UA113948C2 - DOWN-REGULATION OF GENE EXPRESSION BY ARTIFICIAL MICRO-RNA - Google Patents

Abstract

The invention relates to isolated nucleic acid fragments, including precursor mRNAs, artificial siRNAs, and their use in down-regulation of gene expression.

Description

This application claims priority to prior US patent application No. 61/014510, filed Dec. 18, 2007, the disclosure of which is hereby incorporated by reference in its entirety.

The field of this invention relates generally to plant molecular biology. In particular, it relates to constructs and methods for down-regulating the expression of target sequences.

MicroRNAs (migRNAs) were first identified only a few years ago, but it has already become clear that they play an important role in the regulation of gene activity. These 20-22 nucleotide non-coding RNAs have the ability to hybridize through base pairing with specific target mRNAs and down-regulate the expression of these transcripts, mediating either RNA cleavage or translational repression.

Recent studies have shown that miRNAs have important functions in development. In plants, they have been shown to control a number of developmental processes, including flowering period, leaf morphology, organ polarity, flower morphology, and root development (reviewed in

Maiogu and MaisNegei (2006) Mai Sepei 38: 531-36). Given the established regulatory role of miRNAs, it is likely that they are also involved in the control of some major crop traits, such as drought tolerance and disease resistance.

miRNAs are transcribed by RNA polymerase I! as polyadenylated and capped signals known as pri-miRNA (primary miRNA). Such prime-miRNAs are translated into smaller transcripts known as pre-miRNAs, and such precursors have the ability to form stable hairpin structures (reviewed by Vagei! (2004) Cell 116: 281-297; dopez-Wpoade5 MM, Vapei! OR,

Limestone! In city! MA5 apa iNeig tedshiayyugu goyez ip riapi5. Appi Vem Riapi Vioi. 2006; 57:19-53.) Despite the fact that prim-miRNAs are converted to pre-miRNAs by Ogov5pa in the nucleus, and

Oiseg cleaves pre-miRNA in the cytoplasm of multicellular organisms, the maturation of miRNA in plants differs from the metabolic pathway in animals, since plants lack the Ogo5Na homologue.

Instead, the RNase II enzyme OISEV-PIKE 1 (OSI), which is homologous to animal biseg, may have a function of UO5pa in addition to its known function in hairpin processing (Kipinaga and M/aiapabe (2004) Rhos Mai! Asad 5a 101: 12753- 12758).

Agaridorz5 has recently described artificial miRNAs (shmiRNAs) targeting viral mRNA sequences (Mie et al. (2006) Mayte Bioespoiiodou 24:1420-1428) or endogenous genes (Zspuga et al. (2006) Riapi Sei! 18: 1121-1133). The shmiRNA construct can be expressed under

With the control of different promoters to change the spatial structure of silencing (Zspugaye a!. (2006)

Riapi Sei! 18:1121-1133). Artificial miRNAs replace the miRNA and its complementary sequence, indicated by a dash, in the precursor miRNA and replace the sequences that target the mRNA to be silenced. Silencing by endogenous miRNAs can be found in a number of spatial, temporal and developmentally related expression patterns (Ragigotso ei a!. (2007) Sepev5

Rem 18:2237-2242; Amage? you a (2006) Journal of Medicine 18:1134-51). Artificial miRNAs can be constructed to both capture and expand diversity and specificity in silencing patterns.

So far, the use of miRNAs in cultivated plants has not been reported.

MO 2004/009779, published on January 29, 2004, discloses compositions and methods for modulating gene expression in plants.

Assignee's US Patent Application 2005/0138689, published June 23, 2005, describes miRNAs and their use in targeting sequence silencing.

The present invention may be more fully understood from the following Detailed Description and the accompanying Sequence Listing, which forms a part of this application.

The Sequence Description summarizes the Sequence List attached to the document. The list of sequences includes one-letter codes for symbols of nucleotide sequences and one- and three-letter codes for amino acids, as defined in ISRAS-IV standards disclosed in Mysiv and

Acid5 Nezeagsi 13:3021-3030 (1985) and in Viospetisai! Uoigpai 219(2):345-373 (1984).

ZEO IU MO: 1-10 correspond to the primers used for the amplification of maize genomic microRNA (miRNA) precursors.

ZEO IUO MO: 11-15 correspond to the sequences of the maize miRNA precursor for 159c, 164p, 168va, 169g and 3961, respectively.

ZEO IO MO: 16 correspond to the sequence of an artificial miRNA (shmiRNA) used for silencing the corn phytoene desaturase (ROB) transcript.

ZEO IU MO: 17-21 correspond to "dashed sequences" contained in the precursors of smiRNAs for 159c-ROB5, 1640-ROB5, 16va-ROB5, 1691I-ROB5 and 3961-ROB, respectively.

The dashed sequences are mostly complementary sequences in the miRNA precursor forming a duplex with the miRNA.

ZEO IU MO: 22-26 correspond to precursors of shmiRNA for 1590-ROB, 1641-ROB, 16v8a-ROB, 1691-ROB and 3961-ROB, respectively. These precursors, if expressed in maigs, control the silencing of the endogenous ROB transcript.

ZEO IU MO: 27-30 correspond to the truncated precursors of shmiRNA 1691/-ROB-5pi, 1691-ROB-tey, 3960-ROB-5p and 396-ROB-tedy, respectively. 169I-РО5 predecessor (ZEO IO MO: 25) was shortened to 11 95 of its length (1691-РО5-5НИ, ZEO IO MO: 27) and 35 95 of its length (169g-

ROB-tey, 5EO IO MO: 28) compared to 169/-ROB5, 396p-ROB5 predecessor (5EO IO MO: 26) was shortened to 18 95 of its length (396p-ROB-5PII, ZEO IYUO MO: 29) and 46 95 of its length (396n-

ROB-tey, ZEO IO MO: 30) in comparison with 396p-ROB. All of the truncated precursors contained miRNAs and the sequences indicated by a dashed line.

ZEO IU MO: 31-34 correspond to precursors of smiRNAs for 159C-EAB, 168a-EAO, 169I-EAO and 396p-RAO, respectively. These precursors, when expressed in maize, drive the silencing of the endogenous Tad2-1 (fatty acid desaturase responsible for the conversion of oleic acid to linoleic acid) transcript.

ZEO IO MO: 35-38 correspond to miRNA targets and dashed sequences for lethal leaf spot.

ZEO IO MO: 39-41 correspond to miRNA targets and dashed sequences for the multidrug resistance protein, which is a transport protein.

ZEO IU MO: 42-43 correspond to the sequences of the precursors of shmiR NK for 16va-MAR and 396n-

MAR, respectively. These progenitors, if expressed in maize, control silencing

MAP, resulting in reduced levels of phytic acid.

The present invention relates to an isolated nucleic acid fragment that includes a miRNA-precursor, and the specified miRNA-precursor largely corresponds to the deoxyribonucleotide sequence set forth in 5EO IU MO: 11, (i) where nucleotides 430 - 450 in 5EO IU MO: 11 are replaced by the first variable a nucleotide subsequence that varies in size from about 19 to about 30 nucleotides depending on the target sequence whose expression is to be reduced, and (ii) further, wherein nucleotides 244-264 of 5EO IO MO: 11 are replaced by a second variable nucleotide subsequence that varies in size from about 7 p.m. to about

30 nucleotides, and the specified second variable nucleotide subsequence is able to hybridize with the first variable subsequence of the precursor miRNA.

Other isolated nucleic acid fragments that are also of interest include the following: a) an isolated nucleic acid fragment comprising a precursor miRNA, wherein said precursor miRNA substantially corresponds to the deoxyribonucleotide sequence set forth in 5EO IU MO: 12, (| where nucleotides 94 -114 in the 5EO IUO MO: 12 is replaced by a first variable nucleotide subsequence, the size of which varies from about 19 to about 30 nucleotides depending on the target sequence whose expression is to be reduced, and (ii) except where nucleotides 163-183 in the 5EO IUO MO : 12 replaced by a second variable nucleotide subsequence, the size of which varies from about 19 to about 30 nucleotides, and said second variable nucleotide subsequence is capable of hybridizing with the first variable subsequence of the precursor miRNA;

B) an isolated fragment of nucleic acid, which includes an miRNA-precursor, and the specified miRNA-precursor largely corresponds to the deoxyribonucleotide sequence set forth in 5EO IU MO: 13, (| where nucleotides 53-73 in 5EO IU MO: 13 are replaced by the first variable nucleotide subsequence , the size of which varies from about 19 to about 30 nucleotides depending on the target sequence whose expression is to be reduced, and (ii) further, wherein nucleotides 97-117 of 5EO IU MO: 13 are replaced by a second variable nucleotide subsequence, the size of which varies from about 19 to about 30 nucleotides, wherein said second variable nucleotide subsequence is capable of hybridizing with the first variable subsequence of the precursor miRNA; c) an isolated nucleic acid fragment comprising the precursor miRNA, wherein said precursor miRNA substantially corresponds to the deoxyribonucleotide sequence set forth in 5EO IO MO: 14, (i) where nucleotides 110-130 in 5EO IO MO: 14 are replaced by the first

BO variable nucleotide subsequence, the size of which varies from about 19 to about 30 nucleotides depending on the target sequence whose expression is to be reduced, and (ii) further, wherein nucleotides 184 203 in 5EO IJU MO: 14 are replaced by a second variable nucleotide subsequence, the size of which varies from about 19 to about 30 nucleotides, and said second variable nucleotide subsequence is capable of hybridizing with the first variable subsequence of the precursor miRNA; and

I) an isolated nucleic acid fragment that includes a precursor miRNA, and said precursor miRNA largely corresponds to the deoxyribonucleotide sequence set forth in 5EO IU MO: 15, () where nucleotides 83-103 in 5EO IUO MO: 15 are replaced by the first variable nucleotide subsequence , the size of which varies from about 19 to about 30 60 nucleotides depending on the target sequence whose expression is to be reduced, and (ii) further, wherein nucleotides 172-192 of 5EO IU MO: 15 are replaced by a second variable nucleotide subsequence, the size of which varies from about 19 to about 30 nucleotides, and said second variable nucleotide subsequence is capable of hybridizing with the first variable subsequence of the precursor miRNA.

Any of these selected nucleic acid fragments can be used to create a recombinant construct that includes these selected nucleic acid fragments functionally linked to at least one regulatory sequence.

These constructs can be transformed into a plant cell such that the transformed plant cell will include the recombinant construct in its genome.

In another aspect, the present invention relates to a method of reducing the expression of a target gene in a plant cell, which: (a) transforms at least one plant cell with a nucleic acid construct that includes each of the selected nucleic acid fragments described herein; and (5) selecting the transformed plant cell(s) whose expression level of the target sequence is reduced compared to the expression level of the target gene in the wild-type plant cell.

Information relevant to this application can be found in US Patent Applications MoMo 10/963238 and 10/963394, filed October 12, 2004. The entire contents of the above applications are incorporated herein by reference.

Other references that may be used in the context of this invention include US patent application Ser. No. 10/883374, filed July 1, 2004; US Patent Application No. 10/913288, filed Aug. 6, 2004, and US Patent Application No. Mo. 11/334776, filed Jan. 6, 2006.

The disclosure of each reference set forth herein is hereby incorporated by reference in its entirety.

As used herein and in the appended Claims, singular forms include plural references unless the context clearly dictates otherwise. Thus, for example, references to a "plant" include a plurality of such plants, references to a "cell" include one or more cells and their equivalents known to one skilled in the art, etc.

