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: Provided are novel gene regulatory elements for use in plants, recombinant DNA molecule constructs comprising regulatory elements. Also provided are recombinant DNA molecules comprising regulatory elements, as well as methods of making and using transgenic plant cells, plants, and seeds comprising regulatory elements operably linked to transcribable DNA molecules.SELECTED DRAWING: None

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 790,570, filed January 10, 2019. No. 60 / 699,997, filed on Oct. 1, 2007, which claims the benefit of the same application, which is incorporated herein by reference in its entirety. Incorporating a sequence listing

[0002] The sequence listing contained in the file named "MONS468WO_ST25.txt" is , 33,896 bytes (measured on Microsoft Windows (registered trademark)) , prepared on January 9, 2020, and submitted electronically with this specification. and is incorporated herein by reference.

[0003] Technical Field 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. These elements are genetic elements that regulate gene activity by , promoter, leader, introns, and 3' untranslated region, These are useful in the fields of plant molecular biology and plant genetic engineering. Summary of the Invention

[0005] The present invention provides novel gene regulatory elements for use in plants. Also provided are recombinant DNA molecule constructs comprising regulatory elements. Also provided are transgenic plant cells, plants, and seeds comprising the gene. In certain embodiments, the regulatory element is operably linked to the transcribable DNA molecule. In this embodiment, the transcribable DNA molecule may be heterologous to the regulatory sequence. Thus, the regulatory element sequences provided by the present invention may, in certain embodiments, can be defined as being operably linked to a heterologous transcribable DNA molecule. In addition, the regulatory elements are used to produce recombinant DNA molecules containing the regulatory elements, as well as transcribable and a transgenic plant cell line comprising a regulatory element operably linked to a DNA molecule capable of expressing the gene. Methods for making and using the 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 of any of numbers 2-8, 12-14, and 16-19 (b) a sequence having sequence identity with any of SEQ ID NOs: 2 to 8, 12 to 14, and 16 to 19 and (c) a sequence comprising SEQ ID NOs: 2 to 8, 12 to 14, and The DNA sequence is selected from the group consisting of any one of fragments 16 to 19, A recombinant DNA molecule in which the sequence is operably linked to a heterologous transcribable DNA molecule. A "heterologous transcribable DNA molecule" is a DNA molecule that is operably transcribable. It means that the polynucleotide sequence is heterologous to the polynucleotide sequence to which it is linked. The recombinant DNA molecule is a DNA sequence of any one of SEQ ID NOs: 2 to 8, 12 to 14, and 16 to 19. For NA sequences, at least about 85 percent, at least about 86 percent, At least about 87 percent, at least about 88 percent, at least about 89 percent, at least At least about 90 percent, at least 91 percent, at least 92 percent, at least at least 93 percent, at least 94 percent, at least 95 percent, at least At least 96 percent, at least 97 percent, at least 98 percent, or at least In certain embodiments, the DNA sequences have at least 99 percent sequence identity. In some embodiments, the DNA sequence comprises a regulatory element. The element comprises a promoter operably linked to an intron. In yet another embodiment, the regulatory element comprises a 3'UTR. The transcribable DNA molecule of the seed contains a gene of agronomic interest, e.g., herbicide resistance in plants. or a gene capable of providing plant pest resistance in a plant. In other embodiments, the heterologous transcribable DNA molecule is a small RNA (e.g., ds In yet another embodiment, the nucleic acid sequence includes a sequence encoding the nucleic acid sequence (e.g., RNA, miRNA, or siRNA). In accordance with the present invention, a construct comprising a recombinant DNA molecule provided herein is provided.

[0007] In another aspect, provided herein is a transgenic plant cell comprising: (a) a sequence At least about 85 percent of any of numbers 2-8, 12-14, and 16-19 (b) a sequence having sequence identity with any of SEQ ID NOs: 2 to 8, 12 to 14, and 16 to 19 and (c) a sequence comprising SEQ ID NOs: 2 to 8, 12 to 14, and The DNA sequence is selected from the group consisting of any one of fragments 16 to 19, Recombinant DNA, in which a DNA sequence is operably linked to a heterologous transcribable DNA molecule In certain embodiments, a transgenic plant cell is provided, the transgenic plant cell comprising the molecule. The transgenic plant cell is a monocotyledonous plant cell. The plant cell may be a monocotyledonous plant cell or a dicotyledonous plant cell.

