Z. mays
miR444	MADS-box	1073	MRT4577_553467C.1	Z. mays
miR444	MADS-box	1074	MRT4577_613242C.1	Z. mays
miR444	MADS-box	1075	MRT4577_695496C.1	Z. mays
miR472	ATP binding	1076	MRT4577_110498C.5	Z. mays
miR472	ATP binding	1077	MRT4577_251486C.3	Z. mays
miR472	NBS-LRR type disease resistance	1078	MRT4577_320221C.4	Z. mays
	protein
miR475	PPR	1079	MRT4577_110120C.3	Z. mays
miR475	PPR	1080	MRT4577_205728C.3	Z. mays
miR475	PPR	1081	MRT4577_664698C.1	Z. mays
miR477	GRAS	1082	MRT4577_278714C.7	Z. mays
miR477	GRAS	1083	MRT4577_401721C.2	Z. mays
miR477	GRAS	1084	MRT4577_463199C.2	Z. mays
miR477	GRAS	1085	MRT4577_526548C.1	Z. mays
miR477	GRAS	1086	MRT4577_569010C.1	Z. mays
miR482	disease resistance	1087	MRT4577_204880C.4	Z. mays
miR482	disease resistance	1088	MRT4577_285745C.3	Z. mays
miR482	disease resistance	1089	MRT4577_537326C.2	Z. mays
miR482	disease resistance	1090	MRT4577_642390C.1	Z. mays
miR482	disease resistance	1091	MRT4577_647253C.1	Z. mays
miR482	disease resistance	1092	MRT4577_700169C.1	Z. mays
miR776	IRE	1093	MRT4577_475418C.2	Z. mays
miR776	IRE	1094	MRT4577_569446C.1	Z. mays
miR776	IRE	1095	MRT4577_668929C.1	Z. mays
miR827	SYG1/Pho81/XPR1	1096	MRT4577_565044C.1	Z. mays
miR844	protein kinase	1097	MRT4577_34878C.9	Z. mays
miR844	protein kinase	1098	MRT4577_469768C.2	Z. mays
miR857	LAC	1099	MRT4577_447458C.4	Z. mays
miR858	MYB	1100	MRT4577_230084C.4	Z. mays
miR858	MYB	1101	MRT4577_28298C.7	Z. mays
miR858	MYB	1102	MRT4577_365133C.3	Z. mays
miR858	MYB	1103	MRT4577_691552C.1	Z. mays
miR858	myb-like	1104	MRT4577_237723C.3	Z. mays
miR858	myb-like DNA-binding	1105	MRT4577_204899C.4	Z. mays
miR858	myb-like DNA-binding	1106	MRT4577_229676C.2	Z. mays
miR858	myb-like DNA-binding	1107	MRT4577_303539C.6	Z. mays
miR858	myb-like DNA-binding	1108	MRT4577_330816C.1	Z. mays
miR858	myb-like DNA-binding	1109	MRT4577_340919C.6	Z. mays
miR858	myb-like DNA-binding	1110	MRT4577_549954C.1	Z. mays
miR858	myb-like DNA-binding	1111	MRT4577_585620C.1	Z. mays
miR858	myb-like DNA-binding	1112	MRT4577_665482C.1	Z. mays
miR858	myb-like DNA-binding	1113	MRT4577_704749C.1	Z. mays
miR904	AGO	1114	MRT4577_374929C.6	Z. mays

Example 4

This example provides additional embodiments of target genes identified as “validated miRNA targets” (i.e., containing a validated miRNA recognition site) and representative uses of validated miRNA recognition sites, e.g., for the design of artificial sequences useful in making recombinant DNA constructs, including, but not limited to, transgenes with an exogenous miRNA recognition site added, transgenes with a native miRNA recognition site modified or deleted, decoys, cleavage blockers, or translational inhibitors as taught and claimed by Applicants. Recombinant DNA constructs of this invention are useful for modulating expression of such target genes and for making non-natural transgenic plant cells, plant tissues, and plants (especially non-natural transgenic crop plants) having improved yield or other desirable traits.
Table 3 provides a list of miRNAs and miRNA targets containing miRNA recognition sites that were identified in various plants using techniques similar to those described in Example 2. The miRNA targets were identified by gene name, protein domain, function, location, or simply as a gene having a miRNA recognition site; this information is sufficient for designing artificial sequences including miRNA-unresponsive transgenes, cleavage blockers, 5′-modified cleavage blockers, translational inhibitors, and miRNA decoys. Table 3 further provides a list of miRNA precursors (designed to be processed to a native mature miRNA), as well as artificial sequences including miRNA precursors designed to be processed to a synthetic mature miRNA, miRNA decoys, miRNA-unresponsive transgenes, and miRNA cleavage blockers, all of which are especially useful in making recombinant DNA constructs of this invention. One of ordinary skill in the art, informed by the teachings of this application and provided with the nucleotide sequence of a miRNA or a miRNA recognition site in a target gene, would be readily able to devise such artificial sequences. Such a person of ordinary skill would further recognize that knowledge of the target gene itself is not required, merely the sequence of the mature miRNA sequence or of a miRNA precursor that is processed to the mature miRNA—or, alternatively, knowledge of the miRNA recognition site sequence—in combination with the teachings of this application, in order to devise a cleavage blocker (or 5′-modified cleavage blocker) to inhibit the target gene silencing effects of a given miRNA. Table 3 also provides examples of recombinant DNA constructs which, when transgenically expressed in a crop plant (preferably, but not limited to, maize or corn, soybean, canola, cotton, alfalfa, sugarcane, sugar beet, sorghum, and rice), results in improved yield by that crop plant, when compared to the crop plant in which the construct is not expressed. Techniques for making transgenic plants are described under the heading “Making and Using Transgenic Plant Cells and Transgenic Plants”. “Improved yield” can be increased intrinsic yield; in other embodiments, improved yield is yield increased under a particular growing condition, such as abiotic or biotic stress conditions (e.g., heat or cold stress, drought stress, or nutrient stress), when compared to a crop lacking expression of the recombinant DNA construct of this invention.
With the above information about miRNA targets, one of ordinary skill in the art is able to make and use various additional embodiments of aspects of this invention, including a recombinant DNA construct transcribable in a plant cell, including a promoter that is functional in the plant cell and operably linked to at least one polynucleotide selected from: (a) DNA encoding a cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (b) DNA encoding a 5′-modified cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (c) DNA encoding a translational inhibitor to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (d) DNA encoding a decoy to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (e) DNA encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of at least one miRNA target identified in Tables 2 or 3, wherein a miRNA recognition site in the native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage; (f) DNA encoding a miRNA precursor which is processed into a miRNA for suppressing expression of at least one miRNA target identified in Tables 2 or 3; (g) DNA encoding double-stranded RNA which is processed into siRNAs for suppressing expression of at least one miRNA target identified in Tables 2 or 3; and (h) DNA encoding a ta-siRNA which is processed into siRNAs for suppressing expression of at least one miRNA target identified in Tables 2 or 3. Specifically claimed are embodiments wherein the recombinant DNA construct is stably integrated into a plastid or a chromosome of the plant cell. Also specifically claimed are methods to improve yield in a plant, wherein the recombinant DNA construct is transgenically expressed in a crop plant (preferably, but not limited to, maize or corn, soybean, canola, cotton, alfalfa, sugarcane, sugar beet, sorghum, and rice), resulting in improved yield by that crop plant, when compared to the crop plant in which the construct is not expressed.
Embodiments within the scope of this invention include a recombinant DNA construct transcribable in a plant cell, including a promoter that is functional in the plant cell and operably linked to at least one polynucleotide selected from: (a) DNA encoding a cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target; (b) DNA encoding a 5′-modified cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target; (c) DNA encoding a translational inhibitor to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target; (d) DNA encoding a decoy to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target; (e) DNA encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of at least one miRNA target, wherein a miRNA recognition site in the native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage; (f) DNA encoding a miRNA precursor which is processed into a miRNA for suppressing expression of at least one miRNA target; (g) DNA encoding double-stranded RNA which is processed into siRNAs for suppressing expression of at least one miRNA target; and (h) DNA encoding a ta-siRNA which is processed into siRNAs for suppressing expression of at least one miRNA target—wherein the at least one miRNA target is at least one selected from the group consisting of a miR156 target, a miR160 target, a miR164 target, a miR166 target, a miR167 target, a miR169 target, a miR171 target, a miR172 target, a miR319 target, miR395 target, a miR396 target, a miR398 target, a miR399 target, a miR408 target, a miR444 target, a miR528 target, a miR167g target, a miR169g target, COP1 (constitutive photomorphogenesis1), GA2ox (gibberellic acid 2 oxidase), GA20ox (gibberellic acid 20 oxidase), HB2 (homeobox 2), HB2-4 (homeobox 2 and homeobox 4), HB4 (homeobox 4), LG1 (liguleless1), SPX (SYG1, PH081 and XPR1 domain; PFAM entry PF03105 at www.sanger.ac.uk), VIMla (variant in methlylation 1a), DHS1 (deoxyhypusine synthase), DHS2 (deoxyhypusine synthase), DHS3 (deoxyhypusine synthase), DHS4 (deoxyhypusine synthase), DHS5 (deoxyhypusine synthase), DHS6 (deoxyhypusine synthase), DHS7 (deoxyhypusine synthase), DHS8 (deoxyhypusine synthase), CRF (corn RING finger; RNF169), G1543a (maize orthologue of Arabidopsis thaliana homeobox 17), G1543b (maize orthologue of Arabidopsis thaliana homeobox 17), GS3 (grain size 3), and GW2 (grain weight 2). Particular embodiments that are specifically claimed by this invention include a recombinant DNA construct transcribable in a plant cell, including a promoter that is functional in the plant cell and operably linked to at least one polynucleotide selected from the group consisting of DNA encoding a nucleotide sequence selected from SEQ ID NOs: 1120, 1121, 1122, 1248, 1257, 1313, 1314, 1364, 1387, 1478, 1489, 1490, 1491, 1492, 1493, 1585, 1597, 1598, 1599, 1713, 1752, 1753, 1801, 1802, 1820, 1927, 1929, 1931, 1971, 2006, 2007, 2008, 2010, 2012, 2014, 2016, 2018, 2022, 2023, 2025, 2027, 2029, 2031, 2033, 2035, 2037, 2039, 2041, 2043, 2045, 2047, 2049, 2051, 2053, 2055, 2056, 2057, 2059, 2060, 2061, and 2063; also specifically claimed are embodiments wherein the recombinant DNA construct is stably integrated into a plastid or a chromosome of the plant cell.
Further embodiments are methods to improve yield in a plant, wherein a recombinant DNA construct transcribable in a plant cell, including a promoter that is functional in the plant cell and operably linked to at least one polynucleotide selected from: (a) DNA encoding a cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target; (b) DNA encoding a 5′-modified cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target; (c) DNA encoding a translational inhibitor to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target; (d) DNA encoding a decoy to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target; (e) DNA encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of at least one miRNA target, wherein a miRNA recognition site in the native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage; (f) DNA encoding a miRNA precursor which is processed into a miRNA for suppressing expression of at least one miRNA target; (g) DNA encoding double-stranded RNA which is processed into siRNAs for suppressing expression of at least one miRNA target; and (h) DNA encoding a ta-siRNA which is processed into siRNAs for suppressing expression of at least one miRNA target—wherein the at least one miRNA target is at least one selected from the group consisting of a miR156 target, a miR160 target, a miR164 target, a miR166 target, a miR167 target, a miR169 target, a miR171 target, a miR172 target, a miR319 target, miR395 target, a miR396 target, a a miR398 target, a miR399 target, a miR408 target, a miR444 target, a miR528 target, a miR167g target, a miR169g target, COP1 (constitutive photomorphogenesis1), GA2ox (gibberellic acid 2 oxidase), GA20ox (gibberellic acid 20 oxidase), HB2 (homeobox 2), HB2-4 (homeobox 2 and homeobox 4), HB4 (homeobox 4), LG1 (liguleless1), SPX (SYG1, PH081 and XPR1 domain; PFAM entry PF03105 at www.sanger.ac.uk), VIMla (variant in methlylation 1a), DHS1 (deoxyhypusine synthase), DHS2 (deoxyhypusine synthase), DHS3 (deoxyhypusine synthase), DHS4 (deoxyhypusine synthase), DHS5 (deoxyhypusine synthase), DHS6 (deoxyhypusine synthase), DHS7 (deoxyhypusine synthase), DHS8 (deoxyhypusine synthase), CRF (corn RING finger; RNF169), G1543a (maize orthologue of Arabidopsis thaliana homeobox 17), G1543b (maize orthologue of Arabidopsis thaliana homeobox 17), GS3 (grain size 3), and GW2 (grain weight 2)—is transgenically expressed in a crop plant (preferably, but not limited to, maize or corn, soybean, canola, cotton, alfalfa, sugarcane, sugar beet, sorghum, and rice), resulting in improved yield by that crop plant, when compared to the crop plant in which the construct is not expressed. Specifically claimed are methods to improve yield in a plant, wherein a recombinant DNA construct transcribable in a plant cell, including a promoter that is functional in the plant cell and operably linked to at least one polynucleotide selected from the group consisting of DNA encoding a nucleotide sequence selected from SEQ ID NOs: 1120, 1121, 1122, 1248, 1257, 1313, 1314, 1364, 1387, 1478, 1489, 1490, 1491, 1492, 1493, 1585, 1597, 1598, 1599, 1713, 1752, 1753, 1801, 1802, 1820, 1927, 1929, 1931, 1971, 2006, 2007, 2008, 2010, 2012, 2014, 2016, 2018, 2022, 2023, 2025, 2027, 2029, 2031, 2033, 2035, 2037, 2039, 2041, 2043, 2045, 2047, 2049, 2051, 2053, 2055, 2056, 2057, 2059, 2060, 2061, and 2063 is transgenically expressed in a crop plant (preferably, but not limited to, maize or corn, soybean, canola, cotton, alfalfa, sugarcane, sugar beet, sorghum, and rice), resulting in improved yield by that crop plant, when compared to the crop plant in which the construct is not expressed.
Additional aspects of this invention include a non-natural transgenic plant cell including a stably integrated recombinant DNA construct transcribable in the non-natural transgenic plant cell, wherein the recombinant DNA construct includes a promoter functional in the non-natural transgenic plant cell and operably linked to at least one polynucleotide selected from DNA encoding at least one miRNA target identified in Tables 2 or 3; the recombinant DNA construct can be stably integrated into a plastid, a chromosome, or the genome of the plant cell. Embodiments include a non-natural transgenic plant cell including a stably integrated recombinant DNA construct transcribable in the non-natural transgenic plant cell, wherein the recombinant DNA construct includes a promoter functional in the non-natural transgenic plant cell and operably linked to at least one polynucleotide including a DNA sequence selected from SEQ ID NOS: 15-2064.