A number of terms and abbreviations are used in the context of this disclosure. The following definitions are presented. "MicroRNA or miRNA" means an oligoribonucleic acid that regulates the expression of a polynucleotide comprising a target sequence. MicroRNA (miRNA) is a non-coding RNA from about 19 to about 24 nucleotides (nc) in length that has been identified in both animals and plants (Iadoz-Otsipapa et al., bsiepse 294:853-858 2001, Iadoz-Otsipapa et al. ., St. Vioi. 12:735-739 2002; I a ei ai., Zsiepse 294:858-862 2001; I ee and Atbrgo5, Zsiepse 294:862-864 2001; Pame ei ai.,

Riapi SciI 14:1605-1619 2002; Moigeiaios eyi a!., Sepe5. JuOey 16:720-728 2002; Cancer of her ai., Sy. Vio!. 12:1484-1495 2002; Veiipnapy ei aYi., Sepe5. JuOem 16:1616-1626 2002) that regulate the expression of a polynucleotide that includes a target sequence. They are derived from longer precursor transcripts that range in size from about 70 to 2000 nc or more, and these precursor transcripts have the ability to form stable hairpin structures. In animals, the enzyme involved in the processing of miRNA precursors is called biseg, RNase PI-like protein (Stivnok ei ai!., Sei! 106:23-34 2001; Nyimadpeg ei a!., 5siepse 293:834-838 2001; Ketsipo e ai.,

Sepev YuOyem 15:2654-2659 2001). Plants also have an Oiseg-like enzyme, OSI 1 (previously named SAVREI EASTOVU/5NOVT IMGTEBOMEMT51/ BOZREMZOVNI), and recent evidence shows that it, like Oiseg, is involved in the processing of hairpin precursors to form mature miRNAs (RagkK ei ai!., Sit . Vioi. 12:1484-1495 2002; Veiipnag ei a!Yi., Sepev5. Yuvyemkh. 16:1616-1626 2002). In addition, recent work makes it clear that at least some hairpin precursors of miRNAs are formed as longer polyadenylated transcripts, and several different miRNAs and associated hairpins can be present in a single transcript (I ados-Otsipiapa et al.,

Zsiepse 294:853-858 2001; I ee ei ai., EMVO 4) 21:4663-4670 2002). In the last work, the selection of the miRNA strand from the double-stranded RNA product, which comes from hairpin processing with the help of RISEV, was also considered (5sPUagia ei ai!., 2003, SeiI 115:199-208). It appears that the stability (ie, content 2: vs. A and/or mismatches) of the two ends of the treated double-stranded

RNA influences strand choice, with the low-stability end being more easily unwound by helicase activity. A strand with a 5' end at the end with low stability is incorporated into the VI5ZS complex, while the other strand is degraded. "Prime miRNAs" or "primary miRNAs" are long polyadenylated RNAs transcribed by RNA polymerase II and which encode miRNAs. "Pre-miR NC" are primary miRNAs that are processed to form a shorter sequence capable of forming a stable hairpin, and further processed to release miRNA. In plants, both stages of processing are performed by an enzyme similar to aiseg, and therefore it is difficult to functionally differentiate "prime-miRNA" and "pre-miR NC". Therefore, miRNA-precursor, or primary miRNA, is functionally defined in this document as a nucleotide sequence capable of producing miRNA. In view of this functional definition, and as will be clear from the Examples and discussion herein, the precursor miRNA, primary miRNA and/or miRNA of the present invention can be represented as a ribonucleic acid or, alternatively, in a deoxyribonucleic acid form, which "corresponds substantially " miRNA precursor, primary miRNA and/or miRNA. It is clear that

DNA in its double-stranded form will include a strand capable of being transcribed into the described miRNA precursor. Expressing constructs, recombinant DNA constructs, and transgenic organisms including miRNA encoding DNA that results in the expression of described miRNA precursors are described. "Variable nucleotide subsequence" means a part of a nucleotide sequence that replaces a part of a pre-miRNA sequence, provided that this subsequence differs from the sequence being replaced, that is, it cannot be the same sequence. "Target gene" means the gene encoding the target RNA, ie, the gene from which the target RNA is transcribed. A gene can encode mRNA, tRNA, small RNA, etc. "Target sequence" means an RNA whose expression is to be modulated, eg, down-regulated. The target sequence can be part of the open reading frame, 5' or 3 untranslated region, exon(s), intron(s), flanking region, etc. A "dashed sequence" is a complementary sequence in the miRNA precursor that forms a duplex with the miRNA. The complementarity of the dashed sequence does not have to be complete. Sometimes there are non-helical breaking substitutions (ie, C base pairs, etc.), as well as 1-3 false pairings.

The term "genome" means the following: (1) the complete complement of genetic material (genes and non-coding sequences) present in each cell of an organism or in a virus or organelle; (2) an entire set of chromosomes inherited as a (haploid) unit from one parent. "Offspring" includes any subsequent generation of a plant. The offspring will inherit

Zo and stably segregate genes and transgenes from their parent plant(s).

Units, prefixes and symbols may be expressed in an acceptable 5I (International System of Units) form. Unless otherwise noted, nucleic acids are written from left to right in a 5-3 orientation; amino acid sequences are written from left to right in the amino - carboxyl orientation, respectively. The number ranges listed in the specification cover the numbers that define the range and include every integer in the specified range. Amino acids can be designated in this document either by well-known three-letter symbols or by single-letter symbols recommended by the Commission on Biochemical Nomenclature of ISRAS-

IOV Nucleotides, similarly, can be designated by their generally accepted one-letter codes. Unless otherwise specified, the terms software, electrical, and electronic used in this document are defined in IEEE Tem.

Ziapaaga Oisiopagu oi Eiesigis! apa EIesigopisv Teptv (5!" eaiiop, 1993). The terms defined below are more fully defined by reference to the specification as a whole.

The terms "recombinant construct," "expression construct," "chimeric construct," "construct," and "recombinant DNA construct" are used interchangeably herein.

A recombinant construct includes an artificial combination of nucleic acid fragments, such as regulatory and coding sequences, that do not occur together in nature.

For example, a chimeric construct may include regulatory sequences and coding sequences that originate from different sources, or regulatory sequences and coding sequences that originate from the same source but are arranged in a manner different from that found in nature. Such a construct can be used alone or can be used in conjunction with a vector. If a vector is used, the choice of vector depends on the method to be used to transform the host cells, as is well known to those skilled in the art. For example, a plasmid vector can be used. Those skilled in the art are well aware of the genetic elements that must be present in a vector for successful transformation, selection and propagation of host cells comprising any of the selected nucleic acid fragments of the present invention. The skilled person also recognizes that different independent transformation events will result in different levels and patterns of expression (opev ei ai.,

EMVO 3. 4:2411-2418 (1985); Oe AIteida eyi aiYi., Moi. Sept. Sepheisv 218:78-86 (1989)) and thus that multiple events must be screened to obtain lines that reflect the desired level and pattern of expression. Such an examination, among others, can be performed by Southern analysis

DNA, northern analysis of mRNA expression, immunoblotting analysis of protein expression or phenotypic analysis.

Such a construct may include any combination of deoxyribonucleotides, ribonucleotides, and/or modified nucleotides. The construct can be transcribed to form RNA, where the RNA can be capable of forming a double-stranded RNA and/or a hairpin structure. Such a construct can be expressed in a cell, or isolated, or obtained synthetically. The construct may additionally include a promoter or other sequences that facilitate manipulation or expression of the construct.

As used herein, "suppression," or "silencing," or "inhibition" are used interchangeably to refer to the down-regulation of expression of a target sequence product relative to its normal level of expression in a wild-type organism. Suppression involves expression decreasing by about 10 905, 15 90, 20 95, 25 95, 30 95, 35 95, 40 95, 45 95, 50 95, 55

Fo, 60 95, 65 90, 70 90, 75 905, 80 95, 85 90, 90 95, 95 95 or 100 95 relative to the wild-type expression level.

As used herein, "encodes" or "encodes" means a DNA sequence that can be processed to form an RNA and/or a polypeptide.

As used herein, "expression" or "expressing" means the production of a functional product, for example, the formation of an RNA transcript from an introduced construct, endogenous DNA, or a stably incorporated heterologous DNA sequence. The expression can also mean a polypeptide derived from mRNA formed from each of the aforementioned DNA precursors. Thus, expression of a nucleic acid fragment may mean transcription of the nucleic acid fragment (eg, transcription resulting in mRNA or other functional RNA) and/or translation of the RNA into a precursor or mature protein (polypeptide).

As used herein, "heterologous" with respect to a sequence means a sequence derived from a foreign species or, if from the same species, substantially modified by deliberate human intervention from its native form in composition and/or genomic locus. For example, with respect to a nucleic acid, it may be a nucleic acid derived from a foreign species, or synthetically engineered, or, if from the same

Of the species, it is significantly modified by deliberate human intervention from its native form in terms of composition and/or genomic locus. A heterologous protein may come from a foreign species or, if from the same species, be significantly modified by human intervention from its original form.

The term "host cell" means a cell that includes or has been introduced into a nucleic acid construct and that supports the replication and/or expression of the construct.

The host cells can be prokaryotic cells, such as E. soy, or eukaryotic cells, such as fungal, yeast, insect, amphibian, nematode, or mammalian cells.

Alternatively, the host cells are plant cells of monocotyledonous or dicotyledonous plants. An example of a monocot host cell is a corn host cell. "Plant" includes references to whole plants, plant organs, plant tissues, seeds and plant cells and their progeny. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.

The term "plant parts" includes differentiated and undifferentiated tissues including, but not limited to, the following: roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various cell forms and culture (eg, single cells, protoplasts, embryos and callus tissue), plant tissue can be in a plant or in a plant organ, tissue or cell culture.

The term "plant organ" means a plant tissue or a group of tissues that is a morphologically and functionally separate part of a plant.

The term "introduced" means delivery of a nucleic acid (eg, an expression construct) or protein into a cell. Entered includes reference to the incorporation of nucleic acid into a eukaryotic or prokaryotic cell, where the nucleic acid may be incorporated into the genome of the cell, and includes reference to the transient provision of nucleic acid or protein to the cell. Introduced includes references to methods of stable or transient transformation, as well as sexual crossing. Thus, "introduced" in the context of inserting a nucleic acid fragment (eg, a recombinant DNA construct/expression construct) into a cell means "transfection", or "transformation", or "transduction" and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell, where a fragment of nucleic acid can be included in the genome of the cell (for example, chromosomal,

plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon or transiently expressed (eg, transfected mRNA).

The term "genome" as applied to plant cells covers more than just the chromosomal

DNA found in the nucleus, but also DNA of organelles found in subcellular components (for example, mitochondrial, plastid) of the cell.

The term "isolated" means material, such as nucleic acid or protein, that is: (1) substantially or substantially free of components that normally accompany or interact with the material as it is found in its natural environment, or (2) ) if the material is in its natural environment, the material has been altered by intentional human intervention in the composition and/or placed in a locus in the cell different from the locus native to the material.

As used herein, "domain" or "functional domain" refers to a nucleic acid sequence(s) capable of eliciting a biological response in plants. The present invention relates to miRNA, consisting of at least 21 nucleotide sequences, acting either alone or together with other miRNA sequences, so the domain can mean either individual miRNAs or groups of miRNAs. Also, miRNA sequences linked to their backbone sequences can be considered domains used for miRNA processing in its active form. As used herein, "subdomains" or "functional subdomains" refer to subsequences of domains capable of causing a biological response in plants. miRNA can be considered a subdomain of the backbone sequence. "Contiguous" sequences or domains mean sequences that are sequentially linked without added nucleotides located between domains. An example of a contiguous domain thread is found in 5EO IU MO: 7957, which is 5EO IU MO: 1-2652, as a continuous thread that can be represented as 2652 miRNA sequences linked together in a serial connection in the form of a chain.

RNA interference means the process of sequence-specific post-transcriptional gene silencing in animals, mediated by short interfering RNAs (siRNAs) (Rige et al., Maishgte 391:806 1998). The corresponding process in plants is usually called post-transcriptional gene silencing (RT) or RNA silencing, and it is also called repression in fungi. The process of post-transcriptional gene silencing is considered

It is an evolutionary-conservative cellular defense mechanism used to prevent the expression of foreign genes, and is usually shared by a variety of flora and taxonomic types (Brige et al., Ttepd5 Sepei. 15:358 1999). Such protection against the expression of foreign genes could have developed in response to the production of double-stranded RNAs (dnRNAs) obtained from viral infection or from the accidental integration of transposon elements into the host genome by a cellular response that specifically destroys the homologous single-stranded

RNA viral genomic RNA. The presence of double-stranded RNA in cells causes an RNA interference response through a mechanism that has not yet been fully characterized.