[0008] In yet another aspect, the present invention further provides a transgenic plant or a) a site having at least one of SEQ ID NOs: 2 to 8, 12 to 14, and 16 to 19 b) sequences having at least about 85% sequence identity with SEQ ID NOs: 2 to 8, 12 to 14, and and c) a sequence having gene regulatory activity and including any one of SEQ ID NOs. a fragment of any of Nos. 2 to 8, 12 to 14, and 16 to 19; a DNA sequence that is operably linked to a heterologous transcribable DNA molecule, The present invention provides a transgenic plant or part thereof comprising a recombinant DNA molecule comprising: In certain embodiments, the transgenic plant is any plant containing a recombinant DNA molecule. and transgenic seeds that, when grown, produce the progeny of the plant. Transgenic plants containing recombinant DNA molecules that produce transgenic plants such as Seeds are also provided herein.

[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 transformed plant cell from the transformed plant cell. and regenerating the plant.

[0011] A brief description of arrays SEQ ID NO: 1 is the sequence of Setaria italica (Foxtail millet) 3'UT derived from the S-adenosylmethionine synthetase 1 protein gene of R, DNA sequence of T-SETit.Ams1:1.

[0012] SEQ ID NO: 2 is the name of Coix lacryma-jobi (Job's tea) ars)) Hsp16.9 (heat shock protein 16.9) protein gene The DNA sequence of the 3'UTR, T-Cl.Hsp16.9_2:1, is shown.

[0013] SEQ ID NO: 3 is a putative heat shock protein of Eragrostis tef (Tef) The 3'UTR derived from the gene, T-ERIra.Hsp17.9_3:1 DNA sequence be.

[0014] SEQ ID NO: 4 is Saccharum ravennae (hardy grass) Hsp16.9 (heat shock protein 16.9) protein from pampas grass 3'UTR from the protein gene, DNA of T-ERIra.Hsp16.9_3:1 It is an A array.

[0015] SEQ ID NO: 5 is the sequence of Andropogon gerardii (big bluestem g bluestem) and Sorghum bicolor (sorghum) 3'UTR derived from the protein gene, T-ANDge.Hsp / Sb.Hsp, Figure 1 shows the DNA sequence of the Mela 3'UTR.

[0016] SEQ ID NO: 6 is the promoter and the actin 4 gene of Setaria italica a Zea mays-derived lipid fragment operably linked 5' to an intron derived therefrom; EXP, EXP-Z, containing 5'UTR derived from the transcriptional transfer protein-like protein gene This is the DNA sequence of m.LTP-SETit.Act4.

[0017] SEQ ID NO: 7 is a promoter derived from a Zea mays lipid transfer protein-like protein. The DNA sequences of the promoter and P-Zm.Ltp-1:1:2 are shown.

[0018] SEQ ID NO: 8 is a 5' U sequence derived from a lipid transfer protein-like protein of Zea mays. The DNA sequences are TR (leader) and L-Zm.Ltp-1:1:3.

[0019] SEQ ID NO: 9 is an intron derived from the actin 4 gene of Setaria italica. This is the DNA sequence of Ron I-SETit.Act4:2.

[0020] SEQ ID NO: 10 is derived from the cauliflower mosaic virus 35S promoter in the reverse complement orientation. This is the DNA sequence of the enhancer E-CaMV.35S-RC.

[0021] SEQ ID NO: 11 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).

[0022] SEQ ID NO: 12 is Saccharum ravennae (Ravenna grass (hard grass) Hsp16.9 (heat shock protein 16.9) from pampas grass The 3'UTR derived from the protein gene, T-Cl.Hsp16.9:2 DNA sequence be.

[0023] SEQ ID NO: 13 is the sequence of Coix lacryma-jobi (Job's t The Hsp16.9 (heat shock protein 16.9) protein gene of The DNA sequence of the 3'UTR, T-Cl.Hsp16.9:2, is shown.

[0024] SEQ ID NO: 14 is a chimeric promoter derived from multiple published dahlia mosaic virus promoters. This is the DNA sequence of the reconstructed enhancer, E-DaMV.H-Flt:1. The promoter of the DaMV-Holland (DaMV-H) strain, Genban This is derived from accession EU090957, nucleotides 1177-1494. The fragment is then transcribed from the DaMV-H promoter, a second fragment, the nucleotide operably linked to residues 1003-1176 of the native DaMV promoter In this configuration, the second fragment precedes the first fragment. Within this, nucleotides 287-288 and nucleotides 319-322 are Genban k sequence at a similar position in the DaMV promoter in accession JX272320 Changed.