miRNA	miR156	1115			Zea mays
miRNA	miR156	1116			Zea mays
miR156 target	Squamosa Promoter	1117			Zea mays
	Binding Protein
miR156 target	Squamosa Promoter	1118			Zea mays
	Binding Protein
miR156 target	Squamosa Promoter	1119			Zea mays
	Binding Protein
Decoy (artificial	miR156 decoy	1120			Artificial	Improved
sequence)					sequence	yield*
Decoy (artificial	miR156 decoy	1121			Artificial	Improved
sequence)					sequence	yield*
miRNA-	Squamosa Promoter	1122			Artificial	Improved
unresponsive	Binding Protein				sequence	yield*
	(miR156-unresponsive)
miR156 target	Squamosa Promoter	1123	MRT4577_564644C.1	478-497	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1124	MRT4577_23629C.7	1001-1020	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1125	MRT4577_188360C.6	1571-1590	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1126	MRT4577_205098C.7	1658-1677	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1127	MRT4577_565057C.1	980-999	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1128	MRT4577_137984C.6	2097-2116	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1129	MRT4577_532824C.3	1136-1155	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1130	MRT4577_122478C.6	767-786	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1131	MRT4577_31704C.9	1125-1144	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1132	MRT4577_26483C.7	1503-1522	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1133	MRT4577_295538C.7	1433-1452	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1134	MRT4577_644419C.1	962-981	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1135	MRT4577_619443C.1	914-933	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1136	MRT4577_341149C.6	1807-1826	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1137	MRT4577_78773C.8	1202-1221	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1138	MRT4577_42534C.9	1935-1954	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1139	MRT4577_270892C.4	978-997	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1140	MRT4577_571545C.1	623-642	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1141	MRT4577_181019C.5	788-807	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1142	MRT4577_537670C.2	575-594	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1143	MRT4577_535297C.2	1840-1859	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1144	MRT4577_334372C.5	477-496	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1145	MRT4577_568647C.1	1004-1023	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1146	MRT4577_383301C.4	896-915	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1147	MRT4577_427964C.4	991-1010	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1148	MRT4577_240798C.6	769-788	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1149	MRT4577_38044C.8	951-970	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1150	MRT4577_461098C.3	469-488	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1151	MRT4577_333683C.4	643-662	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1152	MRT4577_396357C.4	647-666	Zea mays
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1153	MRT3635_15393C.1	98-117	Gossypium
	Binding-domain				hirsutum
	protein
miR156 target	Squamosa Promoter	1154	MRT3635_15791C.2	990-1009	Gossypium
	Binding-domain				hirsutum
	protein
miR156 target	miR156 target	1155	MRT3635_23851C.2	233-252	Gossypium
					hirsutum
miR156 target	Squamosa Promoter	1156	MRT3635_28051C.1	213-232	Gossypium
	Binding-domain				hirsutum
	protein
miR156 target	Squamosa Promoter	1157	MRT3635_30369C.2	1511-1530	Gossypium
	Binding-domain				hirsutum
	protein
miR156 target	Squamosa Promoter	1158	MRT3635_30868C.2	652-671	Gossypium
	Binding-domain				hirsutum
	protein
miR156 target	Squamosa Promoter	1159	MRT3635_36657C.2	555-574	Gossypium
	Binding-domain				hirsutum
	protein
miR156 target	Squamosa Promoter	1160	MRT3635_48230C.2	857-876	Gossypium
	Binding-domain				hirsutum
	protein
miR156 target	Squamosa Promoter	1161	MRT3635_54380C.2	21-40	Gossypium
	Binding-domain				hirsutum
	protein
miR156 target	Squamosa Promoter	1162	MRT3635_59825C.1	50-69	Gossypium
	Binding-domain				hirsutum
	protein
miR156 target	Squamosa Promoter	1163	MRT3635_65765C.1	709-728	Gossypium
	Binding-domain				hirsutum
	protein
miR156 target	miR156 target	1164	MRT3635_69088C.1	1238-1257	Gossypium
					hirsutum
miR156 target	Squamosa Promoter	1165	MRT3635_69159C.1	892-911	Gossypium
	Binding-domain				hirsutum
	protein
miR156 target	miR156 target	1166	MRT3635_71102C.1	294-313	Gossypium
					hirsutum
miR156 target	Squamosa Promoter	1167	MRT3635_72531C.1	612-631	Gossypium
	Binding-domain				hirsutum
	protein
miR156 target	Squamosa Promoter	1168	MRT3702_110108C.4	1253-1272	Arabidopsis
	Binding-domain				thaliana
	protein
miR156 target	Squamosa Promoter	1169	MRT3702_113039C.2	757-776	Arabidopsis
	Binding-domain				thaliana
	protein
miR156 target	Squamosa Promoter	1170	MRT3702_115945C.3	2609-2628	Arabidopsis
	Binding-domain				thaliana
	protein
miR156 target	Squamosa Promoter	1171	MRT3702_11947C.6	680-699	Arabidopsis
	Binding-domain				thaliana
	protein
miR156 target	Squamosa Promoter	1172	MRT3702_120785C.3	1157-1176	Arabidopsis
	Binding-domain				thaliana
	protein
miR156 target	Squamosa Promoter	1173	MRT3702_141151C.3	1073-1092	Arabidopsis
	Binding-domain				thaliana
	protein
miR156 target	Squamosa Promoter	1174	MRT3702_141152C.2	1172-1191	Arabidopsis
	Binding-domain				thaliana
	protein
miR156 target	miR156 target	1175	MRT3702_147696C.3	1186-1205	Arabidopsis
					thaliana
miR156 target	Squamosa Promoter	1176	MRT3702_147811C.3	1446-1465	Arabidopsis
	Binding-domain				thaliana
	protein
miR156 target	Squamosa Promoter	1177	MRT3702_148347C.1	1118-1137	Arabidopsis
	Binding-domain				thaliana
	protein
miR156 target	Squamosa Promoter	1178	MRT3702_148348C.3	1121-1140	Arabidopsis
	Binding-domain				thaliana
	protein
miR156 target	Squamosa Promoter	1179	MRT3702_15197C.5	785-804	Arabidopsis
	Binding-domain				thaliana
	protein
miR156 target	Squamosa Promoter	1180	MRT3702_177137C.1	2477-2496	Arabidopsis
	Binding-domain				thaliana
	protein
miR156 target	Squamosa Promoter	1181	MRT3702_179579C.1	1149-1168	Arabidopsis
	Binding-domain				thaliana
	protein
miR156 target	Squamosa Promoter	1182	MRT3702_23035C.6	1358-1377	Arabidopsis
	Binding-domain				thaliana
	protein
miR156 target	Squamosa Promoter	1183	MRT3702_23765C.7	1036-1055	Arabidopsis
	Binding-domain				thaliana
	protein
miR156 target	Squamosa Promoter	1184	MRT3702_4036C.6	804-823	Arabidopsis
	Binding-domain				thaliana
	protein
miR156 target	Squamosa Promoter	1185	MRT3702_5396C.6	1297-1316	Arabidopsis
	Binding-domain				thaliana
	protein
miR156 target	Squamosa Promoter	1186	MRT3702_9141C.7	829-848	Arabidopsis
	Binding-domain				thaliana
	protein
miR156 target	Squamosa Promoter	1187	MRT3702_94277C.3	781-800	Arabidopsis
	Binding-domain				thaliana
	protein
miR156 target	Squamosa Promoter	1188	MRT3702_9951C.4	781-800	Arabidopsis
	Binding-domain				thaliana
	protein
miR156 target	miR156 target	1189	MRT3708_10628C.4	459-478	Brassica
					napus
miR156 target	Squamosa Promoter	1190	MRT3708_22559C.1	330-349	Brassica
	Binding-domain				napus
	protein
miR156 target	Squamosa Promoter	1191	MRT3708_53675C.1	290-309	Brassica
	Binding-domain				napus
	protein
miR156 target	miR156 target	1192	MRT3708_58630C.1	407-426	Brassica
					napus
miR156 target	miR156 target	1193	MRT3847_14683C.5	1677-1696	Glycine max
miR156 target	miR156 target	1194	MRT3847_167543C.1	486-505	Glycine max
miR156 target	Squamosa Promoter	1195	MRT3847_197471C.3	295-314	Glycine max
	Binding-domain
	protein
miR156 target	miR156 target	1196	MRT3847_206274C.4	117-136	Glycine max
miR156 target	Squamosa Promoter	1197	MRT3847_207934C.2	547-566	Glycine max
	Binding-domain
	protein
miR156 target	miR156 target	1198	MRT3847_213855C.7	701-720	Glycine max
miR156 target	Squamosa Promoter	1199	MRT3847_217782C.3	851-870	Glycine max
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1200	MRT3847_218322C.4	109-128	Glycine max
	Binding-domain
	protein
miR156 target	Squamosa Promoter	1201	MRT3847_235081C.4	1980-1999	Glycine max
	Binding-domain
	protein
miR156 target	miR156 target	1202	MRT3847_235082C.6	915-934	Glycine max
miR156 target	miR156 target	1203	MRT3847_237444C.4	582-601	Glycine max
miR156 target	miR156 target	1204	MRT3847_252038C.4	515-534	Glycine max
miR156 target	miR156 target	1205	MRT3847_268305C.4	396-415	Glycine max
miR156 target	miR156 target	1206	MRT3847_289291C.3	961-980	Glycine max
miR156 target	Squamosa Promoter	1207	MRT3847_329752C.1	933-952	Glycine max
	Binding-domain
	protein
miR156 target	miR156 target	1208	MRT3847_334134C.1	1239-1258	Glycine max
miR156 target	miR156 target	1209	MRT3847_335568C.1	1747-1766	Glycine max
miR156 target	miR156 target	1210	MRT3847_338602C.1	1070-1089	Glycine max
miR156 target	miR156 target	1211	MRT3847_341315C.1	47-66	Glycine max
miR156 target	miR156 target	1212	MRT3847_341402C.1	978-997	Glycine max
miR156 target	miR156 target	1213	MRT3847_350831C.1	1280-1299	Glycine max
miR156 target	Squamosa Promoter	1214	MRT3880_19943C.1	633-652	Medicago
	Binding-domain				truncatula
	protein
miR156 target	miR156 target	1215	MRT3880_49046C.1	98-117	Medicago
					truncatula
miR156 target	Squamosa Promoter	1216	MRT3880_54023C.1	527-546	Medicago
	Binding-domain				truncatula
	protein
miR156 target	Squamosa Promoter	1217	MRT3880_59834C.1	726-745	Medicago
	Binding-domain				truncatula
	protein
miR156 target	Squamosa Promoter	1218	MRT3880_62151C.1	1070-1089	Medicago
	Binding-domain				truncatula
	protein
miR156 target	Squamosa Promoter	1219	MRT4513_19757C.1	529-548	Hordeum
	Binding-domain				vulgare
	protein
miR156 target	Squamosa Promoter	1220	MRT4513_41849C.1	439-458	Hordeum
	Binding-domain				vulgare
	protein
miR156 target	Squamosa Promoter	1221	MRT4513_4449C.1	221-240	Hordeum
	Binding-domain				vulgare
	protein
miR156 target	Squamosa Promoter	1222	MRT4513_52153C.1	523-542	Hordeum
	Binding-domain				vulgare
	protein
miR156 target	miR156 target	1223	MRT4530_11398C.3	696-715	Oryza
					sativa
miR156 target	Squamosa Promoter	1224	MRT4530_118092C.3	821-840	Oryza
	Binding-domain				sativa
	protein
miR156 target	Squamosa Promoter	1225	MRT4530_135991C.4	710-729	Oryza
	Binding-domain				sativa
	protein
miR156 target	Squamosa Promoter	1226	MRT4530_142142C.4	1074-1093	Oryza
	Binding-domain				sativa
	protein
miR156 target	Squamosa Promoter	1227	MRT4530_195506C.2	981-1000	Oryza
	Binding-domain				sativa
	protein
miR156 target	Squamosa Promoter	1228	MRT4530_199837C.4	2401-2420	Oryza
	Binding-domain				sativa
	protein
miR156 target	miR156 target	1229	MRT4530_219862C.2	146-165	Oryza
					sativa
miR156 target	Squamosa Promoter	1230	MRT4530_220364C.2	1764-1783	Oryza
	Binding-domain				sativa
	protein
miR156 target	Squamosa Promoter	1231	MRT4530_230201C.3	265-284	Oryza
	Binding-domain				sativa
	protein
miR156 target	miR156 target	1232	MRT4530_230404C.3	2222-2241	Oryza
					sativa
miR156 target	Squamosa Promoter	1233	MRT4530_236277C.1	728-747	Oryza
	Binding-domain				sativa
	protein
miR156 target	Squamosa Promoter	1234	MRT4530_257640C.1	956-975	Oryza
	Binding-domain				sativa
	protein
miR156 target	Squamosa Promoter	1235	MRT4530_44605C.5	1148-1167	Oryza
	Binding-domain				sativa
	protein
miR156 target	Squamosa Promoter	1236	MRT4530_53217C.5	858-877	Oryza
	Binding-domain				sativa
	protein
miR156 target	Squamosa Promoter	1237	MRT4530_6964C.4	2113-2132	Oryza
	Binding-domain				sativa
	protein
miR156 target	miR156 target	1238	MRT4530_95203C.4	994-1013	Oryza
					sativa
miR156 target	Squamosa Promoter	1239	MRT4558_12680C.1	78-97	Sorghum
	Binding-domain				bicolor
	protein
miR156 target	Squamosa Promoter	1240	MRT4558_27285C.1	130-149	Sorghum
	Binding-domain				bicolor
	protein
miR156 target	Squamosa Promoter	1241	MRT4558_6587C.1	516-535	Sorghum
	Binding-domain				bicolor
	protein
miR156 target	Squamosa Promoter	1242	MRT4558_8644C.2	866-885	Sorghum
	Binding-domain				bicolor
	protein
miR156 target	miR156 target	1243	MRT4565_169464C.2	296-315	Triticum
					aestivum
miR156 target	Squamosa Promoter	1244	MRT4565_212647C.1	523-542	Triticum
	Binding-domain				aestivum
	protein
miR156 target	Squamosa Promoter	1245	MRT4565_239085C.1	1565-1584	Triticum
	Binding-domain				aestivum
	protein
miR156 target	Squamosa Promoter	1246	MRT4565_259386C.1	339-358	Triticum
	Binding-domain				aestivum
	protein
miR156 target	Squamosa Promoter	1247	MRT4565_272025C.1	954-973	Triticum
	Binding-domain				aestivum
	protein
Decoy (artificial	miR160 decoy	1248			Artificial	Improved
sequence)					sequence	yield*
miR160 target	Auxin Response Factor	1249	MRT4577_429671C.3	1429-1449	Zea mays
	10-like protein
miR160 target	Auxin Response Factor	1250	MRT4577_400043C.4	1894-1914	Zea mays
	10-like protein
miR160 target	Auxin Response Factor	1251	MRT4577_385317C.3	863-883	Zea mays
	10-like protein
miR160 target	Auxin Response Factor	1252	MRT4577_41620C.6	756-776	Zea mays
	10-like protein
miR160 target	Auxin Response Factor	1253	MRT4577_258637C.2	1353-1373	Zea mays
	10-like protein
miR160 target	Auxin Response Factor	1254	MRT4577_448022C.1	421-442	Zea mays
	10-like protein
miRNA	miR164	1255			Zea mays
miR164 target	NAC1; No Apical	1256			Zea mays
	Meristem, ATAF, Cup
	Shaped Cotyledon
	(NAC) domain protein
miRNA-	NAC1 (miR164-	1257			Artificial	Improved
unresponsive	unresponsive)				sequence	yield*
miR164 target	miR164 target	1258	MRT3635_6393C.2	135-155	Gossypium
					hirsutum
miR164 target	miR164 target	1259	MRT3635_64345C.1	925-945	Gossypium
					hirsutum
miR164 target	No Apical Meristem,	1260	MRT3702_105151C.5	843-863	Arabidopsis
	ATAF, Cup Shaped				thaliana
	Cotyledon (NAC)
	domain protein
miR164 target	CUC1; No Apical	1261	MRT3702_11937C.6	651-671	Arabidopsis
	Meristem, ATAF, Cup				thaliana
	Shaped Cotyledon
	(NAC) domain protein
miR164 target	NAC1; No Apical	1262	MRT3702_180541C.1	762-782	Arabidopsis
	Meristem, ATAF, Cup				thaliana
	Shaped Cotyledon
	(NAC) domain protein
miR164 target	NAC1; No Apical	1263	MRT3702_180670C.1	785-805	Arabidopsis
	Meristem, ATAF, Cup				thaliana
	Shaped Cotyledon
	(NAC) domain protein
miR164 target	No Apical Meristem,	1264	MRT3702_20256C.5	651-671	Arabidopsis
	ATAF, Cup Shaped				thaliana
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1265	MRT3702_22669C.4	765-785	Arabidopsis
	ATAF, Cup Shaped				thaliana
	Cotyledon (NAC)
	domain protein
miR164 target	CUC2; No Apical	1266	MRT3702_24103C.6	856-876	Arabidopsis
	Meristem, ATAF, Cup				thaliana
	Shaped Cotyledon
	(NAC) domain protein
miR164 target	No Apical Meristem,	1267	MRT3702_24851C.6	809-829	Arabidopsis
	ATAF, Cup Shaped				thaliana
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1268	MRT3708_39966C.1	192-212	Brassica
	ATAF, Cup Shaped				napus
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1269	MRT3708_51022C.1	803-823	Brassica
	ATAF, Cup Shaped				napus
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1270	MRT3712_8777C.1	316-336	Brassica
	ATAF, Cup Shaped				oleracea
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1271	MRT3847_244824C.2	290-310	Glycine max
	ATAF, Cup Shaped
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1272	MRT3847_259513C.2	719-739	Glycine max
	ATAF, Cup Shaped
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1273	MRT3847_270117C.3	784-804	Glycine max
	ATAF, Cup Shaped
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1274	MRT3847_46332C.2	714-734	Glycine max
	ATAF, Cup Shaped
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1275	MRT3847_46333C.6	731-751	Glycine max
	ATAF, Cup Shaped
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1276	MRT3847_48464C.4	1140-1160	Glycine max
	ATAF, Cup Shaped
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1277	MRT3847_48465C.6	777-797	Glycine max
	ATAF, Cup Shaped
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1278	MRT3880_18003C.2	705-725	Medicago
	ATAF, Cup Shaped				truncatula
	Cotyledon (NAC)
	domain protein
miR164 target	miR164 target	1279	MRT3880_33685C.1	278-298	Medicago
					truncatula
miR164 target	No Apical Meristem,	1280	MRT3880_44619C.1	781-801	Medicago
	ATAF, Cup Shaped				truncatula
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1281	MRT4513_26199C.1	809-829	Hordeum
	ATAF, Cup Shaped				vulgare
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1282	MRT4513_37185C.1	17-37	Hordeum
	ATAF, Cup Shaped				vulgare
	Cotyledon (NAC)
	domain protein
miR164 target	Salicylic acid-induced	1283	MRT4513_4722C.1	251-271	Hordeum
	protein 19				vulgare
miR164 target	No Apical Meristem,	1284	MRT4513_7890C.1	687-707	Hordeum
	ATAF, Cup Shaped				vulgare
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1285	MRT4530_141528C.5	890-910	Oryza
	ATAF, Cup Shaped				sativa
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1286	MRT4530_147737C.4	912-932	Oryza
	ATAF, Cup Shaped				sativa
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1287	MRT4530_157393C.3	923-943	Oryza
	ATAF, Cup Shaped				sativa
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1288	MRT4530_178256C.3	954-974	Oryza
	ATAF, Cup Shaped				sativa
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1289	MRT4530_211705C.4	1929-1949	Oryza
	ATAF, Cup Shaped				sativa
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1290	MRT4530_221769C.1	159-179	Oryza
	ATAF, Cup Shaped				sativa
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1291	MRT4530_224181C.2	790-810	Oryza
	ATAF, Cup Shaped				sativa
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1292	MRT4558_11465C.1	13-33	Sorghum
	ATAF, Cup Shaped				bicolor
	Cotyledon (NAC)
	domain protein
miR164 target	Salicylic acid-induced	1293	MRT4558_31046C.1	256-276	Sorghum
	protein 19, regulation				bicolor
	of transcription, DNA
	binding
miR164 target	No Apical Meristem,	1294	MRT4558_41467C.1	1230-1250	Sorghum
	ATAF, Cup Shaped				bicolor
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1295	MRT4558_43081C.1	344-364	Sorghum
	ATAF, Cup Shaped				bicolor
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1296	MRT4558_43436C.1	853-873	Sorghum
	ATAF, Cup Shaped				bicolor
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1297	MRT4558_4564C.2	691-711	Sorghum
	ATAF, Cup Shaped				bicolor
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1298	MRT4565_235741C.1	849-869	Triticum
	ATAF, Cup Shaped				aestivum
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1299	MRT4565_241295C.1	1062-1082	Triticum
	ATAF, Cup Shaped				aestivum
	Cotyledon (NAC)
	domain protein
miR164 target	SIAH1 protein-like,	1300	MRT4565_246008C.1	696-716	Triticum
	ubiquitin-dependent				aestivum
	protein catabolism,
	nucleus, zinc ion
	binding
miR164 target	No Apical Meristem,	1301	MRT4565_250946C.1	675-695	Triticum
	ATAF, Cup Shaped				aestivum
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1302	MRT4565_269060C.1	730-750	Triticum
	ATAF, Cup Shaped				aestivum
	Cotyledon (NAC)
	domain protein
miR164 target	Salicylic acid-induced	1303	MRT4565_272391C.1	765-785	Triticum
	protein 19, regulation				aestivum
	of transcription, DNA
	binding
miR164 target	No Apical Meristem,	1304	MRT4565_279043C.1	945-965	Triticum
	ATAF, Cup Shaped				aestivum
	Cotyledon (NAC)
	domain protein
miR164 target	No Apical Meristem,	1305	MRT4577_16045C.7	927-947	Zea mays
	ATAF, Cup Shaped
	Cotyledon (NAC)
	domain protein
miR164 target	miR164 target	1306	MRT4577_205444C.5	524-544	Zea mays
miR164 target	hypothetical protein;	1307	MRT4577_325166C.3	868-888	Zea mays
	putative role in
	boundary specification;
	nam2
miR164 target	hypothetical protein;	1308	MRT4577_78918C.6	893-913	Zea mays
	putative role in SAM
	initiation and boundary
	specification; nam1
miR164 target	miR164 target	1309	MRT4577_98755C.5	942-962	Zea mays
miR164 target	miR164 target	1310	MRT4577_9951C.8	930-950	Zea mays
miRNA	miR166	1311			Zea mays
miR166 target	Revoluta	1312			Zea mays
miRNA-	Revoluta (miR166-	1313			Artificial	Improved
unresponsive	unresponsive)				sequence	yield*
miRNA-	Revoluta (miR166-	1314			Artificial	Improved
unresponsive	unresponsive)				sequence	yield*
miR166 target	miR166 target	1315	MRT3635_23433C.2	197-217	Gossypium
					hirsutum
miR166 target	miR166 target	1316	MRT3635_50942C.2	298-318	Gossypium
					hirsutum
miR166 target	interfascicular fiberless	1317	MRT3702_104431C.5	1262-1282	Arabidopsis
	1; IFL1; HDZIPIII				thaliana
	domain protein
miR166 target	homeodomain-leucine	1318	MRT3702_104605C.6	915-935	Arabidopsis
	zipper protein				thaliana
miR166 target	homeodomain-leucine	1319	MRT3702_113325C.3	1268-1288	Arabidopsis
	zipper protein; ATHB-				thaliana
	15
miR166 target	homeodomain-leucine	1320	MRT3702_120571C.3	1281-1301	Arabidopsis
	zipper protein 14;				thaliana
	ATHB-14
miR166 target	homeodomain-leucine	1321	MRT3702_18869C.5	934-954	Arabidopsis
	zipper protein 8; hb-8				thaliana
miR166 target	Glycosyl transferase	1322	MRT3702_24778C.3	2793-2813	Arabidopsis
					thaliana
miR166 target	CORONA; START	1323	MRT3708_45624C.1	210-230	Brassica
	domain; HDZIPIII				napus
	domain transcription
	factor
miR166 target	HD-Zip protein	1324	MRT3708_5493C.1	79-99	Brassica
	(Homeodomain-leucine				napus
	zipper protein);
	START domain
miR166 target	Homeodomain-leucine	1325	MRT3712_4770C.1	229-249	Brassica
	zipper protein; START				oleracea
	domain
miR166 target	miR166 target	1326	MRT3847_209034C.4	506-526	Glycine max
miR166 target	miR166 target	1327	MRT3847_233286C.5	730-750	Glycine max
miR166 target	miR166 target	1328	MRT3847_248020C.5	298-318	Glycine max
miR166 target	miR166 target	1329	MRT3847_251781C.4	950-970	Glycine max
miR166 target	miR166 target	1330	MRT3847_288367C.4	1562-1582	Glycine max
miR166 target	Class III HD-Zip	1331	MRT3847_296736C.1	869-889	Glycine max
	protein 4
miR166 target	Class III HD-Zip	1332	MRT3847_326691C.1	910-930	Glycine max
	protein 4
miR166 target	miR166 target	1333	MRT3847_348410C.1	912-932	Glycine max
miR166 target	Class III HD-Zip	1334	MRT3880_12194C.1	788-808	Medicago
	protein 8				truncatula
miR166 target	Class III HD-Zip	1335	MRT3880_30145C.1	560-580	Medicago
	protein 1				truncatula
miR166 target	Class III HD-Zip	1336	MRT3880_37546C.1	819-839	Medicago
	protein 6				truncatula
miR166 target	Class III HD-Zip	1337	MRT3880_39764C.1	536-556	Medicago
	protein 6				truncatula
miR166 target	homeodomain-leucine	1338	MRT4530_10527C.4	959-979	Oryza
	zipper protein				sativa
miR166 target	Homeodomain-leucine	1339	MRT4530_107863C.5	880-900	Oryza
	zipper protein; START				sativa
	domain
miR166 target	Homeodomain leucine-	1340	MRT4530_160340C.3	1031-1051	Oryza
	zipper protein Hox10;				sativa