The presence of long dRNAs in cells stimulates the activity of the enzyme ribonuclease HER, called "aiiseg!". Cyseg is involved in the processing of dnRNA into short pieces of dnRNA, known as short interfering RNAs (siRNAs) (Vegziwip et al., May 409:363 2001), and/or pre-miRNAs into miRNAs. Short interfering RNAs produced by adiseg activity are typically from about 21 to about 23 nucleotides in length and comprise duplexes of about 19 base pairs (Eyrabvpig eyi a!., sepev

Omg 15:188 2001). Oiseg is also involved in excision of 21- and 22-nucleotide small transients

RNA (mtRNA) from the RNA-precursor of a conservative structure involved in translational control (Niimadpeg et al., 2001, Zsiepse 293:834). The RNA interference response is also characterized by an endonuclease complex, commonly called the RNA-induced silencing (RIS) complex, which mediates the cleavage of single-stranded RNA that has a sequence complementary to the antisense strand of the kiRNA duplex. Cleavage of the target RNA occurs in the middle of the site, the complementary antisense strand of the kiRNA duplex (EiIraznik Yei AI., Sepe5 YuOeem. 15:188 2001). In addition, RNA interference may also involve small RNA (e.g., miRNA or miRNA) mediated gene silencing, presumably through cellular mechanisms that regulate chromatin structure and thereby prevent transcription of target gene sequences (see, e.g., AIb5Nigue, 5cipse 297:1818 -1819 2002; Moiire et al., Science 297:1833-1837 2002; Uepimeip, Science 297:2215-2218 2002; and Nai! Thus, the miRNA molecules of the present invention can be used to mediate gene silencing through interaction with RNA transcripts or otherwise by interaction with specific gene sequences, where such interaction results in gene silencing either at the transcriptional or post-transcriptional level.

RNA interference has been studied in a number of systems. Rige her ai. (Maishte 391:806 1998) were the first to observe RNA interference in S. eijedape. MUiappu and Sboei; (Maishte Sei! Vioi. 2:70 1999) describe double-stranded RNA-mediated RNA interference in mouse embryos. Nattopa ei ai. (Maishte 404:293 2000) describe RNA interference in Ogozorpia cells transfected with double-stranded RNA. Eyirabpig eyi ai. (Mayige 411:494 2001) describe RNA interference induced by the introduction of synthetic 21-nucleotide RNA duplexes in cultured mammalian cells, including primary human kidney and Nei a cells.

Small RNAs play an important role in controlling gene expression. The regulation of many developmental processes, including flowering, is controlled by small RNAs. It is now possible to engineer changes in the gene expression of plant genes by using transgenic constructs that produce small RNAs in the plant.

Small RNAs appear to function by base pairing with complementary RNA or

DNA target sequences. Upon binding to RNA, small RNAs either trigger RNA cleavage or translational inhibition of the target sequence. When binding to DNA target sequences, it is believed that small RNAs can mediate DNA methylation of the target sequence. The consequence of these events, regardless of the specific mechanism, is that gene expression is inhibited.

MicroRNAs (miRNAs) are non-coding RNAs of about 19 to about 24 nucleotides (nt) in length that have been identified in both animals and plants (I ado5-Ocypiapa eyi a!., Zsivepse 294:853-858 2001,

Yi adoz-Otsipiapa ei ai., Sit. Vioi 12:735-739 2002; Gai eyi aiI., Zsiepse 294:858-862 2001; And ee and

Atrbgo5, Zsiepse 294:862-864 2001; Pame ei ai., Riapi Sei! 14:1605-1619 2002; Moishgeiaios ei ai.,

Sepevz. Omg 16:720-728 2002; RaikK evy ai., Sy. VIOII. 12:1484-1495 2002; Veiipnag eyi aiYi., Sepev.

OEM 16:1616-1626 2002). They are derived from longer precursor transcripts, ranging in size from about 70 to 200 nt, and these precursor transcripts have the ability to form stable hairpin structures. In animals, the enzyme involved in the processing of miRNA precursors is called biseg, RNase PI-like protein (StiznokK ei a!., Seii! 106:23-34 2001; Niimadpeg vy ai.,

Zsiepse 293:834-838 2001; Ketsipa ei a!., sSepevz. YuOeu. 15:2654-2659 2001). Plants also have an Oiseg-like enzyme, OSI1 (previously called SAVREI EGASTOVU/5NOVT IMTESOMEMT51/

ZY5BREMBONVI), and recent data show that, like Oiseg, he is involved in processing

From hairpin precursors for the formation of mature miRNAs (RagkK ei ai., Sy. Visi. 12:1484-1495 2002;

Veipna! ей айЙ., Sepe5. JuOem 16:1616-1626 2002). In addition, recent work makes it clear that at least some hairpin precursors of miRNAs are formed as longer polyadenylated transcripts, and some other miRNAs and associated hairpins can be present in a single transcript (I adoz-Otsipapa ei a!., Zsieepse 294: 853-858 2001; I ee vii aI., EMVO U 21:4663-4670 2002). Recent work also addressed the selection of an miRNA strand from a double-stranded RNA product derived from hairpin processing by OISEV (Zspumagtl ei ai!., 2003, Sei! 115:199-208). It is becoming clear that the stability (ie, SS vs. AS content and/or mismatches) of the two ends of the processed double-stranded RNA affects strand choice, with the end with low stability being more easily unwound by helicase activity. The strand with the 5' end of low stability is incorporated into the VZS complex, while the other strand is degraded.

In animals, there is direct evidence for the role of specific miRNAs in development. It was found that Iip-4 and Ieii-7 miRNAs in S. eideap5 control temporal development on the basis of phenotypes obtained by mutation of the genes producing Iip-4 and Ieii-7 miRNAs (I ee ei ai., Seii! 75:843-854 1993; Veipnap ei ai.,

Maishte 403-901-906 2000). In addition, both miRNAs display a temporal pattern of expression consistent with their roles in the temporal pattern of development. Other animal miRNAs display developmentally regulated expression patterns both temporal and tissue-specific (I adov-

Optsipapa eyi a!., Zsiepse 294:853-853 2001, I adoz-Otsipiapa eyi ai!., Sit. Axes 12:735-739 2002; I ay ei ai., Zsiepse 294:858-862 2001; Yee and Atbrgo5, Zsiepse 294:862-864 2001), which led to the hypothesis that miRNAs can in many cases be involved in the regulation of important developmental processes. In addition, in plants, differential expression patterns of many miRNAs predict a role in development (IP iame ei ai., Riapi Sei! 14:1605-1619 2002; Rak ei a!., Sit. Vioi. 12:1484-1495 2002; Veipnap essay AI., September 5. YuOeu. 16:1616-1626 2002). However, a developmental role for miRNAs has not been directly demonstrated in plants, as so far there have been no reports of a developmental phenotype associated with a specific plant miRNA.

Apparently, miRNAs regulate target genes by binding to complementary sequences located in the transcripts produced by these genes. In the case of Iip-4 and 6-7, the target sites are located in the 3 TAs of target mRNAs (i ee vi ai., Seyi! 75:843-854 1993;

M/idnitap ei ai., Seii! 75:855-862 1993; Veiippap ei ai., Maite 403:901-906 2000; 5iaskK ei ai., My.

Sow! 5:659-669 2000), and there is little mispairing between IIP-4 and IIP-7 miRNAs and their target sites. Apparently, the binding of IIp-4 or IeIi-7 miRNA causes the down-regulation of steady-state levels of the protein encoded by the target mRNA, without affecting the transcript itself (Oisep and Atbrgoz, Yueu.

Vio!. 216:671-680 1999). On the other hand, recent data indicate that miRNAs can in some cases cause specific RNA cleavage of the target transcript at the target site (Niimadpeg and 7atoge, bsiepse 297:2056-2060 2002; Tspame et al., Riapi Sei! 14:1605- 1619 2002).

It is likely that miRNAs can participate in at least two metabolic pathways of target gene regulation: protein down-regulation and RNA cleavage. MicroRNAs involved in the metabolic pathway of RNA cleavage are included in the RNA-induced silencing (VI5ZS) complex, which is similar or identical to that observed for RNA interference.

The present invention relates to an isolated nucleic acid fragment that includes a miRNA-precursor, and the specified miRNA-precursor largely corresponds to the deoxyribonucleotide sequence set forth in 5EO IU MO: 11, (i) where nucleotides 430 - 450 in 5EO IU MO: 11 are replaced by the first variable a nucleotide subsequence that varies in size from about 19 to about 30 nucleotides depending on the target sequence whose expression is to be reduced, and (ii) further, wherein nucleotides 244-264 of 5EO IO MO: 11 are replaced by a second variable nucleotide subsequence that varies in size from about 19 to about 30 nucleotides, and said second variable nucleotide subsequence is capable of hybridizing with the first variable subsequence of the precursor miRNA.

This isolated fragment of nucleic acid, which includes the miRNA precursor, can also be called "miRNA core". A person skilled in the art is well aware that if the transcript is a full-length prime-miRNA or pre-miRNA, it is difficult to differentiate. Therefore, the miRNA precursor is functionally defined as a nucleotide sequence capable of producing miRNA.

Other isolated nucleic acid fragments of interest include the following: a) transcribed from an isolated nucleic acid fragment comprising a precursor miRNA, and said precursor miRNA largely corresponds to the deoxyribonucleotide sequence set forth in 5EO I MO: 12, (i) where nucleotides 94-114 in

ZEO IU MO: 12 is replaced by a first variable nucleotide subsequence, the size of which varies from about 19 to about 30 nucleotides depending on the target sequence whose expression is to be reduced, and (iii) in addition, where nucleotides 163 -183 in 5EO IU MO: 12 are substituted the second

Ko) a variable nucleotide subsequence, the size of which varies from about 19 to about 30 nucleotides, and the specified second variable nucleotide subsequence is able to hybridize with the first variable subsequence of the precursor miRNA;

B) an isolated nucleic acid fragment that includes an miRNA-precursor, and the specified miRNA-precursor largely corresponds to the deoxyribonucleotide sequence set forth in 5EO IU MO: 13, (and where nucleotides 53 - 73 in 5EO IO MO: 13 are replaced by the first variable nucleotide subsequence , the size of which varies from about 19 to about 30 nucleotides depending on the target sequence whose expression is to be reduced, and (ii) further, wherein nucleotides 97 - 117 of 5EO IO MO: 13 are replaced by a second variable nucleotide subsequence, the size of which varies from about 19 to about 30 nucleotides, wherein said second variable nucleotide subsequence is capable of hybridizing with the first variable subsequence of the precursor miRNA; c) an isolated nucleic acid fragment comprising the precursor miRNA, wherein said precursor miRNA substantially corresponds to the deoxyribonucleotide sequence set forth in 5EO IO MO: 14, (i) where nucleotides 110-130 in 5EO IO MO: 14 are replaced by the first variable a nucleotide subsequence that varies in size from about 19 to about 30 nucleotides depending on the target sequence whose expression is to be reduced, and (iii) further, wherein nucleotides 184-203 of 5EO IUO MO: 14 are replaced by a second variable nucleotide subsequence that varies in size from about 19 to about 30 nucleotides, and the specified second variable nucleotide subsequence is capable of hybridizing with the first variable subsequence of the precursor miRNA; and

I) an isolated nucleic acid fragment that includes a precursor miRNA, and said precursor miRNA largely corresponds to the deoxyribonucleotide sequence set forth in 5EO IU MO: 15, () where nucleotides 83-103 in 5EO IUO MO: 15 are replaced by the first variable nucleotide subsequence , the size of which varies from about 19 to about 30 nucleotides depending on the target sequence whose expression is to be reduced, and (ii) further, wherein nucleotides 172-192 of 5EO IU MO: 15 are replaced by a second variable nucleotide subsequence, the size of which varies from about 19 to about 30 nucleotides, and the specified second variable nucleotide subsequence is capable of hybridizing with the first variable subsequence of the precursor miRNA.