[0025] SEQ ID NO: 15 is Genbank accession EF513491, nucleotides 1 to 322, the dahlia mosaic virus promoter-derived enhancer, E-DaMV.F This is the DNA sequence of LT:2.

[0026] SEQ ID NO: 16 is a chimeric recombination operably linked 5' to the promoter Enhancer, E-DaMV.H-Flt:1 (SEQ ID NO: 14) and Setaria i talica actin 4 gene-derived intron 5' to the Concatenated 5' from the lipid transfer protein-like protein gene from Zea mays Consists of UTR, EXP, EXP-DaMV.H-Flt+Zm.Ltp+SET It is the DNA sequence of Act4:1.

[0027] SEQ ID NO: 17 is a chimeric recombination operably linked 5' to the promoter Enhancer, E-DaMV.H-Flt:1 (SEQ ID NO: 14) and Setaria i talica actin 4 gene-derived intron 5' to the Concatenated 5' from the lipid transfer protein-like protein gene from Zea mays Consists of UTR, EXP, EXP-DaMV.H-Flt+Zm.Ltp+SET The DNA sequence of the chimeric reconstituted E-DaMV.H-Flt:1 gene is shown. Enhancer is EXP-DaMV.H-Flt+Zm.Ltp+SETit.Act4: 1 (SEQ ID NO: 16) in the opposite orientation.

[0028] SEQ ID NO: 18 is an enhancer operably linked to the 5' side of the promoter. , E-DaMV.H-FLT:2 and 14-3- from Setaria italica operably linked 5' to an intron derived from the 3C gene, It consists of the 5'UTR from the RCc3 gene from cum dactyloides , EXP, EXP-DaMV.FLT+Td.RCc3_1+SETit.14-3-3 This is the DNA sequence of C-5:1.

[0029] SEQ ID NO: 19 is an enhancer operably linked to the 5' side of the promoter. , E-DaMV.H-FLT:2 and 14-3- from Setaria italica operably linked 5' to an intron derived from the 3C gene, It consists of the 5'UTR from the RCc3 gene from cum dactyloides , EXP, EXP-DaMV.FLT+Td.RCc3_1+SETit.14-3-3 The DNA sequence of C-5:1 is shown. The E-DaMV.FLT:2 enhancer is EXP-D aMV.FLT+Td.RCc3_1+SETit.14-3-3C-5:1 (SEQ ID NO: 18) is cloned in the opposite orientation to DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention provides regulatory elements that have gene regulatory activity in plants. The nucleotide sequences of the regulatory elements are shown as SEQ ID NOs: 2-8, 12-14 and 16-19. These regulatory elements are expressed as operably linked transcriptionally active genes in plant tissue. can affect the expression of a DNA molecule that is functional in the transgenic plant. The present invention also provides a method for regulating gene expression of a transgene linked to the gene. Methods for modifying, producing, and using recombinant DNA molecules containing the elements are also provided. The present invention relates to transgenic plant cells, plants, and plant components comprising the recombinant DNA molecules of the present invention. Compositions containing the parts and seeds, as well as methods for preparing and using the same, are also provided.

[0031] The following definitions and methods are provided to better define the present invention and to guide those skilled in the art in practicing the present 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.

[0032] 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. refers to a molecule (i.e., a polymer of deoxyribonucleotide bases or a DNA molecule). As used herein, the term "DNA sequence" refers to the nucleotide sequence of a DNA molecule. The nomenclature used herein corresponds to that of 37 CFR 1.822. and are set out in Tables 1 and 3 of Annex 2 of WIPO Standard ST.25(1998).

[0033] 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 otherwise occur. For example, a recombinant DNA molecule A DNA molecule is a DNA molecule that is composed of at least two DNA molecules of different species. DNA molecules containing DNA sequences that deviate from existing DNA sequences, including synthetic DNA sequences NA molecule, or incorporated into the DNA of the host cell by genetic transformation or gene editing It may also be a DNA molecule embedded therein.

[0034] References in this application to "isolated DNA molecules" or equivalent terms or phrases are This DNA molecule may exist alone or in combination with other compositions, but may not be in its natural environment. It means a DNA molecule that does not exist within a cell. 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. A gene is "isolated" within the scope of this disclosure unless its location in the genome is such that it is not isolated. Similarly, an insecticidal protein or any naturally occurring insecticidal variant of that protein. The nucleotide sequence encoding the protein is An isolated nucleotide sequence is not considered an isolated nucleotide sequence unless the sequence is within the DNA of the bacterium in which it is found in nature. The synthetic nucleotides encoding the amino acid sequences of naturally occurring insecticidal proteins are It is believed that a sequence of the invention may be considered isolated for the purposes of this disclosure. In this respect, any transgenic nucleotide sequence, i.e., plant or bacterial Nucleotides of DNA inserted into the genome of a cell or present in an extrachromosomal vector The nucleotide sequence may be 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 Even if present in detectable amounts in tissues, progeny, biological samples, or commercial products containing It is considered to be a separate nucleotide sequence.

