Plant regulatory elements and uses thereof

Abstract

To provide recombinant DNA molecules and constructs, as well as their nucleotide sequences, useful for modulating gene expression in plants.SOLUTION: The present invention provides novel synthetic gene regulatory elements for use in plants. The invention also provides recombinant DNA molecule constructs comprising the regulatory elements. The invention further provides transgenic plant cells, plants, and seeds comprising the regulatory elements. In one embodiment, the regulatory elements are operably linked to a transcribable DNA molecule. In certain embodiments, the transcribable DNA molecule may be heterologous with respect to the regulatory sequence. The invention also provides methods of using the regulatory elements, and making and using recombinant DNA molecules comprising the regulatory elements, as well as transgenic plant cells, plants, and seeds comprising the regulatory elements operably linked to a transcribable DNA molecule.SELECTED DRAWING: None

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 714,228, filed August 3, 2018. No. 60 / 699,999, filed on Oct. 1, 2003, which application is hereby incorporated by reference in its entirety.

[0002] Incorporating a sequence listing Name: "38-21-62691-0001_Seqlist_ST25.txt" The sequence listing contained in this file is 31,060 bytes (MS-Windows (registered trademark) This sequence listing is based on the data obtained on July 2, 2019. No. 60 / 699,999, filed herewith by way of a prosecution application and incorporated herein by reference.

[0003] The present invention relates to the fields of plant molecular biology and plant genetic engineering. More specifically, the present invention The invention relates to DNA molecules useful for modulating gene expression in plants. [Background technology]

[0004] Regulatory elements regulate the transcription of an operably linked transcribable DNA molecule. Thus, they are genetic elements that regulate gene activity. Such elements include: These include promoters, leaders, introns, and 3' untranslated regions. are useful in the fields of plant molecular biology and plant genetic engineering. Summary of the Invention

[0005] The present invention provides novel synthetic gene regulatory elements for use in plants. The present invention also provides recombinant DNA molecule constructs that include regulatory elements. Transgenic plant cells, plants, and seeds containing the elements are also provided. In this configuration, the regulatory element is operably linked to the transcribable DNA molecule. In certain embodiments, the transcribable DNA molecule may be heterologous to the regulatory sequence. Therefore, the regulatory element sequences provided by the present invention may, in certain embodiments, can be defined as operably linked to a heterologous transcribable DNA molecule. The present invention also provides a recombinant DNA molecule comprising a regulatory element, and a transgenic vector containing a regulatory element operably linked to the transcribable DNA molecule. Methods for making and using the nicked plant cells, plants, and seeds are also provided.

[0006] Thus, in one aspect, the present invention provides a recombinant DNA molecule comprising: (a) the sequence At least about 85 percent sequence identity to any of SEQ ID NOs: 1-19 and SEQ ID NO: 26 (b) a sequence including any one of SEQ ID NOs: 1 to 19 and SEQ ID NO: 26; and (c) a fragment of any one of SEQ ID NOs: 1 to 19 and 26, which has gene regulatory activity. a heterologous transcribable fragment of a DNA sequence selected from the group consisting of: A recombinant DNA molecule operably linked to a heterologous transcription-competent DNA molecule is provided. A "transcribable DNA molecule" is a polynucleotide sequence to which a transcribable DNA molecule is operably linked. In certain embodiments, a recombinant DNA molecule refers to a molecule that is heterologous to a sequence. At least about 85% of the DNA sequence of any of SEQ ID NOs. 1 to 19 and SEQ ID NO: 26 cents, at least about 86 percent, at least about 87 percent, at least about 88 percent percent, at least about 89 percent, at least about 90 percent, at least 9 1 percent, at least 92 percent, at least 93 percent, at least 94 percent, at least 95 percent, at least 96 percent, at least 97 percent percent, at least 98 percent, or at least 99 percent sequence identity In certain embodiments, the DNA sequence comprises a regulatory element. In some embodiments, the regulatory element comprises a promoter. In some embodiments, the regulatory element comprises an intron. The element comprises a 3'UTR. In yet another embodiment, the heterologous transcribable DNA The molecules may be genes of agronomic interest, for example genes capable of providing herbicide resistance in plants, or a gene capable of providing plant pest resistance in a plant. Therefore, the heterologous transcribable DNA molecule can be a small RNA (e.g., dsRNA, miRNA, In yet another embodiment, the present invention provides a method for the preparation of a medicament for the treatment of a medicament comprising administering to a patient a medicament for the treatment of ... Constructs are provided that include the recombinant DNA molecules provided herein.

[0007] In another aspect, provided herein is a transgenic plant cell comprising: (a) a sequence At least about 85 percent sequence identity to any of SEQ ID NOs: 1-19 and SEQ ID NO: 26 (b) a sequence including any one of SEQ ID NOs: 1 to 19 and SEQ ID NO: 26; and (c) a fragment of any one of SEQ ID NOs: 1 to 19 and 26, which has gene regulatory activity. a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: a transgenic vector in which the DNA sequence is operably linked to a heterologous transcribable DNA molecule; In certain embodiments, the transgenic plant cell is In another embodiment, the transgenic plant cell is a dicotyledonous plant cell. It is a biological cell.

[0008] In yet another aspect, the present invention further provides a transgenic plant or a) at least 8 of any of SEQ ID NOs: 1 to 19 and SEQ ID NO: 26 a) a sequence having 5% sequence identity with any of SEQ ID NOs: 1 to 19 and SEQ ID NO: 26; c) a sequence comprising SEQ ID NOs: 1 to 19 and SEQ ID NO: 10, which have gene regulatory activity; A recombinant DNA comprising a DNA sequence selected from the group consisting of any one of 26 fragments. A molecule, said sequence being operably linked to a heterologous transcribable DNA molecule. In certain embodiments, transgenic plants or parts thereof are provided. A transgenic plant is a progeny plant of any generation that contains a recombinant DNA molecule. transgenic seeds which, when grown, produce such transgenic plants. Also provided herein are transgenic seeds comprising the recombinant DNA molecules.

[0009] In another aspect, the present invention provides a method for producing a commodity product, comprising the steps of: Obtaining transgenic plants or parts thereof containing NA molecules and producing them commercially. and producing a commodity product. In one embodiment, the commodity product comprises: Seeds, processed seeds, protein concentrates, protein isolates, starches, grains, plant parts, seed oil, biomass, flour, and meal.

[0010] In yet another aspect, the present invention provides a transgene comprising a recombinant DNA molecule of the present invention. A method for producing a genetically modified plant comprising transforming a plant cell with a recombinant DNA molecule of the present invention. and producing a transgenic plant from the transformed plant cell. and regenerating the

[0011] A brief description of arrays SEQ ID NO:1 is coupled to a synthetic leader (L-Zm.GSP850.nno:3) at the 5' end. a synthetic promoter (P-Zm.GSP850.nno:4) operably linked to the 1 shows the DNA sequence of the regulatory expression element group (EXP) EXP-Zm.GSP850.

[0012] SEQ ID NO:2 is the DNA sequence of the synthetic promoter P-Zm.GSP850.nno:4. be.

[0013] SEQ ID NO:3 is the DNA sequence of the synthetic leader L-Zm.GSP850.nno:3 .

[0014] SEQ ID NO:4 is a sequence consisting of a synthetic intron (I-Zm.GSI153.nno:1) and a 5'-side sequence. The 5'-terminal fragment was constructed with a synthetic leader (L-Zm.GSP850.nno:3) movably linked to the 5'-terminal fragment. operably linked to a synthetic promoter (P-Zm.GSP850.nno:4), Synthetic EXP, EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 It is a DNA sequence.

[0015] SEQ ID NO:5 is the DNA sequence of the synthetic intron I-Zm.GSI153.nno:1. do.

[0016] SEQ ID NO:6 is coupled to a synthetic leader (L-Zm.GSP990.nno:1) at the 5' end. a synthetic promoter (P-Zm.GSP990.nno:2) operably linked to the DNA sequences of EXP and EXP-Zm.GSP990.

[0017] SEQ ID NO: 7 is the DNA sequence of the synthetic promoter P-Zm.GSP990.nno:2. be.

[0018] SEQ ID NO:8 is the DNA sequence of the synthetic leader L-Zm.GSP990.nno:1 .

[0019] SEQ ID NO: 9 is a sequence consisting of a synthetic intron (I-Zm.GSI197.nno:1) and a 5'-side sequence. The 5'-terminal fragment was constructed with a synthetic leader (L-Zm.GSP990.nno:1) movably linked to the 5'-terminal fragment. operably linked to a synthetic promoter (P-Zm.GSP990.nno:2), Synthetic EXP, EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 It is a DNA sequence.

[0020] SEQ ID NO: 10 is the DNA sequence of synthetic intron I-Zm.GSI197.nno:1 be.

[0021] SEQ ID NO: 11 is a sequence consisting of a synthetic intron (I-Zm.GSI140.nno:1) and a 5' sequence. operably linked to a synthetic leader (L-Zm.GSP850.nno:3) on the 5' side operably linked to a synthetic promoter (P-Zm.GSP850.nno:4) , composite EXP, EXP-Zm.GSP850.nno+Zm.GSI140.nno:1 This is the DNA sequence.

[0022] SEQ ID NO: 12 is the DNA sequence of synthetic intron I-Zm.GSI140.nno:1 be.

[0023] SEQ ID NO: 13 is the DNA sequence of the synthetic 3'UTR, T-Zm.GST9.nno:2. do.

[0024] SEQ ID NO: 14 is the DNA sequence of the synthetic 3'UTR, T-Zm.GST18.nno:2. be.

[0025] SEQ ID NO: 15 is operably linked to the intron (I-Zm.DnaK:1) on the 5' side. It is operably linked to the synthetic leader (L-Zm.GSP850.nno:3) at the 5' end. In addition, synthetic EXP, E, containing a synthetic promoter (P-Zm.GSP850.nno:4) This is the DNA sequence of XP-Zm.GSP850.nno+Zm.DnaK:1.

[0026] SEQ ID NO: 16 is operably linked to the intron (I-Zm.DnaK:1) on the 5' side. It is operably linked to the synthetic leader (L-Zm.GSP990.nno:1) at the 5' end. In addition, synthetic EXP, E, containing a synthetic promoter (P-Zm.GSP990.nno:2) This is the DNA sequence of XP-Zm.GSP990.nno+Zm.DnaK:1.

[0027] SEQ ID NO: 17 is a synthetic promoter derived from P-Zm.GSP850.nno:4 1 is the DNA sequence of the gene enhancer E-Zm.GSP850.

[0028] SEQ ID NO: 18 is a synthetic promoter derived from P-Zm.GSP990.nno:2 1 is the DNA sequence of the gene enhancer E-Zm.GSP990.

[0029] SEQ ID NO: 19 identifies NLTP4 (non-specific lipid transport protein 4) of Sorghum bicolor. 3'UTR from the protein 4 gene, T-Sb.Nltp4-1:1:2 DNA It is an array.

[0030] SEQ ID NO: 20 is the potato light-inducible tissue-specific ST-LS1 gene (Genbank β- with a processible intron derived from A synthetic coding sequence optimized for plant expression of glucuronidase (GUS).

[0031] SEQ ID NO: 21 is the 35S promoter and linker from the cauliflower mosaic virus. The DNA sequences of EXP and EXP-CaMV.35S, including the promoter, are shown.

[0032] SEQ ID NO: 22 is the heat shock protein 70 (Hsp70) gene of Zea mays This is the DNA sequence of intron I-Zm.DnaK:1 derived from (DnaK).

[0033] SEQ ID NO: 23 is the lipid transfer protein-like gene (LTP) of Oryza sativa This is the DNA sequence of the 3'UTR, T-Os.LTP:1, derived from

[0034] SEQ ID NO: 24 is the potato light-inducible tissue-specific ST-LS1 gene (Genbank β- with a processible intron derived from This is the coding sequence for glucuronidase (GUS).

[0035] SEQ ID NO: 25 is the sequence of the NanoLuc® luciferase fluorescent protein (Pro Mega, Madison, WI 53711). Nluc is a coding sequence of the deep-sea Directed evolution of shrimp (Oplophorus gacilirostris) luciferase It was operated by.

[0036] SEQ ID NO: 26 is the DNA sequence of the synthetic 3'UTR, T-Zm.GST43.nno:1 be. DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention provides synthetic regulatory elements that have gene regulatory activity in plants. The nucleotide sequences of such synthetic regulatory elements are shown in SEQ ID NOs: 1 to 18 and SEQ ID NO: 26. Such synthetic regulatory elements are provided as operably linked It can affect the expression of transcribable DNA molecules and therefore transgenic The gene expression of an operably linked transgene can be regulated in the plant. The present invention relates to a novel intracellular nucleotide sequence having gene regulatory activity in plants and provided as SEQ ID NO: 19. The present invention also provides synthetic and endogenous regulatory elements. Also provided are methods for modifying, producing, and using recombinant DNA molecules containing the compounds of the present invention. transgenic plant cells, plants, plant parts, Also provided are compositions containing the and seeds, and methods for preparing and using the same.