Any of these selected nucleic acid fragments can be used to create a recombinant construct that includes these selected nucleic acid fragments functionally linked to at least one regulatory sequence.

Such constructs can be transformed into a plant cell so that the transformed plant cell will include the recombinant construct in its genome. Preferably, the plant cell may be a plant cell of a monocot plant. Examples of monocot plant cells include, but are not limited to, corn, sorghum, wheat, rice, oat, rye, barley, sugarcane, millet, bamboo, banana, and orchid cells.

The most preferred plant cell of a monocot is the corn cell.

In another aspect, the present invention relates to a method of reducing the expression of a target sequence in a plant cell, and this method includes: (a) transforming at least one plant cell with a nucleic acid construct that includes any of the selected nucleic acid fragments described herein; and (5) selecting such transformed plant cell(s) whose level of expression of the target sequence is reduced compared to the level of expression of the target sequence in the wild-type plant cell.

Bioinformatic approaches are successfully used to predict targets for plant miRNAs (IPame ei ai., Riapi Sei! 14:1605-1619 2002; Rak evi a!., Sit. Visi. 12:1484-1495 2002; Vpoadez ei ai., Seii! 110:513-520 2002), and thus plant miRNAs apparently have a higher overall complementarity with their putative targets than animal miRNAs.

Most of these predicted plant miRNA target transcripts encode members of transcription factor families involved in plant developmental patterning or cellular differentiation.

General categories of sequences of interest include, for example, genes involved in regulation or information, such as zinc fingers, transcription factors, homeosis genes, or modulators of the cell cycle and cell death, involved in communication, such as kinases, and involved in cell-general functions , such as heat shock proteins.

Target sequences can include coding regions and non-coding regions, such as

From promoters, enhancers, terminators, introns and the like.

The target sequence may be an endogenous sequence or may be an introduced heterologous sequence or transgene. For example, methods can be used to alter the regulation or expression of a transgene, or to remove a transgene or other introduced sequence, such as an introduced site-specific recombination site. The target sequence may also be a sequence from a pathogen, for example, the target sequence may be from a plant pathogen such as a virus, mold or fungus, insect or nematode. The miRNA can be expressed in the plant which, upon infection or infestation, will target the pathogen and add some degree of resistance to the plant.

In plants, other categories of target sequences include genes affecting agronomic traits, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and commercial products. Genes of interest also include those involved in the metabolism of oil, starch, carbohydrate or nutrients, and also affect, for example, grain size, sucrose content, and the like. Grain quality is reflected in traits such as levels and types of oils, saturated and unsaturated, quality and quantity of essential amino acids, and cellulose levels. For example, genes in the biosynthetic pathway of phytic acid metabolism can be suppressed to produce a high phosphorus availability phenotype. See, for example, phytic acid biosynthesis enzymes including inositol polyphosphate kinase-2 polynucleotides disclosed in MO 02/059324, inositol 1,3,4-triphosphate-5/6-kinase polynucleotides disclosed in UMO 03/027243 , and myo-inositol-1-phosphate synthase polynucleotides and other phytate biosynthesis polynucleotides disclosed in M/O 99/05298, all of which are incorporated herein by reference. Genes in the lignification pathway can be suppressed to enhance digestibility or energy availability. Genes affecting the cell cycle or cell death can be suppressed to affect growth or stress response. Genes affecting DNA repair and/or recombination can be suppressed to increase genetic variability. Genes affecting the flowering period may be suppressed, as well as genes affecting fertility. Any target sequence can be suppressed to evaluate or confirm its role in a particular trait or phenotype, or to analyze a molecular, regulatory, biochemical or proteomic metabolic pathway or network.

A number of promoters can be used. These promoters can be chosen depending on the desired outcome. It is recognized that different applications will be expanded by the use of different promoters in plant expression cassettes to modulate the temporal pattern, location and/or level of miRNA expression. Such plant expression cassettes may also include, if desired, a promoter regulatory region (eg, one discussed inducible, constitutive, environmentally or developmentally regulated or cell- or tissue-specific/selective expression), a transcription initiation site, a site of ribosome binding, RNA processing signal, transcription termination site and/or polyadenylation signal.

Constitutive, tissue-preferred, or inducible promoters can be used.

Examples of constitutive promoters include the Cauliflower mosaic virus (Samu) transcription initiation site 355, the 1" or 2' T-DNA-derived promoter, the ibBidiyip 1 promoter, the tau promoter, the cinnamic alcohol dehydrogenase promoter (US Pat. No. 5,683,439 ), Mo5 promoter, pEty promoter, hybrivso promoter (ribulose-1,5-birosphosphate carboxylase oxygenase), SVR1-8 promoter and other transcription initiation sites from various plant genes known to the skilled worker. If a low level of expression is desired, a weak promoter can be used (promoters ). Weak constitutive promoters include, for example, the core Nvzup7 promoter (M/O 99/43838 and US Patent No. 6,072,050), the core 355 Samu promoter, and the like. Other constitutive promoters include, for example, US Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5569597; 5466785; 5399680; 5268463 and 5608142.

See also, US Patent No. 6,177,611, incorporated herein by reference.

Examples of inducible promoters are AiNy! a promoter inducible by hypoxia or cold stress, a Hep70 promoter inducible by heat stress, a PPOK promoter and a pepcarboxylase promoter, both of which are inducible by light. Chemically inducible promoters are also used, such as the Ip2-2 promoter inducible by safener (patent

US Mo 5364780), ENE estrogen-inducible promoter, and Achid! promoter inducible by auxin and tapetum specific, but also active in callus (PCT 0О0501/22169).

Examples of developmentally controlled promoters include promoters that initiate transcription preferentially in specific tissues such as leaves, roots, fruit, seeds, or flowers.

An illustrative promoter is the anther-specific promoter 5126 (US Patent Nos. 5,689,049 and 5,689,051). Examples of seed-preferred promoters include, but are not limited to, the 27 kDa datta 2yip promoter and the dreaded promoter, Vogopai, A. eyi ai. (1986) Riapi zsi. 47:95-102; Veiipa, M. yei ai. capes Asia5 Rev. 18(21):6426; and Kioezdep, V. V. Yei a. (1986) Mine. Sept.

Sepoys 203:237-244. Promoters expressed in the embryo, pericarp, and endosperm are disclosed in US Patent No. 6,225,529 and PCT Publication UMO 00/12733. The disclosures of which are incorporated herein by reference in their entirety.

In some embodiments, it will be useful to express the gene from an inducible promoter, especially from a pathogen-induced promoter. Such promoters include promoters from pathogenesis-related proteins (RA proteins) that are induced after infection with a pathogen; example,

RE proteins, ZAV proteins, beta-1,3-glucanase, chitinase, etc. See, for example, VNedoiii eii ai. (1983)

May in. Riapi Raiyoi. 89:245-254; Okpez you ai. (1992) Riapi Sei! 4:645-656; and Map I oop (1985) Riapi

MY. Mikoi 4:111-116. See also UUO 99/43819, included in this document by reference.

Of interest are promoters expressed locally at or near the site of pathogenic infection. See, for example, Magipeai eyi ai. (1987) Riapi Moi. Vioi 9:335-342; Matsop ey a). (1989) Moiesshiag Riapi-Mistore Ipiegasiyup5 2:325-331; 5 gave her a!. (1986) Rgos. Mai! Asai. 5

BA 83:2427-2430; 5otvivsp her a. (1988) Mine. Sept. S1epei. 2:93-98; and Mapu (1996) Rgos. Mai! Asad from the U.S. BOTH 93:14972-14977. See also, Spep ei ai. (1996) Riapi U). 10:955-966; 2Napao ei ai. (1994)

Rgos. May Asad axis OBA 91:2507-2511; Mateg ei ai. (1993) Riapi 9. 3:191-201; bierepia ei ai. (1989) Ryapi Sei! 1:961-968; US Patent No. 5,750,386 (Nematode-induced) and references cited herein. Of particular interest is the inducible promoter for the PAt5 gene of corn, the expression of which is induced by the pathogen Rizagisht topiiytogte (see, for example, Sogdego et al. (1992) РНузиой. Мой. Рапи Райб. 41:189-200).

Additionally, because pathogens gain entry into plants through wounds or insect damage, a wound-inducible promoter can be used in polynucleotide constructs.

Such wound-inducible promoters include the potato proteinase inhibitor (rip Ii) gene (Vuap (1990) App. Neum. Rnuiorain. 28:425-449; Yutsap ei ai. (1996) Mashge Vioyesi. 14:494-498); myp' and mypg, US patent Me 5428148; m/p and m/ip2 (Zapioga ei ai. (1989) Moi. Sep. Sepei. 215:200-208); zuvietip (MesSyt! eyi ai. (1992) Zsiepse 225:1570-1573); MIRI (Voptvivg eyi a. (1993) Riapi Mo). Vio!. 22:183-792; EsKeIKatr ei ai. (1993) REV5 And hey. 323:73-76); MRI gene (Sogaegok ei ai. (1994) Riapi U. 60 6(2):141-150) and the like, incorporated in this document by reference.

Chemically regulated promoters can be used to modulate gene expression in a plant by applying an exogenous chemical regulator. Depending on the purpose, the promoter can be a chemically inducible promoter, where application of a chemical induces gene expression, or a chemically repressible promoter, where application of a chemical represses gene expression.

Chemically inducible promoters are known in the art and include, but are not limited to, the Ip2-2 maize promoter activated by benzenesulfonamide herbicide safeners, the maize 51 promoter activated by hydrophobic electrophilic compounds used as preemergence herbicides, and the tobacco RA-ia promoter activated salicylic acid. Other chemically regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in 5spepai et al. (1991) Rgos. Mai). Asai. 5

BA 88:10421-10425 and MeMeiiyiv yei a. (1998) Riapi 9. 14(2):247-257), a tetracycline-inducible promoter and a tetracycline-repressible promoter (see, e.g., Szai et al. (1991) Mo).

Sept. Sepoys 227:229-237 and US MoMo patents 5,814,618 and 5,789,156), incorporated herein by reference.

Tissue-preferred promoters can be used to target enhanced expression of a sequence of interest in a specific plant tissue. Tissue-preferred promoters include Ouatatoio and AiY. (1997) Riapi U. 12(2):255-265; Kamataiyya ei ai. (1997) Riapi Sei

РНузиой!. 38(7):792-803; Napzep ei ai. (1997) Mine. Sep Sepei. 254(3):337-343; Wizzy! oh yeah (1997)

Tkgapzdepis Rev. 6(2):157-168; VipeNan ei ai. (1996) Riapi Rnuzioi. 112(3): 1331 - 1341; Map of Satria and I. (1996) Riapi Rnuvzio!i. 112(2):525-535; Sapemavsipi ei ai. (1996) Riapi RNuvzioi. 112(2):513-524;

Uatato ei ai. (1994) Ryapi Sei! Rnuvzioi 35(5):773-778; at (1994) Veziiv Rhori. Sow! Oiyeg. 20:181-196; Ogo2so her ai. (1993) Riapi Moi. Vioi 23(6):1129-1138; Maizzoka evy a!. (1993) Rgos Mai)!.

Asad All together. OBA 90(20):9586-9590; and Sshpemaga-Sagsia yei ai). (1993) Riapi U. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression.

Preferred promoters for leaves are known in the art. See, for example, Matatoiyu ei ai. (1997) Riapi 9. 12(2):255-265; Kmop ey ay. (1994) Riapi Rnuzioi. 105:357-67; Matatoiyo ei ai. (1994)

Riapi Seyi Rpuzioi. 35(5):773-778; (z01og ei ai. (1993) Riapi 9). 3:509-18; Ogo2so ei ai. (1993) Riapi

MY. Cheers! 23(6): 1129-1138; and MaiziokKa ei ai. (1993) Rgos. Mai! Asad 5 BOTH 90(20):9586-9590.

In addition, you can also use the sab and gibrizzo promoters. See, for example, bitrzop of the 6th ai.