[0035] 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, are identical. Manually align the sequence with another sequence to remove any appropriate internal nucleotide insertions, deletions, or deletions. is achieved by maximizing the number of nucleotide matches in a gapped sequence alignment. As used herein, the term "reference sequence" refers to any of SEQ ID NOS: 2 to 8. , 12-14 and 16-19.

[0036] 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 entire full-length reference sequence. divided by the total number of nucleotides in the sequence. Optimal for the reference sequences shown in the specification as SEQ ID NOs: 2-8, 12-14 and 16-19 At least about 85 percent identity to a reference sequence when aligned to at least about 86 percent identity, at least about 87 percent identity, at least at least about 88 percent identity, at least about 89 percent identity, at least about 90 percent identity percent identity, at least about 91 percent identity, at least about 92 percent at least about 93 percent identity; at least about 94 percent identity Sex, at least about 95 percent identity, at least about 96 percent identity, at least about 97 percent identity, at least about 98 percent identity, at least about Sequences with 99 percent identity, or at least about 100 percent identity, A DNA molecule having a certain percentage of sequence identity to a reference molecule is provided. The A molecule may exhibit the activity of the reference sequence.

[0037] Regulatory elements Regulatory elements, such as promoters, leaders (also known as 5'UTRs), endogenous The transcriptional regulators, introns, and transcription termination regions (or 3'UTRs) regulate the transcription of genes in living cells. As used herein, "regulatory elements" refer to elements that play an essential role in the overall expression of a gene. The term "ment" refers to a DNA molecule that has gene regulatory activity. In this case, the term "gene regulatory activity" refers to, for example, the activity of an operably linked transcribable DNA. A molecule by affecting transcription and / or translation of the operably linked transcriptionally active molecule. Regulatory elements that function in plants, e.g., the ability to affect the expression of a gene that can encode a gene encoding ... For example, promoters, leaders, enhancers, introns, and 3'UTRs are components of a gene. It is useful for modifying plant phenotypes through engineering.

[0038] As used herein, a "group of regulatory expression elements" or "EXP" sequence refers to an expression sequence that acts as a Optionally linked regulatory elements, such as enhancers, promoters, leaders, For example, the regulatory expression elements may refer to, for example, an intron a leader sequence operably linked 5' to the sequence; EXP useful in practicing the present invention may be composed of a promoter such as that shown in SEQ ID NO:6. , 16, 17, 18, and 19.

[0039] Regulatory elements can be characterized by their gene expression patterns, e.g., positive and negative. / or negative effects, e.g., constitutive expression or temporal, spatial, developmental, tissue, environmental, Physiological, pathological, cell cycle, and / or chemical responsiveness manifestations, and their role The term "compound" may be used to refer to a compound or a mixture of compounds or a compound that is a compound of a given type. In this case, the "pattern of gene expression" refers to the transcription of RNA from operably linked DNA molecules. A transcribed RNA molecule is a molecule that is translated into a protein molecule. In some cases, antisense or other regulatory RNA molecules, e.g., double-stranded RNA, may be generated. (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA It may also result in miRNAs, small interfering RNAs (miRNAs), and small interfering RNAs (siRNAs).

[0040] As used herein, the term "protein expression" refers to the protein expression of a transcribed RNA molecule. Protein expression is any pattern of translation into a protein molecule. Characterized by developmental or morphological properties and by quantitative or qualitative indicators It can be done.

[0041] A promoter is a molecule that regulates the expression of an operably linked transcribable DNA molecule. As used herein, the term "promoter" is used to refer to a gene that is useful as a regulatory element. The term generally refers to the process by which RNA polymerase II and other proteins ( It refers to a DNA molecule involved in the recognition and binding of promoters (e.g., trans-acting transcription factors). The target was initially 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 is a promoter that contains two or more Promoters useful in practicing the present invention include: , a promoter shown as SEQ ID NO: 7 or contained in SEQ ID NO: 18, or a fragment or variant thereof. The DNA molecules described and claimed herein and any variants or Derivatives are further defined as those containing promoter activity, i.e., transgenes. The promoters can act in host cells such as vegetative plants. In embodiments, a fragment contains the promoter sequence of the starting promoter molecule from which it is derived. In some cases, fragments are defined as those that exhibit transcriptional activity, while in others, fragments are defined as those that exhibit basal levels of transcriptional activity. RNA polymerase II complex recognizes and binds to the nucleus to initiate transcription. It contains a "minimal promoter" consisting of a TATA box or equivalent DNA sequence for This may also occur.