[0038] The following definitions and methods are provided to better define the present invention and to guide those skilled in the art in practicing the invention. Unless otherwise specified, the terms are provided for ease of understanding by those skilled in the relevant art. It shall be understood according to conventional usage.

[0039] dna molecule As used herein, the term "DNA" or "DNA molecule" refers to the 5' (upstream A double-stranded DNA fragment of genomic or synthetic origin that is read from the 3' (downstream) end to the 3' (downstream) end. "DNA molecule" refers to a polymer of deoxyribonucleotide bases (i.e., a DNA molecule). As used herein, the term "DNA sequence" refers to the nucleotide sequence of a DNA molecule. The nomenclature used herein is in accordance with the nomenclature of 37 CFR 1.822. The table in Annex 2, Tables 1 and 3 of WIPO Standard ST.25(1998) corresponds to the Act. are.

[0040] As used herein, a "recombinant DNA molecule" refers to a molecule that is naturally occurring without human intervention. A DNA molecule that contains a combination of DNA molecules that would not occur otherwise. For example, a recombinant DNA A molecule is a DNA molecule composed of at least two DNA molecules of different species. DNA molecules containing DNA sequences that deviate from those found in DNA molecules or those incorporated into the DNA of a host cell by genetic transformation or gene editing It can be an embedded DNA molecule.

[0041] As used herein, a "synthetic nucleotide sequence" or "artificial nucleotide sequence" refers to a nucleotide sequence that is not known to occur in nature or that does not occur in nature. The gene regulatory element of the present invention comprises a synthetic nucleotide sequence. Synthetic nucleotide sequences share little or no stretch of homology with natural sequences An extension of homology in this context generally extends beyond about 25 nucleotides of the contiguous sequence. The greater the extension, the greater the 100% sequence identity.

[0042] References in this application to "isolated DNA molecules" or equivalent terms or phrases are The DNA molecule may exist alone or in combination with other components, but not in its natural environment. For example, a coding sequence, such as intron sequences, untranslated leader sequences, promoter sequences, transcription termination sequences, etc. A nucleic acid element that is naturally found within the DNA of an organism's genome is one that is unique to that organism. As long as the element is in the genome and in the location in the genome where it is found in nature, However, each of these elements and the The subpart of the element is that the element is not in the genome of the organism and the element is not found in nature. is considered "isolated" within the scope of this disclosure unless the location within the genome is such that Similarly, an insecticidal protein or any naturally occurring insecticidal variant of that protein A nucleotide sequence encoding a protein is a sequence of nucleotides encoding the protein. Unless the corresponding sequence is within the DNA of a naturally occurring bacterium, it is considered an isolated nucleotide sequence. Synthetic nucleotide sequences encoding the amino acid sequences of naturally occurring insecticidal proteins A string would be considered isolated for the purposes of this disclosure. In this context, any transgenic nucleotide sequence, i.e., Nucleotides of DNA inserted into the genome of a cell or present in an extrachromosomal vector The code sequence is determined by whether it is present in a plasmid or similar construct used to transform cells. whether present in the genome of a plant or bacterium, or derived from a plant or bacterium Although present in detectable amounts in tissues, progeny, biological samples, or commercial products, The nucleotide sequence is considered to be the sequence of the sequence.

[0043] As used herein, the term "sequence identity" refers to the percentage of two optimally aligned sequences. The polynucleotide sequences or two optimally aligned polypeptide sequences are Optimal sequence alignment refers to the degree to which two sequences, e.g., a reference sequence, and another sequence and manually align them to remove any appropriate internal nucleotide insertions, deletions, or or maximizing the number of nucleotide matches in a gapped sequence alignment. As used herein, the term "reference sequence" refers to SEQ ID NO: 1. 19 and the DNA sequence provided as SEQ ID NO:26.

[0044] As used herein, "percent sequence identity" or "percent identity" or The term "% identity" refers to the percentage of identity multiplied by 100. The "percent identity" in optimally aligned sequences is the percentage of the sequence identity within the optimal alignment. The number of nucleotide matches is calculated based on the total number of nucleotides in the reference sequence, e.g., the total number of nucleotides in the entire full-length reference sequence. divided by the total number of nucleotides. Optimally aligned to the reference sequences shown in the specification as SEQ ID NOs: 1 to 19 and SEQ ID NO: 26 When the sequence is modified, it has at least about 85 percent identity to the reference sequence, at least about 86 percent identity, at least about 87 percent identity, at least about 88 percent identity percent identity, at least about 89 percent identity, at least about 90 percent identity, at least about 91 percent identity, at least about 92 percent identity , at least about 93 percent identity, at least about 94 percent identity, at least about 95 percent identity, at least about 96 percent identity, at least about 9 7 percent identity, at least about 98 percent identity, at least about 99 percent DNA containing a sequence that has at least about 100 percent identity to the target gene A DNA molecule having a certain percentage of sequence identity to a reference molecule is provided. The activity of the reference sequence may be indicated.

[0045] Regulatory elements Regulatory elements, e.g., promoter, leader (also known as 5'UTR) The transcriptional termination region (or 3'UTR) is a region that is expressed in living cells. As used herein, a gene plays an essential role in the overall expression of genes in the organism. The term "regulatory element" refers to a DNA molecule that has gene regulatory activity. As used herein, the term "gene regulatory activity" refers to, for example, the activity of an operably linked transcription factor. operably linked by affecting the transcription and / or translation of a replicable DNA molecule The term "regulatory" refers to the ability of a gene to affect the expression of a transcribable DNA molecule. Elements such as promoters, leaders, enhancers, introns, and 3'U TRs are useful for modifying plant phenotypes through genetic engineering.

[0046] As used herein, a "group of regulatory expression elements" or "EXP" sequence refers to an expression sequence that acts as a operably linked regulatory elements, such as enhancers, promoters, leaders, and For example, the regulatory expression elements may refer to, for example, a leader sequence and The promoter may be operably linked to the 5' end of the gene. Suitable EXPs include SEQ ID NOs: 1, 4, 6, 9, 11, 15, and 16.

[0047] Regulatory elements can be characterized by their gene expression patterns, e.g. Positive and / or negative effects, e.g., constitutive expression or temporal, spatial, developmental, tissue, environmental environmental, physiological, pathological, cell cycle, and / or chemical responsiveness manifestations, and The present invention can be characterized by any combination of these, as well as quantitative or qualitative indicators. As used herein, "gene expression pattern" refers to a sequence of operably linked DNA molecules. A transcriptional pattern is an arbitrary pattern that is transcribed into a transcribed RNA molecule. A transcriptional RNA molecule is translated into a protein. These may produce protein molecules, or antisense or other regulatory RNA molecules, e.g., double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), May also result in microRNA (miRNA), small interfering RNA (siRNA), etc. be.

[0048] As used herein, the term "protein expression" refers to the conversion of a transcribed RNA molecule into a protein. Protein expression is any pattern of translation into protein molecules. Characterized by developmental or morphological properties and by quantitative or qualitative indicators It is possible.

[0049] A promoter is a regulatory molecule that regulates the expression of an operably linked transcribable DNA molecule. As used herein, the term "promoter" is useful as a nodal element. Generally, RNA polymerase II and other proteins (e.g., Promoter refers to a DNA molecule involved in the recognition and binding of a specific transcription factor (e.g., a trans-acting transcription factor). The promoter is first isolated from the 5' untranslated region (5'UTR) of the genomic copy of the gene. Alternatively, the promoter may be a synthetically produced or engineered DNA molecule. The promoter may also be chimeric. A chimeric promoter may be The promoters useful in practicing the present invention are produced by the fusion of two or more heterologous DNA molecules. The vectors are provided as SEQ ID NOs: 2 and 7, or as SEQ ID NOs: 1, 4, 6, 9, 11, 15, and 16, or a promoter element contained in any of these In certain embodiments of the present invention, the present invention provides a method for the preparation of a medicament for the treatment of a medicament comprising administering to a patient a medicament for the treatment of ... The claimed DNA molecules and any variants or derivatives thereof described in the document are also Furthermore, it is defined as containing promoter activity, i.e., transgenic plants. In still further specific embodiments, the promoter can act in host cells such as In this embodiment, the fragment retains the promoter activity of the starting promoter molecule from which it was derived. In some cases, fragments are defined as those that exhibit basal levels of transcription. and TA for recognition and binding by the RNA polymerase II complex to initiate transcription. May contain a "minimal promoter" consisting of a TA box or equivalent DNA sequence be.

[0050] In one embodiment, the EXP sequence or promoter sequence disclosed herein The promoter fragments are provided as promoter fragments having the promoter activity as described above. and may be used alone or (e.g., when constructing chimeric promoters) in combination with other promoters and promoter fragments, or with other expression elements In certain embodiments, the expression element fragments may be useful in combination with the At least about 50, at least about 75, at least about 95, at least about 100 , at least about 125, at least about 150, at least about 175, at least about 200 , at least about 225, at least about 250, at least about 275, at least about 300 , at least about 500, at least about 600, at least about 700, at least about 750 , at least about 800, at least about 900, or at least about 1000 consecutive sequences nucleotide or more of DNA having promoter activity as disclosed herein. A fragment of a promoter is provided, comprising a molecule. Methods for producing such fragments are well known in the art.

[0051] In further embodiments, the enhancer or intron sequences disclosed herein The enhancer or intron fragments are provided as they are It can contain the activity of the base molecule from which it is derived, alone or in combination with other regulatory elements (e.g., promoters). Motors, leaders, other enhancers, other introns, or fragments of these In certain embodiments, at least about 50, At least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least at least about 600, at least about 700, at least about 750, at least about 800, at least about 900, or at least about 1000 contiguous nucleotides, or more The present invention also provides a DNA molecule having enhancer or intron activity as disclosed herein. , enhancer or intron fragments are provided. Methods for producing fragments are well known in the art.

[0052] In other embodiments, fragments of the 3'UTR sequences disclosed herein are provided. 3'UTR fragments contain the activity of the base 3'UTR molecule from which they are derived. It can be used alone or in combination with other regulatory elements (promoter, leader, intron, or fragments thereof). at least about 50, at least about 75, at least about 95, at least about 10 0, at least about 125, at least about 150, at least about 175, at least about 20 0, at least about 225, at least about 250, at least about 275, at least about 30 0, at least about 500, at least about 600, at least about 700, at least about 75 0, at least about 800, at least about 900, or at least about 1000 consecutive nucleotides, or more, of DNA having 3'UTR activity as disclosed herein. A fragment of the intron containing the starting 3'UTR molecule is provided. Methods for producing such fragments are well known in the art.

[0053] provided as SEQ ID NOs: 2 and 7, or SEQ ID NOs: 1, 4, 6, 9, 11, 15, and and 16 (e.g., internal or 5' deleted) promoter elements Compositions derived from any of the above methods may be used to improve or modify expression. The methods described herein can be used to identify, for example, elements that have either a positive or negative effect on expression. deletion, duplication of elements that have a positive or negative effect on expression, and / or tissue-specific expression by methods involving duplication or removal of elements that exert heterologous or cell-specific effects. , can be produced. TATA box element included in any of 6, 9, 11, 15, and 16 A promoter constructed from a 3' deletion in which the downstream sequence has been removed or its equivalent sequence Compositions derived from any of the elements can be used to create, for example, enhancer elements. Further deletions can be made to alter expression in a tissue-specific, cell-specific, or positive manner. have a specific or time-specific (e.g., but not limited to, circadian rhythm) effect. The sequences provided as SEQ ID NOs: 2 and 7, or SEQ ID NO: a promoter element included in any one of 1, 4, 6, 9, 11, 15, and 16; and chimeric transcriptional regulators using fragments or enhancers derived therefrom. An element composition can be made.

[0054] In the present invention, a promoter or a promoter fragment is a known promoter. TATA elements, i.e., TATA boxes and other known transcription factor binding site motifs The DNA sequence can be analyzed for the presence of such known promoter elements. The identity of the promoter can be used by those skilled in the art to identify promoters that have expression patterns similar to those of the original promoter. These can be used to design variants of the promoter.