	START domain
miR166 target	Homeodomain-leucine	1341	MRT4530_21619C.2	563-583	Oryza
	zipper protein; START				sativa
	domain
miR166 target	Homeodomain-leucine	1342	MRT4530_253068C.2	957-977	Oryza
	zipper protein; START				sativa
	domain
miR166 target	Homeodomain-leucine	1343	MRT4558_27560C.1	750-770	Sorghum
	zipper protein; START				bicolor
	domain
miR166 target	Homeodomain-leucine	1344	MRT4565_226777C.1	285-305	Triticum
	zipper protein; START				aestivum
	domain
miR166 target	Homeodomain-leucine	1345	MRT4565_232172C.1	168-188	Triticum
	zipper protein; START				aestivum
	domain
miR166 target	Homeodomain-leucine	1346	MRT4565_264759C.1	954-973	Triticum
	zipper protein; START				aestivum
	domain
miR166 target	miR166 target	1347	MRT4577_141500C.4	839-859	Zea mays
miR166 target	miR166 target	1348	MRT4577_200605C.3	788-808	Zea mays
miR166 target	rolled leaf1; RLD1;	1349	MRT4577_229497C.6	1098-1118	Zea mays
	class III homeodomain-
	leucine zipper (HD-
	ZIPIII)
miR166 target	Rolled leaf1;	1350	MRT4577_312384C.3	563-583	Zea mays
	Homeobox: Homeobox
	domain; START
	domain
miR166 target	miR166 target	1351	MRT4577_320718C.6	963-983	Zea mays
miR166 target	miR166 target	1352	MRT4577_342259C.4	1092-1112	Zea mays
miR166 target	miR166 target	1353	MRT4577_442838C.4	1159-1179	Zea mays
miR166 target	miR166 target	1354	MRT4577_535676C.2	560-580	Zea mays
miR166 target	miR166 target	1355	MRT4577_535928C.2	1142-1162	Zea mays
miR166 target	miR166 target	1356	MRT4577_566770C.1	545-565	Zea mays
miR166 target	miR166 target	1357	MRT4577_568616C.1	801-821	Zea mays
miR166 target	miR166 target	1358	MRT4577_586718C.1	572-592	Zea mays
miR166 target	miR166 target	1359	MRT4577_659410C.1	788-808	Zea mays
miR166 target	miR166 target	1360	MRT4577_673351C.1	161-181	Zea mays
miRNA	miR167b	1361			Zea mays
miRNA	miR167b	1362			Zea mays
miR167 target	ARF8	1363			Zea mays
miRNA-	ARF8 (mir167-	1364			Artificial	Improved
unresponsive	unresponsive)				sequence	yield*
miR167 target	auxin response factor	1365	MRT3702_22410C.4	4382-4402	Arabidopsis
	8; ARF8;				thaliana
miR167 target	auxin response factor	1366	MRT3708_50323C.1	89-109	Brassica
	domain; ARF8-like				napus
miR167 target	miR167 target	1367	MRT3847_305421C.4	1358-1378	Glycine max
miR167 target	miR167 target	1368	MRT3847_340154C.1	1586-1606	Glycine max
miR167 target	auxin response factor	1369	MRT3847_41926C.6	1489-1509	Glycine max
	domain; ARF8-like
miR167 target	auxin response factor	1370	MRT3880_12926C.1	365-385	Medicago
	domain; ARF8-like				truncatula
miR167 target	auxin response factor	1371	MRT3880_25270C.1	1758-1778	Medicago
	domain; ARF8-like				truncatula
miR167 target	miR167 target	1372	MRT4513_29483C.2	564-584	Hordeum
					vulgare
miR167 target	miR167 target	1373	MRT4530_178528C.2	2219-2239	Oryza
					sativa
miR167 target	auxin response factor	1374	MRT4530_86291C.3	2659-2679	Oryza
	domain; ARF8-like				sativa
miR167 target	auxin response factor	1375	MRT4558_37108C.1	147-167	Sorghum
	domain; ARF8-like				bicolor
miR167 target	miR167 target	1376	MRT4577_306050C.5	647-667	Zea mays
miR167 target	miR167 target	1377	MRT4577_339989C.4	2584-2604	Zea mays
miR167 target	miR167 target	1378	MRT4577_377896C.4	244-264	Zea mays
miR167 target	miR167 target	1379	MRT4577_521851C.2	1595-1615	Zea mays
miR167 target	miR167 target	1380	MRT4577_650810C.1	1618-1638	Zea mays
miR167 target	miR167 target	1381	MRT4577_680014C.1	208-228	Zea mays
miR167 target	miR167 target	1382	MRT4577_681995C.1	230-250	Zea mays
miR167 target	miR167 target	1383	MRT4577_683953C.1	442-462	Zea mays
miRNA	miR169	1384			Zea mays
miRNA	miR169	1385			Zea mays
miR169 target	NFY family of TFs	1386			Zea mays
miRNA-	NFY family of TFs	1387			Artificial	Improved
unresponsive	(miR169-unresponsive)				sequence	yield*
miR169 target	HAP2, CCAAT-	1388	MRT3635_18720C.2	1123-1143	Gossypium
	binding transcription				hirsutum
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1389	MRT3635_24490C.1	345-365	Gossypium
	binding transcription				hirsutum
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1390	MRT3635_60547C.1	1610-1630	Gossypium
	binding transcription				hirsutum
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1391	MRT3635_63203C.1	1353-1373	Gossypium
	binding transcription				hirsutum
	factor (CBF-B/NF-YA)
miR169 target	miR169 target	1392	MRT3635_63602C.1	692-712	Gossypium
					hirsutum
miR169 target	HAP2, CCAAT-	1393	MRT3635_751C.2	1156-1176	Gossypium
	binding transcription				hirsutum
	factor (CBF-B/NF-YA)
miR169 target	miR169 target	1394	MRT3635_7843C.2	302-322	Gossypium
					hirsutum
miR169 target	HAP2/CCAAT	1395	MRT3702_11008C.6	1183-1203	Arabidopsis
	transcription factor;				thaliana
	At3g05690
miR169 target	HAP2A, CCAAT-	1396	MRT3702_145277C.3	1122-1142	Arabidopsis
	binding transcription				thaliana
	factor (CBF-B/NF-YA)
	family protein;
	ATHAP2A, EMBRYO
	DEFECTIVE 2220
miR169 target	miR169 target	1397	MRT3702_145278C.1	1870-1890	Arabidopsis
					thaliana
miR169 target	HAP2, CCAAT-	1398	MRT3702_1608C.8	1254-1274	Arabidopsis
	binding transcription				thaliana
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1399	MRT3702_167062C.2	1489-1509	Arabidopsis
	binding transcription				thaliana
	factor (CBF-B/NF-YA)
miR169 target	HAP2C, CCAAT-	1400	MRT3702_175138C.1	1412-1432	Arabidopsis
	binding transcription				thaliana
	factor (CBF-B/NF-YA)
	family protein;
	At1g17590
miR169 target	HAP2A, CCAAT-	1401	MRT3702_176968C.1	1037-1057	Arabidopsis
	binding transcription				thaliana
	factor (CBF-B/NF-YA)
	family protein;
	ATHAP2A, EMBRYO
	DEFECTIVE 2220
miR169 target	HAP2, CCAAT-	1402	MRT3702_180826C.1	1610-1630	Arabidopsis
	binding transcription				thaliana
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1403	MRT3702_20139C.6	1305-1325	Arabidopsis
	binding transcription				thaliana
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1404	MRT3702_20659C.7	1428-1448	Arabidopsis
	binding transcription				thaliana
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1405	MRT3702_4133C.5	1308-1328	Arabidopsis
	binding transcription				thaliana
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1406	MRT3702_5699C.6	1504-1524	Arabidopsis
	binding transcription				thaliana
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1407	MRT3708_42756C.1	928-948	Brassica
	binding transcription				napus
	factor (CBF-B/NF-YA)
miR169 target	miR169 target	1408	MRT3708_45516C.2	1074-1094	Brassica
					napus
miR169 target	HAP2, CCAAT-	1409	MRT3708_46224C.1	1017-1037	Brassica
	binding transcription				napus
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1410	MRT3708_56325C.1	670-690	Brassica
	binding transcription				napus
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1411	MRT3711_4547C.1	157-177	Brassica
	binding transcription				rapa
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1412	MRT3712_6671C.1	481-501	Brassica
	binding transcription				oleracea
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1413	MRT3847_251095C.3	995-1015	Glycine max
	binding transcription
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1414	MRT3847_25786C.5	1208-1228	Glycine max
	binding transcription
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1415	MRT3847_278998C.2	722-742	Glycine max
	binding transcription
	factor (CBF-B/NF-YA)
miR169 target	miR169 target	1416	MRT3847_305217C.3	1028-1048	Glycine max
miR169 target	HAP2, CCAAT-	1417	MRT3847_312701C.1	803-823	Glycine max
	binding transcription
	factor (CBF-B/NF-YA)
miR169 target	miR169 target	1418	MRT3847_335193C.1	1452-1472	Glycine max
miR169 target	HAP2, CCAAT-	1419	MRT3847_51286C.6	801-821	Glycine max
	binding transcription
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1420	MRT3847_53466C.6	1490-1510	Glycine max
	binding transcription
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1421	MRT3847_53467C.5	902-922	Glycine max
	binding transcription
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1422	MRT3847_54010C.4	1403-1423	Glycine max
	binding transcription
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1423	MRT3880_16272C.2	1496-1516	Medicago
	binding transcription				truncatula
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1424	MRT3880_21811C.2	1054-1074	Medicago
	binding transcription				truncatula
	factor (CBF-B/NF-YA)
miR169 target	miR169 target	1425	MRT3880_36579C.1	90-110	Medicago
					truncatula
miR169 target	miR169 target	1426	MRT3880_48656C.1	73-94	Medicago
					truncatula
miR169 target	miR169 target	1427	MRT3880_55431C.1	145-166	Medicago
					truncatula
miR169 target	HAP2, CCAAT-	1428	MRT3880_59679C.1	1268-1288	Medicago
	binding transcription				truncatula
	factor (CBF-B/NF-YA)
miR169 target	miR169 target	1429	MRT3880_9392C.1	182-202	Medicago
					truncatula
miR169 target	HAP2, CCAAT-	1430	MRT4513_27452C.1	721-741	Hordeum
	binding transcription				vulgare
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1431	MRT4513_38912C.1	1037-1057	Hordeum
	binding transcription				vulgare
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1432	MRT4513_51394C.1	631-651	Hordeum
	binding transcription				vulgare
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1433	MRT4530_156068C.3	1715-1735	Oryza
	binding transcription				sativa
	factor (CBF-B/NF-YA)
miR169 target	miR169 target	1434	MRT4530_16169C.4	1389-1409	Oryza
					sativa
miR169 target	HAP2, CCAAT-	1435	MRT4530_196466C.4	2027-2047	Oryza
	binding transcription				sativa
	factor (CBF-B/NF-YA)
miR169 target	miR169 target	1436	MRT4530_223395C.1	653-673	Oryza
					sativa
miR169 target	RAPB protein; rapB	1437	MRT4530_225972C.3	867-887	Oryza
					sativa
miR169 target	miR169 target	1438	MRT4530_238300C.1	220-240	Oryza
					sativa
miR169 target	HAP2, CCAAT-	1439	MRT4530_267924C.1	1002-1022	Oryza
	binding transcription				sativa
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1440	MRT4530_268072C.1	756-776	Oryza
	binding transcription				sativa
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1441	MRT4530_52650C.3	1391-1411	Oryza
	binding transcription				sativa
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1442	MRT4530_67920C.7	1637-1657	Oryza
	binding transcription				sativa
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1443	MRT4530_98042C.6	1170-1190	Oryza
	binding transcription				sativa
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1444	MRT4558_11671C.2	530-550	Sorghum
	binding transcription				bicolor
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1445	MRT4558_13240C.2	880-900	Sorghum
	binding transcription				bicolor
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1446	MRT4558_19368C.2	726-746	Sorghum
	binding transcription				bicolor
	factor (CBF-B/NF-YA)
miR169 target	Transcription factor	1447	MRT4558_8287C.2	346-366	Sorghum
					bicolor
miR169 target	miR169 target	1448	MRT4565_219265C.1	936-956	Triticum
					aestivum
miR169 target	HAP2, CCAAT-	1449	MRT4565_224073C.1	1081-1101	Triticum
	binding transcription				aestivum
	factor (CBF-B/NF-YA)
miR169 target	miR169 target	1450	MRT4565_232474C.1	1040-1060	Triticum
					aestivum
miR169 target	miR169 target	1451	MRT4565_236768C.1	1284-1304	Triticum
					aestivum
miR169 target	HAP2, CCAAT-	1452	MRT4565_240119C.1	934-954	Triticum
	binding transcription				aestivum
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1453	MRT4565_250357C.1	1230-1250	Triticum
	binding transcription				aestivum
	factor (CBF-B/NF-YA)
miR169 target	HAP2, CCAAT-	1454	MRT4565_270644C.1	1050-1070	Triticum
	binding transcription				aestivum
	factor (CBF-B/NF-YA)
miR169 target	miR169 target	1455	MRT4565_271488C.1	1032-1052	Triticum
					aestivum
miR169 target	HAP2, CCAAT-	1456	MRT4565_271817C.1	2171-2191	Triticum
	binding transcription				aestivum
	factor (CBF-B/NF-YA)
miR169 target	miR169 target	1457	MRT4565_278167C.1	895-915	Triticum
					aestivum
miR169 target	miR169 target	1458	MRT4577_136204C.6	573-593	Zea mays
miR169 target	miR169 target	1459	MRT4577_192239C.6	1297-1317	Zea mays
miR169 target	miR169 target	1460	MRT4577_270253C.7	1375-1395	Zea mays
miR169 target	miR169 target	1461	MRT4577_321589C.4	1051-1071	Zea mays
miR169 target	miR169 target	1462	MRT4577_35015C.6	1679-1699	Zea mays
miR169 target	miR169 target	1463	MRT4577_40749C.8	1361-1381	Zea mays
miR169 target	miR169 target	1464	MRT4577_411247C.4	1445-1465	Zea mays
miR169 target	miR169 target	1465	MRT4577_428392C.4	1583-1603	Zea mays
miR169 target	miR169 target	1466	MRT4577_434247C.4	671-691	Zea mays
miR169 target	miR169 target	1467	MRT4577_536961C.2	920-940	Zea mays
miR169 target	miR169 target	1468	MRT4577_536962C.2	1836-1856	Zea mays
miR169 target	miR169 target	1469	MRT4577_540147C.2	1327-1347	Zea mays
miR169 target	miR169 target	1470	MRT4577_556372C.2	1417-1437	Zea mays
miR169 target	miR169 target	1471	MRT4577_570253C.1	340-360	Zea mays
miR169 target	miR169 target	1472	MRT4577_570254C.1	1391-1411	Zea mays
miR169 target	miR169 target	1473	MRT4577_668660C.1	1292-1312	Zea mays
miR169 target	miR169 target	1474	MRT4577_693949C.1	400-420	Zea mays
miR169 target	miR169 target	1475	MRT4577_701125C.1	471-491	Zea mays
miR169 target	miR169 target	1476	MRT4577_72313C.1	262-282	Zea mays
miRNA	miR171b	1477			Zea mays
miRNA	osa-MIR171b	1478			Oryza	Improved
precursor for	(precursor)				sativa	yield*
overexpression
of mature
miR171
miR171 target	Scarecrow-like Scl1	1479	MRT4577_520619C.1	106-126	Zea mays
	protein (3e−37); GRAS
	family transcription
	factor
miR171 target	Scarecrow-like Scl1	1480	MRT4577_139132C.5	1336-1356	Zea mays
	protein (3e−37); GRAS
	family transcription
	factor
miR171 target	Scarecrow-like Scl1	1481	MRT4577_75777C.7	640-660	Zea mays
	protein (3e−37); GRAS
	family transcription
	factor
miR171 target	Scarecrow-like Scl1	1482	MRT4577_234039C.5	771-791	Zea mays
	protein (3e−37); GRAS
	family transcription
	factor
miR171 target	Scarecrow-like Scl1	1483	MRT4577_57336C.8	1274-1294	Zea mays
	protein (3e−37); GRAS
	family transcription
	factor
miR171 target	Scarecrow-like Scl1	1484	MRT4577_140896C.5	507-527	Zea mays
	protein (3e−37); GRAS
	family transcription
	factor
miR171 target	Scarecrow-like Scl1	1485	MRT4577_30852C.5	800-820	Zea mays
	protein (3e−37); GRAS
	family transcription
	factor
miRNA	miR172	1486			Zea mays
miRNA	miR172	1487			Zea mays
miR172 target	Glossy15	1488			Zea mays
Decoy	miR172 decoy	1489			Artificial	Improved
					sequence	yield*
Decoy	miR172 decoy	1490			Artificial	Improved
					sequence	yield*
Decoy	miR172 decoy	1491			Artificial	Improved
					sequence	yield*
miRNA	miRMON18	1492			Zea mays
Cleavage	mirR172 cleavage	1493			Artificial	Improved
blocker	blocker				sequence	yield*
miR172 target	AP2 domain	1494	MRT3635_50596C.2	622-642	Gossypium
	transcription factor;				hirsutum
	SCHNARCHZAPFEN;
	SNZ
miR172 target	AP2 domain	1495	MRT3635_64291C.1	246-266	Gossypium
	transcription factor;				hirsutum
	SCHNARCHZAPFEN;
	SNZ
miR172 target	AP2 domain	1496	MRT3635_64989C.1	1102-1122	Gossypium
	transcription factor;				hirsutum
	SCHNARCHZAPFEN;
	SNZ
miR172 target	miR172 target	1497	MRT3635_65450C.1	241-261	Gossypium
					hirsutum
miR172 target	miR172 target	1498	MRT3635_70864C.1	646-666	Gossypium
					hirsutum
miR172 target	AP2 domain	1499	MRT3635_8244C.2	1657-1677	Gossypium
	transcription factor;				hirsutum
	SCHNARCHZAPFEN;
	SNZ
miR172 target	AP2 domain	1500	MRT3702_103726C.5	1044-1064	Arabidopsis
	transcription factor;				thaliana
	SCHNARCHZAPFEN;
	SNZ
miR172 target	AP2 domain containing	1501	MRT3702_103748C.5	1560-1580	Arabidopsis
	protein RAP2.7				thaliana
miR172 target	AP2 domain	1502	MRT3702_14904C.2	1095-1115	Arabidopsis
	transcription factor;				thaliana
	SCHLAFMUTZE;
	SMZ
miR172 target	AP2 domain	1503	MRT3702_150241C.1	947-967	Arabidopsis
	transcription factor-like				thaliana
miR172 target	AP2 domain	1504	MRT3702_156728C.3	1030-1050	Arabidopsis
	transcription factor-like				thaliana
miR172 target	APETALA2; AP2	1505	MRT3702_168284C.1	1271-1291	Arabidopsis
					thaliana
miR172 target	AP2 domain-	1506	MRT3702_175574C.1	1630-1650	Arabidopsis
	containing transcription				thaliana
	factor RAP2.7
miR172 target	AP2 domain	1507	MRT3702_179746C.1	263-283	Arabidopsis
	transcription factor;				thaliana
	SCHNARCHZAPFEN;
	SNZ
miR172 target	AP2 domain	1508	MRT3702_19267C.5	1368-1388	Arabidopsis
	transcription factor-like				thaliana
miR172 target	elongation factor 2-like	1509	MRT3702_4319C.8	1045-1065	Arabidopsis
					thaliana
miR172 target	AP2 domain	1510	MRT3702_76733C.6	1663-1683	Arabidopsis
	transcription factor;				thaliana
	SCHNARCHZAPFEN;
	SNZ
miR172 target	AP2 domain	1511	MRT3708_36942C.2	411-431	Brassica
	transcription factor-like				napus
miR172 target	AP2 domain	1512	MRT3708_39387C.1	366-386	Brassica
	transcription factor-like				napus
miR172 target	AP2 domain	1513	MRT3711_6838C.1	137-157	Brassica
	transcription factor-like				rapa
miR172 target	miR172 target	1514	MRT3847_196945C.3	667-687	Glycine max
miR172 target	AP2 domain	1515	MRT3847_202930C.3	1630-1650	Glycine max
	transcription factor-like
miR172 target	AP2 domain	1516	MRT3847_235857C.3	1789-1809	Glycine max
	transcription factor-like
miR172 target	miR172 target	1517	MRT3847_257655C.4	1984-2004	Glycine max
miR172 target	AP2 domain	1518	MRT3847_289890C.3	2213-2233	Glycine max
	transcription factor-like
miR172 target	miR172 target	1519	MRT3847_289891C.3	529-549	Glycine max
miR172 target	AP2 domain	1520	MRT3847_295726C.1	1539-1559	Glycine max
	transcription factor-like
miR172 target	AP2 domain	1521	MRT3847_326790C.1	1269-1289	Glycine max
	transcription factor-like
miR172 target	AP2 domain	1522	MRT3847_329301C.1	775-795	Glycine max
	transcription factor-like
miR172 target	miR172 target	1523	MRT3847_344570C.1	564-584	Glycine max
miR172 target	AP2 domain	1524	MRT3847_43925C.7	811-831	Glycine max
	transcription factor-like
miR172 target	AP2 domain	1525	MRT3847_46007C.5	1544-1564	Glycine max
	transcription factor-like
miR172 target	AP2 domain	1526	MRT3847_51633C.3	910-930	Glycine max
	transcription factor-like
miR172 target	miR172 target	1527	MRT3847_59804C.6	2369-2389	Glycine max
miR172 target	AP2 domain	1528	MRT3880_19283C.1	558-578	Medicago
	transcription factor-like				truncatula
miR172 target	AP2 domain	1529	MRT3880_32459C.1	311-331	Medicago
	transcription factor-like				truncatula
miR172 target	AP2 domain	1530	MRT3880_36568C.1	1424-1444	Medicago
	transcription factor-like				truncatula
miR172 target	AP2 domain	1531	MRT3880_39959C.1	1689-1709	Medicago
	transcription factor-like				truncatula
miR172 target	AP2 domain	1532	MRT3880_55789C.1	1241-1261	Medicago
	transcription factor-like				truncatula
miR172 target	AP2 domain	1533	MRT4513_42015C.1	1464-1484	Hordeum
	transcription factor-like				vulgare
miR172 target	AP2 domain	1534	MRT4513_6417C.1	632-652	Hordeum
	transcription factor-like				vulgare
miR172 target	miR172 target	1535	MRT4530_140532C.4	1358-1378	Oryza
					sativa
miR172 target	AP2 domain	1536	MRT4530_146548C.4	669-689	Oryza
	transcription factor;				sativa
	SCHNARCHZAPFEN;
	SNZ
miR172 target	AP2 domain	1537	MRT4530_160275C.3	1405-1425	Oryza
	transcription factor-like				sativa
miR172 target	miR172 target	1538	MRT4530_16723C.7	804-824	Oryza
					sativa
miR172 target	AP2 domain	1539	MRT4530_209082C.4	1976-1996	Oryza
	transcription factor;				sativa
	SCHNARCHZAPFEN;
	SNZ
miR172 target	AP2 domain	1540	MRT4530_212672C.3	187-207	Oryza
	transcription factor-like				sativa
miR172 target	miR172 target	1541	MRT4530_238241C.2	1481-1501	Oryza
					sativa
miR172 target	AP2 domain	1542	MRT4530_263068C.2	1768-1788	Oryza
	transcription factor;				sativa
	SCHNARCHZAPFEN;
	SNZ
miR172 target	miR172 target	1543	MRT4530_266671C.1	2391-2411	Oryza
					sativa
miR172 target	miR172 target	1544	MRT4530_272652C.1	378-398	Oryza
					sativa
miR172 target	miR172 target	1545	MRT4530_274692C.1	236-256	Oryza
					sativa
miR172 target	AP2 domain	1546	MRT4530_56773C.3	1148-1168	Oryza
	transcription factor-like				sativa
miR172 target	Zinc finger (C3HC4-	1547	MRT4530_57252C.7	41-61	Oryza
	type RING				sativa
	finger)protein-like,
	transport, nucleus,
	metal ion binding
miR172 target	miR172 target	1548	MRT4558_24999C.3	298-318	Sorghum
					bicolor
miR172 target	AP2 domain	1549	MRT4558_25704C.2	512-532	Sorghum
	transcription factor;				bicolor
	SCHNARCHZAPFEN;
	SNZ
miR172 target	miR172 target	1550	MRT4565_108668C.1	220-240	Triticum
					aestivum
miR172 target	AP2 domain	1551	MRT4565_118657C.1	354-374	Triticum
	transcription factor-like				aestivum
miR172 target	AP2 domain	1552	MRT4565_235388C.1	572-592	Triticum
	transcription factor-like				aestivum
miR172 target	AP2 domain	1553	MRT4565_245146C.1	1148-1168	Triticum
	transcription factor-like				aestivum
miR172 target	AP2 domain	1554	MRT4565_247090C.1	1462-1482	Triticum
	transcription factor-like				aestivum
miR172 target	miR172 target	1555	MRT4565_249252C.1	551-571	Triticum
					aestivum
miR172 target	AP2 domain	1556	MRT4565_256056C.1	810-830	Triticum
	transcription factor-like				aestivum
miR172 target	AP2 domain	1557	MRT4565_273183C.1	1152-1172	Triticum
	transcription factor-like				aestivum
miR172 target	AP2 domain	1558	MRT4565_279009C.1	1155-1175	Triticum
	transcription factor-like				aestivum
miR172 target	miR172 target	1559	MRT4565_83602C.3	26-46	Triticum
					aestivum
miR172 target	Glycosyltransferase	1560	MRT4565_88032C.3	361-381	Triticum
					aestivum
miR172 target	miR172 target	1561	MRT4577_12523C.7	2414-2434	Zea mays
miR172 target	miR172 target	1562	MRT4577_243746C.1	140-160	Zea mays
miR172 target	miR172 target	1563	MRT4577_27478C.9	1546-1566	Zea mays
miR172 target	miR172 target	1564	MRT4577_304712C.4	1326-1346	Zea mays
miR172 target	miR172 target	1565	MRT4577_307553C.7	1508-1528	Zea mays
miR172 target	AP2 domain	1566	MRT4577_39951C.8	1611-1631	Zea mays
	transcription factor-like
miR172 target	miR172 target	1567	MRT4577_431122C.3	1359-1379	Zea mays
miR172 target	miR172 target	1568	MRT4577_431125C.4	824-844	Zea mays
miR172 target	miR172 target	1569	MRT4577_455774C.3	963-983	Zea mays
miR172 target	miR172 target	1570	MRT4577_468762C.3	2414-2434	Zea mays
miR172 target	miR172 target	1571	MRT4577_49516C.9	408-428	Zea mays
miR172 target	AP2 domain	1572	MRT4577_49517C.8	1652-1672	Zea mays
	transcription factor-like
miR172 target	miR172 target	1573	MRT4577_548310C.2	1451-1471	Zea mays
miR172 target	miR172 target	1574	MRT4577_556612C.2	1352-1372	Zea mays
miR172 target	miR172 target	1575	MRT4577_597136C.1	551-571	Zea mays
miR172 target	miR172 target	1576	MRT4577_616573C.1	670-690	Zea mays
miR172 target	miR172 target	1577	MRT4577_668951C.1	270-290	Zea mays
miR172 target	miR172 target	1578	MRT4577_669210C.1	1031-1051	Zea mays