[0042] 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 contain other promoters, either alone or in combination with other promoters (e.g., in constructing chimeric promoters). In combination with promoters and promoter fragments, or with other expression elements and and fragments of expression elements. , at least about 50, at least about 75, at least about 95, at least about 100, at least at least about 125, at least about 150, at least about 175, at least about 200, at least at least about 225, at least about 250, at least about 275, at least about 300, at least at least about 350, at least about 400, at least about 450, at least about 500, at least at least about 550, at least about 600, at least about 650, at least about 700, at least at least about 750, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 14 00, at least about 1500, at least about 1600, at least about 1700, at least at least about 1800, at least about 1900, or at least about 2000 contiguous nucleotides DNA molecules having promoter activity as disclosed herein, A fragment of a promoter is provided, which comprises the starting promoter molecule. Methods for producing fragments are well known in the art.

[0043] The promoter provided as SEQ ID NO: 7 or contained in SEQ ID NO: 18 (e.g., Compositions derived from promoters (internal or 5' deleted) can improve or alter expression, for example. To alter the expression of a gene, one may use methods known in the art to determine whether the gene has a positive or negative effect on expression. removal of elements that have a positive or negative effect on expression; and duplication of elements that have a positive or negative effect on expression. and / or overlapping or overlapping elements that exert tissue- or cell-specific effects on expression. The promoter provided as SEQ ID NO: 7, or or the promoter contained in SEQ ID NO: 18 (TATA box element or its equivalent) and a 3' deletion in which the downstream sequence was removed. Further deletions may be made to enhance expression. Positive or negative tissue-specific, cell-specific, or time-specific (e.g., limited Elements that exert a circadian rhythm (but not a circadian rhythm) effect may be removed. or the promoter contained in SEQ ID NO: 18, and Chimeric transcriptional regulatory element compositions using derived fragments or enhancers You can also create things.

[0044] According to the present invention, the promoter or promoter fragment may be any known promoter. -elements, i.e., TATA boxes and other known transcription factor binding site motifs The promoter elements can be analyzed for the presence of any of the DNA sequence features. The identity of the promoter can be determined by one skilled in the art to identify promoters with expression patterns similar to those of the original promoter. It can be used to design variants of the motor.

[0045] 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. The leader may be a synthetically produced or engineered DNA element. A 5' regulatory element for regulating expression of an operably linked transcribable DNA molecule. The leader molecule may be used as a promoter from a heterologous promoter or its native promoter. Leaders useful in practicing the present invention include SEQ ID NO: 8 and In certain embodiments, the leader is provided as follows: Such DNA sequences can be expressed in host cells (including, for example, transgenic plant cells). In one embodiment, such a sequence may be defined as , is decoded as containing leader activity.

[0046] The leader sequence (also referred to as 5'UTR) presented as SEQ ID NO: 8, or SEQ ID NO: The reader included in No. 18 may consist of a regulatory element or an actuating A secondary structure that can affect the transcription or translation of operably linked transcribable DNA molecules The leader sequence presented as SEQ ID NO: 8, or SEQ ID NO: 18 This leader, contained in Chimeric regulatory elements may be created that affect the transcription or translation of the NA molecule.

[0047] As used herein, the term "intron" refers to a region of interest that has been isolated or identified from a gene. can be spliced ​​out during pre-translational messenger RNA (mRNA) processing Alternatively, an intron may refer to a region of a DNA molecule that can be generally defined as a segment of a DNA molecule. Introns may be DNA elements that are genetically generated or engineered. The introductory sequence may contain an enhancer element that confers transcription of the gene linked to it. The gene contains regulatory elements for regulating the expression of an operably linked transcribable DNA molecule. The construct may contain an intron, and this intron may be used as a The gene may or may not be heterologous to the transcribable DNA molecule. Examples of introns include rice actin intron and maize HSP70 intron. Examples include:

[0048] In plants, including some introns in a gene construct can significantly reduce the number of intron-free This effect is due to the increased accumulation of mRNA and protein compared to the untransfected construct. This is called "intron-mediated enhancement" (IME). Known introns are found in maize genes (e.g., tubA1, Adh1, Sh1, and Ubi1), rice genes (e.g., tpi), and petunia (e.g., rbcS) , from potato (e.g., st-ls1), and Arabidopsis thali Identified in dicotyledonous genes such as those from ana (e.g., ubq3 and pat1) Deletions or mutations within intron splice sites reduce gene expression This suggests that splicing may be required for IME. However, the IME in dicotyledonous plants is This is shown by a point mutation within the splice site of the t1 gene. The multiple use of introns has been shown to present disadvantages. Contains a set of basic control elements for constructing suitable recombinant DNA elements Exemplary introns useful in practicing the present invention are SEQ ID NO: 9, and and is presented as a leader contained within the sequence.

[0049] In one embodiment, a fragment of the intron sequence disclosed herein is provided. The intron fragment may contain intron activity as described above and may be used alone. or in combination with other expression elements and fragments of expression elements. 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 350, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1200, less At least about 1300, at least about 1400, at least about 1500, at least about 1600 , at least about 1700, at least about 1800, at least about 1900, or less The presently disclosed A fragment of an intron is provided, including a DNA molecule having intron activity. Methods for producing such fragments from introne molecules are well known in the art. is.

[0050] As used herein, "3' transcription terminator," "3' untranslated region," or "3' The term "UTR" is used during transcription into the untranslated region of the 3' portion of the mRNA molecule. The 3' untranslated region of an mRNA molecule is a region of DNA that undergoes specific cleavage and 3' polyadenylation. The 3'UTR can be generated by transcribing a nucleotide sequence (also known as a polyA tail). A molecule, and may be located downstream thereof, and may be involved in transcription, mRNA processing, and contains polyadenylation signals and other regulatory signals that can affect gene expression. The poly(A) tail is thought to function in mRNA stability and translation initiation. Examples of 3' transcription termination molecules in the art include the nopaline synthase 3' region region, wheat hsp17 3' region, endo-urbisco small subunit 3' region, cotton E6 3' region, and coixin 3'UTR.

[0051] 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 This may allow for efficient transcription termination (the transfer of RNA polymerase II from DNA). release) is a prerequisite for transcription resumption, which directly affects global transcription levels. Following transcription termination, mature mRNA is released from the synthesis site and the template is transported to the cytoplasm. Eukaryotic mRNAs accumulate in vivo in the poly(A) form, and are This makes it difficult to detect the transcription termination site using this method.

[0052] Regulation of gene function through 3'UTRs is a relatively new field, as recent sequencing Only this technique provided a comprehensive picture of 3'UTRs across species and cell types. Before the availability of sequencing technology, detailed functional and mechanistic studies were performed on a few These model 3'UTRs were only performed with the 3'UTR Although they have contributed greatly to our understanding of biology, the conclusions drawn about their regulatory functions are limited. The focus was on mRNA stability. (Mayr, Christine (2 017)Regulation by 3'-Untranslated Region s.Annual Review of Genetics,51:171-194) Genome-wide in silico analysis revealed that motifs in the 3'UTR are primarily involved in gene regulation at the post-transcriptional level. Conserved on one strand, which corresponds to the 3'UTR that acts to regulate expression. It was revealed (Xie, X. et. al., (2005) Systematic di discovery of regulatory motifs in human pr omoters and 3' UTRs by comparison of sev (European mammals, Nature 434:338-345). The 3'UTR is mRNA stability and translation mediated primarily by AU-rich elements and miRNAs The 3'UTR also regulates mRNA localization. The length of the 3'UTR allows for alternative cleavage and polyadenylation. The 3'UTR mediates protein-protein interactions (PPIs), which , which have widespread effects on protein complex formation, protein localization, and protein function. The 3'UTR regulates gene expression through the binding of RNA-binding proteins (RBPs). RBPs bind to 3'UTR cis-elements and mediate the expression of effector proteins. RBPs mediate 3'UTR function through recruitment of RBPs. To achieve this, RBPs cooperate with other RBPs bound to the 3'UTR at specific moments. The composition of RBPs is dynamic, e.g., due to the addition of post-translational modifications, the local expression of other RBPs, and These may change depending on the local environment, such as interactions with membranes and cytoskeletal filaments. Binding may also be mediated by the formation of secondary and tertiary RNA structures that regulate the accessibility of the 3'UTR. Mayr, Christine (2017) Regulation by 3'-Untranslated Regions.Annual Review of Genetics,51:171-194).