Zo (1958) EMVO U 4:2723-2729 and Titko eyi ai!. (1988) Maishte 318:57-58.

Root-preferred promoters are known and may be selected from many available in the literature or isolated de pomo from various compatible species. See, for example, Nige ei ai. (1992)

Riapi Moi. Vioi 20(2):207-218 (soybean root-specific glutamine synthetase gene); KeiPe" and

Washitaoagpeg (1991) Riapi Sei! 3(10):1051-1061 (root-specific control in

SOVAR 1.8 genes of common beans); zapdeg her ai (1990) Riapi Moi. Vioi 14(3):433-443 (root-specific mannopin synthase gene promoter (MA5) Adgobaciel and Ishtegasiep5); and Miao ei ai. (1991)

Riapi Sci. 3(1):11-22 (a full-length cDNA clone encoding a cytosolic glutamine synthetase ((5) expressed in soybean roots and root nodules). See also Vodyov et al. (1990) Riapi

This! 2(7):633-641, where two root-specific promoters are described, isolated from the hemoglobin genes of the nitrogen-fixing non-legume Ragazropia apaegsopia and the related non-nitrogen-fixing non-legume Tteta Eutepiosa. The promoters of these genes were linked to the p-glucuronidase reporter gene and introduced into the non-legume Misoiyapa yabasit and the legume Ioiy5 sogpissiaiv5, and in both cases the activity of the root-specific promoter was preserved. Iasi and Asouadi (1991) describe their analysis of the promoters of the root-inducible and highly expressed genes, Adhorasiagis and Pigodepe5 (see Riapi Zsiepse (Gitegisk) 79(1):69-76). They concluded that the enhancer and tissue-preferred DNA determinants are dissociated in these promoters. Teeeeti yei a!. (1989) used a gene fusion with Ias7 to show that the T-DNA gene

Adhorasieghist, which encodes octopine synthase, is particularly active in the root tip epidermis, and that

The TA2" gene is specific for the root in a healthy plant and is stimulated by wounding in leaf tissue, a particularly desirable combination of characteristics for use with an insecticidal or larvicidal gene (see EMVO 3. 8(2):343-350). The TVA gene fused to pri ( neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the promoter of the M'EMOY-SVARZ gene (Kiwiei ei a). (1995) Riapi Moi. Vio). 29(4):759-772); and "0ЙВ promoter (Sarapa ei ai. (1994) Riapi Moi. Vioi. 25(4):681-691. See also US Patent MoMo 5837876; 5750386; 5633363; 5459252; 5401836; 5110732 and 5023179. Geneolin (Mygai ei ai! (1983) bsiepse 23:476-482 and Zepdoria-Ssoraiep evy ai. (1988) RMAB 82:3320-3324.

Transformation protocols, as well as protocols for introducing nucleotide sequences into plants, may vary depending on the type of plant or plant cell, i.e., monocotyledonous or dicotyledonous, intended for transformation. Suitable methods of introducing the DNA construct include microinjection (Stob5mau et al. (1986) Bioespidiez 4:320-334 and US patent Mo 6300543), sexual crossing, electroporation (Vidaoz sei al!. (1986) Rhos. May). Asai. 5 OBA 83:5602-5606), Adgobasiegist-mediated transformation (Tompzepa et al., US patent No. 5563055 and US patent No. 5981840), directed gene transfer (Raz2Komuzvki et al. (1984) EMVO

U. 3:2717-2722) and ballistic particle acceleration (see, e.g., Dapioga et al., U.S. Pat.

MO 4945050; Totez Yei AI., US patent No. 5879918; Totez Evyi AI., US patent No. 5886244; In response to her AI.,, US patent No. 5932782; Totez ei ai. (1995) "Oigesi OMA Ttapvteg ipio Ipiasi Riapi SeiIIv mia

Mistorgo|esiiye Votbragatepi," in Riapi SeiI, Tivvye, apa Ogdap Sipyige: Hypdatepia! Meipodv, ea.

Satboga apa RnNiyiire (Zrhipdeg-Mepad, Vepip); and MeSabe ei ai. (1988) Journal of Biochemistry 6:923-926).

Also see M/eizzipdeg eyi ai. (1988) App. German Sepoys 22:421-477; Zapioga ei ai. (1987) Rapisciate

Zsiepse apa Thesppoiodu 5:27-37 (onion); СНызиой ей яй. (1988) Riapi Rnuzio! 87:671-674 (soy);

Ripeg ii MemMiyep (1991) Ip Myo SeiI Oyem. Axes 27R:175-182 (soy); 5ipop ei ai. (1998) Tneog. Arri.

Sepoys 96:319-324 (soy); Wow! (1990) Journal of Biochemistry 8:736-740 (fig); Kiyip ey a!. (1988) Rhos.

Maya Asad si. BOTH 85:4305-4309 (May); KiIvip heri a. (1988) Journal of Biochemistry 6:559-563 (Maize); Totev, US patent Mo 5240855; U.S. Patent No. 5,322,783 and 5,324,646; Kiwip sei a. (1988)

Riapi Rpuzioi. 91:440-444 (May); Gegott ei ai. (1990) Physiology 8:833-839 (Maize); Nosoukaa5-Map

Ziodietep ei ai. (1984) Maishte (Hopdop) 311:763-764; Vozhep eyi AI., US patent Me 5736369 (cereals); Vuiebieg ei ai. (1987) Rhos. Mai). Asad 5 OBA 84:5345-5349 (lilies); Oe M/ei ei a). (1985) in Te Ehregitepia! Mapiriyop oi Omyshe Tizztsev, widow. Spartap hey ay. (Hopdtap, Mem/ Mo), 197-209 (pollen); Kaerrieg heri a. (1990) Ryapi Sei! Verogov 9:415-418 and Kaerrieg ei a)i. (1992)

Theog. Arri. Sepoys 84:560-566 (mpizKeg-mediated transformation); O'Nainip ei ai. (1992)

Riapi Sei! 4:1495-1505 (electroporation); And her ai. (1993) Riapi Seyi Verohiv 12:250-255 and Singing and

Eoga (1995) Appai5 oi Voiapu 75:407-413 (fig); Ozioda evy a!. (1996) Maize Violiesnpoioduh 4:745-750 (maize using Aagobasiegist Iishteiasiepo) and US Patent Mo 5,736,369 (meristem transformation), all of which are incorporated herein by reference.

Nucleotide constructs can be introduced into plants by contacting the plant with a virus or viral nucleic acids. In general, such methods include embedding the nucleotide construct of this invention into a viral DNA or RNA molecule. In addition, it is recognized that suitable promoters include promoters used for transcription

With viral RNA polymerases. Methods of introducing nucleotide constructs into plants and expressing the protein encoded in them, including viral DNA or RNA molecules, are known in this field. See, e.g., US Patent Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, and 5,316,931; incorporated herein by reference.

In some embodiments, transient expression may be desired. In these cases, standard methods of transient transformation can be used. Such methods include, but are not limited to, methods of viral transformation and microinjection of DNA or RNA, as well as other methods well known in the art.

Cells from plants having a stably incorporated nucleotide sequence can be grown in plants according to conventional methods. See, for example, MeSoptisk ei ai. (1986) Riapi Sei! Berohiv 5:81-84. These plants can then be grown and pollinated with either the same transformed variety or other varieties, and the resulting hybrid will have constitutive expression of the desired phenotypic characteristic provided by the nucleotide sequence of interest and/or genetic markers contained in the target site or transfer cassette. To ensure stable maintenance and inheritance of the expression of the desired phenotypic characteristic, two or more generations can be grown and then the seeds harvested to ensure that the expression of the desired phenotypic characteristic is achieved.

Examples

Example 1

Isolation of genes of precursors of genomic microRNAs

The sequences coding for the microRNA genes of maize are as described in 7Napo B, ei a. (2006) RGEV5

And en. 580:3753-62, were used as queries for VI AT analysis of the collection of Riopeg Opisogp 6.0 marker sequences that are expressed. Approximately 30 95 queries had exact matches in the Description 6.0 collection. The following primers (purchased at MU/S-BIOTESN Ips.) were designed to amplify a selection of five of these sequences (see Table 1).

Table 1

Primers for amplification of genomic microRNA precursors

Saturday 00 | ossntrnoyetyaneorovsvekonvvovetveve i 0202016 blveya 00 fBsesveoyueevvssvyaveoetkenaiya 000018

So3ebne 00 5osoovkssokssstsmny eni 200719 159 sense primer (159c5, BEO IYUO MO: 1) included nucleotides encoding Moso I site. 159 antisense primer (159sa, 5EO IO MO: 2) included nucleotides encoding the Wat NI site. The other four sense primers (ZEO IO MO: 3, 5, 7 and 9) included nucleotides encoding the Wat NI site. The other four antisense primers (5EO IYUO MO: 4, 6, 8 and 10) included nucleotides encoding the Meo I site.

Seeds 7/ea tau5 su. B7Z were germinated and genomic DNA was obtained from seedling tissue using the Oiadep OMeavzu Riapi Mahi kit according to the manufacturer's instructions. DNA products were amplified using genomic DNA as a template and the above primer pairs with ExTad polymerase (TaKaVa Vio Ips.). The obtained DNA products were cloned in pCH2.1 (Ipmygodep) and completely sequenced. Characterized miRNA precursors are listed in Table 2.

Table 2

Sequences of miRNA precursors

Example 2

Construction of artificial microRNA sequences Artificial microRNAs (shmiRNAs) that will have the ability to silence the phytoendesaturase gene of corn (MSVI number 037285) were constructed mainly according to the rules described in 5spmar B, ey ai. (2005) Yuoi Sei! 8: 517-27. In summary, the miRNA sequences are 21 nucleotides long, start at their 5' end with "OO", exhibit 5' instability with respect to their dashed sequence achieved by the inclusion of C or C at position 19, and their 10th nucleotide is or "A" or "0".

An additional requirement for the artificial miRNA design was that the artificial miRNA should have a high free energy delta Si, calculated using the 7irgEoia algorithm (MaikNnat, M.

A. a 2yKeg, M. (2005) Mysivys Asia5 Nev5. 33: M/577-M/581). Optionally, the replacement of one base pair was added to the 5th part of the shmiRNA so that the sequence differed from the target sequence by one nucleotide. The shmiRNA used for phytoendesaturase silencing was 5'-isadsadsaaishshisassadda-3" (the DNA sequence corresponding to this shmiRNA is presented in 5EO IO MO: 16). Two additional shmiRNAs were constructed for phytoendesaturase silencing. Such sequences are ROB 2, 5- dsaaaaaaaassisasoaca-3 (DNA

Ko) the sequence corresponding to this smiRNA is presented in 5EO IU MO: 35) and ROB 3, 5- chasysdsaaasaisisidad-3' (the DNA sequence corresponding to this smiRNA is presented in 5EO

IO MO: 36).

Example C

Construction of artificial sequences marked with a dash "Sequences marked with a dash" are such that base pairs with smiRNA sequences, in the RNA precursor, form imperfect stem structures. To form a perfect stem structure, the dashed sequence will be the exact reverse complement of the smiRNA. Maize precursor sequence as described in 7Napod ei ai. in Table 51 of the supplementary material, was folded with the help of thioiy (M. 2yKeg (2003) Mysieis Asiyb5 Ne5. 31: 3406-15; and Yu. N. MashShemuye, 5. ei ai. (1999) U). My. Vioi 288: 911-940). The miRNA sequences were then replaced by the smiRNA sequence, and the endogenous sequence indicated by a dash was replaced by the exact reverse complement of the artificial miRNA. Substitutions in the artificial sequence indicated by a dash were introduced so that the stem structure remained the same as the endogenous structure. The modified sequence was then collapsed using thioiy, and the original and modified structures were compared visually. If necessary, additional changes to the artificial sequence, indicated by a dash, were introduced to maintain the original structure. The DNA sequences corresponding to the dashed artificial sequences used to silence the phytoendesaturase are:

Table C

Artificial miRNA sequences marked with a dash

Example 4

Conversion of precursors of genomic miRNAs into precursors of artificial miRNAs

Precursor genes of genomic siRNAs were converted to smiRNAs using overlapping PCR (polymerase chain reaction), and the resulting DNA was completely sequenced.