miR172 target	miR172 target	1579	MRT4577_676464C.1	1308-1328	Zea mays
miR172 target	miR172 target	1580	MRT4577_679724C.1	157-177	Zea mays
miR172 target	miR172 target	1581	MRT4577_700043C.1	147-167	Zea mays
miR172 target	miR172 target	1582	MRT4577_701524C.1	136-156	Zea mays
miR172 target	miR172 target	1583	MRT4577_708079C.1	540-560	Zea mays
miRNA	miR319	1584			Zea mays
miRNA	osa-MIR319	1585			Oryza	Improved
precursor for	(precursor)				sativa	yield*
overexpression
of mature
miR319
miR319 target	TCP family	1586	MRT4577_275782C.5	1673-1692	Zea mays
	transcription factor
miR319 target	TCP family	1587	MRT4577_558102C.1	949-968	Zea mays
	transcription factor
miR319 target	TCP family	1588	MRT4577_30525C.5	1316-1335	Zea mays
	transcription factor
miR319 target	TCP family	1589	MRT4577_275060C.2	818-836	Zea mays
	transcription factor
miR319 target	TCP family	1590	MRT4577_22397C.4	943-961	Zea mays
	transcription factor
miR319 target	TCP family	1591	MRT4577_275063C.5	1247-1265	Zea mays
	transcription factor
miR319 target	TCP family	1592	MRT4577_480991C.1	150-169	Zea mays
	transcription factor
miR319 target	TCP family	1593	MRT4577_427906C.3	1557-1576	Zea mays
	transcription factor
miR319 target	TCP family	1594	MRT4577_213173C.3	1594-1613	Zea mays
	transcription factor
miRNA	miR396	1595			Zea mays
miR396 target	Zm-GRF1	1596			Zea mays
Decoy	miR396 decoy	1597			Artificial	Improved
					construct	yield*
Decoy	miR396 decoy	1598			Artificial	Improved
					sequence	yield*
Decoy	miR396 decoy	1599			Artificial	Improved
					sequence	yield*
miR396 target	miR396 target	1600	MRT3635_67262C.1	6-25	Gossypium
					hirsutum
miR396 target	miR396 target	1601	MRT3635_70418C.1	147-166	Gossypium
					hirsutum
miR396 target	miR396 target	1602	MRT3635_71272C.1	414-433	Gossypium
					hirsutum
miR396 target	miR396 target	1603	MRT3635_71696C.1	37-56	Gossypium
					hirsutum
miR396 target	ATP-dependent RNA	1604	MRT3702_15262C.6	1141-1160	Arabidopsis
	helicase-like protein				thaliana
miR396 target	subtilase family	1605	MRT3702_17628C.6	1886-1905	Arabidopsis
	protein, contains Pfam				thaliana
	profile: PF00082
	subtilase family
miR396 target	miR396 target	1606	MRT3702_18069C.6	2763-2782	Arabidopsis
					thaliana
miR396 target	miR396 target	1607	MRT3702_2454C.7	1387-1406	Arabidopsis
					thaliana
miR396 target	miR396 target	1608	MRT3708_59476C.1	194-213	Brassica
					napus
miR396 target	miR396 target	1609	MRT3708_61891C.1	236-255	Brassica
					napus
miR396 target	Cysteine proteinase	1610	MRT3847_115000C.2	180-199	Glycine max
	precursor, proteolysis;
	cysteine-type
	endopeptidase activity
miR396 target	miR396 target	1611	MRT3847_249313C.3	1165-1184	Glycine max
miR396 target	Putative fimbriata,	1612	MRT3847_260044C.4	1337-1356	Glycine max
	ubiquitin cycle,
	nucleus, protein
	binding
miR396 target	miR396 target	1613	MRT3847_282324C.5	578-597	Glycine max
miR396 target	Microsomal	1614	MRT3847_32554C.3	245-264	Glycine max
	cytochrome b5,
	electron transport,
	mitochondrial inner
	membrane, iron ion
	binding
miR396 target	BRASSINOSTEROID	1615	MRT3847_60193C.5	1967-1986	Glycine max
	INSENSITIVE 1-
	associated receptor
	kinase 1 precursor (EC
	2.7.11.1) (BRI1-
	associated receptor
	kinase 1) (Somatic
	embryogenesis
	receptor-like kinase 3),
	protein amino acid
	phosphorylation,
	integral to membrane,
	protein serine/threonine
	kinase activity
miR396 target	miR396 target	1616	MRT3847_72393C.1	34-53	Glycine max
miR396 target	Putative AFG1-like	1617	MRT4513_2056C.1	294-313	Hordeum
	ATPase				vulgare
miR396 target	Putative fimbriata, cell	1618	MRT4513_23211C.1	721-740	Hordeum
	differentiation, nucleus,				vulgare
	protein binding
miR396 target	Cryptochrome 2, DNA	1619	MRT4513_24452C.1	19-38	Hordeum
	repair, DNA				vulgare
	photolyase activity
miR396 target	miR396 target	1620	MRT4513_32857C.1	621-640	Hordeum
					vulgare
miR396 target	S-locus protein 5	1621	MRT4513_48780C.1	84-103	Hordeum
					vulgare
miR396 target	miR396 target	1622	MRT4530_139664C.5	2371-2390	Oryza
					sativa
miR396 target	Putative RNA	1623	MRT4530_171648C.2	1063-1082	Oryza
	polymerase III,				sativa
	RNA_pol_Rpb2_1:
	RNA polymerase beta
	subunit,
	RNA_pol_Rpb2_3:
	RNA polymerase
	Rpb2, domain 3,
	RNA_pol_Rpb2_4:
	RNA polymerase
	Rpb2, domain 4,
	RNA_pol_Rpb2_5:
	RNA polymerase
	Rpb2, domain 5,
	RNA_pol_Rpb2_6:
	RNA polymerase
	Rpb2, domain 6,
	RNA_pol_Rpb2_7:
	RNA polymerase
	Rpb2, domain 7;
	transcription; nucleus;
	metal ion binding
miR396 target	miR396 target	1624	MRT4530_267934C.1	467-486	Oryza
					sativa
miR396 target	miR396 target	1625	MRT4530_268027C.1	95-114	Oryza
					sativa
miR396 target	miR396 target	1626	MRT4530_27400C.6	682-701	Oryza
					sativa
miR396 target	miR396 target	1627	MRT4530_59122C.7	573-591	Oryza
					sativa
miR396 target	miR396 target	1628	MRT4530_62393C.7	2341-2360	Oryza
					sativa
miR396 target	miR396 target	1629	MRT4530_81835C.6	1243-1262	Oryza
					sativa
miR396 target	Hypothetical protein	1630	MRT4530_98651C.4	271-290	Oryza
	P0698A04.3; GRP:				sativa
	Glycine rich protein
	family
miR396 target	Putative fimbriata, F-	1631	MRT4558_11973C.2	1234-1253	Sorghum
	box: F-box domain				bicolor
miR396 target	Methyltransferase,	1632	MRT4558_29180C.1	101-120	Sorghum
	putative, cell wall				bicolor
	(sensu Magnoliophyta),
	methyltransferase
	activity
miR396 target	miR396 target	1633	MRT4558_34091C.1	266-285	Sorghum
					bicolor
miR396 target	Putative receptor-like	1634	MRT4558_9324C.2	375-394	Sorghum
	kinase; Pkinase_Tyr:				bicolor
	Protein tyrosine kinase,
	protein amino acid
	phosphorylation,
	integral to membrane,
	protein-tyrosine kinase
	activity
miR396 target	Acyl-CoA	1635	MRT4565_127266C.2	27-46	Triticum
	dehydrogenase,				aestivum
	putative
miR396 target	miR396 target	1636	MRT4565_162831C.1	1134-1153	Triticum
					aestivum
miR396 target	Ribulose-1,5-	1637	MRT4565_200090C.1	1047-1066	Triticum
	bisphosphate				aestivum
	carboxylase/oxygenase
	small subunit
miR396 target	Putative fimbriata	1638	MRT4565_230957C.1	450-469	Triticum
					aestivum
miR396 target	Dirigent-like protein	1639	MRT4565_234418C.1	1427-1446	Triticum
					aestivum
miR396 target	putative F-box protein	1640	MRT4565_242541C.1	1472-1491	Triticum
					aestivum
miR396 target	Putative	1641	MRT4565_244837C.1	918-937	Triticum
	folylpolyglutamate				aestivum
	synthetase, folic acid
	and derivative
	biosynthesis,
	extracellular space,
	ATP binding
miR396 target	miR396 target	1642	MRT4565_248632C.1	625-644	Triticum
					aestivum
miR396 target	miR396 target	1643	MRT4565_249453C.1	108-127	Triticum
					aestivum
miR396 target	Folylpolyglutamate	1644	MRT4565_253149C.1	616-635	Triticum
	synthetase, putative,				aestivum
	folic acid and
	derivative biosynthesis,
	ATP binding (4e−99)
miR396 target	Phytochrome/protein	1645	MRT4565_253747C.1	894-913	Triticum
	kinase-like, protein				aestivum
	amino acid
	phosphorylation,
	protein-tyrosine kinase
	activity
miR396 target	Putative fimbriata	1646	MRT4565_259298C.1	1362-1381	Triticum
					aestivum
miR396 target	Putative fimbriata	1647	MRT4565_260134C.1	414-433	Triticum
					aestivum
miR396 target	miR396 target	1648	MRT4565_273137C.1	137-156	Triticum
					aestivum
miR396 target	Putative	1649	MRT4577_130243C.1	12-31	Zea mays
	dihydrolipoamide S-
	acetyltransferase;
	Biotin_lipoyl: Biotin-
	requiring enzyme,
	metabolism,
	mitochondrion,
	dihydrolipoyllysine-
	residue
	acetyltransferase
	activity
miR396 target	miR396 target	1650	MRT4577_165771C.1	95-114	Zea mays
miR396 target	miR396 target	1651	MRT4577_213750C.1	60-79	Zea mays
miR396 target	miR396 target	1652	MRT4577_26483C.7	805-824	Zea mays
miR396 target	miR396 target	1653	MRT4577_341149C.6	1110-1129	Zea mays
miR396 target	miR396 target	1654	MRT4577_355112C.1	159-177	Zea mays
miR396 target	Putative gag-pol	1655	MRT4577_406214C.1	376-395	Zea mays
miR396 target	beta-keto acyl	1656	MRT4577_416676C.5	1463-1482	Zea mays
	reductase; cuticular
	wax biosynthesis;
	glossy8
miR396 target	miR396 target	1657	MRT4577_521629C.3	555-574	Zea mays
miR396 target	miR396 target	1658	MRT4577_540304C.2	1355-1374	Zea mays
miR396 target	miR396 target	1659	MRT4577_540948C.2	1095-1114	Zea mays
miR396 target	miR396 target	1660	MRT4577_548836C.1	467-486	Zea mays
miR396 target	Retrotransposon	1661	MRT4577_555855C.1	148-167	Zea mays
	protein, putative,
	unclassified;
	Retrotrans_gag:
	Retrotransposon gag
	protein, RNA-
	dependent DNA
	replication, nucleus,
	RNA-directed DNA
	polymerase activity
miR396 target	miR396 target	1662	MRT4577_557678C.2	344-363	Zea mays
miR396 target	miR396 target	1663	MRT4577_561121C.1	956-975	Zea mays
miR396 target	miR396 target	1664	MRT4577_564288C.1	290-309	Zea mays
miR396 target	miR396 target	1665	MRT4577_56429C.8	1315-1334	Zea mays
miR396 target	miR396 target	1666	MRT4577_595828C.1	63-82	Zea mays
miR396 target	miR396 target	1667	MRT4577_613832C.1	1029-1048	Zea mays
miR396 target	miR396 target	1668	MRT4577_619443C.1	394-413	Zea mays
miR396 target	miR396 target	1669	MRT4577_635169C.1	602-621	Zea mays
miR396 target	miR396 target	1670	MRT4577_638921C.1	172-191	Zea mays
miR396 target	miR396 target	1671	MRT4577_664914C.1	581-600	Zea mays
miRNA	miR393	1672			Zea mays
miR393 target	TIR1-like transport	1673	MRT3635_18188C.2	746-766	Gossypium
	inhibitor response-like				hirsutum
	protein
miR393 target	TIR1-like transport	1674	MRT3635_18850C.2	171-191	Gossypium
	inhibitor response-like				hirsutum
	protein
miR393 target	TIR1-like transport	1675	MRT3635_35639C.2	1049-1069	Gossypium
	inhibitor response-like				hirsutum
	protein
miR393 target	TIR1-like transport	1676	MRT3635_49076C.2	373-393	Gossypium
	inhibitor response-like				hirsutum
	protein
miR393 target	TIR1-like transport	1677	MRT3635_68504C.1	1996-2016	Gossypium
	inhibitor response-like				hirsutum
	protein
miR393 target	TIR1-like transport	1678	MRT3702_13118C.8	2015-2035	Arabidopsis
	inhibitor response-like				thaliana
	protein; At3g26830
miR393 target	TIR1-like transport	1679	MRT3702_145409C.1	1508-1528	Arabidopsis
	inhibitor response-like				thaliana
	protein
miR393 target	TIR1-like transport	1680	MRT3702_15703C.8	1738-1758	Arabidopsis
	inhibitor response-like				thaliana
	protein
miR393 target	TIR1-like transport	1681	MRT3702_16076C.7	1587-1607	Arabidopsis
	inhibitor response-like				thaliana
	protein
miR393 target	TIR1-like transport	1682	MRT3702_92498C.6	1898-1918	Arabidopsis
	inhibitor response-like				thaliana
	protein; At1g12820
miR393 target	TIR1-like transport	1683	MRT3708_31301C.1	259-280	Brassica
	inhibitor response-like				napus
	protein
miR393 target	TIR1-like transport	1684	MRT3708_52518C.1	250-270	Brassica
	inhibitor response-like				napus
	protein
miR393 target	TIR1-like transport	1685	MRT3708_55951C.1	93-113	Brassica
	inhibitor response-like				napus
	protein
miR393 target	TIR1-like transport	1686	MRT3711_1771C.1	103-123	Brassica
	inhibitor response-like				rapa
	protein
miR393 target	TIR1-like transport	1687	MRT3847_238705C.4	1172-1192	Glycine max
	inhibitor response-like
	protein
miR393 target	TIR1-like transport	1688	MRT3847_27973C.7	1339-1359	Glycine max
	inhibitor response-like
	protein
miR393 target	miR393 target	1689	MRT3847_313402C.3	958-978	Glycine max
miR393 target	miR393 target	1690	MRT3847_329954C.2	1740-1760	Glycine max
miR393 target	miR393 target	1691	MRT3847_335477C.1	1715-1735	Glycine max
miR393 target	miR393 target	1692	MRT3847_338734C.1	1474-1494	Glycine max
miR393 target	TIR1-like transport	1693	MRT3847_44371C.6	2345-2365	Glycine max
	inhibitor response-like
	protein
miR393 target	miR393 target	1694	MRT3880_18564C.2	3116-3136	Medicago
					truncatula
miR393 target	TIR1-like transport	1695	MRT3880_38847C.1	139-159	Medicago
	inhibitor response-like				truncatula
	protein
miR393 target	TIR1-like transport	1696	MRT4513_12741C.1	197-217	Hordeum
	inhibitor response-like				vulgare
	protein
miR393 target	TIR1-like transport	1697	MRT4513_38675C.1	419-439	Hordeum
	inhibitor response-like				vulgare
	protein
miR393 target	miR393 target	1698	MRT4530_113561C.5	5590-5610	Oryza
					sativa
miR393 target	TIR1-like transport	1699	MRT4530_237446C.2	2221-2241	Oryza
	inhibitor response-like				sativa
	protein
miR393 target	TIR1-like transport	1700	MRT4530_241313C.2	1706-1726	Oryza
	inhibitor response-like				sativa
	protein
miR393 target	TIR1-like transport	1701	MRT4558_1226C.2	167-187	Sorghum
	inhibitor response-like				bicolor
	protein
miR393 target	TIR1-like transport	1702	MRT4558_20000C.2	412-432	Sorghum
	inhibitor response-like				bicolor
	protein
miR393 target	TIR1-like transport	1703	MRT4565_141193C.1	43-63	Triticum
	inhibitor response-like				aestivum
	protein
miR393 target	TIR1-like transport	1704	MRT4565_226582C.1	486-506	Triticum
	inhibitor response-like				aestivum
	protein
miR393 target	TIR1-like transport	1705	MRT4565_247449C.1	28-48	Triticum
	inhibitor response-like				aestivum
	protein
miR393 target	TIR1-like transport	1706	MRT4565_274399C.1	1499-1519	Triticum
	inhibitor response-like				aestivum
	protein
miR393 target	miR393 target	1707	MRT4577_262597C.7	2373-2393	Zea mays
miR393 target	miR393 target	1708	MRT4577_39097C.9	1716-1736	Zea mays
miR393 target	miR393 target	1709	MRT4577_546333C.2	1349-1369	Zea mays
miR393 target	miR393 target	1710	MRT4577_656737C.1	1325-1345	Zea mays
miRNA	miR395	1711			Zea mays
miR395 target	ATP sulfurylase	1712			Zea mays
	domain protein
Decoy	miR395 decoy	1713			Artificial	Improved
					sequence	yield*
miR395 target	ATP sulfurylase	1714	MRT3635_15903C.2	410-429	Gossypium
	domain protein				hirsutum
miR395 target	ATP sulfurylase	1715	MRT3635_48567C.2	480-499	Gossypium
	domain protein				hirsutum
miR395 target	ATP sulfurylase	1716	MRT3702_166264C.1	202-221	Arabidopsis
	domain protein				thaliana
miR395 target	Sulfate transporter	1717	MRT3702_169467C.1	107-126	Arabidopsis
					thaliana
miR395 target	ATP sulfurylase	1718	MRT3702_17054C.8	470-489	Arabidopsis
	domain protein				thaliana
miR395 target	ATP sulfurylase	1719	MRT3702_177422C.1	340-359	Arabidopsis
	domain protein				thaliana
miR395 target	Sulfate transporter	1720	MRT3702_20451C.6	125-144	Arabidopsis
					thaliana
miR395 target	ATP sulfurylase	1721	MRT3702_23086C.8	544-563	Arabidopsis
	domain protein				thaliana
miR395 target	ATP sulfurylase	1722	MRT3702_57141C.1	331-350	Arabidopsis
	domain protein				thaliana
miR395 target	ATP sulfurylase	1723	MRT3708_36129C.1	403-422	Brassica
	domain protein				napus
miR395 target	ATP sulfurylase	1724	MRT3708_4492C.1	316-335	Brassica
	domain protein				napus
miR395 target	ATP sulfurylase	1725	MRT3708_55043C.1	400-419	Brassica
	domain protein				napus
miR395 target	ATP sulfurylase	1726	MRT3711_3394C.1	356-375	Brassica
	domain protein				rapa
miR395 target	ATP sulfurylase	1727	MRT3711_4165C.1	383-402	Brassica
	domain protein				rapa
miR395 target	ATP sulfurylase	1728	MRT3711_4313C.1	384-403	Brassica
	domain protein				rapa
miR395 target	Sulfate transporter	1729	MRT3712_1686C.1	124-143	Brassica
					oleracea
miR395 target	Sulfate transporter	1730	MRT3847_10451C.5	125-144	Glycine max
miR395 target	Sulfate transporter	1731	MRT3847_131987C.4	153-172	Glycine max
miR395 target	ATP sulfurylase	1732	MRT3847_14792C.7	641-660	Glycine max
	domain protein
miR395 target	Sulfate transporter	1733	MRT3847_245035C.3	64-83	Glycine max
miR395 target	ATP sulfurylase	1734	MRT3847_331787C.1	381-400	Glycine max
	domain protein
miR395 target	ATP sulfurylase	1735	MRT4530_16384C.4	560-579	Oryza
	domain protein				sativa
miR395 target	Sulfate transporter	1736	MRT4530_33633C.6	746-765	Oryza
					sativa
miR395 target	ATP sulfurylase	1737	MRT4558_11861C.1	474-493	Sorghum
	domain protein				bicolor
miR395 target	Sulfate transporter	1738	MRT4558_24400C.2	275-294	Sorghum
					bicolor
miR395 target	Sulfate transporter	1739	MRT4565_219452C.1	259-278	Triticum
					aestivum
miR395 target	ATP sulfurylase	1740	MRT4565_223839C.1	541-560	Triticum
	domain protein				aestivum
miR395 target	ATP sulfurylase	1741	MRT4565_232080C.1	462-481	Triticum
	domain protein				aestivum
miR395 target	ATP sulfurylase	1742	MRT4565_236093C.1	542-561	Triticum
	domain protein				aestivum
miR395 target	ATP sulfurylase	1743	MRT4565_254783C.1	482-501	Triticum
	domain protein				aestivum
miR395 target	miR395 target	1744	MRT4565_35429C.3	207-226	Triticum
					aestivum
miR395 target	ATP sulfurylase	1745	MRT4577_118322C.5	455-474	Zea mays
	domain protein
miR395 target	ATP sulfurylase	1746	MRT4577_386324C.4	465-484	Zea mays
	domain protein
miR395 target	ATP sulfurylase	1747	MRT4577_57434C.9	528-547	Zea mays
	domain protein
miR395 target	miR395 target	1748	MRT4577_644561C.1	27-46	Zea mays
miR395 target	miR395 target	1749	MRT4577_694623C.1	449-468	Zea mays
miRNA	miR398	1750			Zea mays
miR398 target	SODs and cytochrome	1751			Zea mays
	c oxidase
Decoy	miR398 decoy	1752			Artificial	Improved
					sequence	yield*
Decoy	miR398 decoy	1753			Artificial	Improved
					sequence	yield*
miR398 target	miR398 target	1754	MRT3702_118804C.3	1651-1671	Arabidopsis
					thaliana
miR398 target	Copper/zinc superoxide	1755	MRT3708_22683C.2	117-137	Brassica
	dismutase (SODC)				napus
	domain protein
miR398 target	Las1-like	1756	MRT3847_22858C.5	2306-2326	Glycine max
miR398 target	Copper/zinc superoxide	1757	MRT3847_235546C.3	112-132	Glycine max
	dismutase (SODC)
	domain protein
miR398 target	Copper/zinc superoxide	1758	MRT4530_151653C.4	66-86	Oryza
	dismutase (SODC)				sativa
	domain protein
miR398 target	miR398 target	1759	MRT4530_201873C.4	1720-1740	Oryza
					sativa
miR398 target	Copper/zinc superoxide	1760	MRT4530_20521C.4	152-172	Oryza
	dismutase (SODC)				sativa
	domain protein
miR398 target	Copper/zinc superoxide	1761	MRT4558_3896C.2	103-123	Sorghum
	dismutase (SODC)				bicolor
	domain protein
miR398 target	Copper/zinc superoxide	1762	MRT4558_9962C.2	176-196	Sorghum
	dismutase (SODC)				bicolor
	domain protein
miR398 target	miR398 target	1763	MRT4565_118267C.1	66-86	Triticum
					aestivum
miR398 target	miR398 target	1764	MRT4565_122618C.1	14-34	Triticum
					aestivum
miR398 target	Copper/zinc superoxide	1765	MRT4565_123037C.3	94-114	Triticum
	dismutase (SODC)				aestivum
	domain protein
miR398 target	miR398 target	1766	MRT4565_129871C.1	54-74	Triticum
					aestivum
miR398 target	Copper/zinc superoxide	1767	MRT4565_133338C.1	172-192	Triticum
	dismutase (SODC)				aestivum
	domain protein
miR398 target	Copper/zinc superoxide	1768	MRT4565_162003C.1	144-164	Triticum
	dismutase (SODC)				aestivum
	domain protein
miR398 target	miR398 target	1769	MRT4565_16358C.1	66-86	Triticum
					aestivum
miR398 target	miR398 target	1770	MRT4565_187852C.1	194-214	Triticum
					aestivum
miR398 target	Copper/zinc superoxide	1771	MRT4565_201143C.1	93-113	Triticum
	dismutase (SODC)				aestivum
	domain protein
miR398 target	Copper/zinc superoxide	1772	MRT4565_201144C.1	85-105	Triticum
	dismutase (SODC)				aestivum
	domain protein
miR398 target	Cytochrome c oxidase	1773	MRT4565_221067C.1	153-173	Triticum
	subunit Vb				aestivum
miR398 target	Cytochrome c oxidase	1774	MRT4565_223829C.1	139-159	Triticum
	subunit Vb				aestivum
miR398 target	Cytochrome c oxidase	1775	MRT4565_230710C.1	303-323	Triticum
	subunit Vb				aestivum
miR398 target	Copper/zinc superoxide	1776	MRT4565_236346C.1	91-111	Triticum
	dismutase (SODC)				aestivum
	domain protein
miR398 target	Copper/zinc superoxide	1777	MRT4565_244294C.1	69-89	Triticum
	dismutase (SODC)				aestivum
	domain protein
miR398 target	Cytochrome c oxidase	1778	MRT4565_246005C.1	160-180	Triticum
	subunit Vb				aestivum
miR398 target	Copper/zinc superoxide	1779	MRT4565_248858C.1	69-89	Triticum
	dismutase (SODC)				aestivum
	domain protein
miR398 target	Copper/zinc superoxide	1780	MRT4565_72209C.2	105-125	Triticum
	dismutase (SODC)				aestivum
	domain protein
miR398 target	Copper/zinc superoxide	1781	MRT4577_19020C.8	92-112	Zea mays
	dismutase (SODC)
	domain protein
miR398 target	Copper/zinc superoxide	1782	MRT4577_211709C.6	85-105	Zea mays
	dismutase (SODC)
	domain protein
miR398 target	Copper/zinc superoxide	1783	MRT4577_329847C.3	89-109	Zea mays
	dismutase (SODC)
	domain protein
miR398 target	Copper/zinc superoxide	1784	MRT4577_329851C.4	114-134	Zea mays
	dismutase (SODC)
	domain protein
miR398 target	Copper/zinc superoxide	1785	MRT4577_335011C.2	7-27	Zea mays
	dismutase (SODC)
	domain protein
miR398 target	Copper/zinc superoxide	1786	MRT4577_339810C.4	174-194	Zea mays
	dismutase (SODC)
	domain protein
miR398 target	Copper/zinc superoxide	1787	MRT4577_339813C.4	233-253	Zea mays
	dismutase (SODC)
	domain protein
miR398 target	Copper/zinc superoxide	1788	MRT4577_358061C.1	120-140	Zea mays
	dismutase (SODC)
	domain protein
miR398 target	Copper/zinc superoxide	1789	MRT4577_388896C.4	200-220	Zea mays
	dismutase (SODC)
	domain protein
miR398 target	Copper/zinc superoxide	1790	MRT4577_401904C.1	49-69	Zea mays
	dismutase (SODC)
	domain protein
miR398 target	Copper/zinc superoxide	1791	MRT4577_54564C.7	147-167	Zea mays
	dismutase (SODC)
	domain protein
miR398 target	miR398 target	1792	MRT4577_561629C.1	222-242	Zea mays
miR398 target	miR398 target	1793	MRT4577_570532C.1	129-149	Zea mays
miR398 target	Copper/zinc superoxide	1794	MRT4577_571443C.1	184-204	Zea mays
	dismutase (SODC)
	domain protein
miR398 target	miR398 target	1795	MRT4577_648609C.1	83-103	Zea mays
miRNA	miR399	1796			Zea mays
miRNA	miR399	1797			Zea mays
miRNA	miR399	1798			Zea mays
miRNA	miR399	1799			Zea mays
miR399 target	pho2 and inorganic	1800			Zea mays
	phosphate transporter
Decoy	miR399 decoy	1801			Artificial	Improved
					sequence	yield*
Cleavage	miR399 cleavage	1802			Artificial	Improved
blocker	blocker (in miRMON1				sequence	yield*
	backbone)
miR399 target	E2, ubiquitin-	1803	MRT3702_9137C.7	607-627	Arabidopsis
	conjugating enzyme;				thaliana
	At2g33770 PHO2
miR399 target	PHO2-like (phosphate)	1804	MRT3847_4521C.5	139-159	Glycine max
	E2 ubiquitin-
	conjugating enzyme
miR399 target	Phosphate transporter	1805	MRT3847_51499C.6	381-401	Glycine max
miR399 target	PHO2-like (phosphate)	1806	MRT3880_39637C.1	33-53	Medicago
	E2 ubiquitin-				truncatula
	conjugating enzyme
miR399 target	miR399 target	1807	MRT3880_45031C.1	512-532	Medicago
					truncatula
miR399 target	miR399 target	1808	MRT3880_48872C.1	5-25	Medicago
					truncatula
miR399 target	miR399 target	1809	MRT3880_54972C.1	5-25	Medicago
					truncatula
miR399 target	Phosphate transporter	1810	MRT3880_64645C.1	245-265	Medicago
					truncatula
miR399 target	miR399 target	1811	MRT4530_189375C.1	502-522	Oryza
					sativa
miR399 target	Phosphate transporter	1812	MRT4530_40506C.4	292-312	Oryza
					sativa
miR399 target	miR399 target	1813	MRT4530_53090C.4	821-841	Oryza