[0055] As used herein, the term "leader" refers to the untranslated 5' region of a gene (5' isolated from the UTR and located between the transcription start site (TSS) and the protein-coding sequence start site. It refers to a DNA molecule, generally defined as a segment of nucleotides. A leader may be a synthetically produced or engineered DNA element. 5' regulatory element for regulating expression of an operably linked transcribable DNA molecule The leader molecule can be a heterologous promoter or its native promoter. Leaders useful in practicing the present invention can be used in conjunction with active promoters. SEQ ID NOs: 3 and 8, or SEQ ID NOs: 1, 4, 6, 9, 11, 15, and 16 Any reader element contained in one of these fragments In certain embodiments, such DNA sequences are capable of acting as a leader in cells (including, for example, transgenic plant cells) In one embodiment, such a sequence can be defined as a leader sequence. It is decoded as including activity.

[0056] the leader sequences (also referred to as 5'UTR) set out as SEQ ID NOs: 3 and 8, or A leader element contained in any one of SEQ ID NOs: 1, 4, 6, 9, 11, 15, and 16 Either of these may consist of regulatory elements or may be operably linked transcription factors. A DNA molecule may also adopt secondary structures that can affect the transcription or translation of the resulting DNA molecule. The leader sequences presented as SEQ ID NOs: 3 and 8, or SEQ ID NOs: 1, 4, 6, 9, 11 , 15, and 16 according to the present invention. to affect the transcription or translation of an operably linked transcribable DNA molecule. Mera regulatory elements can be created.

[0057] As used herein, the term "intron" refers to a region of interest that is isolated or identified from a gene. can be spliced during pre-translational messenger RNA (mRNA) processing. The term "DNA molecule" refers to a region of a DNA molecule that can generally be defined as a region that has been depleted. Additionally, an intron may be a synthetically produced or engineered DNA element. Introns contain enhancer elements that effect transcription of operably linked genes. Introns regulate the expression of transcribable DNA molecules to which they are operably linked. The construct can be used as a regulatory element for The intron may or may not be heterologous to the transcribable DNA molecule. Examples of introns in the art include the rice actin intron and the maize actin intron. Examples include HSP70 introns.

[0058] In plants, including several introns in a gene construct can Compared to constructs that do not contain the nucleotide, the accumulation of mRNA and protein is increased. The result is called "intron-mediated enhancement" (IME) of gene expression. Introns known to function in maize genes (e.g., tubA1, Ad h1, Sh1, and Ubi1), rice genes (e.g., tpi), and petunia ( e.g., rbcS), potato (e.g., st-ls1), and Arabidopsis Similar genes are found in dicotyledonous plants such as S. thaliana (e.g., ubq3 and pat1). Deletions or mutations within intron splice sites result in reduced gene expression. It has been shown that IME reduces the number of splicing events, suggesting that splicing may be required for IME. However, the IME in dicotyledonous plants is similar to that of A. thaliana. This is indicated by a point mutation within the splice site of the at1 gene. The multiple use of the same intron has been shown to have disadvantages. , which contains a set of basic control elements for constructing suitable recombinant DNA elements. Exemplary introns useful in practicing the present invention are those set forth in SEQ ID NO:5, They are presented as 10 and 12.

[0059] As used herein, "3' transcription terminator," "3' untranslated region," or "3' The term "UTR" is used during transcription to describe the untranslated region in the 3' portion of an mRNA molecule. The 3' untranslated region of an mRNA molecule is a region that undergoes specific cleavage and a 3' polyadenylation. The 3'UTR can be generated by 3'-nucleotide poly(A) tailing (also known as poly(A) tailing). can be operably linked to and located downstream of the transcribable DNA molecule, and Polyadenylation, which can affect transcription, mRNA processing, or gene expression The poly(A) tail can contain transcription signals and other regulatory signals. It is believed that 3' transcription factors play a role in the stability of the nucleotide sequence and in the initiation of translation. Examples of transcription terminators include the nopaline synthase 3' region, the wheat hsp17 3' region, Endourbisco small subunit 3' region, cotton E6 3' region, and coixin (co ixin) 3'UTR.

[0060] The 3'UTR typically finds beneficial use in the recombinant expression of a particular DNA molecule. A weak 3'UTR has the potential to cause readthrough, which can lead to adjacent This may affect the expression of the DNA molecule contained in the expression cassette. By controlling the transcription of the gene, it is possible to control the transcription of the gene to a downstream DNA sequence (e.g., another expression cassette). Efficient RNA polymerase synthesis to prevent overexpression and improve gene expression It can enable efficient transcription termination (RNA polymerase chain reaction from DNA). The release of ATPase II is a prerequisite for the resumption of transcription, which in turn affects the overall transcription level. Directly affected. Following transcription termination, mature mRNA is released from the site of synthesis and the template is released into the cell. Eukaryotic mRNAs accumulate in vivo in the poly(A) form. Therefore, it is difficult to detect the transcription termination site using conventional methods. Functional and efficient 3'UTR prediction using informatics methods is a key step in effective 3' Difficult due to lack of conserved DNA sequences that allow for easy prediction of UTRs .

[0061] From a practical standpoint, typically, the 3'UTR used in an expression cassette should have the following properties: First, the 3'UTR must be able to efficiently and effectively transfect the transgene. Multiple expression vectors that should be able to terminate and are present within a single transfer DNA (T-DNA) Any adjacent DNA sequences that may be comprised of another expression cassette, such as in the case of a cassette Alternatively, it can prevent the read-through of transcripts into the adjacent chromosomal DNA where the T-DNA is inserted. Second, the 3'UTR is a promoter used to drive expression of the DNA molecule. This results in a reduction in the transcriptional activity conferred by motors, leaders, enhancers, and introns. Finally, in plant biotechnology, the 3'UTR is essential for transformation. It is often used to prime the amplification reaction of reverse-transcribed RNA extracted from plants ( 1) Evaluation of transcriptional activity or expression of expression cassettes once integrated into plant chromosomes; (3) evaluation of the zygosity of the seeds obtained after breeding. The 3'UTR is also used to characterize the integrity of the inserted cassette. It is also used in the amplification reaction of DNA extracted from transformed plants in order to carry out the present invention. 3'UTRs useful above are provided as SEQ ID NOs: 13, 14, 19, and 26.

[0062] As used herein, the term "enhancer" or "enhancer element" The term refers to a cis-acting regulatory element (also known as a cis-element). The expression of a transcriptionally active gene results in one aspect of the overall expression pattern of a transcriptionally active DNA molecule to which it is linked, but typically Enhancer elements, unlike promoters, are usually insufficient to drive transcription. The nucleotide sequence contains a transcription start site (TSS) or TATA box, or equivalent DNA sequence. The promoter or promoter fragment does not contain a naturally occurring, operable Contains one or more enhancer elements that affect transcription of a DNA sequence to which it is linked Alternatively, an enhancer element can be fused to a promoter to form a chimeric promoter. They can also produce motor cis elements, which allow them to play a role in the global regulation of gene expression. The aspect is provided.

[0063] Many promoter-enhancer elements bind to DNA-binding proteins and affecting DNA topology and / or DNA accessibility of RNA polymerase to the DNA template. Local conformations that selectively permit or restrict transcriptional access, or duplexes at the transcription start site It is believed that this enhancer effect creates a local conformation that promotes the selective release of cancer. The element can function to bind to transcription factors that regulate transcription. The enhancer element binds two or more transcription factors, which bind with different affinities. Can interact with two or more enhancer domains. Enhancer element Identification of the promoter sequence can be achieved by deletion analysis (i.e., one or more deletions from the 5' end or internal to the promoter). (deleting the nucleotide above) or DNA using DNase I footprinting Binding protein analysis, methylation interference, electrophoretic mobility shift assay, ligation media In vivo genomic footprinting by polymerase chain reaction (PCR), and other traditional assays, or by conventional methods such as BLAST. The use of known cis elements as target sequences or target motifs in conjunction with conventional DNA sequence comparison methods This is done by DNA sequence similarity analysis using promoter motifs or enhancer elements. The fine structure of the enhancer domain can be determined by mutagenesis of one or more nucleotides. (or substitution), or by other conventional methods known in the art. Enhancer elements can be synthesized chemically or by These can be obtained by isolation from regulatory elements containing elements such as Additional flanking nucleotides containing useful restriction enzyme sites to facilitate manipulation of the subsequence. Thus, the development of operably linked transcribable DNA molecules is possible. Design of enhancer elements according to the methods disclosed herein to modulate expression The construction and use of such an enhancer is encompassed by the present invention. The sensors are presented as SEQ ID NOs: 17 and 18.

[0064] As used herein, the term "chimera" refers to a first DNA molecule that is fused to a second DNA molecule. means a single DNA molecule produced by fusing a first DNA molecule with a second DNA molecule; Neither the first nor the second DNA molecule is normally found in this configuration (i.e., fused to the other). Therefore, chimeric DNA molecules are novel molecules not otherwise normally found in nature. As used herein, the term "chimeric promoter" refers to a DNA molecule A promoter produced by the manipulation of such DNA molecules is called a chimeric promoter. The promoter combines two or more DNA fragments (e.g., a promoter and an endogenous Thus, operably linked transcription factors can be fused to transcription factors. Chimeric promoters according to the methods disclosed herein for modulating expression of a gene encoding ... The design, construction, and use of promoters are encompassed by the present invention.

[0065] Chimeric regulatory elements can be synthesized by various methods known in the art, for example, by restriction enzyme digestion. ligation, ligation-independent cloning, and monitoring of PCR products during amplification. Direct chemical synthesis of regulatory elements, as well as methods known in the art Various components that can be operably linked by other means as described herein. The resulting various chimeric regulatory elements can be designed to contain the same structure. It may consist of elements or variants of the same constituent elements, but the constituent parts A DNA sequence that contains a linking DNA sequence(s) that allows it to be operably linked In the present invention, SEQ ID NOs: 1 to 19 and SEQ ID NO: 26 are different. The DNA sequence provided may provide a regulatory element reference sequence, where The components that make up the reference sequence are linked by methods known in the art. and one or more nucleotides that occur naturally in the transformation of bacterial and plant cells. The sequences may include substitutions, deletions, and / or insertions or mutations of the sequences.

[0066] As used herein, the term "variant" refers to a variant of a DNA molecule that is similar in composition to a first DNA molecule. means a second DNA molecule that is similar but not identical, e.g., a regulatory element, The second DNA molecule, for example, can transduce the first DNA molecule with roughly equal transcription activity. still maintain the general functionality of the gene, i.e., the same or similar expression pattern. A variant is a shorter or truncated version of the first DNA molecule, or a variant of the first DNA molecule. Modified versions of the molecule's sequence, e.g., different restriction enzyme sites and / or internal deletions A "variant" may be a version of a reference sequence that has a sequence, substitutions, or insertions. having a nucleotide sequence containing one or more nucleotide substitutions, deletions, or insertions in the sequence Regulatory elements may also be included, where the derivative regulatory element is a It has greater, lesser, or equal transcription or translation activity than the regulatory molecule. Sectional element "variants" arise from naturally occurring mutations during transformation of bacterial and plant cells. In the present invention, SEQ ID NOs: 1 to 19 and SEQ ID NO: 26 include variants thereof. Using the provided polynucleotide sequences, the DNA sequence and composition of the original regulatory elements can be determined. Similar but not identical, while retaining the general functionality of the original control element, i.e., the same Alternatively, variants can be created that still maintain a similar expression pattern. The production of such variants of the present invention is well within the ordinary skill of one in the art in light of this disclosure. and are included within the scope of the present invention.

[0067] The desired expression of a particular transgene in the modifications, duplications, or deletions described herein Validity for the present embodiment can be demonstrated in stable, transient plant assays, e.g., as described in the work herein. The assays described in the examples can be empirically tested to verify the results. The modifications made to the expressed DNA molecule and the purpose of the modifications can vary.

[0068] construct As used herein, the term "construct" refers to a nucleic acid sequence derived from any source, capable of genomic integration or self-replication, with at least one DNA molecule replicating to another Any DNA molecule, including a DNA molecule functionally linked, i.e., operably linked, to Any recombinant DNA molecule, such as a plasmid, cosmid, virus, phage, or lineage. As used herein, "vector" refers to a circular or oval DNA or RNA molecule. The term "transfector" refers to the process of transformation, i.e., the introduction of heterologous DNA or RNA into a host cell. Construct means any construct that can be used for the purpose of introducing Typically, the vector contains one or more expression cassettes. As used herein, "expression cassette" refers to a vector containing one or more expression cassettes. A "set" refers to one or more regulatory elements, typically at least a promoter and a 3' U a DNA molecule comprising at least one transcribable DNA molecule operably linked to a TR; means.

[0069] As used herein, the term "operably linked" means that a first DNA molecule Links to a second DNA molecule, and the first DNA molecule affects the function of the second DNA molecule This means that the first and second DNA molecules are arranged in such a way that the two DNA molecules , which may or may not be part of a single, continuous DNA molecule and may be adjacent This is not always the case. For example, a promoter may be used to encode a target transcribable DNA sequence within a cell. A promoter is operably linked to a transcribable DNA molecule when it regulates transcription of the gene. For example, a leader may be capable of influencing the transcription or translation of a DNA sequence. , operably linked to the DNA sequence.