[0053] The poly(A) tail is the result of the addition of a series of adenosine bases to the 3' end of an RNA molecule. This results in the regulation of gene expression, including mRNA export, stability and decay, and translation. The expression of a regulatory factor called poly(A)-binding protein (PABP) plays a role in the regulation of The 5' cap structure and poly(A) tail of the mRNA provide a binding site for Ras. They function synergistically to regulate mRNA translation. Binding of PABP to poly(A) tails promotes the interaction with eIF4F bound to the 5' cap structure, resulting in translation initiation This promotes ribosome recycling and results in mRNA circularization, ensuring efficient translation. This interaction also prevents the inhibition of translation by inhibitor proteins bound to the 3'UTR. It is also possible (Barrett, L et.al. (2012) Regulation f eukaryotic gene expression by the untr anslated regions and other non-coding regions elements.Cell.Mol.Life Sci.69:3613-3634).

[0054] From a practical standpoint, typically, the 3'UTR used in an expression cassette should have the following characteristics: First, the 3'UTR is essential for efficient and effective transcription of 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, as in the case of a cassette or preventing transcript read-through into adjacent chromosomal DNA where the T-DNA is inserted. Second, the 3'UTR is used to drive expression of the DNA molecule. Reduce the transcriptional activity conferred by promoters, leaders, enhancers, and introns ( (unless this is a desired outcome). Driving expression using promoter, leader, and intron sequences Data are provided showing that the 3'UTRs shown as SEQ ID NOS: 1 to 5 are capable of reversing expression. These genes are differentially regulated in different plants and exhibit tissue-specific effects on protein expression. In biotechnology, the 3'UTR is extracted from reverse-transcribed RNA extracted from transformed plants. They are often used to prime amplification reactions, and (1) once integrated into plant chromosomes, (2) assessing the transcriptional activity or expression of the inserted expression cassette within the plant DNA; (3) assessing the zygosity of the seeds obtained after breeding. The 3'UTR also characterizes the integrity of the inserted cassette. For this purpose, it is also used in the amplification reaction of DNA extracted from transformed plants. 3'UTRs useful for this purpose are presented as SEQ ID NOS: 1 to 5 and 12 to 13.

[0055] In one embodiment, fragments of the 3'UTR sequences disclosed herein are provided. The 3'UTR fragment may contain 3'UTR activity as described above and may also be used alone. or in combination with other expression elements and fragments of expression elements. 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 1 75, at least about 200, at least about 225, at least about 250, at least about 2 75, at least about 300, at least about 350, at least about 400, at least about 4 50, at least about 500, at least about 550, at least about 600, or at least At least about 650 or more DNA molecules with 3'UTR activity as disclosed herein A fragment of the 3'UTR containing the starting 3'UTR molecule is provided. Methods for producing fragments are well known in the art.

[0056] As used herein, the term "enhancer" or "enhancer element" The term refers to a cis-acting regulatory element (also known as a cis-element), which is a regulatory element that The overall expression pattern of operably linked transcribable DNA molecules (although usually not alone) Unlike promoters, Hanser elements include a transcription start site (TSS) or TATA box, or The promoter or promoter fragment does not contain any DNA sequence. Naturally, one or more enhancer elements that affect the transcription of an operably linked DNA sequence. Alternatively, an enhancer element may be fused to the promoter to Chimeric promoter cis-elements may be generated, which allow for the overall regulation of gene expression. This provides a form of adaptive regulation.

[0057] Many promoter-enhancer elements bind to DNA-binding proteins or and / or affect DNA topology, thereby allowing RNA polymerase to target the DNA template. Local conformations that selectively allow or restrict access, or duplexes at transcription start sites It is thought to generate a local conformation that promotes selective helix opening. The nucleotide sequence of a transcription factor can function to bind to a transcription factor that regulates transcription. The promoter element binds two or more transcription factors, which bind with different affinities. The identification of enhancer elements is crucial for the deletion analysis. analysis (i.e., one or more nucleotides from the 5' end or internal to the promoter) and DNA-binding protein analysis using DNase I footprinting. analysis, methylation interference, electrophoretic mobility shift assay, ligation-mediated polymerase chain reaction In vivo genomic footprinting by PCR and other conventional methods These can be performed by multiple techniques, including quantitative assays, or by traditional D Known sis-elements as target sequences or target motifs in conjunction with NA sequence comparison methods By DNA sequence similarity analysis using motifs or enhancer elements The fine structure of the enhancer domain can be determined by mutagenesis of one or more nucleotides (if Further investigation may be carried out by further modification (e.g., by cleavage or substitution) or by other conventional methods known in the art. Enhancer elements can be chemically synthesized or can be prepared by incorporating such elements. These can be obtained by isolation from regulatory elements containing the nucleotide sequence, which facilitates manipulation of the partial sequence. The fragments may be synthesized with additional flanking nucleotides that contain useful restriction enzyme sites for the synthesis of the fragments. Thus, the present invention provides a method for modulating the expression of an operably linked transcribable DNA molecule. The design, construction, and use of enhancer elements according to the methods disclosed in Enhancers useful in practicing the present invention include those set forth in SEQ ID NOs: 10, 14, and 15. Available as 5.