					sativa
miR399 target	miR399 target	1814	MRT4530_7904C.4	1144-1164	Oryza
					sativa
miR399 target	miR399 target	1815	MRT4558_16475C.1	693-713	Sorghum
					bicolor
miR399 target	miR399 target	1816	MRT4558_34625C.1	171-191	Sorghum
					bicolor
miR399 target	miR399 target	1817	MRT4565_160343C.1	481-501	Triticum
					aestivum
miRNA	miR408	1818			Zea mays
miR408 target	laccase and	1819			Zea mays
	plantacyanin
Decoy	miR408 decoy	1820			Artificial	Improved
					sequence	yield*
miR408 target	Laccase (Diphenol	1821	MRT3635_36078C.2	61-80	Gossypium
	oxidase); Multicopper				hirsutum
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1822	MRT3635_36080C.2	61-80	Gossypium
	oxidase); Multicopper				hirsutum
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1823	MRT3702_153631C.1	42-61	Arabidopsis
	oxidase); Multicopper				thaliana
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1824	MRT3702_20027C.5	108-127	Arabidopsis
	oxidase); Multicopper				thaliana
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1825	MRT3702_20202C.5	99-118	Arabidopsis
	oxidase); Multicopper				thaliana
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1826	MRT3702_6668C.5	71-90	Arabidopsis
	oxidase); Multicopper				thaliana
	oxidase Plantacyanin
miR408 target	miR408 target	1827	MRT3708_48434C.2	137-156	Brassica
					napus
miR408 target	Laccase (Diphenol	1828	MRT3711_7108C.1	9-28	Brassica
	oxidase); Multicopper				rapa
	oxidase Plantacyanin
miR408 target	miR408 target	1829	MRT3847_133008C.1	25-44	Glycine max
miR408 target	miR408 target	1830	MRT3847_166855C.1	17-36	Glycine max
miR408 target	Laccase (Diphenol	1831	MRT3847_261984C.4	181-200	Glycine max
	oxidase); Multicopper
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1832	MRT3847_273040C.3	702-721	Glycine max
	oxidase); Multicopper
	oxidase Plantacyanin
miR408 target	miR408 target	1833	MRT3847_273288C.3	114-133	Glycine max
miR408 target	Laccase (Diphenol	1834	MRT3847_296270C.2	189-208	Glycine max
	oxidase); Multicopper
	oxidase Plantacyanin
miR408 target	miR408 target	1835	MRT3847_31127C.7	232-251	Glycine max
miR408 target	miR408 target	1836	MRT3847_329905C.2	137-156	Glycine max
miR408 target	miR408 target	1837	MRT3847_336704C.1	58-77	Glycine max
miR408 target	miR408 target	1838	MRT3847_343250C.1	286-305	Glycine max
miR408 target	miR408 target	1839	MRT3847_346770C.1	38-57	Glycine max
miR408 target	miR408 target	1840	MRT3847_349900C.1	68-87	Glycine max
miR408 target	miR408 target	1841	MRT3847_66506C.8	33-52	Glycine max
miR408 target	miR408 target	1842	MRT3847_66508C.1	12-31	Glycine max
miR408 target	miR408 target	1843	MRT3880_52991C.2	96-115	Medicago
					truncatula
miR408 target	Laccase (Diphenol	1844	MRT3880_53025C.1	96-115	Medicago
	oxidase); Multicopper				truncatula
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1845	MRT3880_58299C.2	659-678	Medicago
	oxidase); Multicopper				truncatula
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1846	MRT3880_5838C.1	37-56	Medicago
	oxidase); Multicopper				truncatula
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1847	MRT3880_61178C.1	715-734	Medicago
	oxidase); Multicopper				truncatula
	oxidase Plantacyanin
miR408 target	miR408 target	1848	MRT4513_31098C.2	106-125	Hordeum
					vulgare
miR408 target	Laccase (Diphenol	1849	MRT4513_36864C.1	93-112	Hordeum
	oxidase); Multicopper				vulgare
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1850	MRT4513_43046C.1	113-132	Hordeum
	oxidase); Multicopper				vulgare
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1851	MRT4513_47240C.1	630-649	Hordeum
	oxidase); Multicopper				vulgare
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1852	MRT4513_8677C.1	71-90	Hordeum
	oxidase); Multicopper				vulgare
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1853	MRT4530_137979C.3	929-948	Oryza
	oxidase); Multicopper				sativa
	oxidase Plantacyanin
miR408 target	miR408 target	1854	MRT4530_148564C.5	1091-1110	Oryza
					sativa
miR408 target	Laccase (Diphenol	1855	MRT4530_160612C.2	220-239	Oryza
	oxidase); Multicopper				sativa
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1856	MRT4530_169405C.1	105-124	Oryza
	oxidase); Multicopper				sativa
	oxidase Plantacyanin
miR408 target	miR408 target	1857	MRT4530_247839C.2	360-379	Oryza
					sativa
miR408 target	Laccase (Diphenol	1858	MRT4530_260849C.1	658-677	Oryza
	oxidase); Multicopper				sativa
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1859	MRT4530_26787C.5	611-630	Oryza
	oxidase); Multicopper				sativa
	oxidase Plantacyanin
miR408 target	miR408 target	1860	MRT4530_274369C.1	112-131	Oryza
					sativa
miR408 target	miR408 target	1861	MRT4530_275579C.1	108-127	Oryza
					sativa
miR408 target	miR408 target	1862	MRT4530_36958C.6	99-118	Oryza
					sativa
miR408 target	Laccase (Diphenol	1863	MRT4530_40477C.6	182-201	Oryza
	oxidase); Multicopper				sativa
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1864	MRT4530_69716C.6	162-181	Oryza
	oxidase); Multicopper				sativa
	oxidase Plantacyanin
miR408 target	miR408 target	1865	MRT4558_23167C.3	713-732	Sorghum
					bicolor
miR408 target	Laccase (Diphenol	1866	MRT4558_2496C.2	104-123	Sorghum
	oxidase); Multicopper				bicolor
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1867	MRT4558_26802C.1	87-106	Sorghum
	oxidase); Multicopper				bicolor
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1868	MRT4558_37109C.1	109-128	Sorghum
	oxidase); Multicopper				bicolor
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1869	MRT4558_40844C.1	217-236	Sorghum
	oxidase); Multicopper				bicolor
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1870	MRT4558_5019C.2	102-121	Sorghum
	oxidase); Multicopper				bicolor
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1871	MRT4558_8981C.2	180-199	Sorghum
	oxidase); Multicopper				bicolor
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1872	MRT4565_100542C.3	91-110	Triticum
	oxidase); Multicopper				aestivum
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1873	MRT4565_130135C.1	10-29	Triticum
	oxidase); Multicopper				aestivum
	oxidase Plantacyanin
miR408 target	Hsp70 domain protein	1874	MRT4565_198220C.1	1221-1240	Triticum
					aestivum
miR408 target	miR408 target	1875	MRT4565_202586C.1	51-70	Triticum
					aestivum
miR408 target	Laccase (Diphenol	1876	MRT4565_216408C.1	206-225	Triticum
	oxidase); Multicopper				aestivum
	oxidase Plantacyanin
miR408 target	Ammonium	1877	MRT4565_219732C.1	742-761	Triticum
	transporter; basic helix-				aestivum
	loop-helix domain
	(bHLH)
miR408 target	Laccase (Diphenol	1878	MRT4565_229783C.1	98-117	Triticum
	oxidase); Multicopper				aestivum
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1879	MRT4565_235378C.1	116-135	Triticum
	oxidase); Multicopper				aestivum
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1880	MRT4565_250808C.1	652-671	Triticum
	oxidase); Multicopper				aestivum
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1881	MRT4565_257176C.1	91-110	Triticum
	oxidase); Multicopper				aestivum
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1882	MRT4565_263239C.1	102-121	Triticum
	oxidase); Multicopper				aestivum
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1883	MRT4565_263949C.1	94-113	Triticum
	oxidase); Multicopper				aestivum
	oxidase Plantacyanin
miR408 target	miR408 target	1884	MRT4565_267955C.1	84-103	Triticum
					aestivum
miR408 target	Laccase (Diphenol	1885	MRT4565_274907C.1	720-739	Triticum
	oxidase); Multicopper				aestivum
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1886	MRT4565_276632C.1	172-191	Triticum
	oxidase); Multicopper				aestivum
	oxidase Plantacyanin
miR408 target	miR408 target	1887	MRT4565_278866C.1	365-384	Triticum
					aestivum
miR408 target	Laccase (Diphenol	1888	MRT4565_66211C.2	36-55	Triticum
	oxidase); Multicopper				aestivum
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1889	MRT4565_67059C.3	133-152	Triticum
	oxidase); Multicopper				aestivum
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1890	MRT4565_87146C.2	314-333	Triticum
	oxidase); Multicopper				aestivum
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1891	MRT4577_137208C.1	94-113	Zea mays
	oxidase); Multicopper
	oxidase Plantacyanin
miR408 target	miR408 target	1892	MRT4577_191445C.5	696-715	Zea mays
miR408 target	miR408 target	1893	MRT4577_234909C.4	331-350	Zea mays
miR408 target	miR408 target	1894	MRT4577_245033C.8	117-136	Zea mays
miR408 target	Laccase (Diphenol	1895	MRT4577_264839C.3	102-121	Zea mays
	oxidase); Multicopper
	oxidase Plantacyanin
miR408 target	miR408 target	1896	MRT4577_30771C.9	282-301	Zea mays
miR408 target	miR408 target	1897	MRT4577_325201C.6	619-638	Zea mays
miR408 target	Laccase (Diphenol	1898	MRT4577_325458C.1	59-78	Zea mays
	oxidase); Multicopper
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1899	MRT4577_327865C.2	113-132	Zea mays
	oxidase); Multicopper
	oxidase Plantacyanin
miR408 target	miR408 target	1900	MRT4577_341887C.5	132-151	Zea mays
miR408 target	miR408 target	1901	MRT4577_37590C.9	800-819	Zea mays
miR408 target	miR408 target	1902	MRT4577_380413C.6	208-227	Zea mays
miR408 target	miR408 target	1903	MRT4577_387021C.4	151-170	Zea mays
miR408 target	miR408 target	1904	MRT4577_388860C.4	117-136	Zea mays
miR408 target	miR408 target	1905	MRT4577_427804C.4	729-748	Zea mays
miR408 target	Laccase (Diphenol	1906	MRT4577_446604C.1	67-86	Zea mays
	oxidase); Multicopper
	oxidase Plantacyanin
miR408 target	miR408 target	1907	MRT4577_456053C.1	66-85	Zea mays
miR408 target	miR408 target	1908	MRT4577_461451C.3	463-482	Zea mays
miR408 target	miR408 target	1909	MRT4577_46308C.7	273-292	Zea mays
miR408 target	Laccase (Diphenol	1910	MRT4577_517561C.1	883-902	Zea mays
	oxidase); Multicopper
	oxidase Plantacyanin
miR408 target	Laccase (Diphenol	1911	MRT4577_528699C.2	636-655	Zea mays
	oxidase); Multicopper
	oxidase Plantacyanin
miR408 target	miR408 target	1912	MRT4577_536494C.2	151-170	Zea mays
miR408 target	Laccase (Diphenol	1913	MRT4577_550892C.1	659-678	Zea mays
	oxidase); Multicopper
	oxidase Plantacyanin
miR408 target	miR408 target	1914	MRT4577_572693C.1	101-120	Zea mays
miR408 target	miR408 target	1915	MRT4577_602288C.1	5-24	Zea mays
miR408 target	miR408 target	1916	MRT4577_603948C.1	206-225	Zea mays
miR408 target	miR408 target	1917	MRT4577_603999C.1	226-245	Zea mays
miR408 target	miR408 target	1918	MRT4577_610458C.1	111-130	Zea mays
miR408 target	miR408 target	1919	MRT4577_623809C.1	153-172	Zea mays
miR408 target	miR408 target	1920	MRT4577_625157C.1	254-273	Zea mays
miR408 target	miR408 target	1921	MRT4577_629379C.1	269-288	Zea mays
miR408 target	miR408 target	1922	MRT4577_645720C.1	236-255	Zea mays
miR408 target	miR408 target	1923	MRT4577_650403C.1	788-807	Zea mays
miR408 target	miR408 target	1924	MRT4577_686202C.1	160-179	Zea mays
miR408 target	miR408 target	1925	MRT4577_710942C.1	48-67	Zea mays
miR444	miR444	1926			Zea mays
miRNA	miR444	1927			Zea mays	Improved
precursor						yield*
miR444 target	Os.ANR1	1928			Oryza
					sativa
miRNA-	Os.ANR1 (miR444	1929			Artificial	Improved
unresponsive	unresponsive)				construct	yield*
miR444 target	AGL17, AGL21,	1930			Zea mays
	ANR1
Decoy	miR444 decoy	1931			Artificial	Improved
					construct	yield*
miR444 target	MADS-box	1932	MRT3847_247970C.2	471-491	Glycine max
	transcription factor
	protein
miR444 target	MADS-box	1933	MRT3847_259952C.3	453-473	Glycine max
	transcription factor
	protein
miR444 target	MADS-box	1934	MRT3880_12754C.1	75-95	Medicago
	transcription factor				truncatula
	protein
miR444 target	miR444 target	1935	MRT4513_18691C.1	73-93	Hordeum
					vulgare
miR444 target	miR444 target	1936	MRT4513_36208C.1	320-340	Hordeum
					vulgare
miR444 target	miR444 target	1937	MRT4530_101813C.4	1164-1184	Oryza
					sativa
miR444 target	MADS-box	1938	MRT4530_196636C.3	539-559	Oryza
	transcription factor				sativa
	protein
miR444 target	miR444 target	1939	MRT4530_197829C.2	585-605	Oryza
					sativa
miR444 target	miR444 target	1940	MRT4530_223119C.3	610-630	Oryza
					sativa
miR444 target	miR444 target	1941	MRT4530_244375C.1	208-228	Oryza
					sativa
miR444 target	miR444 target	1942	MRT4530_251481C.2	1234-1254	Oryza
					sativa
miR444 target	miR444 target	1943	MRT4530_272160C.1	571-591	Oryza
					sativa
miR444 target	miR444 target	1944	MRT4530_274638C.1	337-357	Oryza
					sativa
miR444 target	miR444 target	1945	MRT4530_275771C.1	97-117	Oryza
					sativa
miR444 target	MADS-box	1946	MRT4530_78475C.3	305-325	Oryza
	transcription factor				sativa
	protein
miR444 target	MADS-box	1947	MRT4558_10090C.1	400-420	Sorghum
	transcription factor				bicolor
	protein
miR444 target	MADS-box	1948	MRT4558_11440C.2	434-454	Sorghum
	transcription factor				bicolor
	protein
miR444 target	miR444 target	1949	MRT4558_3598C.3	1024-1044	Sorghum
					bicolor
miR444 target	miR444 target	1950	MRT4558_37372C.1	1355-1375	Sorghum
					bicolor
miR444 target	MADS-box	1951	MRT4565_247066C.1	375-395	Triticum
	transcription factor				aestivum
	protein
miR444 target	MADS-box	1952	MRT4565_39318C.3	416-436	Triticum
	transcription factor				aestivum
	protein
miR444 target	miR444 target	1953	MRT4565_98921C.1	352-372	Triticum
					aestivum
miR444 target	miR444 target	1954	MRT4577_166928C.8	1146-1166	Zea mays
miR444 target	miR444 target	1955	MRT4577_204116C.4	475-495	Zea mays
miR444 target	miR444 target	1956	MRT4577_296919C.6	475-495	Zea mays
miR444 target	MADS-box	1957	MRT4577_321664C.4	1029-1049	Zea mays
	transcription factor
	protein
miR444 target	miR444 target	1958	MRT4577_417091C.4	1757-1777	Zea mays
miR444 target	miR444 target	1959	MRT4577_502196C.3	468-488	Zea mays
miR444 target	miR444 target	1960	MRT4577_537511C.2	364-384	Zea mays
miR444 target	miR444 target	1961	MRT4577_538474C.2	451-471	Zea mays
miR444 target	miR444 target	1962	MRT4577_5433C.4	473-493	Zea mays
miR444 target	miR444 target	1963	MRT4577_543434C.2	377-397	Zea mays
miR444 target	MADS-box	1964	MRT4577_553467C.1	17-37	Zea mays
	transcription factor
	protein
miR444 target	miR444 target	1965	MRT4577_581326C.1	388-408	Zea mays
miR444 target	miR444 target	1966	MRT4577_590710C.1	509-529	Zea mays
miR444 target	miR444 target	1967	MRT4577_613242C.1	18-38	Zea mays
miR444 target	miR444 target	1968	MRT4577_672581C.1	430-450	Zea mays
miRNA	miR528	1969			Zea mays
miR528 target	SOD	1970			Zea mays
Decoy	miR528 decoy	1971			Artificial	Improved
					construct	yield*
miR528 target	Salicylic acid-binding	1972	MRT3847_26249C.5	98-118	Glycine max
	protein
miR528 target	Laccase (Diphenol	1973	MRT4513_36138C.1	838-858	Hordeum
	oxidase); Multicopper				vulgare
	oxidase Plantacyanin
miR528 target	Laccase (Diphenol	1974	MRT4513_39686C.1	35-55	Hordeum
	oxidase); Multicopper				vulgare
	oxidase Plantacyanin
miR528 target	Laccase (Diphenol	1975	MRT4513_5560C.1	506-525	Hordeum
	oxidase); Multicopper				vulgare
	oxidase Plantacyanin
miR528 target	Laccase (Diphenol	1976	MRT4530_128077C.2	269-289	Oryza
	oxidase); Multicopper				sativa
	oxidase Plantacyanin
miR528 target	Laccase (Diphenol	1977	MRT4530_139238C.4	2152-2172	Oryza
	oxidase); Multicopper				sativa
	oxidase Plantacyanin
miR528 target	Laccase (Diphenol	1978	MRT4530_155994C.3	247-267	Oryza
	oxidase); Multicopper				sativa
	oxidase Plantacyanin
miR528 target	VIP2-like protein;	1979	MRT4530_237311C.1	632-652	Oryza
	PHD-zinc finger				sativa
miR528 target	Laccase (Diphenol	1980	MRT4530_275240C.1	24-44	Oryza
	oxidase); Multicopper				sativa
	oxidase Plantacyanin
miR528 target	Laccase (Diphenol	1981	MRT4530_68465C.5	687-706	Oryza
	oxidase); Multicopper				sativa
	oxidase Plantacyanin
miR528 target	VIP2-like protein;	1982	MRT4530_85016C.5	215-235	Oryza
	PHD-zinc finger				sativa
miR528 target	Laccase (Diphenol	1983	MRT4558_8881C.1	101-121	Sorghum
	oxidase); Multicopper				bicolor
	oxidase Plantacyanin
miR528 target	Laccase (Diphenol	1984	MRT4565_204482C.1	212-231	Triticum
	oxidase); Multicopper				aestivum
	oxidase Plantacyanin
miR528 target	Laccase (Diphenol	1985	MRT4565_219247C.1	923-943	Triticum
	oxidase); Multicopper				aestivum
	oxidase Plantacyanin
miR528 target	Laccase (Diphenol	1986	MRT4565_22497C.4	806-826	Triticum
	oxidase); Multicopper				aestivum
	oxidase Plantacyanin
miR528 target	Major Facilitator	1987	MRT4565_260315C.1	584-604	Triticum
	Superfamily				aestivum
miR528 target	Laccase (Diphenol	1988	MRT4565_276632C.1	219-239	Triticum
	oxidase); Multicopper				aestivum
	oxidase Plantacyanin
miR528 target	miR528 target	1989	MRT4565_278866C.1	412-432	Triticum
					aestivum
miR528 target	Laccase (Diphenol	1990	MRT4565_6214C.4	548-567	Triticum
	oxidase); Multicopper				aestivum
	oxidase Plantacyanin
miR528 target	Laccase (Diphenol	1991	MRT4577_302078C.5	115-135	Zea mays
	oxidase); Multicopper
	oxidase Plantacyanin
miR528 target	Laccase (Diphenol	1992	MRT4577_327865C.2	163-183	Zea mays
	oxidase); Multicopper
	oxidase Plantacyanin
miR528 target	Laccase (Diphenol	1993	MRT4577_338803C.6	189-209	Zea mays
	oxidase); Multicopper
	oxidase Plantacyanin
miR528 target	miR528 target	1994	MRT4577_574203C.1	48-68	Zea mays
miRNA	miR827	1995			Zea mays
miR827 target	SPX	1996	MRT3702_118660C.4	258-278	Arabidopsis
	(SYG1/Pho81/XPR1)				thaliana
	domain-containing
	protein; RING domain
	ubiquitin ligase
miR827 target	SPX	1997	MRT3702_165543C.2	253-273	Arabidopsis
	(SYG1/Pho81/XPR1)				thaliana
	domain-containing
	protein; MFS_1: Major
	Facilitator Superfamily
miR827 target	SPX	1998	MRT3702_4781C.6	153-173	Arabidopsis
	(SYG1/Pho81/XPR1)				thaliana
	domain-containing
	protein; MFS_1: Major
	Facilitator Superfamily
miR827 target	SPX	1999	MRT3708_29390C.1	32-52	Brassica
	(SYG1/Pho81/XPR1)				napus
	domain-containing
	protein; RING domain
	ubiquitin ligase
miR827 target	miR827 target	2000	MRT3711_10064C.1	155-175	Brassica
					rapa
miR827 target	SPX	2001	MRT3712_6456C.1	96-116	Brassica
	(SYG1/Pho81/XPR1)				oleracea
	domain-containing
	protein
miR827 target	SPX	2002	MRT4530_236774C.2	395-415	Oryza
	(SYG1/Pho81/XPR1)				sativa
	domain-containing
	protein; MFS_1: Major
	Facilitator Superfamily
miR827 target	SPX	2003	MRT4530_45193C.6	335-355	Oryza
	(SYG1/Pho81/XPR1)				sativa
	domain-containing
	protein; MFS_1: Major
	Facilitator Superfamily
miR827 target	miR827 target	2004	MRT4577_197256C.1	135-155	Zea mays
miR827 target	miR827 target	2005	MRT4577_235663C.3	559-579	Zea mays
miRNA	miRCOP1_1227-1247	2006			Artificial	Improved
					sequence	yield*
miRNA	miRCOP1_653-673	2007			Artificial	Improved
					sequence	yield*
miRNA	miRCOP1_1417-1437	2008			Artificial	Improved
					sequence	yield*
miRCOP1 target	COP1 (constitutive	2009			Zea mays
	photomorphogenesis 1)
miRNA	miRGA2_945-965	2010			Artificial	Improved
					sequence	yield*
miRGA2 target	zm-GA2ox (gibberellic	2011			Zea mays
	acid 2 oxidase)
miRNA	miRGA20_852-872	2012			Artificial	Improved
					sequence	yield*
miRGA20 target	zm-GA20ox (gibberellic	2013			Zea mays
	acid 20 oxidase)
miRNA	miRHB2-4_700-720	2014			Artificial	Improved
					sequence	yield*
miRHB2-4 target	ZmHB2-4 (homeobox	2015			Zea mays
	2 and homeobox 4)
miRNA	miRHB4_84-104	2016			Artificial	Improved
					sequence	yield*
miRHB4 target	ZmHB-4 (homeobox 4)	2017			Zea mays
miRNA	miRLG1_899-919	2018			Artificial	Improved
					sequence	yield*
miRLG1 target	LG1 (Liguleless1)	2019			Zea mays
miRNA	miRMON18	2020			Glycine max
miRMON18	SPX	2021			Zea mays
target	(SYG1, PHO81
	and XPR1 domain;
	PFAM entry PF03105
	at www.sanger.ac.uk)
Decoy	miRMON18 decoy	2022			Artificial	Improved
					sequence	yield*
miRNA	miRVIM1a	2023			Artificial	Improved
precursor					sequence	yield*
(synthetic)
miRVIM1a	VIM1a (Variant in	2024			Zea mays
target	Methylation1a)
miRNA	miRDHS1	2025			Artificial	Improved
precursor					sequence	yield*
(synthetic)
miRDHS1	DHS1 (Deoxyhypusine	2026			Zea mays
target	synthase)
miRNA	miRDHS2	2027			Artificial	Improved
precursor					sequence	yield*
(synthetic)
miRDHS2	DHS2 (Deoxyhypusine	2028			Zea mays
target	synthase)
miRNA	miRDHS3	2029			Artificial	Improved
precursor					sequence	yield*
(synthetic)
miRDHS3	DHS3 (Deoxyhypusine	2030			Zea mays
target	synthase)
miRNA	miRDHS4	2031			Artificial	Improved
precursor					sequence	yield*
(synthetic)
miRDHS4	DHS4 (Deoxyhypusine	2032			Zea mays
target	synthase)
Synthetic	DHS5 ta-siRNA	2033			Artificial	Improved
tasiRNA					sequence	yield*
DHS5 ta-siRNA	DHS5 (Deoxyhypusine	2034			Zea mays
target	synthase)
Synthetic	DHS6 ta-siRNA	2035			Artificial	Improved
tasiRNA					sequence	yield*
DHS6 ta-siRNA	DHS6 (Deoxyhypusine	2036			Zea mays
target	synthase)
Synthetic	DHS7 ta-siRNA	2037			Artificial	Improved
tasiRNA					sequence	yield*
DHS7 ta-siRNA	DHS7 (Deoxyhypusine	2038			Zea mays
target	synthase)
Synthetic	DHS8 ta-siRNA	2039			Artificial	Improved
tasiRNA					sequence	yield*
DHS8 ta-siRNA	DHS8 (Deoxyhypusine	2040			Zea mays
target	synthase)
Synthetic	DHS ta-siRNA	2041			Artificial	Improved
tasiRNA					sequence	yield*
DHS ta-siRNA	DHS (Deoxyhypusine	2042			Zea mays
target	synthase)
miRNA	miRCRF_804-824	2043			Artificial	Improved
precursor					sequence	yield*
(synthetic)
miRCRF target	CRF (corn RING	2044			Zea mays
	ringer; also RNF169)
miRNA	miRMON18	2045			Zea mays	Improved
precursor						yield*
miRMON18	SPX	2046			Zea mays
target
miRNA	miRZmG1543a	2047			Artificial	Improved
precursor					sequence	yield*
(synthetic)
miRZmG1543a	ZmG1543a (maize	2048			Zea mays
target	orthologue of
	Arabidopsis thaliana
	homeobox 17)
miRNA	miRZmG1543	2049			Artificial	Improved
precursor					sequence	yield*
(synthetic)
miRZmG1543	ZmG1543a (maize	2050			Zea mays
target	orthologue of
	Arabidopsis thaliana
	homeobox 17)
miRNA	miRZmG1543b	2051			Artificial	Improved
precursor					sequence	yield*
(synthetic)
miRZmG1543b	ZmG1543b (maize	2052			Zea mays
target	orthologue of
	Arabidopsis thaliana
	homeobox 17)
miRNA	miRHB2	2053			Artificial	Improved
precursor					sequence	yield*
(synthetic)
miRHB2 target	HB2 (homeobox 2)	2054			Zea mays
miRNA	Os.MIR169g	2055			Oryza	Improved
precursor					sativa	yield*
miRNA	Zm.MIR167g	2056			Artificial	Improved
precursor					sequence	yield*
miRNA	miRGS3	2057			Artificial	Improved
precursor					sequence	yield*
(synthetic)
miRGS3 target	GS3 (grain size 3)	2058			Zea mays
miRNA	Zm_GW2_miR1	2059			Artificial	Improved
precursor					sequence	yield*
(synthetic)
miRNA	Zm_GW2_miR2	2060			Artificial	Improved
precursor					sequence	yield*
(synthetic)
miRNA	Zm_GW2_miR3	2061			Artificial	Improved
precursor					sequence	yield*
(synthetic)
GW2_miR1/2/3	GW2 (grain weight 2)	2062			Zea mays
target
miRNA	miR-IPS	2063			Artificial	Improved
precursor					construct	yield*
(synthetic)
miR-IPS target	Zm_2-isopropylmalate	2064			Zea mays
	synthase