[0070] In one embodiment, the constructs of the present invention are derived from A. tumefaciens cells. Together with the transfer molecules carried by the vesicle, T allows the T-DNA to integrate into the genome of the plant cell. -DNA isolated from Agrobacterium tumefaciens The right border (RB or AGRtu.RB) and left border of the double tumor-inducing (Ti) plasmid Ti plasmid border constrains containing the lateral border (LB or AGRtu.LB) region tracts (see, e.g., U.S. Pat. No. 6,603,061). The construct also contains a promoter that provides replication function and antibiotic selection in bacterial cells. Plasmid backbone DNA segments, e.g., Escherichia coli replication origin, broad host range replication origins such as oriV or oriRi, and Tn7 aminoglycosides conferring resistance to tyrannycin or streptomycin Selection of Spec / Strp, which encodes adenyltransferase (aadA) Selectable marker or gentamicin (Gm, Gent) selectable marker gene For plant transformation, the host bacterial strain is often At umefaciens ABI, C58, or LBA4404, but not plant transformation Other strains known to those skilled in the art may also function in the present invention.

[0071] Transcribable DNA molecules are transformed into functional mRNA molecules that are translated and expressed as proteins. Methods for assembling and introducing constructs into cells in a transcribed manner are known in the art. For the practice of the present invention, the constructs and host cells are prepared and Conventional compositions and methods for their use are well known to those skilled in the art. Exemplary vectors useful for expressing nucleic acids in mammals are well known in the art and include those described in The vector is derived from the Ti plasmid of Agrobacterium tumefaciens. The vectors include the pCaMVCN transposition control vector.

[0072] A variety of regulatory elements, including any of the regulatory elements provided herein, can be used in constructs. Any such regulatory element may be included in a nucleotide sequence other than the nucleotide sequence of the other regulatory elements. Such a combination may provide the desired regulatory function. In one embodiment, the constructs of the present invention can be designed or modified to The tract was operably linked to a transcribable DNA molecule operably linked to a 3'UTR , comprising at least one regulatory element.

[0073] Constructs of the invention may be prepared using any of the methods provided herein or known in the art. For example, the promoters of the present invention can be any promoter or leader that can - a heterologous untranslated 5' leader (e.g., from a heat shock protein gene) Alternatively, the leaders of the invention can be operably linked to a heterologous promoter. operably coupled with a promoter (e.g., the cauliflower mosaic virus 35S transcript promoter) can be combined.

[0074] The expression cassette is used to express an operably linked protein in a plant, particularly in chloroplasts, leucoplasts, or other plant cells. in plastid organelles, mitochondria, peroxisomes, vacuoles, or extracellular locations In contrast, transport peptide codes that encode peptides useful for subcellular targeting are also available. Many chloroplast-localized proteins are expressed as precursors from nuclear genes. These single proteins are expressed in the chloroplast and targeted to the chloroplast by a chloroplast transit peptide (CTP). Examples of isolated chloroplast proteins include, but are not limited to, ribulose-1, Small subunit (SSU) of 5-bisphosphate carboxylase, ferredoxin, ferre Doxin oxidoreductase, light-harvesting complex protein I and protein II, thioredoxin oxin F and related enzymes for enolpyruvylshikimate phosphate synthase (EPSPS) Chloroplast transit peptides include those described in, for example, U.S. Patent No. 7,193,133. Non-chloroplast proteins are expressed by transgenes encoding non-chloroplast proteins. By expressing a heterologous CTP operably linked to has been proven.

[0075] transcribable DNA molecule As used herein, the term "transcribeable DNA molecule" refers to a molecule capable of being transcribed into an RNA molecule. "DNA" refers to any DNA molecule capable of being transcribed into a protein, including but not limited to: Molecules that produce RNA molecules with coding sequences or sequences useful for gene silencing The types of DNA molecules include, but are not limited to, DNA molecules from different plants, DNA molecules from different organisms, or synthetic DNA molecules, e.g., DNA molecules containing antisense messages of genes, or artificial DNA molecules encoding synthetic or otherwise modified versions of the transgene are listed. Exemplary transcribable DNA for incorporation into constructs of the invention The molecule may be, for example, a DNA molecule or gene from a species other than the species into which the DNA molecule is to be incorporated. offspring, or those arising from or existing in the same species but derived from genetic material rather than classical breeding techniques Examples include genes that are incorporated into recipient cells by genetic engineering methods.

[0076] A "transgene" is a gene that is heterologous to a host cell, at least with respect to its location within the host cell genome. a transcribable DNA molecule that is a It refers to a transcribable DNA molecule that has been artificially integrated into the genome of a host cell.

[0077] The regulatory element, e.g., the promoter of the present invention, is heterologous to the regulatory element. As used herein, two The term "heterologous" refers to a combination of DNA molecules not normally found in nature. This refers to a combination of two or more such DNA molecules. For example, the two DNA molecules may be different. The two DNA molecules may be derived from different species and / or may be derived from different genes. (e.g., they may be derived from different genes from the same species, or from the same gene from different species. Thus, regulatory elements have the ability to regulate the transcription of operably linked transcribable DNA molecules. In cases where such combinations are not normally found in nature, i.e., where the transcriptional activity of a DNA fragment is not sufficient, An element is heterologous if it does not naturally occur in operably linked to the regulatory element.

[0078] A transcribable DNA molecule is generally any DNA molecule from which expression of a transcript is desired. Expression of such transcripts results in the translation of the resulting mRNA molecules, and thus the transcription factor. Alternatively, for example, a transcribable DNA molecule can ultimately result in protein expression. , may be designed to cause reduced expression of a specific gene or protein. In one embodiment, this uses a transcribable DNA molecule oriented in the antisense direction. This can be achieved by the use of antisense technology. Any gene can be negatively regulated in this manner, and in one embodiment In this context, the transcribable DNA molecule is capable of producing dsRNA, siRNA, or miRNA molecules. These can be designed to suppress specific genes through expression.

[0079] Thus, one embodiment of the present invention is a regulatory element of the present invention (e.g., SEQ ID NO: 1-19 and SEQ ID NO: 26), When the construct is integrated into the genome of a transgenic plant cell, it produces a transcriptionally active Heterologous transcription can be used to regulate transcription of DNA molecules at desired levels or in desired patterns. In one embodiment, the recombinant DNA molecule is operably linked to a recombinant DNA molecule. In one embodiment, the transcribable DNA molecule comprises the protein coding region of a gene, and in another embodiment, In this case, the transcribable DNA molecule comprises the antisense region of the gene.

[0080] Genes of agricultural interest The transcribable DNA molecule may be a gene of agricultural interest. The term "gene of agronomic interest" refers to a gene that is expressed in a particular plant tissue, cell, or cell type. A gene for agricultural purposes refers to a transcribable DNA molecule that, when transcribed, confers a desired characteristic. Gene products affect plant morphology, physiology, growth, development, yield, grain composition, nutritional profile, Causes effects on disease or pest resistance and / or environmental or chemical tolerance They may act within the plant to remove bacteria or as insecticides to feed on plant-feeding pests. In one embodiment of the present invention, the regulatory element of the present invention may be The element is operably linked to a transcribable DNA molecule that is a gene of agricultural interest. The transgene containing such a construct is In nicked plants, expression of genes of agronomic interest can confer beneficial agronomic traits. Beneficial agronomic traits include, but are not limited to, herbicide tolerance, Insect control, altered yield, disease resistance, pathogen resistance, altered plant growth and development, Modified starch content, modified oil content, modified fatty acid content, modified protein Protein content, modified fruit ripening, animal and human nutritional enrichment, biopolymer production, environmental stress resistance, medicinal peptides, improved processing quality, improved flavor, useful for hybrid seed production , improved fiber production, as well as desirable biofuel production.

[0081] Non-limiting examples of genes of agronomic interest known in the art include genes encoding herbicide resistance genes. (U.S. Patent Nos. 6,803,501; 6,448,476; 6,248,876 ; No. 6,225,114; No. 6,107,549; No. 5,866,775; No. 5, Nos. 804,425; 5,633,435; and 5,463,175), yield increase (U.S. Patent Nos. USRE38,446; 6,716,474; 6,663,906 No. 6,476,295; No. 6,441,277; No. 6,423,828; No. 6 ,399,330;No.6,372,211;No.6,235,971;No.6,222 ,098; and 5,716,837), insect control (U.S. Patent Nos. 6,809,078 No. 6,713,063; No. 6,686,452; No. 6,657,046; No. 6 ,645,497;No.6,642,030;No.6,639,054;No.6,620 ,988;No.6,593,293;No.6,555,655;No.6,538,109 No. 6,537,756; No. 6,521,442; No. 6,501,009; No. 6 ,468,523;No.6,326,351;No.6,313,378;No.6,284 ,949;No.6,281,016;No.6,248,536;No.6,242,241 No. 6,221,649; No. 6,177,615; No. 6,156,573; No. 6 ,153,814;No.6,110,464;No.6,093,695;No.6,063 ,756;No.6,063,597;No.6,023,013;No.5,959,091 No. 5,942,664; No. 5,942,658; No. 5,880,275; No. 5, Nos. 7,763,245; and 5,763,241), fungal disease resistance (U.S. Pat. Nos. 6,6 No. 53,280; No. 6,573,361; No. 6,506,962; No. 6,316,4 No. 07; No. 6,215,048; No. 5,516,671; No. 5,773,696; Nos. 6,121,436; 6,316,407; and 6,506,962), Virus resistance (U.S. Patent Nos. 6,617,496; 6,608,241; 6,01 Nos. 5,940; 6,013,864; 5,850,023; and 5,304, 730), nematode resistance (U.S. Patent No. 6,228,992), bacterial disease resistance (U.S. Patent No. No. 5,516,671), Plant Growth and Development (U.S. Patent No. 6,723,897 and No. 6,518,488), starch production (U.S. Pat. No. 6,538,181; No. 8,179; No. 6,538,178; No. 5,750,876; No. 6,476,29 No. 5), modified oil production (U.S. Patent Nos. 6,444,876; 6,426,447 ;6,380,462), high oil production (U.S. Patent No. 6,495,739; Nos. 08,149; 6,483,008; and 6,476,295), modified Fatty acid content (U.S. Patent No. 6,828,475; No. 6,822,141; No. 6,770 ,465;No.6,706,950;No.6,660,849;No.6,596,538 No. 6,589,767; No. 6,537,750; No. 6,489,461; No. 6 ,459,018), high protein production (U.S. Patent No. 6,380,466), fruit maturation (U.S. Patent No. 5,512,466), animal and human nutritional fortification (U.S. Patent No. 6,7 No. 23,837; No. 6,653,530; No. 6,5412,59; No. 5,985,6 05; No. 6,171,640), biopolymers (U.S. Patent No. USRE37,543; Nos. 6,228,623; and Nos. 5,958,745 and 6,946,588) , environmental stress resistance (U.S. Patent No. 6,072,103), medicinal peptides and secretory peptides Petide (U.S. Patent Nos. 6,812,379; 6,774,283; 6,140,0 75; and 6,080,560), improved processing traits (U.S. Pat. Nos. 6,477,487; 6,295), improved digestibility (U.S. Patent No. 6,531,648), low raffinose (U.S. Patent No. 6,166,292), industrial enzyme production (U.S. Patent No. 5,543,576 No. 6,011,199), flavor improvement (U.S. Patent No. 6,011,199), nitrogen fixation (U.S. Patent No. 5,222 9,114), hybrid seed production (U.S. Patent No. 5,689,041), fiber production (U.S. Patent Nos. 6,576,818; 6,271,443; 5,981,834 and 5,869,720), and biofuel production (U.S. Pat. Nos. 5,998,7 00).

[0082] Alternatively, genes of agronomic interest can be expressed by targeted modulation of gene expression of endogenous genes. For example, antisense (see, e.g., U.S. Pat. No. 5,629,663) can be used by encoding an RNA molecule that induces No. 5,107,065), inhibitory RNA ("RNAi"; see, e.g., published application U.S. Pat. No. 5,107,065), 2006 / 0200878 and US2008 / 0066206, and U.S. Patent No. As described in application Ser. No. 11 / 974,469, miRNA, siRNA, trans Regulation of gene expression by phase-acting siRNA and phase-sRNA-mediated mechanisms affect the plant traits or phenotypes mentioned above through mechanisms mediated by co-suppression. RNA can also be engineered to cleave a desired endogenous mRNA product. catalytic RNA molecules (e.g., ribozymes or riboswitches; e.g., US200 It is believed that the transcribable DNA molecule may be genetically modified. Constructs are constructed in a way that results in transcription into molecules that can induce gene suppression. Methods for introducing the vector into cells are known in the art.