Then these DNAs were cloned in the 5 - 3 direction of a suitable promoter into a vector capable of transforming corn. Then the resulting plasmids were co-integrated into the Adgobasiegiit strain

And BA4404 could plasmid PNR10523 (PCT publication Mo MMO2002/004649, published on January 17, 2002) and can be used for corn transformation.

Alternatively, smiRNAs can be synthesized commercially, for example by

Sodop Remisez (Satgidde, MA). Then the synthesized DNA is cloned in the 5' - 3' direction of a suitable promoter into a vector capable of transforming corn. Then the resulting plasmids are co-integrated into the Adhorasiyit I BA4404 strain with the PHP10523 plasmid and can be used for

From the transformation of corn.

Example 5

Conversion of precursors of genomic miRNAs into precursors of artificial miRNAs

Genomic siRNA precursor genes were converted to smiRNA precursors using overlapping PCR as described in example 4, and the resulting DNA was completely sequenced. Received the following five predecessors of ShmiR NK:

Table 4

Precursor sequences of artificial miRNAs targeting phytoendesaturase

Then these DNAs were cloned in the 5' - 3' direction of the ibidiyip promoter-intron of PHP23576. PHP23576 includes Saiemzhau (Ipmitodep) 11 and 12 sites. Then the resulting plasmids were recombined with the plasmid

РНР20622 (PCT publication Me M/O2006/107931, published on October 12, 2006). Then the resulting plasmids were co-integrated into the strain Adgobrasiegisht IVA4404 with the plasmid PHP1O0523 and used for corn transformation.

Example 6

Maize transformation

A. Particle-mediated DNA delivery in maize

The DNA construct can be introduced into corn cells that are capable of growing on a suitable corn culture medium. Such competent cells can be from maize suspension culture, callus culture on solid medium, freshly isolated immature embryos or meristematic cells. Immature embryos of the No-HER genotype can be used as target cells. Heads are harvested approximately 10 days after pollination, 1.2-1.5 mm immature embryos are separated from the grains and placed with the embryo shield down on the corn culture medium.

Immature embryos are bombarded within 18-72 hours after collection from the cob. Between 6 and 18 hours before bombardment, immature embryos are placed on a medium with an additional osmotically active substance (basic medium M, Myzavzpide and 5Ko09, 1962, RnNuzio).

Riapi 15:473-497, with 0.25 M sorbitol) Embryos on highly osmotic medium are used as targets for bombardment and left on this medium for another 18 hours after bombardment.

For particle bombardment, plasmid DNA (described above) is deposited on 1.8 mm tungsten particles using standard CaCl-spermidine chemistry (see, e.g.,

KiIvip eyi ai., 1987, Mayte 327:70-73). Each tablet is bombarded once at 600 PSI with a Yuyropi Neiisht Syp (I ouge ei ai!., 1995, Vio/Tesppoi! 13:677-682). For typical media compounds used for isolation of immature maize embryos, callus initiation, callus proliferation, and plant regeneration, see Agtvigopa, S., 1994, in "Pe Maige Naparosok",

M. Egeiiipd apa U. UaiBoi, edv5. Zrgipdeg Mepad, MU, 663-671.

Within 1-7 days after bombardment with particles, the embryos are transferred to the culture room

A medium based on the MB medium, which includes 3 mg/l of the selective agent bialofos.

Embryos and late callus are transferred to fresh plates for selection every 2 weeks. The callus, which develops from immature embryos, is examined for the desired phenotype. After 6-8 weeks, the transformed callus is restored.

B. Maize transformation using Adgobasivetgiit

Mediated Adgobasiegsht transformation of corn is performed mainly as described in 7pao ei ai., in Meiy. My. VioI. 318:315-323 (2006) (see also 7pao ei ai!., Moi. Vgeed. 8:323-333 (2001) and US Pat. No. 5,981,840, issued Nov. 9, 1999, incorporated herein by reference). The transformation process includes inoculation with bacteria, co-cultivation, rest, selection and recovery of the plant. 1. Preparation of immature embryos:

Immature maize germs are excised from the kernels and placed in a 2 ml microtube containing 2 ml of RNI-A medium. 2. Adgobasiegtiitis infection and co-cultivation of immature embryos: 2.1 Stage of infection:

1 ml of RNI-A medium (1) is removed with a micropipette and 1 ml of suspension is added

Adhobrasiegist. The tube is gently inverted to mix. The mixture is incubated for 5 minutes at room temperature. 2.2 Stage of joint cultivation:

Adgobrasieghsht suspension is removed from the stage of infection with a 1 ml micropipette. With the help of a sterile spatula, the embryos are scraped from the test tube and transferred to a plate of RNI-B medium in a 100x15 mm Petri dish. Embryos orient the axis of the embryo downward on the surface of the medium. Embryo plates were cultured at 20 °C in the dark for three days. In the co-cultivation phase, I-cysteine can be used. With the standard binary vector, co-culture medium supplemented with 100-400 mg/L I-cysteine is critical for the recovery of stable transgenic 3. Selection of putative transgenic events:

10 embryos are transferred to each plate of RNI-O medium in a 100x15 mm Petri dish, maintaining the orientation, and the dishes are sealed with Parafil. Plates are incubated in the dark at 28°C. Actively growing presumptive events, such as pale yellow embryo tissue, are expected to be visible after six to eight weeks. Non-event embryos may be brown and necrotic, and a pronounced small growth of loose tissue. The expected transgenic germ tissue is transplanted onto fresh RNI-Y plates at two- to three-week intervals depending on the growth rate. Events are recorded. 4. Regeneration of plant tissues:

Germ tissue propagated on RNI-O medium is transplanted to RNI-E medium (medium for maturation of somatic embryos) in 100x25 mm Petri dishes and incubated at 28 "C in the dark until maturation of somatic embryos, for about ten to eighteen days.

Individual mature somatic embryos with a well-defined scutellum and coleoptile are transferred to RNI-E embryo germination medium and incubated at 28 C in light (about 80 NE from cool-white or equivalent fluorescent lamps). On the seventh-tenth day, regenerated plants, about 10 cm tall, are planted in a pot in a horticultural mixture and hardened using standard horticultural methods.

Medium for plant transformation: 1. RNI-A: 4 g/l CHO basic salts, 1.0 mol/l 1000X vitamin mixture of Erickson, 0.5 mg/l thiamine NCI, 1.5 mg/l 2,4-O , 0.69 g/l I - proline, 68.5 g/l sucrose, 36 g/l glucose, pH 5.2. Add 100 rm of acetosyringone (sterilized by filtration). 2. RNI-B: RNI-A without glucose, increase 2,4-Y0 to 2 mg/l, reduce sucrose to 30 g/l, supplemented with 0.85 mg/l silver nitrate (sterilized by filtration), 3.0 g/ l Seinyet, 100 rm of acetosyringone (sterilized by filtration), pH 5.8. 3. RNI-C: RNI-B without Seipef and acetosyringone, reduce 2,4-O to 1.5 mg/l and supplemented with 8.0 g/l agar, 0.5 g/l buffer 2-IM-morpholino| ethanesulfonic acid (ME5), 100 mg/l carbenicillin (sterilized by filtration). 4. RNI-O: RNI-C supplemented with 3 mg/l bialofos (sterilized by filtration). 5. RNI-E: 4.3 g/l Murasige-Skug salts (M5), (Sibso, BB 11117-074), 0.5 mg/l nicotinic acid, 0.1 mg/l thiamine NS, 0.5 mg/l pyridoxine NOSI, 2.0 mg/l glycine, 0.1 g/l myo-inositol, 0.5 mg/l zeatin (Zidta, MO according to catalog 2-0164), 1 mg/l indoleacetic acid (IA ), 26.4 μg/l abscisic acid (ABA), 60 g/l sucrose, 3 mg/l bialofos (sterilized by filtration), 100 mg/l carbenicillin (sterilized by filtration), 8 g/l agar, pH 5.6 . 6. RNI-E: RNI-E without zeatin, IAA, ABA; reduce sucrose to 40 g/l; replacing agar with 1.5 g/l Seishef; pH 5.6.

Plants can be regenerated from transgenic callus by first transferring tissue clusters to Mb medium supplemented with 0.2 mg per liter of 2,4-O0. After two weeks, the tissue can be transferred to a regenerating medium (Egott et al., Bio/Hesppoiodu 8:833-839 (1990)).

It is possible to regenerate transgenic TO plants and determine their phenotype. You can collect T1 seeds.

In addition, the recombinant DNA construct containing the proven Agaridorzi5 gene can be introduced into the maize inbred line either by direct transformation or introgression from a separately transformed line.

Transgenic plants, either inbred or hybrid, can be subjected to more intensive field experiments to study the effects of expression.

Example 7

PO5 phenotype testing and results

Nine to ten days after pollination, the embryos were infected with Adgobasiegisht, as described above, and selected for Vavia. Five different infections were carried out, each with help

An adhorasiegist containing one of the five smiRNAs described in example five. Somatic embryos were regenerated and grouped in a 3x3 grid. One layer of plates containing grouped seedlings was exposed to 114 mEinstein m-2 sec. -1 light. A seedling in the middle of the grid was visually scored by phenotype as either "green," "white," or "light." The percentage of silencing is the summation of the percentage of white plants and the percentage of light plants. Basically, 50 separate events were scored for each construct.

Table 5

The effectiveness of the silencing of the ShmiR NK

RNRZO223 | 7 159S-ROI 177700 | 143 17777714

RNRZO448 | 0 164p-ROII 77/00 | 776 17777778 2

RNRZZOOE | 396p-ROB2 | 4893956 | 41495 2 sh |:/90 727

These results show that some of the precursors of smiRNAs are able to produce smiRNAs that are effective in gene silencing. Two additional phytoendesaturase-targeting miRNAs were cloned into the Z396bpP backbone along with their dashed sequences. Both constructs showed similar silencing efficiency to PHP30452, which is shown in Table 5.

Example 8

Northern blot analysis

RNA was obtained from frozen seedling tissue using Tti20! (Ipmigtodep) according to the protocol provided by the manufacturer. Total RNA was run on 15 95 TVE (trisborate buffer) - urea RACE gel (analysis by electrophoresis in a polyacrylamide gel), run at 200

In 0.5x TVE for approximately 60 - 90 minutes, and then transferred to a positively charged nylon membrane of Vhidpiyag-Riy5 (Atriop) using a Tgape-Vioi 50 chamber. After transfer, the blot was washed with 0.5XTVE, and the RNA was cross-linked to the membrane using one cycle of Ashio-Epegda in 5ighaiinKeg (5ighaiadepe). The blot was pre-hybridized in hybridization buffer OI TVANub-Oiido (Atbiop) for 30 minutes at 42 degrees C, and then hybridized overnight at 42 degrees in hybridization buffer OI TVANur-Oiido with the addition of a biotin-labeled probe. The biotin-labeled probe was a concatemer of two copies of the reverse complement of the smiRNA sequence separated by a 4-bp spacer and was labeled using the Vhydniegag psoralen-biotin nonisotopic labeling kit (Atrbiop).

After hybridization, the blot was washed with MogpipegpMax wash solution (Atriop), and the determination was performed using a non-isotopic determination kit Viognisiag VioOeiesi (Atriop) according to the protocol provided by the manufacturer. In all cases, the presence of a 21-bp long shmiRNA sequence was correlated with the phenotype. In addition, moderate signal levels were correlated with pale plants, while higher signal levels were correlated with white plants.

Example 9

Truncated precursors of artificial microRNAs are capable of gene silencing

In an attempt to determine the gene silencing ability of constructs containing artificial miRNA precursors of less than full length, the following shortened smiRNA precursors were obtained by PCR.