Particularly preferred crop plants are maize, soybean, canola, cotton, alfalfa, sugarcane, sugar beet, sorghum*, and rice

Example 5

This example illustrates various aspects of the invention relating to transgenic plant cells and transgenic plants. More specifically, this example illustrates transformation vectors and techniques useful with different crop plants for providing non-natural transgenic plant cells, plants, and seeds having in their genome any of this invention's recombinant DNA constructs transcribable in a plant cell, including a promoter that is functional in the plant cell and operably linked to at least one polynucleotide as disclosed herein, including: (1) a recombinant DNA construct transcribable in a plant cell, including a promoter that is functional in the plant cell and operably linked to at least one polynucleotide selected from: (a) DNA encoding a cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (b) DNA encoding a 5′-modified cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (c) DNA encoding a translational inhibitor to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (d) DNA encoding a decoy to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3; (e) DNA encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of at least one miRNA target identified in Tables 2 or 3, wherein a miRNA recognition site in the native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage; (f) DNA encoding a miRNA precursor which is processed into a miRNA for suppressing expression of at least one miRNA target identified in Tables 2 or 3; (g) DNA encoding double-stranded RNA which is processed into siRNAs for suppressing expression of at least one miRNA target identified in Tables 2 or 3; and (h) DNA encoding a ta-siRNA which is processed into siRNAs for suppressing expression of at least one miRNA target identified in Tables 2 or 3; (2) a recombinant DNA construct transcribable in a plant cell, including a promoter that is functional in the plant cell and operably linked to at least one polynucleotide selected from: (a) DNA encoding a cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target; (b) DNA encoding a 5′-modified cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target; (c) DNA encoding a translational inhibitor to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target; (d) DNA encoding a decoy to prevent or decrease small RNA-mediated cleavage of the transcript of at least one miRNA target; (e) DNA encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of at least one miRNA target, wherein a miRNA recognition site in the native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage; (f) DNA encoding a miRNA precursor which is processed into a miRNA for suppressing expression of at least one miRNA target; (g) DNA encoding double-stranded RNA which is processed into siRNAs for suppressing expression of at least one miRNA target; and (h) DNA encoding a ta-siRNA which is processed into siRNAs for suppressing expression of at least one miRNA target—wherein the at least one miRNA target is at least one selected from the group consisting of a miR156 target, a miR160 target, a miR164 target, a miR166 target, a miR167 target, a miR169 target, a miR171 target, a miR172 target, a miR319 target, miR395 target, a miR396 target, a miR398 target, a miR399 target, a miR408 target, a miR444 target, a miR528 target, a miR167g target, a miR169g target, COP1 (constitutive photomorphogenesis1), GA2ox (gibberellic acid 2 oxidase), GA20ox (gibberellic acid 20 oxidase), HB2 (homeobox 2), HB2-4 (homeobox 2 and homeobox 4), HB4 (homeobox 4), LG1 (liguleless1), SPX (SYG1, PH081 and XPR1 domain; PFAM entry PF03105 at www.sanger.ac.uk), VIMla (variant in methlylation 1a), DHS1 (deoxyhypusine synthase), DHS2 (deoxyhypusine synthase), DHS3 (deoxyhypusine synthase), DHS4 (deoxyhypusine synthase), DHS5 (deoxyhypusine synthase), DHS6 (deoxyhypusine synthase), DHS7 (deoxyhypusine synthase), DHS8 (deoxyhypusine synthase), CRF (corn RING finger; RNF169), G1543a (maize orthologue of Arabidopsis thaliana homeobox 17), G1543b (maize orthologue of Arabidopsis thaliana homeobox 17), GS3 (grain size 3), and GW2 (grain weight 2); (3) a recombinant DNA construct transcribable in a plant cell, including a promoter that is functional in the plant cell and operably linked to at least one polynucleotide selected from the group consisting of DNA encoding a nucleotide sequence selected from SEQ ID NOs: 1120, 1121, 1122, 1248, 1257, 1313, 1314, 1364, 1387, 1478, 1489, 1490, 1491, 1492, 1493, 1585, 1597, 1598, 1599, 1713, 1752, 1753, 1801, 1802, 1820, 1927, 1929, 1931, 1971, 2006, 2007, 2008, 2010, 2012, 2014, 2016, 2018, 2022, 2023, 2025, 2027, 2029, 2031, 2033, 2035, 2037, 2039, 2041, 2043, 2045, 2047, 2049, 2051, 2053, 2055, 2056, 2057, 2059, 2060, 2061, and 2063; (4) a recombinant DNA construct transcribable in a plant cell, including a promoter functional in the non-natural transgenic plant cell and operably linked to at least one polynucleotide selected from DNA encoding at least one miRNA target identified in Tables 2 or 3; and (5) a recombinant DNA construct transcribable in a plant cell, including a promoter functional in the non-natural transgenic plant cell and operably linked to at least one polynucleotide including a DNA sequence selected from SEQ ID NOS: 15-2064). It is clear that the polynucleotide to be expressed using these recombinant DNA vectors in the non-natural transgenic plant cells, plants, and seeds can encode a transcript that prevents or decreases small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3 (including the specific miRNA targets identified by name in this paragraph), or a transcript that suppresses expression of at least one miRNA target identified in Tables 2 or 3 (including the specific miRNA targets identified by name in this paragraph), or a transcript encoding at least one miRNA target identified in Tables 2 or 3, or encodes DNA sequence selected from SEQ ID NOS: 15-2064.

Transformation Vectors and Protocols

The following sections describe examples of a base vector for preparing transformation vectors including recombinant DNA constructs of this invention for transformation of a specific crop plant. The recombinant DNA constructs are transcribable in a plant cell and include a promoter that is functional in the plant cell and operably linked to at least one polynucleotide, which encodes a transcript that prevents or decreases small RNA-mediated cleavage of the transcript of at least one miRNA target identified in Tables 2 or 3 (including the specific miRNA targets identified by name in this paragraph), or a transcript that suppresses expression of at least one miRNA target identified in Tables 2 or 3 (including the specific miRNA targets identified by name in this paragraph), or a transcript encoding at least one miRNA target identified in Tables 2 or 3, or encodes DNA sequence selected from SEQ ID NOS: 15-2064. Also provided are detailed examples of crop-specific transformation protocols for using these vectors including recombinant DNA constructs of this invention to generate a non-natural transgenic plant cell, non-natural transgenic tissue, or non-natural transgenic plant. Additional transformation techniques are known to one of ordinary skill in the art, as reflected in the “Compendium of Transgenic Crop Plants”, edited by Chittaranjan Kole and Timothy C. Hall, Blackwell Publishing Ltd., 2008; ISBN 978-1-405-16924-0 (available electronically at mrw.interscience.wiley.com/emrw/9781405181099/hpt/toc). Such transformation methods are useful in producing a non-natural transgenic plant cell having a transformed nucleus. Non-natural transgenic plants, seeds, and pollen are subsequently produced from such a non-natural transgenic plant cell having a transformed nucleus, and screened for an enhanced trait (e.g., increased yield, enhanced water use efficiency, enhanced cold tolerance, enhanced nitrogen or phosphate use efficiency, enhanced seed protein, or enhanced seed oil, or any trait such as those disclosed above under the heading “Making and Using Transgenic Plant Cells and Transgenic Plants”).