[0083] Selection Marker A selectable marker transgene may be used in conjunction with the regulatory elements of the present invention. As used, the term "selectable marker transgene" refers to a transgenic plant, The expression or lack of expression in tissues or cells is screened or screened in any way. It means any transcribable DNA molecule that can be cored. Selectable marker genes and their associated selection and screening techniques for use in Methods are known in the art and include, but are not limited to, β-glucuronides. GUS, green fluorescent protein (GFP), and proteins that confer antibiotic resistance and transcribable DNA molecules encoding proteins that confer herbicide resistance. Examples of selectable marker transgenes are provided as SEQ ID NOs: 20 and 24.

[0084] Cell transformation The present invention provides a method for producing transformed cells and plants, comprising the steps of: Also covered are methods comprising one or more regulatory elements operably linked to the gene.

[0085] The term "transformation" means the introduction of a DNA molecule into a recipient host. As used herein, the term "host" means a bacterium, a fungus, or a plant; This includes any cell, tissue, organ, or progeny of a bacterium, fungus, or plant. Particularly target plant tissues and cells include protoplasts, callus, roots, tubers, seeds, etc. , stems, leaves, seedlings, embryos, and pollen.

[0086] As used herein, the term "transformed" refers to the introduction of a foreign DNA molecule (e.g., , construct) into a cell, tissue, organ, or organism. Integrating the DNA molecule into the genomic DNA of a recipient cell, tissue, organ, or organism The introduced DNA molecule can be passed on to subsequent generations. A "geneic" or "transformed" cell or organism includes the progeny of the cell or organism, and produced from breeding programs using such transgenic organisms as parents in crosses The present invention also includes progeny that exhibit phenotypic alterations resulting from the presence of the exogenous DNA molecule. The introduced DNA molecule is transiently introduced into recipient cells, and the introduced DNA molecule It is also possible to prevent the gene from being passed on to future generations. means a bacterium, fungus, or plant that contains one or more heterologous DNA molecules.

[0087] There are many methods for introducing DNA molecules into plant cells and these are well known to those skilled in the art. The process generally involves the steps of selecting a suitable host cell and infecting the host cell with a vector. and obtaining a transformed host cell. In this regard, plant cells are transformed by introducing a plant construct into the plant genome. Methods and materials for the preparation of the cellulose may include any well-known and proven method. Examples include, but are not limited to, bacterial infections (e.g., Agrobacterium um), binary BAC vectors, direct delivery of DNA (e.g., PEG-mediated transformation , Desiccation / Inhibition-Mediated DNA Uptake, Electroporation, and Agitation with Silicon Carbide Fibers and acceleration of DNA-coated particles), gene editing (e.g., CRISPR-Cas systems), stem).

[0088] A host cell can be any cell or organism, for example, a plant cell, an algae cell, an alga, a fungal cell, The host cell may be a fungus, a bacterial cell, or an insect cell. The cells and transformed cells may include cells from crop plants.

[0089] Transgenic plants are then regenerated from the transgenic plant cells of the invention. This transgenic plant can be cultivated using conventional breeding techniques or self-pollination. Such seeds and the products grown from such seeds may be used in the manufacture of The resulting progeny plants contain the recombinant DNA molecules of the present invention and are therefore transgenic. This becomes:

[0090] Transgenic plants of the present invention can be self-pollinated (homozygous for the recombinant DNA molecule) and then grown in a manner similar to that described above. and providing seeds of the homozygous transgenic plants of the present invention (synthetic), and is crossed with a non-transgenic plant or a different transgenic plant to produce a recombinant A heterozygous transgenic plant seed of the present invention (heterozygous for the DNA molecule) Such homozygous and heterozygous transgenic mice can provide offspring. Both the progeny plants and the plants are referred to herein as "progeny plants." Progeny plants are plants that are the same as the original transgenic plants. A transgenic plant derived from a plant of the present invention contains a recombinant DNA molecule of the present invention. Seeds produced using the transgenic plants of the present invention are harvested and transgenic. and a transgenic plant containing the gene and expressing a gene of agricultural interest, i.e., a progeny plant of the present invention. It can be used to grow new generations of plants. It is commonly used in a variety of crops. A description of breeding methods can be found in one of several references. Allard, Principles of Plant Breeding, Jo hn Wiley & Sons,NY,U.of CA,Davis,CA,50-9 8(1960);Simmonds,Principles of Crop Impr. ovement,Longman,Inc.,NY,369-399(1979);Sn eep and Hendriksen,Plant breeding Perspe ctives,Wageningen(ed),Center for Agricul tural Publishing and Documentation(1979) ;Fehr,Soybeans: Improvement,Production a nd Uses,2nd Edition,Monograph,16:249(198 7);Fehr,Principles of Variety Developmentmen t,Theory and Technique,(Vol.1)and Crop S pecies Soybean(Vol.2),Iowa State Univ.,M See acmillan Pub. Co., NY, 360-376 (1987).

[0091] Transformed plants are characterized by the presence of the gene(s) of interest, as well as the regulatory elements of the present invention. The expression levels and / or profiles conferred by the ment can be analyzed. recognizes the many methods available for analyzing transformed plants. For example, methods of plant analysis Examples include, but are not limited to, Southern blot or Northern blot, PCR, -based approaches, biochemical analysis, phenotypic screening methods, field evaluation, and immunodiagnostics The expression of transcribable DNA molecules can be assayed using TaqMan® (A Reagents and materials from Applied Biosystems (Foster City, CA) The method described by the manufacturer, as well as the TaqMan® Testing Matrix x. Alternatively, the PCR cycle time can be determined using In vader (registered trademark) (Third Wave Technologies (Madi Assess transgene expression using the reagents provided and methods described by the manufacturer. It may be worth it.

[0092] The present invention also provides parts of the plants of the present invention. The plant parts include, but are not limited to: Plants of the present invention include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovules, and pollen. The site may be viable, non-viable, regrowable, and / or non-regrowable. The invention also includes and provides transformed plant cells containing the DNA molecules of the invention. Transformed or transgenic plant cells include regenerable and / or non-regenerable plant cells. Contains biological cells.

[0093] The present invention also relates to a transgenic plant or a plant thereof comprising a recombinant DNA molecule of the present invention. Commercial products produced from the strains of the present invention are also provided. and SEQ ID NO: 26. As used herein, "commercial product" includes a recombinant DNA molecule of the present invention. Consisting of material derived from transgenic plants, seeds, plant cells, or plant parts Commercial products include, but are not limited to, any composition or product that is manufactured using a commercial product. However, the commercial products of the present invention include processed seeds, grains, plant parts, and meal. The resulting product will contain a detectable amount of DNA corresponding to a recombinant DNA molecule of the present invention. Detection of one or more of these DNAs in a sample to determine the content or source of a product. Any standard DNA detection method can be used, including the detection methods disclosed herein. A method for detecting the molecule can be used.

[0094] The present invention can be more readily understood by reference to the following examples: The examples are offered by way of illustration and not by way of limitation, unless otherwise specified. Those skilled in the art will appreciate that the techniques disclosed in the following examples are not intended to be limiting. It is understood that the present invention corresponds to techniques discovered that function well in practicing the present invention. However, those skilled in the art will appreciate that in light of this disclosure, many of the specific embodiments disclosed may be and still be within the spirit and scope of the present invention. It should be understood that similar or equivalent results will be obtained. All matters described or shown shall be interpreted as illustrative and in a limiting sense. shall not be interpreted as such. [Example]

[0095] Example 1 Design, synthesis, and cloning of synthetic regulatory elements The novel synthetic transcriptional regulatory elements are synthetic expression elements designed by algorithmic methods. These computationally designed regulatory elements were chemically synthesized and cloned to To this end, we have created a set of synthetic regulatory expression elements (EXPs). The elements were designed and used to transform maize protoplasts and stably transformed maize. and assaying in plants to determine the desired properties (e.g., protein expression levels and expression patterns). The synthetic regulatory elements of the present invention have been identified to confer a number of Various patterns useful for driving the expression of interfering RNAs of different coding sequences and agricultural purposes. This results in constitutive expression of the gene.

[0096] The designed synthetic transcriptional regulatory elements are compatible with any known nucleic acid sequence found in nature. Although the same sex is not expanded, it is still a naturally occurring promoter, leader, and It affects transcription of operably linked coding sequences in the same manner as the 3'UTR and 3'UTR. Synthetic EXP and its corresponding synthetic promoter, leader, intron, and synthetic The 3'UTR is presented in Table 1. Synthetic EXP was synthesized using methods known in the art. , a binary plant trait operably linked to a β-glucuronidase (GUS) coding sequence Cloned into a transformation vector and analyzed for expression in stably transformed maize plants. The signal and pattern were evaluated.

[0097] Analysis of regulatory elements, TSSs, and intron / exon splice junctions This can be done using transformed plant tissue. A plant expression vector comprising a cloned DNA fragment operably linked to a DNA molecule Next, the 5' RACE System for Rapid Amplification of cDNA Ends, Version 2.0(I Invitrogen (Carlsbad, California 92008) was used. By analyzing the DNA sequence of the mRNA transcripts produced, the regulatory element T The SS and intron / exon splice junctions were confirmed. , and were characterized for their effects on gene expression and proper termination of the transcript.

[0098] In addition to the synthetic expression elements, the nonspecific lipid transport protein of Sorghum bicolor was A novel endogenous 3'UTR from the protein 4 gene, T-Sb.Nltp4-1:1: 2 is provided herein and presented as SEQ ID NO: 19. T-Sb.Nltp4-1:1: 2 was characterized in a similar manner to the synthetic 3'UTR. [Table 1]

[0099] Example 2 Analysis of synthetic regulatory elements driving GUS in maize leaf protoplasts. Maize leaf protoplasts were transfected with the vector, specifically, β-glucuronidase (G Transform the cells with an expression vector containing the test regulatory elements that drive expression of the transgene. The resulting transformed corn leaf protoplasts were used to examine GUS protein expression. Analysis was performed to assess the effect of selected regulatory elements on expression.

[0100] Maize protoplasts derived from leaf tissue were transfected with expression vectors containing synthetic expression elements. These synthetic expression elements were transformed into maize protoplasts. The level and pattern of expression of the vectors can be determined by expression element expression methods known in the art. The levels and patterns were compared with those of the current experiment. .GSP850 (SEQ ID NO: 1) and EXP-Zm.GSP990 (SEQ ID NO: 6), For Ron, I-Zm.GSI153.nno:1 (SEQ ID NO: 5) and I-Zm.GSI 197.nno:1 (SEQ ID NO: 10), T-Zm.GST9.nno for 3'UTR The activity of T-Zm.GST18.nno:2 (SEQ ID NO: 13) and T-Zm.GST18.nno:2 (SEQ ID NO: 14) The expression element was cloned into an expression vector and a processible intro GUS coding sequence containing the nucleotide sequence GOI-Ec.uidA+St.LS1:1:1 (SEQ ID NO: 24). The control expression vectors contained known expression elements in different configurations. The expression element includes the type of element being evaluated (EXP, intron, or 3'UTR). A plasmid was also constructed for use in co-transformation of the plasmids and data normalization. The smid is operably linked at the 5' end to the 3'UTR, T-Os.LTP:1 (SEQ ID NO: 23). Conjugated NanoLuc® luciferase fluorescent protein (Promega) A coding sequence encoding Nluc ( EXP, EXP-CaMV, operably linked at the 5' end to EXP (referred to as SEQ ID NO: 25). The transgene cassette consisted of .35S (SEQ ID NO: 21).

[0101] Corn leaf protoplasts were cultured using PEG-based PEG-transferases, similar to those known in the art. Protoplast cells were transformed using the 96-well format. Twelve micrograms of test vector DNA or control vector DNA were used. The NanoLuc® vector DNA was added to 3.2 x 10 cells per well. 5 After transformation, the protoplasts were incubated at 25°C in the dark. After incubation, the protoplasts were lysed and the solution was The lysates were used to measure luciferase and GUS expression. The cells in the tube were pelleted by centrifugation, washed, resuspended in a small volume, and stripped. The tube was centrifuged again, and the supernatant was aspirated to remove the protoplast cells. The pellet was left behind. The cell pellet was then resuspended in QB buffer (100 mM KPO, pH 7.8 ; 1mM EDTA; 1% Triton X-100; 10% glycerol; 1mM DTT) The cells were resuspended in 100 ml of PBS. The cells were pipetted vigorously several times, the tube was vortexed, and the tube was resuspended in 100 ml of PBS. The cells were lysed by incubating the tube on ice for 5 minutes. The lysate was then centrifuged. Cell debris was pelleted by centrifugation, and the resulting lysate was then transferred to a clean plate. did.