Table 6

Truncated precursors of artificial microRNAs targeting phytoendesaturase

These truncated siRNA constructs were fully sequenced and cloned into constructs as described in Example 5. These constructs were transformed into maize embryos and evaluated for their ability to silence ROB.

Table 7

Silencing efficiency of truncated shmiR NCs

RNRZ2347 | 169-РО5-яM | 000 | 00095 | 0 2 the same as JI

RNRZ2346 0 169-РО5-tey/ | 000 2 | 339 2 sh | 339

RNRZ2349 0 396p-ROV-yaY | 000 | 000 | 2 YuyuYuroo

These results show that the truncated constructs are capable of producing siRNA. However, if the construct is too truncated, it is no longer able to produce shmigR NC.

Example 10

Artificial miRNAs to silence Tad2-1

The above examples show ROB silencing of a maize gene, but one skilled in the art knows that smiRNAs can be engineered to silence many genes. As an example of another gene that can be silenced, a shmiRNA targeting Tad2-1 was constructed as described in Example 2, artificial sequences indicated by a dash were constructed as described in

Example 3, and precursor siRNAs were generated as explained in Example 4. These constructs are listed in Table 8.

Table 8

Precursors of artificial miRNAs targeting fatty acid desaturase

Then these DNAs were cloned in the 5'-3' direction of the 16 kDa promoter of the 7/ea tauv5 gene, which includes 5 TA (untranslated region), in the PHP20790 plasmid. РНР20790 includes Saiezhmau (Ipmyktodep) 1/1 and 12 sites. The resulting plasmids were then recombined with the PHP20622 plasmid.

The resulting plasmids were then co-integrated into the strain Adgobasiegsht I BA4404 plasmid

RNRI10523 and used to transform maize embryos from cultivar (53) pollinated with pollen from cultivar НСб69. Plants were allowed to regenerate and crossed using the transgenic plant as female and НСб9 as male. Seeds were collected and analyzed for fatty acid content by standard methods.

GC (gas chromatography) analysis of BAME (fatty acid methyl ether) was used to investigate whether expression of smiRNA changes the fatty acid profile of maize seeds. Approximately 50 seeds per event were analyzed and 18-25 events per construct were analyzed. Hexane, 1.5 mL, was added to the crushed corn in the extraction tray. Hexane with dissolved oil was transferred to 1.8 mL glass GC vessels, to which 100 mL of trimethylsulfonium hydroxide was added for transesterification. Fatty acid methyl esters (1

Hl was injected from the hexane layer, the dilution index was 80 to 1) was separated and quantitatively

Zo was determined with the help of a gas chromatograph Adiyeyepi 6890, equipped with a 15 m capillary column 7ergop 2B-mach, hole diameter 0.25 mm, film thickness 0.25 μm. (According to catalog No. 7ЕС-4007-11, Rpyepotepeh). The oven temperature was isothermal 220 "C for 2.5 minutes.

The carrier gas was helium of a standard grade. The retention time was compared with that for methyl esters of commercially available standards (My-SNek Rger, Ips.). General approaches to altering and analyzing corn oils by down-regulating fatty acid desaturase metabolic pathways can be found in US Patent Application Mo. 10/223646. The results are shown in Table 9.

Constructs with a 168a core and a 396p core provided effective silencing. Constructs made as containing the 159s core and the 169 core did not produce any silencing (90 Tad2-1 silencing; which is the percentage of events showing silencing).

Subsequent generations of transgenic seeds were grown in the field, and T2 and TZ seeds were tested for fatty acid content. As it was shown, the effect of these constructs is hereditary and stable.

When compared to the silencing achieved by siRNA targeting ROB (95 ROB silencing in Table 9), some precursor miRNA backbones were found to be silent in many tissue types (396p and potentially 1641), while others were more tissue-specific, in which they are effective (159c and 169g). All of the five miRNA backbones showed efficacy in at least one tissue type.

Table 9

Precursors of artificial miRNAs showing electoral efficiency 5EO Y MO 95 silencing ROB 95 silencing yad2-1 22.31 ns shin shi 164p o 23, none | (|: 8 7777/7// | about no data7/7/7/ 24.32 25.33 02581110

Job 26,34

Possible explanations for the results presented in Table 9 include, but are not limited to, differential stability of miRNA precursors in temporal, spatial, or organ-specific ways; differential processing of miRNA precursors (the basis for the stages of differential post-transcriptional processing for miRNAs was found in animal systems ObBetovieteg eyi a). (2007) VAMA 12: 1161-1167); or differential competition for a different complex among miRNA populations.

Example 11

Artificial miRNAs to silence lethal leaf spot

The above examples show the silencing of the maize ROB gene and the Tad2 gene, but one skilled in the art knows that smiRNAs can be constructed to silence multiple genes.

As an example of another gene that can be silenced, a miRNA targeting lethal leaf spot was constructed as described in Example 2, the miRNA is 5'- ishidaaaddasddidiaadds-3" (the DNA sequence corresponding to this miRNA is presented in the ZEO

IO MO: 35). Artificial sequences indicated by a hatch were constructed as described in Example 3 and shown in Table 10. Precursor smiRNAs were constructed as explained in Example 4.

Table 10

Sequences are marked with a dash, artificial microRNAs with a dash 168a-1i5 asspaaissvisstisava 69-16 sessiasassivisstshsvas 396p-Й5 esospasavsrissshslyai

Maize plants in which the lethal leaf spot gene was silenced (MOVI pitrbeg, 77345; Stgau Uevyi a. (1997) A pomei zirrgezzog oi sei! ayeaiyy ip riapi5 epsodeid Bu She I I51 depe oi taiie. Seii. Arg 4; 89( 1):25-31.), reveal a phenotype of lethal lesions in all developing leaves, and in some cases plant death. 96 silencing was determined by visual inspection of the plants approximately 8 weeks after the plants were transferred to the greenhouse. All of them

Of the three constructs, effective silencing was achieved (Table 11).

Table 11

Constructs of artificial miRNAs effectively silence the lethal spotting of the leaves

RNRZB155 168a-1Y5

RNR 33788 69I-Yiv 2801

РНР 36154 396p-v

Example 12

Artificial miRNAs for both fatty acid desaturase and multidrug resistance protein

Sometimes it is desirable to silence more than one gene with a given construct.

Individual smiRNA precursors can be functionally linked to the same or to different promoters. Alternatively, two or more smiRNA precursors can be functionally linked to each other, and then linked to a single promoter. Two or more shmiR can be obtained from such a construction? NC.

Constructs comprising artificial miRNAs to silence Tad2g-1 are shown in Example 10. As an example of another gene that can be silenced, an artificial miRNA targeting the multidrug resistance protein (MDR) was constructed as described in

In Example 2, the miRNA is 5'-chaashisasaashsisassasis-3" (the DNA sequence corresponding to this miRNA is presented in 5EO I MO: 39). The artificial sequences marked with a dash were constructed as described in Example 3 and are shown in Table 12 Two precursor MAP miRNAs, 168a-MAR (5EO IO Mo:42) and 3960-MAR (5EO IO Mo:43) were generated, as explained in Example 4. Silencing of MAR leads to a decrease in phytic acid (Pi). (2007).

Table 12

Sequences, marked with a dash, of artificial microRNAs precursor of shmiRNA ZEO IU MO 168а-МАР zavehosivoano mana 396p-МАР zasos

These progenitors were cloned either in the 3-5" direction or in the 5-3 direction of the Tai 2 progenitors (described in Example 10) to generate cassettes, and then cloned into constructs and transferred into

Adhorasiegist to generate the constructs described in Table 13 as previously described.

Maize transformation was performed as described in Example 6, and transgenic seeds will be evaluated for phytate and fatty acid content.

Table 13

Artificial miRNA constructs, including smiRNAs, were constructed to silence as

Then 2-1, these are the MAR numbers of the Russian People's Republic

RNRZ8380 16v Ador2g-1/16va-MA R

RNRZ38381 396p-Rad2-1/168a-MA R

RNRZ38382 16va-MA R/396p-EAO2-1

RNRZ38383 z96p-MAR/ZO6P-EAO2-1

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ЗЕВС поко свист ссдоорнес ес песо ко ост коазос ссвсКоцеяс оз село ечасопцаєво песо аю есе савсЕссоя слово Состав засос песо НУ хори сос своє ссссессекл Собака ко Сеасосакою соч АБО шЕсксчКссо Єесасаеаччє соці ссз сасзвосов опвассенни осо 20 пеасесзессов сасдосзвссаес сбсаосвюа сао счаавсс свои оса БЕК НИ севжусоса сбекксоссоце vosoozaoe soc so sosvy sepacheosoV i staltassoe sok esE sssr ZuKscoKok sot ossatsa pe ao B speku zok pines: sbozaslazo sezdsasext solo oskoz se e 5 vimnsheoskooe black seg VA "2105 23 "2115 739 "212" DNA "213" Artificial "220" "223 » 1641-ROB5 miRNA predecessor 400» 23 ne ss EK EE cesaseoi Vot ches haseh zdo doopeshechsa i: zchikasektes Sesam Zesaoss zosofaoasia oso azs sko sssEsrvovs zaachoseyu poem sozvatchecheok sor sosie peso me zschovieIo sposo peso ie ze ssspcovmoeh esosae va poesosoio pesososoe person nipple 35 zElospevys sec Yevuzsspsssva pososchea ssavsakom opa la zZizksssvovV somy oSK sokosvyevVeh svkksvyke basslezov sova 8 aptozvssazoaes sleepy zopsa ваевсє ззЗеЕсзчсвсй зом Коновомоу р сутханзосввЗи чзеаккодаВек сою оку вуса зо м чес есосвеє сука СІМмменосос сопе чес зов оаЕ ЯНВ всзевсгста чсассскоца звссвваочен зссесосиа защБЕгоєка стос о ко вповні зві соцення поса Клео кеє са ссаесав чзецавВоноса Та асассассце сосни Зо з «2105» 24 «2115 791 "212" DNA "213" Artificial "220" "223" 16va-РО5 miRNA precursor

"400" 24 sheassksksche sposo heart "fan opera lov sixte o 5 zvossschosa zezssschosa syusessovaso sopaosshos sova akko sokah 130 ouvoyeeso hung Zososspese zchasoosshio your sossgu sossice i5o sessoszastye zov sees secho or KoZEsXsSKe oso sova ko ha what stvo oss zo tesosesasichs Nssos ssoz сесессвву Касовоєює засо тес кссваввие БЕЗЕ певосксме сосаззсесее сечеККЕсЗК сечо зов седшсвокя БУК совки воював песзаоюви зоесеацєрся Ка сце ує що сассосленс соаосине спосо Ессе состав ооо ЗУ ставок асувсоЗесто ессобесткас сезЕссвасоЕ Зак У За еКНУи во сЕсотжККК Єстобовасав вассводесо Часто весетвгою дес етоя ва капсочечак засос спаеєссіуя ков ЄВотв zossmaava staff and vychsshsssva szavovsaechs sszososao aceszaodaoe zessnove zvo tvo zussakko TUH

"2105 25 "-2115" 860 "212" DNA "213" Artificial

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B ii zi sisnmh s S Bezh eOYA "223" piza, p, 9, ogi kume et M ee v EMV zh ek vyshch ZK eNUMVV sha shosse SES Sosny shosse pryus vytya Kyki skh m izhizhmh Mk uky MK zvu fk ce pref ek fe uche et du i od Matt NN

SA KINEMATICS EV SA OCE I ож тур чат EI. fedin zhi gte u kiv fetr dra Zhe Kerr teh KN shlamy ma U nI Myum m K lam SMAK UK M UK Ya AK DOAZ drink kuvizh o go tt tugym UK uK ura baku nase s Mito, Ag ke ka U VAMI M sche ke Ge Kama hya EI U pvh Dr Du i MKU KUKI Z MKU xx la phtzhi i yuyu sew kut kit ut ozh zhemetiih zhchechuchiu tity x Z eudy Kt M etet fetr ki ogh YAZ syVshsseminM sausages hearty sesotsochna sword chena YAM e - -. zach Jak erkt putch p tk v zh pi V Ko z aeyuiviu ok M m oche tu pekrs;sachis sososszechs pozchesavis zoseaosvivaye svosakacee aso io "pine spesrsoch pose sava plesa zvo zavshevya Se KUh re vavssssoseyes aabovsovad dzvosae BESHOOsemMaist nipples Vo oVoveo 5 pyhi poppy yukЖ Fu in emu kih pet ож in their ota, o nn NN you o ne a NA YAYA peassochomechev sao ov voVvsooeasoya sao Wow. zako a savse sSzassasaotse sozisaleov papilla sbovayeaaaknyane Bash that judges Kr Mamo td Oky A wl U a