Transformation of Maize

A base transformation vector pMON93039 (SEQ ID NO: 2065), illustrated in Table 4 and FIG. 2, is used in preparing recombinant DNA constructs for Agrobacterium-mediated transformation of maize cells. A transformation vector for expressing each of the recombinant DNA constructs of this invention is constructed by inserting a polynucleotide of this invention into the base vector pMON93039 (SEQ ID NO: 2065) in the gene of interest expression cassette at an insertion site, i.e., between the intron element (coordinates 1287-1766) and the polyadenylation element (coordinates 1838-2780). For example, a transformation vector for expression of a miR399 cleavage blocker is prepared by inserting the DNA of SEQ ID NO: 1802 (see Table 3) into the gene of interest expression cassette at an insertion site between the intron element (coordinates 1287-1766) and the polyadenylation element (coordinates 1838-2780) of pMON93039 (SEQ ID NO: 2065).
For Agrobacterium-mediated transformation of maize embryo cells, maize plants of a transformable line are grown in the greenhouse and ears are harvested when the embryos are 1.5 to 2.0 mm in length. Ears are surface sterilized by spraying or soaking the ears in 80% ethanol, followed by air drying. Immature embryos are isolated from individual kernels from sterilized ears. Prior to inoculation of maize cells, cultures of Agrobacterium each containing a transformation vector for expressing each of the recombinant DNA constructs of this invention are grown overnight at room temperature. Immature maize embryo cells are inoculated with Agrobacterium after excision, incubated at room temperature with Agrobacterium for 5 to 20 minutes, and then co-cultured with Agrobacterium for 1 to 3 days at 23 degrees Celsius in the dark. Co-cultured embryos are transferred to a selection medium and cultured for approximately two weeks to allow embryogenic callus to develop. Embryogenic callus is transferred to a culture medium containing 100 mg/L paromomycin and subcultured at about two week intervals. Multiple events of transformed plant cells are recovered 6 to 8 weeks after initiation of selection.
Transgenic maize plants are regenerated from transgenic plant cell callus for each of the multiple transgenic events resulting from transformation and selection. The callus of transgenic plant cells of each event is placed on a medium to initiate shoot and root development into plantlets which are transferred to potting soil for initial growth in a growth chamber at 26 degrees Celsius, followed by growth on a mist bench before transplanting to pots where plants are grown to maturity. The regenerated plants are self-fertilized. First generation (“R1”) seed is harvested. The seed or plants grown from the seed is used to select seeds, seedlings, progeny second generation (“R2”) transgenic plants, or hybrids, e.g., by selecting transgenic plants exhibiting an enhanced trait as compared to a control plant (a plant lacking expression of the recombinant DNA construct).
The above process is repeated to produce multiple events of transgenic maize plant cells that are transformed with separate recombinant DNA constructs of this invention, i.e., a construct transcribable in a maize plant cell, including a promoter that is functional in the maize plant cell and operably linked to each polynucleotide selected from: (a) DNA encoding a cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of each miRNA target identified in Tables 2 and 3; (b) DNA encoding a 5′-modified cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of each miRNA target identified in Tables 2 and 3; (c) DNA encoding a translational inhibitor to prevent or decrease small RNA-mediated cleavage of the transcript of each miRNA target identified in Tables 2 and 3; (d) DNA encoding a decoy to prevent or decrease small RNA-mediated cleavage of the transcript of each miRNA target identified in Tables 2 and 3; (e) DNA encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of each miRNA target identified in Tables 2 and 3, wherein a miRNA recognition site in the native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage; (f) DNA encoding a miRNA precursor which is processed into a miRNA for suppressing expression of each miRNA target identified in Tables 2 and 3; (g) DNA encoding double-stranded RNA which is processed into siRNAs for suppressing expression of each miRNA target identified in Tables 2 and 3; and (h) DNA encoding a ta-siRNA which is processed into siRNAs for suppressing expression of each miRNA target identified in Tables 2 and 3.
The above process is repeated to produce multiple events of transgenic maize plant cells that are transformed with each of the following recombinant DNA constructs of this invention, i.e., a construct transcribable in a maize plant cell, including a promoter that is functional in the maize plant cell and operably linked to a polynucleotide selected from: (a) DNA encoding a cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of the miRNA target; (b) DNA encoding a 5′-modified cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of the miRNA target; (c) DNA encoding a translational inhibitor to prevent or decrease small RNA-mediated cleavage of the transcript of the miRNA target; (d) DNA encoding a decoy to prevent or decrease small RNA-mediated cleavage of the transcript of the miRNA target; (e) DNA encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of the miRNA target, wherein a miRNA recognition site in the native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage; (f) DNA encoding a miRNA precursor which is processed into a miRNA for suppressing expression of the miRNA target; (g) DNA encoding double-stranded RNA which is processed into siRNAs for suppressing expression the miRNA target; and (h) DNA encoding a ta-siRNA which is processed into siRNAs for suppressing expression of the miRNA target—wherein separate constructs are made for each of the miRNA targets enumerated in Table 5.
The above process is repeated to produce multiple events of transgenic maize plant cells that are transformed with each of the following recombinant DNA constructs of this invention, i.e., a construct transcribable in a maize plant cell, including a promoter that is functional in the maize plant cell and operably linked to each polynucleotide provided in Table 6, wherein separate constructs are made for each polynucleotide.
The above process is repeated to produce multiple events of transgenic maize plant cells that are transformed with each of the following recombinant DNA constructs of this invention, i.e., a construct transcribable in a maize plant cell, including a promoter that is functional in the plant cell and operably linked to a polynucleotide selected from DNA encoding each miRNA target identified in Tables 2 and 3.
The above process is repeated to produce multiple events of transgenic maize plant cells that are transformed with each of the following recombinant DNA constructs of this invention, i.e., a construct transcribable in a maize plant cell, including a promoter that is functional in the plant cell and operably linked to each polynucleotide of SEQ ID NOS: 15-2064.
The regenerated transgenic maize plants, or progeny transgenic maize plants or maize seeds, produced from the regenerated transgenic maize plants, are screened for an enhanced trait (e.g., increased yield), as compared to a control plant or seed (a plant or seed lacking expression of the recombinant DNA construct). From each group of multiple events of transgenic maize plants with a specific recombinant construct of this invention, the event that produces the greatest enhanced trait (e.g., greatest enhancement in yield) is identified and progeny maize seed is selected for commercial development.

TABLE 4

			Coordinates of
			SEQ ID NO:
Function	Name	Annotation		2065

Agrobacterium	B-AGRtu.right	Agro right border sequence, essential for	11364-11720
T-DNA	border	transfer of T-DNA.
transfer
Gene of	E-Os.Act1	Upstream promoter region of the rice actin	19-775
interest		1 gene
expression	E-CaMV.35S.2xA1-	Duplicated35S A1-B3 domain without	788-1120
cassette	B3	TATA box
	P-Os.Act1	Promoter region of the rice actin 1 gene	1125-1204
	L-Ta.Lhcb1	5′ untranslated leader of wheat major	1210-1270
		chlorophyll a/b binding protein
	I-Os.Act1	First intron and flanking UTR exon	1287-1766
		sequences from the rice actin 1 gene
	T-St.Pis4	3′ non-translated region of the potato	1838-2780
		proteinase inhibitor II gene which functions
		to direct polyadenylation of the mRNA
Plant	P-Os.Act1	Promoter from the rice actin 1 gene	2830-3670
selectable	L-Os.Act1	First exon of the rice actin 1 gene	3671-3750
marker	I-Os.Act1	First intron and flanking UTR exon	3751-4228
expression		sequences from the rice actin 1 gene
cassette	TS-At.ShkG-CTP2	Transit peptide region of Arabidopsis	4238-4465
		EPSPS
	CR-AGRtu.aroA-	Coding region for bacterial strain CP4	4466-5833
	CP4.nat	native aroA gene.
	T-AGRtu.nos	A 3′ non-translated region of the nopaline	5849-6101
		synthase gene of Agrobacterium
		tumefaciens Ti plasmid which functions to
		direct polyadenylation of the mRNA.
Agrobacterium	B-AGRtu.left border	Agro left border sequence, essential for	6168-6609
T-DNA		transfer of T-DNA.
transfer
Maintenance	OR-Ec.oriV-RK2	The vegetative origin of replication from	6696-7092
in E. coli		plasmid RK2.
	CR-Ec.rop	Coding region for repressor of primer from	8601-8792
		the ColE1 plasmid. Expression of this gene
		product interferes with primer binding at the
		origin of replication, keeping plasmid copy
		number low.
	OR-Ec.ori-ColE1	The minimal origin of replication from the	9220-9808
		E. coli plasmid ColE1.
	P-Ec.aadA-	Promoter for Tn7 adenylyltransferase	10339-10380
	SPC/STR	(AAD(3″))
	CR-Ec.aadA-	Coding region for Tn7 adenylyltransferase	10381-11169
	SPC/STR	(AAD(3″)) conferring spectinomycin and
		streptomycin resistance.
	T-Ec.aadA-	3′ UTR from the Tn7 adenylyltransferase	11170-11227
	SPC/STR	(AAD(3″)) gene of E. coli.

TABLE 5

miRNA Targets

a miR156 target, a miR160 target, a miR164 target, a miR166 target, a miR167 target, a miR169 target, a

miR171 target, a miR172 target, a miR319 target, miR395 target, a miR396 target, a a miR398 target, a

miR399 target, a miR408 target, a miR444 target, a miR528 target, a miR167g target, a miR169g target,

COP1 (constitutive photomorphogenesis1), GA2ox (gibberellic acid 2 oxidase), GA20ox (gibberellic

acid 20 oxidase), HB2 (homeobox 2), HB2-4 (homeobox 2 and homeobox 4), HB4 (homeobox 4), LG1

(liguleless1), SPX (SYG1, PHO81 and XPR1 domain; PFAM entry PF03105 at www.sanger.ac.uk),

VIM1a (variant in methlylation 1a), DHS1 (deoxyhypusine synthase), DHS2 (deoxyhypusine synthase),

DHS3 (deoxyhypusine synthase), DHS4 (deoxyhypusine synthase), DHS5 (deoxyhypusine synthase),

DHS6 (deoxyhypusine synthase), DHS7 (deoxyhypusine synthase), DHS8 (deoxyhypusine synthase),

CRF (corn RING finger; RNF169), G1543a (maize orthologue of Arabidopsis thaliana homeobox 17),

G1543b (maize orthologue of Arabidopsis thaliana homeobox 17), GS3 (grain size 3), and GW2 (grain

weight 2)

TABLE 6

Polynucleotides Expressed by Constructs of This Invention

SEQ ID NOs: 1120, 1121, 1122, 1248, 1257, 1313, 1314, 1364, 1387,

1478, 1489, 1490, 1491, 1492, 1493, 1585, 1597, 1598, 1599, 1713,

1752, 1753, 1801, 1802, 1820, 1927, 1929, 1931, 1971, 2006, 2007,

2008, 2010, 2012, 2014, 2016, 2018, 2022, 2023, 2025, 2027, 2029,

2031, 2033, 2035, 2037, 2039, 2041, 2043, 2045, 2047, 2049, 2051,

2053, 2055, 2056, 2057, 2059, 2060, 2061, and 2063

Transformation of Soybean

A base transformation vector pMON82053 (SEQ ID NO: 2066), illustrated in Table 7 and FIG. 3, is used in preparing recombinant DNA constructs of this invention for Agrobacterium-mediated transformation into soybean cells or tissue. To construct a transformation vector for expressing any of the recombinant DNA constructs of this invention, nucleotides encoding the at least one polynucleotide are inserted into the base vector pMON82053 (SEQ ID NO: 2066) in the gene of interest expression cassette at an insertion site, i.e., between the promoter element (coordinates 1-613) and the polyadenylation element (coordinates 688-1002). For example, a transformation vector for expression of a miR399 cleavage blocker is prepared by inserting the DNA of SEQ ID NO: 1802 (see Table 3) into the gene of interest expression cassette at an insertion site between the promoter element (coordinates 1-613) and the polyadenylation element (coordinates 688-1002) of pMON82053 (SEQ ID NO: 2066).
For Agrobacterium-mediated transformation, soybean seeds are imbided overnight and the meristem explants excised and placed in a wounding vessel. Cultures of induced Agrobacterium cells each containing a transformation vector for expressing each of the recombinant DNA constructs of this invention are mixed with prepared explants. Inoculated explants are wounded using sonication, placed in co-culture for 2-5 days, and transferred to selection media for 6-8 weeks to allow selection and growth of transgenic shoots. Resistant shoots are harvested at approximately 6-8 weeks and placed into selective rooting media for 2-3 weeks. Shoots producing roots are transferred to the greenhouse and potted in soil.
The above process is repeated to produce multiple events of transgenic soybean plant cells that are transformed with separate recombinant DNA constructs of this invention, i.e., a construct transcribable in a soybean plant cell, including a promoter that is functional in the soybean plant cell and operably linked to each polynucleotide selected from: (a) DNA encoding a cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of each miRNA target identified in Tables 2 and 3; (b) DNA encoding a 5′-modified cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of each miRNA target identified in Tables 2 and 3; (c) DNA encoding a translational inhibitor to prevent or decrease small RNA-mediated cleavage of the transcript of each miRNA target identified in Tables 2 and 3; (d) DNA encoding a decoy to prevent or decrease small RNA-mediated cleavage of the transcript of each miRNA target identified in Tables 2 and 3; (e) DNA encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of each miRNA target identified in Tables 2 and 3, wherein a miRNA recognition site in the native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage; (f) DNA encoding a miRNA precursor which is processed into a miRNA for suppressing expression of each miRNA target identified in Tables 2 and 3; (g) DNA encoding double-stranded RNA which is processed into siRNAs for suppressing expression of each miRNA target identified in Tables 2 and 3; and (h) DNA encoding a ta-siRNA which is processed into siRNAs for suppressing expression of each miRNA target identified in Tables 2 and 3.
The above process is repeated to produce multiple events of transgenic soybean plant cells that are transformed with each of the following recombinant DNA constructs of this invention, i.e., a construct transcribable in a soybean plant cell, including a promoter that is functional in the soybean plant cell and operably linked to a polynucleotide selected from: (a) DNA encoding a cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of the miRNA target; (b) DNA encoding a 5′-modified cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of the miRNA target; (c) DNA encoding a translational inhibitor to prevent or decrease small RNA-mediated cleavage of the transcript of the miRNA target; (d) DNA encoding a decoy to prevent or decrease small RNA-mediated cleavage of the transcript of the miRNA target; (e) DNA encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of the miRNA target, wherein a miRNA recognition site in the native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage; (f) DNA encoding a miRNA precursor which is processed into a miRNA for suppressing expression of the miRNA target; (g) DNA encoding double-stranded RNA which is processed into siRNAs for suppressing expression the miRNA target; and (h) DNA encoding a ta-siRNA which is processed into siRNAs for suppressing expression of the miRNA target—wherein separate constructs are made for each of the miRNA targets enumerated in Table 5.
The above process is repeated to produce multiple events of transgenic soybean plant cells that are transformed with each of the following recombinant DNA constructs of this invention, i.e., a construct transcribable in a soybean plant cell, including a promoter that is functional in the soybean plant cell and operably linked to each polynucleotide provided in Table 6, wherein separate constructs are made for each polynucleotide.
The above process is repeated to produce multiple events of transgenic soybean plant cells that are transformed with each of the following recombinant DNA constructs of this invention, i.e., a construct transcribable in a soybean plant cell, including a promoter that is functional in the plant cell and operably linked to a polynucleotide selected from DNA encoding each miRNA target identified in Tables 2 and 3.
The above process is repeated to produce multiple events of transgenic soybean plant cells that are transformed with each of the following recombinant DNA constructs of this invention, i.e., a construct transcribable in a soybean plant cell, including a promoter that is functional in the plant cell and operably linked to each polynucleotide of SEQ ID NOS: 15-2064.
The regenerated transgenic soybean plants, or progeny transgenic soybean plants or soybean seeds, produced from the regenerated transgenic soybean plants, are screened for an enhanced trait (e.g., increased yield), as compared to a control plant or seed (a plant or seed lacking expression of the recombinant DNA construct). From each group of multiple events of transgenic soybean plants with a specific recombinant construct of this invention, the event that produces the greatest enhanced trait (e.g., greatest enhancement in yield) is identified and progeny soybean seed is selected for commercial development.

Transformation of Canola

A base transformation vector pMON82053 (SEQ ID NO: 2066), illustrated in Table 7 and FIG. 3, is used in preparing recombinant DNA constructs of this invention for Agrobacterium-mediated transformation into canola cells or tissue. To construct a transformation vector for expressing any of the recombinant DNA constructs of this invention, nucleotides encoding the at least one polynucleotide are inserted into the base vector pMON82053 (SEQ ID NO: 2066) in the gene of interest expression cassette at an insertion site, i.e., between the promoter element (coordinates 1-613) and the polyadenylation element (coordinates 688-1002). For example, a transformation vector for expression of a miR399 cleavage blocker is prepared by inserting the DNA of SEQ ID NO: 1802 (see Table 3) into the gene of interest expression cassette at an insertion site between the promoter element (coordinates 1-613) and the polyadenylation element (coordinates 688-1002) of pMON82053 (SEQ ID NO: 2066).
Overnight-grown cultures of Agrobacterium cells each containing a transformation vector for expressing each of the recombinant DNA constructs of this invention are used to inoculate tissues from in vitro-grown canola seedlings. Following co-cultivation with Agrobacterium, the infected tissues are grown on selection to promote growth of transgenic shoots, followed by growth of roots from the transgenic shoots, potting of the selected plantlets in soil, and transfer of the potted plants to the greenhouse. Molecular characterization is performed to confirm the presence of a recombinant DNA construct of this invention and its expression in transgenic plants.
The above process is repeated to produce multiple events of transgenic canola plant cells that are transformed with separate recombinant DNA constructs of this invention, i.e., a construct transcribable in a canola plant cell, including a promoter that is functional in the canola plant cell and operably linked to each polynucleotide selected from: (a) DNA encoding a cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of each miRNA target identified in Tables 2 and 3; (b) DNA encoding a 5′-modified cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of each miRNA target identified in Tables 2 and 3; (c) DNA encoding a translational inhibitor to prevent or decrease small RNA-mediated cleavage of the transcript of each miRNA target identified in Tables 2 and 3; (d) DNA encoding a decoy to prevent or decrease small RNA-mediated cleavage of the transcript of each miRNA target identified in Tables 2 and 3; (e) DNA encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of each miRNA target identified in Tables 2 and 3, wherein a miRNA recognition site in the native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage; (f) DNA encoding a miRNA precursor which is processed into a miRNA for suppressing expression of each miRNA target identified in Tables 2 and 3; (g) DNA encoding double-stranded RNA which is processed into siRNAs for suppressing expression of each miRNA target identified in Tables 2 and 3; and (h) DNA encoding a ta-siRNA which is processed into siRNAs for suppressing expression of each miRNA target identified in Tables 2 and 3.
The above process is repeated to produce multiple events of transgenic canola plant cells that are transformed with each of the following recombinant DNA constructs of this invention, i.e., a construct transcribable in a canola plant cell, including a promoter that is functional in the canola plant cell and operably linked to a polynucleotide selected from: (a) DNA encoding a cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of the miRNA target; (b) DNA encoding a 5′-modified cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of the miRNA target; (c) DNA encoding a translational inhibitor to prevent or decrease small RNA-mediated cleavage of the transcript of the miRNA target; (d) DNA encoding a decoy to prevent or decrease small RNA-mediated cleavage of the transcript of the miRNA target; (e) DNA encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of the miRNA target, wherein a miRNA recognition site in the native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage; (f) DNA encoding a miRNA precursor which is processed into a miRNA for suppressing expression of the miRNA target; (g) DNA encoding double-stranded RNA which is processed into siRNAs for suppressing expression the miRNA target; and (h) DNA encoding a ta-siRNA which is processed into siRNAs for suppressing expression of the miRNA target—wherein separate constructs are made for each of the miRNA targets enumerated in Table 5.
The above process is repeated to produce multiple events of transgenic canola plant cells that are transformed with each of the following recombinant DNA constructs of this invention, i.e., a construct transcribable in a canola plant cell, including a promoter that is functional in the canola plant cell and operably linked to each polynucleotide provided in Table 6, wherein separate constructs are made for each polynucleotide.
The above process is repeated to produce multiple events of transgenic canola plant cells that are transformed with each of the following recombinant DNA constructs of this invention, i.e., a construct transcribable in a canola plant cell, including a promoter that is functional in the plant cell and operably linked to a polynucleotide selected from DNA encoding each miRNA target identified in Tables 2 and 3.
The above process is repeated to produce multiple events of transgenic canola plant cells that are transformed with each of the following recombinant DNA constructs of this invention, i.e., a construct transcribable in a canola plant cell, including a promoter that is functional in the plant cell and operably linked to each polynucleotide of SEQ ID NOS: 15-2064.
The regenerated transgenic canola plants, or progeny transgenic canola plants or canola seeds, produced from the regenerated transgenic canola plants, are screened for an enhanced trait (e.g., increased yield), as compared to a control plant or seed (a plant or seed lacking expression of the recombinant DNA construct). From each group of multiple events of transgenic canola plants with a specific recombinant construct of this invention, the event that produces the greatest enhanced trait (e.g., greatest enhancement in yield) is identified and progeny canola seed is selected for commercial development.