[0102] Nano-Glo® Luciferase Assay in QB Buffer Substrate (Promega, Madison, WI 53711) Briefly, a small volume of lysate, QB buffer, and NaCl were added to the lysate. no-Glo® Luciferase Assay Substrate / Q The B solutions were mixed together in a white 96-well plate. (registered trademark) plate reader (BMG LABTECH Inc., Cary, NC) Fluorescence was measured using a fluorometer.

[0103] The fluorogenic substrate 4-methylumbelliferyl-β- GUS activity was assayed using D-glucuronide (MUG). Umbelliferone (4-MU) is maximally fluorescent at high pH, where the hydroxyl groups The addition of a basic solution of sodium carbonate stops the assay and simultaneously An aliquot of the lysate is added to QB buffer, the pH of which is adjusted to allow for quantification of the fluorescent product. The lysate was mixed with an aliquot of dissolved MUG and incubated at 37°C. Small aliquots of the reaction mixture were removed and analyzed at three different time points: (1) "time zero" Immediately after mixing the lysate / MUG reaction mixture as described above, (2) at 20 minutes, and (3) at 60 minutes. Stop buffer was added. LABTECH Inc. (Cary, NC 27513) with 355 nm excitation Fluorescence was measured at 460 nm emission. Expression levels were derived from an in-plate standard curve. The values are expressed as "nM MUG hydrolysis".

[0104] For each plate, transform each construct into 4-8 wells. An aliquot was removed from each transformation for assay and the "MUG addition" was determined from the on-plate standard curve. The water decomposition nM was derived. Aliquots were also taken from each transformation for RLU. The mean MUG hydrolysis nM / NanoLuc® RLU in the The EXP-CaMV.35S / I-Zm.DnaK:1 / T-Os.LTP:1 control Normalized to the structure.

[0105] Maize leaf protoplasts driven by synthetic EXP, EXP-Zm.GSP850 Analysis of GUS expression in st. An expression element driving GUS expression was constructed using methods known in the art. Maize leaf protoplast cells were transformed with an expression vector containing the nucleotide sequence. The experimental expression vectors included synthetic EXP, EXP-Zm.GSP850 (SEQ ID NO: 1), and The synthetic EXP-Zm.GSP850 contained the synthetic leader LZ m.GSP850.nno:3 (SEQ ID NO: 3) and a synthetic promoter operably linked at the 5' end The first test vector is composed of P-Zm.GSP850.nno:4 (SEQ ID NO:2). The vector is operably linked to the 3'UTR, T-Os.LTP:1 (SEQ ID NO: 23) on the 5' side. The resulting code encoding GUS (SEQ ID NO: 24) contains a processible intron. The second gene contained EXP-Zm.GSP850 operably linked at the 5' end to a nucleotide sequence. The transgene cassette is operably linked to the 3'UTR, T-Os.LTP:1, on the 5' side. operably linked 5' to the GUS coding sequence, :1 (SEQ ID NO:22) operably linked at the 5' end to EXP-Zm.GSP850 I was.

[0106] Three control expression vectors were also constructed and used to transform maize leaf protoplasts. The first control expression vector contained a promoterless transgene cassette and a 3' U TR, operably linked to T-Os.LTP:1 on the 5' side and GUS coding sequence on the 5' side The second consisted of intron I-Zm.DnaK:1 operably linked by The control vector contains an intronless transgene cassette, 3′UTR, T-Os. operably linked 5' to the GUS coding sequence, operably linked 5' to LTP:1 The third construct consisted of EXP and EXP-CaMV.35S (SEQ ID NO: 21). The control vector contained the 3'UTR, T-Os.LTP:1, operably linked at the 5' end to G Introns I-Zm.DnaK:1 and 5 operably linked 5' to the US coding sequence A transgene cassette containing EXP, EXP-CaMV.35S, operably linked at the '-side It contained

[0107] Maize leaf protoplasts were transformed with all five vectors. Transformation and lysis of luciferase cells was performed as described herein. Expression of ATP and GUS was assayed as described herein. Table 2 shows the assay results. The average GUS expression level was measured using EXP-CaMV.35S and I, which drive GUS. -Percentage of expression compared to a third control expression vector containing Zm.DnaK:1; It is expressed as: [Table 2]

[0108] As can be seen in Table 2 above, EXP-Zm.GSP850 (SEQ ID NO: 1) Compare with maize leaf protoplast cells transformed with the target-free construct. and was able to drive GUS transgene expression in maize leaf protoplasts.

[0109] Maize leaf protoplasts driven by synthetic EXP, EXP-Zm.GSP990 Analysis of GUS expression in st. Maize leaf protoplast cells were transfected with constructs containing expression elements that drive GUS expression. The test expression vectors were transformed with the constructed expression vectors. A coding sequence encoding GUS (SEQ ID NO: 20), operably linked 5' to P:1 and intron I-Zm.DnaK:1 (SEQ ID NO: 22) operably linked at the 5' end to operably linked at the 5' end to synthetic EXP, EXP-Zm.GSP990 (SEQ ID NO: 6) The synthetic EXP-Zm.GSP990 (SEQ ID NO: 6) contained a transgene cassette containing: ) operates 5' with the synthetic leader L-Zm.GSP990.nno:1 (SEQ ID NO: 8) operably linked to the synthetic promoter P-Zm.GSP990.nno:2 (SEQ ID NO: 7) Three control expression vectors were also transformed into maize leaf protoplasts. Table 3 shows the GUS-driving EXP-CaMV.35S and The average percent expression compared to a third control expression vector containing I-Zm.DnaK:1 was It shows. [Table 3]

[0110] As can be seen in Table 3, EXP-Zm.GSP990 (SEQ ID NO: 6) contains a promoter Compared with maize leaf protoplast cells transformed with the construct, It was able to drive GUS transgene expression in maize leaf protoplasts.

[0111] Analysis of enhancement of GUS expression by synthetic intron I-Zm.GSI153.nno:1 Maize leaf protoplast cells were transfected with constructs containing expression elements that drive GUS expression. The test expression vector was used to transform CaMV. 35 driven synthetic intron I-Zm.GSI153.nno:1 (SEQ ID NO: The transgene cassette consisted of 3'UTR, T- A coding sequence encoding GUS (sequence number 1) operably linked to Os.LTP:1 on the 5' side operably linked at the 5' end to the synthetic intron I-Zm.GSI153.n EXP operably linked at the 5' end to no:1 (SEQ ID NO:5), EXP-CaMV.35 Two control expression vectors were also constructed and used to transform maize leaf protoplasts. The first control expression vector contained an intronless transgene cassette. 3'UTR, GUS coding sequence operably linked to T-Os.LTP:1 on the 5' side. It consists of EXP and EXP-CaMV.35S, operably linked at the 5' end to the The second control vector was operably linked at the 5' end to the 3'UTR, T-Os.LTP:1. operably linked at the 5' end to the GUS coding sequence, K:1 and EXP, EXP-CaMV.35S, operably linked at the 5' end. Table 4 shows the EXP-CaMV.35S and EXP-CaMV.35S gene cassettes that drive GUS. Average percentage expression compared to a second control expression vector containing both I-Zm.DnaK:1 This shows the point. [Table 4]

[0112] As can be seen in Table 4, the synthetic intron I-Zm.GSI153.nno:1 (SEQ ID NO: 5) was found to be significantly higher in EXP-CaMV.35S than in the intronless control expression vector. Enhanced GUS transgene expression in maize leaf protoplasts driven by α-GUS. .

[0113] Analysis of enhancement of GUS expression by synthetic intron I-Zm.GSI197.nno:1 Maize leaf protoplast cells were transfected with constructs containing expression elements that drive GUS expression. The test expression vector was used to transform CaMV. 35 driven synthetic intron I-Zm.GSI197.nno:1 (SEQ ID NO: The transgene cassette consisted of 3'UTR, T - a coding sequence encoding GUS (sequence number 1) operably linked at the 5' end to Os.LTP:1 operably linked at the 5' end to the synthetic intron I-Zm.GSI197) (column number 24). It contained EXP, EXP-CaMV.35, operably linked at the 5′ end to nno:1. Three control expression vectors were also constructed and used to transform maize leaf protoplasts. The first control expression vector contained a promoterless transgene cassette and a 3' U TR, operably linked to T-Os.LTP:1 on the 5' side and GUS coding sequence on the 5' side The second consisted of intron I-Zm.DnaK:1 operably linked by The control vector contains an intronless transgene cassette, 3′UTR, T-Os. EXP, EXP-, linked 5' to the GUS coding sequence, linked 5' to LTP:1 The third control vector consisted of the 3'UTR, T-Os.L. operably linked 5' to the GUS coding sequence, operably linked 5' to TP:1 In addition, EXP, EXP are operably linked to intron I-Zm.DnaK:1 on the 5' side. -CaMV.35S contained a transgene cassette containing the GUS-driving A third control expression vector containing both EXP-CaMV.35S and I-Zm.DnaK:1 The mean percent expression relative to the target is shown. [Table 5]

[0114] As can be seen in Table 5, the synthetic intron I-Zm.GSI197.nno:1 (SEQ ID NO: 10) compared with the intronless control expression vector EXP-CaMV.35S Enhanced GUS transgene expression in maize leaf protoplasts driven by Enhanced expression was observed in the intron I-Zm.DnaK:1 and the E gene operably linked to the 5' side. Compared with a third control expression vector containing XP, EXP-CaMV.35S, The enhancement was greater than that conferred by I-Zm.DnaK:1.

[0115] Synthetic 3'UTR, T-Zm.GST9.nno:2 and T-Zm.GST18.nno: Analysis of enhancement of GUS expression by 2. Maize leaf protoplast cells were transfected with constructs containing expression elements that drive GUS expression. The two test vectors were 3'UTR, T-Zm.G ST9.nno:2 (SEQ ID NO: 13) and T-Zm.GST18.nno:2 (SEQ ID NO: 14) contains a transgene cassette used for analyzing the enhanced GUS expression conferred by R, T-Zm.GST9.nno:2 (SEQ ID NO: 13) or 3'UTR, T-Zm.G GU operably linked at the 5' end to either ST18.nno:2 (SEQ ID NO: 14) an intron operably linked at the 5' end to the coding sequence encoding S (SEQ ID NO: 24) It consists of EXP-CaMV.35S operably linked to I-Zm.DnaK:1 at the 5' end. As described above, three control expression vectors were also constructed and expressed in maize leaf protoplasts. Table 6 shows the GUS-driving EXP-CaMV.35S and The average expression percentage was compared to a third control expression vector containing both I-Zm.DnaK:1 and I-Zm.DnaK:1. It shows cents. [Table 6]

[0116] As can be seen in Table 6, the 3'UTR, T-Zm.GST9.nno:2 (SEQ ID NO: 13) and T-Zm.GST18.nno:2 (SEQ ID NO: 14) derived from maize leaf protoplasts. Compared with the control, GUS expression was enhanced.

[0117] Example 3 Synthetic EXP, EXP-Zm in stably transformed maize plants of the LH244 variety .GSP850.nno+Zm.GSI153.nno:2 and EXP-Zm.GSP8 Analysis of GUS expression driven by 50.nno+Zm.GSI140.nno:1 Maize plants were transfected with vectors, specifically the β-glucuronidase (GUS) gene. The plants were transformed with a plant expression vector containing the test regulatory element driving the expression of the gene. The plants were analyzed for GUS protein expression to determine whether the selected regulatory elements were involved in the expression. The effects were evaluated.

[0118] Maize plants were transformed with a plant GUS expression construct. Regulatory elements are cloned into the base plant expression vector using standard methods known in the art. The obtained plant expression vector was transformed into Agrobacterium tumefaciens. The left border region (B-AGRtu.left border) from ns and the herbicide The first gene introduced into the plant used to select transformed plant cells that confers resistance to glyphosate. A gene selection cassette and a second transgene cassette for assessing the activity of the synthetic regulatory element. 3' termination region, T-Sb.Nltp4-1:1:2 (SEQ ID NO: 19) and 5' termination region, The potato light-inducible tissue-specific ST-LS1 gene (Gen It contains a processible intron derived from the nucleotide sequence of ... A synthetic coding sequence designed for expression in plant cells encoding a β-glucuronidase. (GUS, GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO:20) and 5' operably linked at the side, synthetic EXP, EXP-Zm.GSP850.nno+Zm.G SI153.nno:2 (SEQ ID NO: 4) or EXP-Zm.GSP850.nno+Z A second transgene cluster containing either m.GSI140.nno:1 (SEQ ID NO: 11) Set and right border region from Agrobacterium tumefaciens It included the area (B-AGRtu.right border). -Zm.GSP850.nno+Zm.GSI153.nno:2 (SEQ ID NO: 4) The synthetic intron I-Zm.GSI153.nno:1 (SEQ ID NO: 5) is operably located 5' to 5' to the synthetic leader L-Zm.GSP850.nno:3 (SEQ ID NO: 3) operably linked to the synthetic promoter P-Zm.GSP850.nno:4 (SEQ ID NO: 2) It consisted of: Synthetic EXP, EXP-Zm.GSP850.nno+Zm.G SI140.nno:1 (SEQ ID NO: 11) is a synthetic intron I-Zm.GSI140. Synthetic leader L-Zm.G operably linked at the 5' end to nno:1 (SEQ ID NO:12) A synthetic promoter operably linked at the 5' end to SP850.nno:3 (SEQ ID NO: 3) -, P-Zm.GSP850.nno:4 (SEQ ID NO: 2).