CHEM KY SEN S re that "2105 27 "2115" 96 "212" DNA "213" Artificial "220" "223" 1691/-ROB-short miRNA precursor "4005" 27 ptatovlsve ss ossovach vssoseodova sSebolates lateral rest from six Ex oZ anew Owls ZE "2105" 28 "-2115 305 "212" DNA "213" Artificial "220" "223" 1691-РО5-medium miRNA precursor "400" 28 ssspssvasno vorde aleaosvoya sovsaceso zossoaeoyyuvi emu Zo chamyssye sossonso nipple Koschevustvya masa kyah sunny 155 ktsvosvoa sposo sposo Cleot tovo robo sm ate pruzhuzheit Mu uFivuze ti Ege uukhin M am sew vy he i a shi ev No KehuzhiMMK sim xx A y NO

Vyko spets srossomos ho mly and SI eat chovyszesev Fe osrache sssr asavoaEsvVie zZoeSssaskio Za aanso 28 coffee 35 "2105" 29 "2115117 212" DNA "213" Artificial

From "220" "223" 3961p-ROB-short miRNA precursor "400" 29 voshshovoetv savVEsesvy zoba sovy boat chom Erevohkive Tu pam tsav soch ossav sazseoaomi sob svaYeszegov so Ya -2105 30 "2115 292

"212" DNA "213" Artificial "223" 3961-ROB-medium miRNA precursor "400" 30 cocoa press it nd z kah Zh M la itiv tszsschiktsiuchichu ve ih bugs fu uh V dv lizh sich zhZyEsososma pos soa Sas shosse sake ZE KV HO started ichchs srossmooeya Sossochevoas aaBseslasse ss vV Savu Ж zo seok ches Ves szavy r resskaze your Zoo yin avsoszsmny sezaKseseya sao chis chao opse tsNe ke IH se s wort EK ONUKA Sokom skh "I -2105 31 "-2115" 684 "212 » DNA "213" Artificial "220" "223" 159s-EAO miRNA precursor "400" 31 poka kralya yav sec a skkih E I kine mly pIPaAOose PO KI I: ge BO pana chi UNK NI en pk vvihi pub pub uyu u yu use them sf them taught by them name lyu write sUu Vinos ns You Sasha ku I drank them PEK KL I On them if In backgrounds about me in uchuchuchischo iz and in gu zh ch u zh M zhim in killed r syh Al chOvstgzhyesu zschsrosgozsyu seasezvless sides aso sheeS Ese TE KO

Or vych du zrm ee i o S m o i Ky U tran ik i u ke izuititi Di i NI ss ss Kon sov palat atsasopahcho es kA en v NN v i NN i KK ef ze keuzhi and wife Ka ethos sososizchaKo posa Uch became his Yerko meh WITHOUT kefsoeisenu osaspivyssva sospevssvimu spra Severoeeyuu ooo zva p feyu info kyu. zhe pky Still EE zhimi Still zo skol Ky yus EE shu fr ry V usu KK pESssocKKe pocha ka comedian peso sope KN sche chn NN NK pkt zh Azha kinu ni NK di ee sne shoBIOkoUye soma sao Be ZNO sesshesoone sssosskoso wssbozaovko soaochevovs obezoasesevo BusSachueusUou Za man cupcakes glass KK weight Beka HE ss asssvEs Odessa seseeooaaya sazsvochea se soyekoyu sister ke pe pn nn kna na pn sho vysh ches Vescha "-2105 32 "2115 790 "212" DNA "213" Artificial "220" "223" 16va -EAO miRNA precursor

From "4005" 32 more sssocis cheshe ooo zeoaasisoe sposo schi aavsozoeshesye Chetsshssosse solo sovo ss sova seo steVokye Mai faavoassopeoya sezschvyssv seoscheososyu sabo cho to semo 15

Kshopsueletse sashe village srsr so o: deka e BE sssr oassist sozsschvseyu about sossoyu ossrzachave sego sossos Z ssssssosos szpossvoVO sSzasopechsos sia sopee ssbrchsoyeshev peso Zp trap bovlasenie sbaksheoktsa oche opaavavishyeyu series same passveklis sas saVso sope chosimcheno tale secho SUS bvospsseatchs sezos sezse zone 4 праска вбшнососеу за саёе sсропвеееении зыпвесе зов r: скссссеекев ссссссвача ааесбвсяссоз зчасесојео зсозосаечос сосвеосто 50 kabekovza: peso zve oseov sopevsvek vu achopskvasvaa se snaE i zaasoeso waza oe ozone en vo ochova sao BUT it av "2105" 33 "21151" 2" 861 " DNA «213» Штучна «220» «223» 1691/-ЕАО міРНК попередник 400» ЗЗ рони зов ЗеНасеєтюня сс Еюююи екЗУеНЯевУ везе а сфе с посох ссасесомов бегмучею есе сус їх зе сокчоцч соссодааєс свинки сосеосеє сс ече сова 185 взбссбсеесув сссссстсоко свосаасвес сзауЕЕсКехе осбоиссвєох аодцссекосВ zha che Gaya g vezh i kodi i NN i tu zpsvoschssov vozoaossasg zesskonass sesesaksvk semokoens stva aeno KN lsvavozasva with iyo fasok ovav Boyle ssasssevis osa Bo paz owl pup aero ovcha aye papoaszwa apa zay shose ves soraka ssannia psa setiV Ve nMh B popes sov sosg seapovVoe baltesrae ZosVzsvVa Frltuih Br kchakochaszav sashe ssKoakome soshsenno zle ovogeaa o gtktichch shpyudi im ee na i in NN Nya shi chchsevet she oasev the term of the son of a social worker

KO AN fax s nya en NN NN uk ххема SSZ tnSkU ap eMaeo lasi bas se kla NE sietdu put vit presitu softu is vich BOM f uzkoviichuchit ti uni sim sv iodide v shumi kore

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YAKA KI O KO al KANA KONKU OM s azs asv sochziaschkv ZHAN "2105" 34 "2115 633 "212" DNA "213" Artificial "220" "223" 3961-EAO miRNA precursor "220" "221" tivzs iyeashte "2225" (594). NI U SN KN em zhu szheh szheh zhuh u iktiv zhu M ekery metu sno o o NN i chechei osa no moshok sce se steoe esse oman I

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When: asso tsovV zo sovno vezoveaaKo om o a od NK i pozhryu m rik fo ku iyu svkh je hu ik S pechu dO soavknis posa sava KOoshchshesassam stos ova poza aAVIVya village BU kmin lm, Ko g feolh tu llyn m dz uz ih ra i koh Z sao Sawa ssv Ks SK a "2105 35 "2115 21 "212" DNA "213" Artificial "220" "223" lethal leaf spot miRNA "4005" 35 skh BUT M KANOM E "AH "2105" 36 "2115 21 "212" DNA "213" Artificial

"020" "223" 168va-115-sequence marked with a dash "400" 36 vssveveaes ssosvEssva in 21 "-2105" 37 "-2115 22 "212" DNA 213" Artificial "020" "223" 69I-115-sequence, marked with a stroke "400" 37 highway perso a de yy "210" 38 "2115 21 "212" DNA 213" Artificial "020" "223" 3961p-115-sequence marked with a stroke "400" 38 posekasas sauce i and

From "210" 39 "2115 21 "212" DNA "213" Artificial "020" "223" MAR miRNA sequence "400" 39

BHaasrsaova soesevosasK is in "-210" 40 "2115 21 "212" DNA "213" Artificial "020" "223" 16va-MAP-sequence, marked with a dash "400" 40 5O of its origin in "2105" 41 "2115 21 "212" DNA "213" Artificial "020" "223" 396p-MEAR-sequence, marked with a dash 60 "400" 41 used wash in the cheeks

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THE LIFE Ege Ed M mu gu sodu Me tuyu rhythms of those tshihA highway cheO chssnst scenes shchen I ie M KMU KE KIM KOH

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Ko a o van o o i oo o a o Ne NI ut o u M feya sim fe here

I x FETA I KATA still es ss pas peruk MO Zhe nin i NN NN KA NK A NN in NK KU gi

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STЕЖшКЕКЕЕК Кобогосваяяв ввастеасоплеч час се весеовео цес коя веб до те дек жор журу ючы и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и и.

AK OO z OA AD S KZ d Uk MY OO UYSATA MY KU. MOMS AKA MM petiyuya s zimi x xx b

Pro i i NK i V NI part of the senate MEZO TKS K cm Hundred day what vky "az rhou Kv etkhv kazan shen "2105 43 "2115 633 "212" DNA "213" Artificial "220" "223" 396p-MAR "220 » "221" tivzs iyeashte "2225 (594)...(594) "223" piza, p, 9, ogi

From "400" 43 shosssssvov BEK ovas seesvosiki sosssvoveyes chuv eschsa veVee ve SCHO sache schee sonu fkavkev sala sepevsoh sabo seoh I5 zhk oKOs sova zasos lavs koasosvayae zaosaschchemaiya ches zp saksvsovah svezskfseo edepeassya des oass dos aoak skosa sha sssassocs chossassos ssio Svapovs sssssozuo sSsrlnmyaza bezos abs forty zap avassmnnI a zalo szpasuoZVya seKosshchanoh vpesosaak ze aome ana secha svoi Vzvizozhov slpachschkasvo all savyUk grief B honors s orav pospoKwasZv doros ska kine GOATS NO rations so BezaschsvVe ze ko ZNM

Claims (5)

FORMULA OF THE INVENTION 1. An isolated nucleic acid fragment comprising a deoxyribonucleotide sequence as described in 5EO IU MO: 14, where (i) nucleotides 110-130 in 5EO IO MO: 14 are replaced by a first variable nucleotide subsequence, the size of which varies from 19 to 24 nucleotides depending from the target sequence whose expression is to be reduced, (ii) nucleotides 184-203 in ZEO IU MO: 14 are replaced by a second variable nucleotide subsequence, the size of which varies from 19 to 24 nucleotides, and the specified second variable nucleotide subsequence is able to hybridize with the first variable subsequence of miRNA -precursor, and (iii) miRNA-precursor, produced by the specified isolated nucleic acid fragment, has the same stem structure as miRNA-precursor, produced by endogenous 5EO IO MO: 14. 2. Recombinant construct, including the selected fragment of nucleic acid according to claim 1, functionally linked to at least one regulatory sequence. 3. A plant cell including the recombinant construct according to claim 2. 4. Plant cell according to claim 3, where the plant cell is a plant cell of a monocot plant. 5. A method of reducing the expression of a target sequence in a plant cell, according to which: (a) at least one plant cell is transformed with a nucleic acid construct that includes a deoxyribonucleotide sequence, as described in 5EO IU MO: 14, where (i) nucleotides 110-130 in 5EO IO MO: 14 replaced by a first variable nucleotide subsequence, the size of which varies from 19 to 24 nucleotides depending on the target sequence whose expression is to be reduced, (ii) nucleotides 184-203 in 5EO IU MO: 14 replaced by a second variable nucleotide subsequence, the size of which varies from 19 to 24 nucleotides, wherein said second variable nucleotide subsequence is capable of hybridizing to the first variable subsequence of the precursor miRNA, and (ii) the precursor miRNA produced by said isolated nucleic acid fragment has the same stem structure as the precursor miRNA produced endogenous 5EO IU MO: 14; and (b) select the transformed plant cell(s) whose expression level of the target sequence is reduced compared to the expression level of the target sequence in the wild-type plant cell.