Transformation of Cotton

A base transformation vector pMON99053 (SEQ ID NO: 2067), illustrated in Table 8 and FIG. 4, is used in preparing recombinant DNA constructs of this invention for Agrobacterium-mediated transformation into maize cells or tissue. To construct a transformation vector for expressing any of the recombinant DNA constructs of this invention, nucleotides encoding the at least one polynucleotide are inserted into the base vector pMON99053 (SEQ ID NO: 2067) in the gene of interest expression cassette at an insertion site, i.e., between the promoter element (coordinates 388-1091) and the polyadenylation element (coordinates 1165-1791).
Methods for transformation of cotton are known in the art, see, for example, the techniques described in U. S. Patent Application Publications 2004/0087030A1 2008/0256667A1, 2008/0280361A1, and 2009/0138985A1, which are incorporated by reference. In an example of a cotton transformation protocol, seeds of transformable cotton genotypes (e.g., nectarless, DP393, 00SO4, 07W610F, STN474, Delta Pearl, DP5415, SureGrow501, or SureGrow747) are surface sterilized, rinsed, and hydrated in CSM medium (containing carbenicillin, cefotaxime, BRAVO, and Captan 50) for 14 to 42 hours in the dark. Meristematic explants are processed from seeds as described in U. S. Patent Application Publications 2008/0256667A1. Cultures of Agrobacterium cells each containing a transformation vector for expressing each of the recombinant DNA constructs of this invention are used to inoculate the explants using sonication. The inoculum is removed and the inoculated explants transferred to INO medium and incubated for 2 to 5 days using a 16-hour light photoperiod. Following co-cultivation, explants are transferred onto semi-solid selection medium (modified Lloyd & McCown Woody Plant Medium supplemented with 200 mg/L cefotaxime, 200 mg/L carbenicillin and 100-200 mg/L spectinomycin) with or without plant growth regulators or other additives to promote multiple shoot formation and growth. The explants are cultured in a 16-hour light photoperiod. After 4 to 6 weeks on the selection medium those explants that have developed green shoots are transferred to plugs and placed in liquid medium containing 0.25 mg/L IBA for shoot growth and rooting under plastic domes for 3 to 4 weeks. Tissues are assayed for molecular characterization by one or more molecular assay methods (e.g., PCR, or Southern blots).
The above process is repeated to produce multiple events of transgenic cotton plant cells that are transformed with separate recombinant DNA constructs of this invention, i.e., a construct transcribable in a cotton plant cell, including a promoter that is functional in the cotton plant cell and operably linked to each polynucleotide selected from: (a) DNA encoding a cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of each miRNA target identified in Tables 2 and 3; (b) DNA encoding a 5′-modified cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of each miRNA target identified in Tables 2 and 3; (c) DNA encoding a translational inhibitor to prevent or decrease small RNA-mediated cleavage of the transcript of each miRNA target identified in Tables 2 and 3; (d) DNA encoding a decoy to prevent or decrease small RNA-mediated cleavage of the transcript of each miRNA target identified in Tables 2 and 3; (e) DNA encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of each miRNA target identified in Tables 2 and 3, wherein a miRNA recognition site in the native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage; (f) DNA encoding a miRNA precursor which is processed into a miRNA for suppressing expression of each miRNA target identified in Tables 2 and 3; (g) DNA encoding double-stranded RNA which is processed into siRNAs for suppressing expression of each miRNA target identified in Tables 2 and 3; and (h) DNA encoding a ta-siRNA which is processed into siRNAs for suppressing expression of each miRNA target identified in Tables 2 and 3.
The above process is repeated to produce multiple events of transgenic cotton plant cells that are transformed with each of the following recombinant DNA constructs of this invention, i.e., a construct transcribable in a cotton plant cell, including a promoter that is functional in the cotton plant cell and operably linked to a polynucleotide selected from: (a) DNA encoding a cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of the miRNA target; (b) DNA encoding a 5′-modified cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of the miRNA target; (c) DNA encoding a translational inhibitor to prevent or decrease small RNA-mediated cleavage of the transcript of the miRNA target; (d) DNA encoding a decoy to prevent or decrease small RNA-mediated cleavage of the transcript of the miRNA target; (e) DNA encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of the miRNA target, wherein a miRNA recognition site in the native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage; (f) DNA encoding a miRNA precursor which is processed into a miRNA for suppressing expression of the miRNA target; (g) DNA encoding double-stranded RNA which is processed into siRNAs for suppressing expression the miRNA target; and (h) DNA encoding a ta-siRNA which is processed into siRNAs for suppressing expression of the miRNA target—wherein separate constructs are made for each of the miRNA targets enumerated in Table 5.
The above process is repeated to produce multiple events of transgenic cotton plant cells that are transformed with each of the following recombinant DNA constructs of this invention, i.e., a construct transcribable in a cotton plant cell, including a promoter that is functional in the cotton plant cell and operably linked to each polynucleotide provided in Table 6, wherein separate constructs are made for each polynucleotide.
The above process is repeated to produce multiple events of transgenic cotton plant cells that are transformed with each of the following recombinant DNA constructs of this invention, i.e., a construct transcribable in a cotton plant cell, including a promoter that is functional in the plant cell and operably linked to a polynucleotide selected from DNA encoding each miRNA target identified in Tables 2 and 3.
The above process is repeated to produce multiple events of transgenic cotton plant cells that are transformed with each of the following recombinant DNA constructs of this invention, i.e., a construct transcribable in a cotton plant cell, including a promoter that is functional in the plant cell and operably linked to each polynucleotide of SEQ ID NOS: 15-2064.
The regenerated transgenic cotton plants, or progeny transgenic cotton plants or cotton seeds, produced from the regenerated transgenic cotton plants, are screened for an enhanced trait (e.g., increased yield), as compared to a control plant or seed (a plant or seed lacking expression of the recombinant DNA construct). From each group of multiple events of transgenic cotton plants with a specific recombinant construct of this invention, the event that produces the greatest enhanced trait (e.g., greatest enhancement in yield) is identified and progeny cotton seed is selected for commercial development.

TABLE 7

			Coordinates of
			SEQ ID NO:
Function	Name	Annotation		2066

Agrobacterium T-	B-AGRtu.left	Agro left border sequence, essential for	6144-6585
DNA transfer	border	transfer of T-DNA.
Plant selectable	P-At.Act7	Promoter from the Arabidopsis actin 7	6624-7861
marker		gene
expression	L-At.Act7	5′UTR of Arabidopsis Act7 gene
cassette	I-At.Act7	Intron from the Arabidopsis actin7 gene
	TS-At.ShkG-CTP2	Transit peptide region of Arabidopsis	7864-8091
		EPSPS
	CR-AGRtu.aroA-	Synthetic CP4 coding region with dicot	8092-9459
	CP4.nno_At	preferred codon usage.
	T-AGRtu.nos	A 3′ non-translated region of the nopaline	9466-9718
		synthase gene of Agrobacterium
		tumefaciens Ti plasmid which functions
		to direct polyadenylation of the mRNA.
Gene of interest	P-CaMV.35S-enh	Promoter for 35S RNA from CaMV	1-613
expression		containing a duplication of the −90 to −350
cassette		region.
	T-Gb.E6-3b	3′ untranslated region from the fiber	688-1002
		protein E6 gene of sea-island cotton.
Agrobacterium T-	B-AGRtu.right	Agro right border sequence, essential for	1033-1389
DNA transfer	border	transfer of T-DNA.
Maintenance in	OR-Ec.oriV-RK2	The vegetative origin of replication from	5661-6057
E. coli		plasmid RK2.
	CR-Ec.rop	Coding region for repressor of primer	3961-4152
		from the ColE1 plasmid. Expression of
		this gene product interferes with primer
		binding at the origin of replication,
		keeping plasmid copy number low.
	OR-Ec.ori-ColE1	The minimal origin of replication from	2945-3533
		the E. coli plasmid ColE1.
	P-Ec.aadA-	Promoter for Tn7 adenylyltransferase	2373-2414
	SPC/STR	(AAD(3″))
	CR-Ec.aadA-	Coding region for Tn7	1584-2372
	SPC/STR	adenylyltransferase (AAD(3″)) conferring
		spectinomycin and streptomycin
		resistance.
	T-Ec.aadA-	3′ UTR from the Tn7 adenylyltransferase	1526-1583
	SPC/STR	(AAD(3″)) gene of E. coli.

TABLE 8

			Coordinates of
			SEQ ID NO:
Function	Name	Annotation		2067

Agrobacterium	B-AGRtu.right	Agro right border sequence, essential for	1-357
T-DNA	border	transfer of T-DNA.
transfer
Gene of	Exp-CaMV.35S-	Enhanced version of the 35S RNA	388-1091
interest	enh + Ph.DnaK	promoter from CaMV plus the petunia
expression		hsp70 5′ untranslated region
cassette	T-Ps.RbcS2-E9	The 3′ non-translated region of the pea	1165-1797
		RbcS2 gene which functions to direct
		polyadenylation of the mRNA.
Plant selectable	Exp-CaMV.35S	Promoter and 5′ untranslated region from	1828-2151
marker		the 35S RNA of CaMV
expression	CR-Ec.nptII-Tn5	Coding region for neomycin	2185-2979
cassette		phosphotransferase gene from transposon
		Tn5 which confers resistance to neomycin
		and kanamycin.
	T-AGRtu.nos	A 3′ non-translated region of the nopaline	3011-3263
		synthase gene of Agrobacterium
		tumefaciens Ti plasmid which functions to
		direct polyadenylation of the mRNA.
Agrobacterium	B-AGRtu.left	Agro left border sequence, essential for	3309-3750
T-DNA	border	transfer of T-DNA.
transfer
Maintenance in	OR-Ec.oriV-RK2	The vegetative origin of replication from	3837-4233
E. coli		plasmid RK2.
	CR-Ec.rop	Coding region for repressor of primer from	5742-5933
		the ColE1 plasmid. Expression of this gene
		product interferes with primer binding at
		the origin of replication, keeping plasmid
		copy number low.
	OR-Ec.ori-ColE1	The minimal origin of replication from the	6361-6949
		E. coli plasmid ColE1.
	P-Ec.aadA-	Promoter for Tn7 adenylyltransferase	7480-7521
	SPC/STR	(AAD(3″))
	CR-Ec.aadA-	Coding region for Tn7 adenylyltransferase	7522-8310
	SPC/STR	(AAD(3″)) conferring spectinomycin and
		streptomycin resistance.
	T-Ec.aadA-	3′ UTR from the Tn7 adenylyltransferase	8311-8368
	SPC/STR	(AAD(3″)) gene of E. coli.

Transformation of Sugarcane

Sugarcane transformation techniques are known in the art; see, for example, the procedures describedfor sugarcane by Brumbley et al. in “Sugarcane” (available electronically at mrw.interscience.wiley.com/emrw/9781405181099/hpt/article/k0701/current/pdf), published in: “Compendium of Transgenic Crop Plants”, edited by Chittaranjan Kole and Timothy C. Hall, Blackwell Publishing Ltd., 2008; ISBN 978-1-405-16924-0 (available electronically at mrw.interscience.wiley.com/emrw/9781405181099/hpt/toc), and in PCT International Patent Application Publications WO2007/003023 (sugarcane) and WO2008/049183 (sugarcane). In one example of sugarcane transformaiton (see Example 3 of PCT International Patent Application Publication W02007003023A2), embryonic sugarcane callus cultures are established from apical meristem and primordial leafs of sugarcane (Saccharum spp. hybrid). Eight-week old calli are co-bombarded with an equimolar mixture of either UBI-1::Bar::NOSpolyA and UBI-1::Oas::NOSpolyA or UBI-1::Bar::NOSpolyA and UBI-1::CPs::NOSpolyA expression cassettes (10 pg DNAI3/mg particle) by particle bombardment as described previously (Sanford (1990) Plant Physiol., 79:206-209). After bombardment, calli are transferred to MS medium containing 1 mg/L PPT and 1 mg/L BAP to promote shoot regeneration and inhibit development of non transgenic tissue. Two weeks later, calli are transferred to MS medium containing 1 mg/L PPT and 1 mg/L Affi for shoot elongation and to induce root formation. After two weeks, plantlets are placed into magenta boxes for acclimatization and 2 weeks later, shoots (10-15 cm) with well developed roots are transferred to potting soil and placed in the greenhouse.
The above process is repeated to produce multiple events of transgenic sugarcane plant cells that are transformed with separate recombinant DNA constructs of this invention, i.e., a construct transcribable in a sugarcane plant cell, including a promoter that is functional in the sugarcane plant cell and operably linked to each polynucleotide selected from: (a) DNA encoding a cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of each miRNA target identified in Tables 2 and 3; (b) DNA encoding a 5′-modified cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of each miRNA target identified in Tables 2 and 3; (c) DNA encoding a translational inhibitor to prevent or decrease small RNA-mediated cleavage of the transcript of each miRNA target identified in Tables 2 and 3; (d) DNA encoding a decoy to prevent or decrease small RNA-mediated cleavage of the transcript of each miRNA target identified in Tables 2 and 3; (e) DNA encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of each miRNA target identified in Tables 2 and 3, wherein a miRNA recognition site in the native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage; (f) DNA encoding a miRNA precursor which is processed into a miRNA for suppressing expression of each miRNA target identified in Tables 2 and 3; (g) DNA encoding double-stranded RNA which is processed into siRNAs for suppressing expression of each miRNA target identified in Tables 2 and 3; and (h) DNA encoding a ta-siRNA which is processed into siRNAs for suppressing expression of each miRNA target identified in Tables 2 and 3.
The above process is repeated to produce multiple events of transgenic sugarcane plant cells that are transformed with each of the following recombinant DNA constructs of this invention, i.e., a construct transcribable in a sugarcane plant cell, including a promoter that is functional in the sugarcane plant cell and operably linked to a polynucleotide selected from: (a) DNA encoding a cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of the miRNA target; (b) DNA encoding a 5′-modified cleavage blocker to prevent or decrease small RNA-mediated cleavage of the transcript of the miRNA target; (c) DNA encoding a translational inhibitor to prevent or decrease small RNA-mediated cleavage of the transcript of the miRNA target; (d) DNA encoding a decoy to prevent or decrease small RNA-mediated cleavage of the transcript of the miRNA target; (e) DNA encoding a miRNA-unresponsive transgene having a nucleotide sequence derived from the native nucleotide sequence of the miRNA target, wherein a miRNA recognition site in the native nucleotide sequence is deleted or otherwise modified to prevent miRNA-mediated cleavage; (f) DNA encoding a miRNA precursor which is processed into a miRNA for suppressing expression of the miRNA target; (g) DNA encoding double-stranded RNA which is processed into siRNAs for suppressing expression the miRNA target; and (h) DNA encoding a ta-siRNA which is processed into siRNAs for suppressing expression of the miRNA target—wherein separate constructs are made for each of the miRNA targets enumerated in Table 5.
The above process is repeated to produce multiple events of transgenic sugarcane plant cells that are transformed with each of the following recombinant DNA constructs of this invention, i.e., a construct transcribable in a sugarcane plant cell, including a promoter that is functional in the sugarcane plant cell and operably linked to each polynucleotide provided in Table 6, wherein separate constructs are made for each polynucleotide.
The above process is repeated to produce multiple events of transgenic sugarcane plant cells that are transformed with each of the following recombinant DNA constructs of this invention, i.e., a construct transcribable in a sugarcane plant cell, including a promoter that is functional in the plant cell and operably linked to a polynucleotide selected from DNA encoding each miRNA target identified in Tables 2 and 3.
The above process is repeated to produce multiple events of transgenic sugarcane plant cells that are transformed with each of the following recombinant DNA constructs of this invention, i.e., a construct transcribable in a sugarcane plant cell, including a promoter that is functional in the plant cell and operably linked to each polynucleotide of SEQ ID NOS: 15-2064.
The regenerated transgenic sugarcane plants, or progeny transgenic sugarcane plants or sugarcane seeds, produced from the regenerated transgenic sugarcane plants, are screened for an enhanced trait (e.g., increased yield), as compared to a control plant or seed (a plant or seed lacking expression of the recombinant DNA construct). From each group of multiple events of transgenic sugarcane plants with a specific recombinant construct of this invention, the event that produces the greatest enhanced trait (e.g., greatest enhancement in yield) is identified and progeny sugarcane seed is selected for commercial development.

Further Embodiments

A miRNA decoy competes with the endogenous target gene to bind to that particular miRNA and thus reduces the effect of the miRNA in the biochemical network or networks involving the miRNA. Decoys include endogenous (native) miRNA decoy sequences, decoys created by manipulating an endogenous sequence (e.g., by chemical or other mutagenesis or site-directed recombination), and synthetic miRNA decoy sequences. A recombinant DNA construct can be designed to express multiple miRNA decoys. The advantages of a miRNA decoy approach include the fact that no protein is expressed, and because miRNAs often belong to multi-gene families (wherein each miRNA gene produces a unique miRNA primary transcript) that a single miRNA decoy is useful for binding to a mature miRNA that is derived from more than one miRNA gene or primary transcript.
However, an alternative to a miRNA decoy is sometimes preferred, as it is possible for a miRNA decoy that binds to mature miRNAs from more than one miRNA gene to unintentionally affect the expression of a non-target gene. Applicants have disclosed herein additional novel approaches for manipulating a miRNA-regulated pathway by interfering with the binding of the mature miRNA to its target. These approaches generally involve the in vivo (e.g., in planta) expression and processing of a recombinant DNA construct of this invention, and are especially useful for regulating the expression of single (or, where desired, multiple) target genes, and in manipulating gene expression in transgenic plants, resulting in improved phenotypes such as increased yield or biotic or abiotic stress tolerance.
One approach is by using a “cleavage blocker” or “5′-modified cleavage blocker” that is transgenically expressed in a eukaryotic cell and that binds to a miRNA recognition site of a target gene's transcript in a manner that does not lead to cleavage, thereby preventing or decreasing miRNA-mediated cleavage of the transcript by competing with the miRNA for binding to the recognition site. This method controls the rate of post-transcriptional suppression of miRNA target genes by protecting them from being cleaved by miRNA-Ago complex, and decreases or prevents down-regulation of the miRNA target gene. The invention includes analogous cleavage blockers that compete with other small RNAs involved in silencing, e.g., si-RNAs, trans-acting siRNAs, phased RNAs, natural antisense transcript siRNAs, natural antisense transcript miRNAs, or indeed any small RNA associated with a silencing complex such as RISC or an Argonaute or Argonaute-like protein.
Another approach is by using a “translational inhibitor” that is transgenically expressed in a eukaryotic cell and that binds to and inhibit translation of the target gene's transcript, thereby decreasing expression of the target gene. The nucleotide sequence of the translational inhibitor is designed so that the hybridized segment formed between the translational inhibitor and the target gene's transcript imparts to the transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of the hybridized segment. Translational inhibitors provide the advantages of reducing the likelihood of transitive small RNAs forming (as can occur in miRNA-mediated degradation of a target gene), and achievement of more controlled regulation of target suppression because the translational inhibitor remains associated with the target gene's transcript (unlike miRNAs, which dissociate from the cleaved transcript and can then bind another transcript molecule). Translational inhibitors can be based on sequences selected from any small RNA associated with a silencing complex such as RISC or an Argonaute or Argonaute-like protein.
One of ordinary skill in the art easily recognizes that the above procedures are equally applicable to situations where the double-stranded RNA that mediates the target gene suppression is other than a miRNA. Thus, various aspects of this invention include analogous recombinant DNA constructs that are processed in vivo or in planta to provide RNA including single-stranded RNA that serve as an “siRNA cleavage blocker”, a “trans-acting siRNA cleavage blocker”, a “phased small RNA cleavage blocker”, a “natural antisense transcript siRNA cleavage blocker”, or a “natural antisense transcript miRNA cleavage blocker” (or, in general terms, a “small RNA cleavage blocker”), according to whether the RNase III ribonuclease cleavage that is inhibited is mediated by, respectively, an siRNA, a trans-acting siRNA, a phased small RNA, a natural antisense transcript siRNA, or a natural antisense transcript miRNA (or, in general terms, any small RNA associated with a silencing complex such as RISC or an Argonaute or Argonaute-like protein).
All of the materials and methods disclosed and claimed herein can be made and used without undue experimentation as instructed by the above disclosure. Although the materials and methods of this invention have been described in terms of preferred embodiments and illustrative examples, it will be apparent to those of skill in the art that variations can be applied to the materials and methods described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Nucleotide

Rationale for plant

Construct type

transformation*

1-10. (canceled)

11. A recombinant DNA construct comprising a promoter operable in a plant cell, operably linked to DNA encoding a single-stranded cleavage blocker RNA that binds in vivo to a target RNA transcript at an miRNA recognition site for an endogenous mature miRNA, and forms, through complementary base-pairing, a hybridized segment of from 19 to 24 nucleotides in length at said miRNA recognition site in said target RNA transcript,

wherein said hybridized segment comprises an A, G, or C in said single-stranded cleavage blocker RNA at a position corresponding to the 5′ terminus of said endogenous mature miRNA, and matches through complementary base-pairing between said single-stranded cleavage blocker RNA and said miRNA recognition site at positions corresponding to positions 9, 10, and 11 in 3′ to 5′ direction of said endogenous mature miRNA, and

wherein said single-stranded cleavage blocker RNA interferes with the binding of said endogenous mature miRNA to said target RNA transcript at said miRNA recognition site.

12. The recombinant DNA construct of claim 11, wherein formation of said hybridized segment inhibits cleavage of said target RNA transcript mediated by said endogenous mature miRNA.

13. A method of modulating expression of at least one target gene, comprising expressing in said plant cell the recombinant DNA construct of claim 11, wherein said at least one target gene encodes said target RNA transcript.

14. The method of claim 13, wherein formation of said hybridized segment inhibits suppression of said at least one target gene by said endogenous mature miRNA.

15. A non-natural plant chromosome or plastid comprising the recombinant DNA construct of claim 11.

16. A non-natural transgenic plant cell having in its genome the recombinant DNA construct of claim 11, or a non-natural transgenic plant or a non-natural transgenic plant seed or a non-natural transgenic pollen grain, each comprising said non-natural transgenic plant cell.

17. A non-natural partially transgenic plant, wherein:

(a) said non-natural partially transgenic plant comprises the non-natural transgenic plant cell of claim 16 and further comprises non-transgenic tissue; or

(b) said non-natural partially transgenic plant comprises a transgenic rootstock comprising the non-natural transgenic plant cell of claim 16 and further comprises a non-transgenic scion.

18. The recombinant DNA construct of claim 11, wherein said target RNA transcript is transcribed from at least one target gene comprising:

(a) coding sequence, non-coding sequence, or both coding and non-coding sequence; or

(b) a single target gene, or multiple target genes; or

(c) one or more of the group consisting of:

(1) an endogenous gene of a eukaryote,

(2) a transgene of a transgenic plant,

(3) an endogenous gene of a pest or pathogen of a plant, and

(4) an endogenous gene of a symbiont associated with a pest or pathogen of a plant.