[0119] Maize cultivar LH244 plant cells were cultured in Agrobacterium tuberculosis, as known in the art. The above binary transformation vector constructs were transformed by Bacillus subtilis-mediated transformation. The resulting transformed plant cells were used to form whole corn plants. was guided to do so.

[0120] in selected plant organs and tissues of transformed plants using qualitative and quantitative GUS analysis. The activity of the expression element in the nucleus was evaluated. For this purpose, whole mount or sectioned tissues were treated with 1 mg / mL of X-Gluc (5-bromo-4-chloro- Ink was then incubated at 37°C for 5 hours with a GUS staining solution containing β-3-indolyl-β-glucuronide. The sections were then incubated and destained with 35% EtOH and 50% acetic acid. GUS expression was confirmed by visual inspection of the blue coloration of selected plant organs or tissues under was determined qualitatively.

[0121] Transformed maize plants for quantitative analysis of GUS expression by enzymatic assay Total protein was extracted from selected tissues. 1-2 micrograms of total protein was extracted from 50 fluorogenic substrate 4-methylumbelliferyl at a concentration of 1 mM in a microliter total reaction volume The cells were incubated with methyl-β-D-glucuronide (MUG) at 37°C for 1 hour. After incubation, 350 microliters of 200 mM sodium bicarbonate solution was added. The reaction product, 4-methylumbelliferone (4-MU), was dissolved in water at high pH. It becomes maximally fluorescent at 1000 kJ / cm2, when the hydroxyl group is ionized. The addition of sodium solution stops the assay and simultaneously quantifies the fluorescent product 4-MU. The pH of the solution is adjusted. The amount of 4-MU formed is measured using the FLUOstar Omega Plate reader (BMG LABTECH) (355 nm excitation, 460 nm emission) GUS activity was estimated by measuring the fluorescence intensity using a 4-MU / hour / mg total protein is provided in nanomoles.

[0122] The following tissues were sampled for GUS expression in the R0 generation: leaves at the V4 stage and Roots; leaves and roots at V7 stage; leaves, roots, flowers / anthers at VT stage; cobs / hairs at R1 stage; 2 from pollination Seed embryos and seed endosperms at R3 stage after 1 day. Table 7 shows the average quantitative GUS expression of each synthetic EXP. The values are shown. [Table 7]

[0123] As can be seen in Table 7, the synthetic GSP850 promoter and leader (P-Zm.GSP 850.nno:4 (SEQ ID NO: 2) and L-Zm.GSP850.nno:3 (SEQ ID NO: 3)) constitutive expression of GUS in stably transformed maize plants of the LH244 variety. Molecular analysis of the transcription start site revealed the GSP850 promoter and leader The synthetic intron I-Zm.GSI153.nno:1 (sequence column number 5) and I-Zm.GSI140.nno:1 (SEQ ID NO: 12) were sampled. The expression was affected differently in different tissues. Molecular analysis of intron splice sites revealed showed consistent processing of the synthetic intron. The overall enhancement of GUS expression was Most tissue samples from plants containing I-Zm.GSI153.nno:1 (SEQ ID NO: 5) The V4 leaf, V7 root, and R3 seed embryo were the exceptions. The GUS expression levels were relatively similar. The enhanced expression conferred by I-Zm.GSI140.nno:1 (SEQ ID NO: 1) 2) Compared to V4 roots, V4 leaves, VT roots, and VT leaves, the results were approximately 7.5 times, 7.7 times, and 6.0 times, respectively. It was 4.3 times higher in flowers / anthers, 3.2 times higher in R1 cobs / hairs, and 2.3 times higher in R3 seed endosperm.

[0124] Example 4 Synthetic EXP, EXP-Zm, was synthesized in stably transformed corn plants of the O1DKD2 variety. .GSP850.nno+Zm.DnaK:1, EXP-Zm.GSP850.nno+ Zm.GSI153.nno:2, and EXP-Zm.GSP850.nno+Zm.G Analysis of GUS expression driven by SI140.nno:1 Maize plants were transfected with vectors, specifically the β-glucuronidase (GUS) gene. The plants were transformed with a plant expression vector containing the test regulatory element driving the expression of the gene. The plants were analyzed for GUS protein expression to determine whether the selected regulatory elements were involved in the expression. The effects were evaluated.

[0125] Maize plants were transformed with a plant GUS expression construct. Regulatory elements are cloned into the base plant expression vector using standard methods known in the art. The obtained plant expression vector was transformed into Agrobacterium tumefaciens. The left border region (B-AGRtu.left border) from ns and the herbicide The first gene introduced into the plant used to select transformed plant cells that confers resistance to glyphosate. A gene selection cassette and a second transgene cassette to assess the activity of regulatory elements 3' termination region, T-Sb.Nltp4-1:1:2 (SEQ ID NO: 19) and 5' side The potato light-inducible tissue-specific ST-LS1 gene (Genba) was operably linked to β containing a processible intron derived from nk accession: X04753 - a synthetic coding sequence designed for expression in plant cells encoding glucuronidase (G US, GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO:20) and the 5' side Operably linked synthetic EXP, EXP-Zm.GSP850.nno+Zm.Dna K:1 (SEQ ID NO: 15), EXP-Zm.GSP850.nno+Zm.GSI153. nno:2 (SEQ ID NO: 4), or EXP-Zm.GSP850.nno+Zm.GSI 140.nno:1 (SEQ ID NO: 11), and a second transgene cassette comprising either The right border region (BA) from Agrobacterium tumefaciens GRtu.right border) and included. Synthetic EXP, EXP-Zm.G SP850.nno+Zm.DnaK:1 (SEQ ID NO: 15) contains intron I-Zm.D Synthetic leader L-Zm.G operably linked at the 5' end to naK:1 (SEQ ID NO: 22) A synthetic promoter operably linked at the 5' end to SP850.nno:3 (SEQ ID NO: 3) The synthetic EX was composed of P-Zm.GSP850.nno:4 (SEQ ID NO:2). P, EXP-Zm.GSP850.nno + Zm.GSI153.nno:2 (SEQ ID NO: 4) and EXP-Zm.GSP850.nno+Zm.GSI140.nno:1 (sequence Number 11) is described in Example 3.

[0126] Corn variety 01DKD2 plant cells were cultured in a manner known in the art using Ag The above binary transformation vector components were transformed by robacterium-mediated transformation. The resulting transformed plant cells were transformed into whole corn plants. Qualitative and quantitative GUS expression was measured as described in Example 3. Table 8 shows the average quantitative GUS expression values for each synthetic EXP. [Table 8]

[0127] As can be seen in Table 8, the synthetic GSP850 promoter and leader (P-Zm.GSP 850.nno:4 (SEQ ID NO: 2) and L-Zm.GSP850.nno:3 (SEQ ID NO: 3)) constitutive expression of GUS in stably transformed maize plants of the O1DKD2 variety. The synthetic intron I-Zm.GSI153.nno:1 (SEQ ID NO: 5) and and I-Zm.GSI140.nno:1 (SEQ ID NO: 12) is the intron I-Zm.Dn Compared to aK:1 (SEQ ID NO: 22), expression was enhanced in all tissues assayed.

[0128] Example 5 Synthetic EXP, EXP-Z, in stably transformed 01DKD2 maize plants m.GSP990.nno+Zm.DnaK:1 and EXP-Zm.GSP990.nn Analysis of GUS expression driven by o+Zm.GSI197.nno:2 Maize plants were transfected with vectors, specifically the β-glucuronidase (GUS) gene. The plants were transformed with a plant expression vector containing the test regulatory element driving the expression of the gene. The plants were analyzed for GUS protein expression to determine whether the selected regulatory elements were involved in the expression. The effects were evaluated.

[0129] Maize plants were transformed with a plant GUS expression construct. Regulatory elements are cloned into the base plant expression vector using standard methods known in the art. The obtained plant expression vector was transformed into Agrobacterium tumefaciens. The left border region (B-AGRtu.left border) from ns and the herbicide The first gene introduced into the plant used to select transformed plant cells that confers resistance to glyphosate. A gene selection cassette and a second transgene cassette to assess the activity of regulatory elements 3' termination region, T-Sb.Nltp4-1:1:2 (SEQ ID NO: 19) and 5' side The potato light-inducible tissue-specific ST-LS1 gene (Genba) was operably linked to β containing a processible intron derived from nk accession: X04753 - a synthetic coding sequence designed for expression in plant cells encoding glucuronidase (G US, GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO:20) and the 5' side Operably linked synthetic EXP, EXP-Zm.GSP990.nno+Zm.Dna K:1 (SEQ ID NO: 16) or EXP-Zm.GSP990.nno+Zm.GSI19 7. A second transgene cassette comprising either nno:2 (SEQ ID NO: 9) or Ag The right border region (B-AGRt) from Bacterium tumefaciens u.right border) and included. Synthetic EXP, EXP-Zm.GSP9 90.nno+Zm.GSI197.nno:2 (SEQ ID NO: 9) is a synthetic intron I- Zm.GSI197.nno:1 (SEQ ID NO: 10) operably linked at the 5' end to a synthetic Operatively linked to leader L-Zm.GSP990.nno:1 (SEQ ID NO: 8) at the 5' end The promoter consists of a synthetic promoter, P-Zm.GSP990.nno:2 (SEQ ID NO: 7). It was. Synthetic EXP, EXP-Zm.GSP990.nno+Zm.DnaK:1( SEQ ID NO: 16) acts 5' to intron I-Zm.DnaK:1 (SEQ ID NO: 22) operably linked to the synthetic leader L-Zm.GSP990.nno:1 (SEQ ID NO: 8) and 5 operably linked at the '-side, a synthetic promoter P-Zm.GSP990.nno:2 (sequence It consists of column number 7).

[0130] Corn variety 01DKD2 plant cells were cultured in a manner known in the art using Ag The above binary transformation vector components were transformed by robacterium-mediated transformation. The resulting transformed plant cells were transformed into whole corn plants. Qualitative and quantitative GUS expression was measured as previously described in Example 3. Table 9 shows the average quantitative GUS expression values for each synthetic EXP. "D" indicates not measured. [Table 9]

[0131] As can be seen here, the synthetic GSP990 promoter and leader (P-Zm.GSP 990.nno:2 (SEQ ID NO: 7) and L-Zm.GSP990.nno:1 (SEQ ID NO: 8)) drove GUS expression. Molecular analysis of the transcription start site revealed that the GSP990 promoter The synthetic intron I-Zm.GSI19 showed consistent TSSs for the marker and leader. 7.nno:1 (SEQ ID NO: 10) is a nucleotide sequence of intron I-Zm.DnaK:1 (SEQ ID NO: 22) ), it weakened expression in some tissues while enhancing expression in others. For example, GUS expression was weakened in leaves at the V4, V7, and VT stages. -Slightly enhanced in roots of V7 and VT compared to Zm.DnaK:1. Flower / anther expression Compared with I-Zm.DnaK:1, I-Zm.GSI197.nno:1 (SEQ ID NO: :10) was enhanced by approximately 2.7-fold. The difference in expression conferred by m.GSI197.nno:1 (SEQ ID NO: 10) is low expression in leaves. This can be very useful when high expression in the flower / anther is desired. Molecular analysis of the position revealed a synthetic intron I-Zm.GSI197.nno:1 (SEQ ID NO: 10) demonstrated consistent processing.

[0132] Example 6 Synthetic 3'UTR, T-Zm.GST9.nno:2, T-Zm.GST18.nno:2 , and T-Zm.GST43.nno:1, as well as the native T-Sb.Ntlp4- 1:1:2 suppresses GUS expression in stably transformed 01DKD2 maize plants. Analysis of the effects on the present Maize plants were transfected with vectors, specifically the β-glucuronidase (GUS) gene. The plants were transformed with a plant expression vector containing the test regulatory element driving the expression of the gene. The plants were analyzed for GUS protein expression to determine whether the selected regulatory elements were involved in the expression. The effects were evaluated.

[0133] Maize plants were transformed with a plant GUS expression construct. Regulatory elements are cloned into the base plant expression vector using standard methods known in the art. The obtained plant expression vector was transformed into Agrobacterium tumefaciens. The left border region (B-AGRtu.left border) from ns and the herbicide The first gene introduced into the plant used to select transformed plant cells that confers resistance to glyphosate. A gene selection cassette and a second transgene to assess the activity of the 3'UTR regulatory element a potato light-inducible promoter cassette operably linked at the 5' end to a 3' termination region; Derived from the tissue-specific ST-LS1 gene (Genbank accession: X04753) In plant cells, a gene encoding a β-glucuronidase containing a processible intron is A synthetic coding sequence (GUS, GOI-Ec.uidA+St.LS) designed for expression in 1.nno:1, SEQ ID NO:20) and intron I-Zm. EXP, EXP-CaM operably linked to DnaK:1 (SEQ ID NO: 22) on the 5' side a second transgene cassette containing V.35S (SEQ ID NO: 21); and The right border region from Rhizobium tumefaciens (B-AGRtu.righ The three test expression vectors contained the GUS coding sequence and movably linked 3'UTR, T-Zm.GST9.nno:2 (SEQ ID NO: 13), T- Zm.GST18.nno:2 (SEQ ID NO: 14), or T-Zm.GST43.nno :1 (SEQ ID NO:26). Additional test expression vectors were engineered to contain the GUS coding sequence. The operably linked native 3'UTR, T-Sb.Nltp4-1:1:2 (SEQ ID NO: 19) and was used to compare expression between native and synthetic 3′UTRs.

[0134] Corn variety 01DKD2 plant cells were cultured in a manner known in the art using Ag The above binary transformation vector components were transformed by robacterium-mediated transformation. The resulting transformed plant cells were transformed into whole corn plants. was induced to form.

[0135] Qualitative and quantitative GUS analysis was used to characterize the expression of GUS in V4 leaf and root tissues of transformed plants. The element activity was assessed and performed as described in Example 3 above. To compare the effect of expression with T-Sb.Nltp4-1:1:2 (SEQ ID NO: 19), The effect of the synthetic 3'UTR was evaluated. The resulting transcripts were analyzed to confirm proper termination. The presence or absence of transcript read-through was assessed. One method corresponds to the portion of the T-DNA border sequence that is 3' to the 3'UTR. This was achieved by amplifying the transcribed cDNA using amplification primers. m.GST9.nno:2 (SEQ ID NO: 13), T-Zm.GST18.nno:2 (SEQ ID NO: No. 14), T-Zm.GST43.nno:1 (SEQ ID NO: 26), and T-Sb.Nl Plants transformed with four constructs containing tp4-1:1:2 (SEQ ID NO: 19) The mean GUS expression is shown in Table 10. [Table 10]

[0136] As can be seen in Table 10, T-Zm.GST9.nno:2 (SEQ ID NO: 13) and TZ m.GST18.nno:2 (SEQ ID NO: 14) contains both the 3'UTR, T-Sb.Nlt Compared with p4-1:1:2, EXP-C operably bound to I-Zm.DnaK:1 Enhanced GUS expression driven by aMV.35S. T-Zm.GST9.nno GUS expression in plants containing T-Zm.GST18.nno:2 (SEQ ID NO: 13) was T-Zm.GST43.nno:1 (SEQ ID NO: 26) was higher than that of plants containing T -Compared to Sb.Nltp4-1:1:2, GUS expression was enhanced in V4 roots but not in V4 leaves. Analysis of GUS transcripts from all four constructs revealed that the The resulting GUS transcript showed proper termination and no evidence of read-through. 3'UTR, T-Zm.GST9.nno:2 (SEQ ID NO: 13), T-Zm.G ST18.nno:2 (SEQ ID NO: 14), and T-Zm.GST43.nno:1 (SEQ ID NO: No. 26) operates in a manner similar to the native 3'UTR, -Sb.Nltp4-1:1:2 showed a down-regulation of GUS expression. The synthesized 3'UTR and an additional native 3'UTR, T-Sb.Nltp4-1:1:2, A range of expression values useful for fine-tuning expression in stably transformed maize plants. bring about.

[0137] Example 7 Enhancer elements derived from regulatory elements The enhancer is derived from the promoter elements presented as SEQ ID NOs: 2 and 7. Enhancer elements can act either 5' or 3' to the promoter element. When bound to a promoter or when bound to an additional enhancer element operably linked to the promoter The level of expression of a transcribable DNA molecule when operably linked at the 5' or 3' end to a or in specific cell types or plant organs, or to direct the expression of a transcribable DNA molecule at a specific time during development or circadian rhythm. The enhancer may be composed of one or more cis-regulatory elements that act as a regulator of the transcription factor. and 7, which are capable of initiating transcription from the promoters or fragments thereof. A TATA box or functionally similar element and any It is created by removing the downstream sequence of

[0138] TATA boxes in plant promoters are not as highly conserved as in some other eukaryotes. Therefore, to define a fragment as an enhancer, first, It is necessary to identify the transcription start site (TSS) of the gene where the UTR is first transcribed. The enhancer derived from the synthetic promoter P-Zm.GSP850.nno:4 (SEQ ID NO: 2) The enhancer can include nucleotides 1 to 418 of SEQ ID NO:2, and the synthetic enhancer E The synthetic promoter P-Zm.GSP is obtained by the procedure of the present invention. The enhancer derived from 990.nno:2 (SEQ ID NO: 7) is and the synthetic enhancer E-Zm.GSP990 (SEQ ID NO: 1). 18) are obtained. Enhancers derived from these promoters are shown in SEQ ID NO: 17 and 18, or a duplication of SEQ ID NOs: 17 and 18, or their respective fragments. The effectiveness of synthetic enhancers derived from synthetic promoters can be , synthetic promoter P-Zm.GSP850.nno:4 (SEQ ID NO: 2) or P-Zm A gene encoding a protein containing a fragment derived from either GSP990.nno:2 (SEQ ID NO: 7) This is determined empirically by constructing a mel transcriptional regulatory element. , operably linked to a promoter and leader, and suitable for stable or transient plant assays. It is used to drive the expression of transcribable DNA molecules such as GUS in plants.

[0139] Further refinement of enhancer elements may be required, which is empirically In addition, other elements of the enhancer element within the chimeric transcriptional regulatory element will be verified. The position relative to the element is also determined empirically. The order of each element may have different effects depending on the relative position of each element. Some promoter elements contain multiple TATA boxes or has a TATA box-like element and possibly multiple transcription start sites. In such a situation, first identify where the first TSS is located, then Start the enhancer design using S to identify potential sites within the putative enhancer element. It may be necessary to prevent unintended transcription initiation from occurring.

[0140] Enhancer elements derived from the promoter elements presented as SEQ ID NOs: 2 and 7 The element is constructed 5' to or within the promoter element. an additional enhancer operably linked to or in operable association with the promoter; operably linked to the 5' or 3' side of the element, as known in the art. Alternatively, enhancer elements can be cloned using methods known in the art. Using methods described in the literature, we have created larger enhancers consisting of two or more copies of the enhancer. The promoter may be cloned to provide an enhancer element, and may be modified by techniques known in the art. operably linked to the promoter at the 5' or 3' end, or or an additional gene operably linked to the promoter resulting in a chimeric transcriptional regulatory element. The promoter may be cloned so that it is operably linked to the 5' or 3' side of the promoter. Enhancer sequences derived from promoters derived from genes from organisms of multiple genera are The element can be operably linked to an enhancer derived from a synthetic promoter. do.

[0141] Constructs described in Example 3 and A similar GUS expression plant transformation vector can be constructed, and the resulting plant expression vector - The left border region from Agrobacterium tumefaciens (B-AGRtu.left border) and resistance to the herbicide glyphosate a first transgene selection cassette used to select transformed plant cells that have received the gene; A second transgene cassette for testing the sarelement, 3 from the tiva lipid transfer protein-like gene (T-Os.LTP:1, SEQ ID NO: 23) The potato light-inducible tissue-specific ST-LS1 gene ( A processable introductory sequence derived from Genbank accession number X04753 The coding sequence for β-glucuronidase containing GOI-Ec.uidA+St.L S1.nno:1, SEQ ID NO:20), operably linked to an intron element and 5' a leader element operably linked 5' to the promoter element; operably linked to a target element on the 5' or 3' side or operably linked to a promoter operably linked to an additional enhancer element operably linked to the 5' or 3' side , a second transgene cassette consisting of an enhancer element and A. tume Right border region from faciens (B-AGRtu.right border) The resulting plasmid can be used to transform corn plants or other plants by the above method. Alternatively, a monocotyledonous plant derived from maize or another monocotyledonous plant may be transformed. The resulting protoplast cells are transformed using methods known in the art to produce A transient assay is performed.

[0142] Stable GUS expression driven by regulatory elements containing one or more enhancers or the effect of enhancers on the expression of transcribable DNA molecules as assessed in transient plant assays. Determine the effect of one or more enhancer elements. The overlap of more than one enhancer element was determined by empirical experiments and by using each regulatory element composition. This can be done based on the resulting gene expression regulation observed in the Altering the relative position of one or more enhancers within a regulatory or chimeric regulatory element This may affect the transcriptional activity or specificity of the regulatory or chimeric regulatory element. and the desired transgene expression profile in corn plants or other plants. The best enhancer for the gene is determined empirically to identify it.

[0143] Having illustrated and described the principles of the present invention, it is understood that the present invention may be practiced without departing from such principles. It will be apparent to those skilled in the art that variations in arrangement and detail may be made. The inventors claim all modifications that come within the spirit and scope of the claims. All publications and published patent documents used are incorporated by reference unless the respective publication or patent application is expressly incorporated by reference. The references are incorporated herein to the same extent as if they were specifically and individually indicated as being incorporated by reference. Incorporated by reference.

Claims

1. 1. A recombinant DNA molecule comprising: a) a sequence having at least 85 percent sequence identity to SEQ ID NO: 26; b) a sequence comprising SEQ ID NO: 26, and c) A fragment of SEQ ID NO: 26 that has gene regulatory activity comprising a DNA sequence selected from the group consisting of: the recombinant DNA sequence is operably linked to a heterologous transcribable DNA molecule. A DNA molecule.

2. The sequence has at least 90 percent sequence identity to the DNA sequence of SEQ ID NO:

26.

2. The recombinant DNA molecule of claim 1, comprising:

3. The sequence has at least 95 percent sequence identity to the DNA sequence of SEQ ID NO:

26.

2. The recombinant DNA molecule of claim 1, comprising:

4. The recombinant DNA molecule of claim 1 , wherein the DNA sequence has gene regulatory activity.

5. 2. The recombinant vector of claim 1, wherein said heterologous transcribable DNA molecule comprises a gene of agricultural interest. A DNA molecule.

6. 6. The method of claim 5, wherein the gene of agronomic interest confers herbicide resistance in plants. Recombinant DNA molecule.

7. 6. The method of claim 5, wherein the gene of agronomic interest confers pest resistance in plants. Recombinant DNA molecule.

8. The heterologous transcribable DNA molecule encodes a dsRNA, miRNA, or siRNA.

2. The recombinant DNA molecule of claim 1 .

9. 1. A transgenic plant cell, comprising: a) a sequence having at least 85 percent sequence identity to SEQ ID NO: 26; b) a sequence comprising SEQ ID NO: 26, and c) A fragment of SEQ ID NO: 26 that has gene regulatory activity a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: the DNA sequence is operably linked to a heterologous transcribable DNA molecule. Transgenic plant cells.

10. The transgenic plant cell of claim 9, wherein the transgenic plant cell is a monocotyledonous plant cell. Transgenic plant cells.

11. The transgenic plant cell of claim 9, wherein the transgenic plant cell is a dicotyledonous plant cell. Transgenic plant cells.

12. A transgenic plant or part thereof comprising the recombinant DNA molecule of claim 1.

13. A progeny plant of the transgenic plant according to claim 12 or a part thereof, The progeny plant or part thereof comprising the recombinant DNA molecule.

14. A transgenic seed comprising the recombinant DNA molecule of claim 1. Lancegenic seeds.

15. 13. A method for producing a commodity product, comprising the steps of: said method comprising obtaining said part and producing said commercial product therefrom.

16. The commodity product may be a seed, a processed seed, a protein concentrate, a protein isolate, a starch, 16. The method of claim 15, wherein the raw material is a grain, a plant part, a seed oil, a biomass, a flour, or a meal. How to do it.

17. 13. A method for expressing a transcribable DNA molecule comprising the steps of: and cultivating said plant, wherein said transcribable DNA is expressed. The method.

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