[0058] In one embodiment, a fragment of the enhancer sequence disclosed herein is provided. The enhancer fragment may contain enhancer activity as described above. and alone or in combination with other expression elements and fragments of expression elements. In certain embodiments, it may be useful to combine 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 350, at least about 400, or at least about 450, or more, enhancer activities as disclosed herein A fragment of an enhancer is provided, comprising a DNA molecule having the following: Methods for producing such fragments from a molecule are well known in the art. .

[0059] As used herein, the term "chimera" refers to a first DNA molecule that is fused to a second DNA molecule. refers to a single DNA molecule produced by fusing a first DNA molecule with a second DNA molecule. The second DNA molecule is also not normally found in this configuration (i.e., fused to the other). Thus, chimeric DNA molecules are novel molecules not otherwise normally found in nature. As used herein, the term "chimeric promoter" refers to a DNA molecule that "Chimera enhancer" refers to a promoter produced by manipulating DNA molecules such as "Chimere intro" refers to an enhancer created by manipulating DNA molecules. "Chimeric 3' UTR" refers to an intron created by engineering a DNA molecule. " refers to a 3'UTR generated by manipulation of a DNA molecule. Chimeric promoter, Enhancers, introns, or 3'UTRs combine two or more DNA fragments. It may also be combined with other elements (e.g., promoter fusion to enhancer elements). Thus, the methods described herein for modulating expression of operably linked transcribable DNA molecules are Chimeric promoters, enhancers, introns or 3'UT according to the disclosed methods The design, construction, and use of R are encompassed by the present invention.

[0060] Chimeric regulatory elements can be synthesized by various methods known in the art, such as by restriction enzyme digestion and and ligation, ligation-independent cloning, and modularization of PCR products during amplification r assembly, or direct chemical synthesis of regulatory elements, as well as methods known in the art Designed to include various components that can be operably linked by other means The resulting various chimeric regulatory elements may be identical or may be identical in structure. It may be composed of variants of the elements, but any part that allows the components to be operably linked. The DNA sequence(s) comprising the linked DNA sequence(s) that enable the Therefore, the DNA sequences provided as SEQ ID NOs: 2-8, 12-14 and 16-19 are A reference sequence may be provided, where the constituent elements that make up the reference sequence The ligation may be carried out by methods known in the art and is also possible in bacterial and plant cell forms. Substitution, deletion, and / or insertion of one or more nucleotides that occur naturally in the transformation, or may contain mutations.

[0061] As used herein, the term "variant" refers to a variant of a DNA molecule that is similar in composition to a first DNA molecule. refers to a second DNA molecule that is similar but not identical, e.g., a regulatory element, The second DNA molecule, for example, has roughly the same transcription activity as the first DNA molecule. The general functionality of the DNA molecule, i.e., the same or similar expression pattern, remains. A variant may be a shorter or truncated version of the first DNA molecule, or is a modified version of the sequence of the first DNA molecule, e.g., with different restriction enzyme sites and / or or versions with internal deletions, substitutions, or insertions. A "variant" is a nucleotide sequence that contains a substitution, deletion, or insertion of one or more nucleotides of the reference sequence. The derivative regulatory element may also include a regulatory element having a corresponding have greater, less, or equal transcriptional or translational activity than the parent regulatory molecule that they In addition, regulatory element "variants" are naturally occurring in bacterial and plant cell transformations. In the present invention, the sequences of SEQ ID NOs: 2 to 8, 12 to 13, and variants resulting from mutations caused by the mutations are also included. The polynucleotide sequences provided in Tables 4 and 16-19 were used to identify the original regulatory elements. The DNA sequence and composition of the original are similar but not identical, while the general structure of the original regulatory element is Variants that are functional, i.e., still maintain the same or similar expression pattern, are The production of such variants of the present invention is within the routine skill of one of ordinary skill in the art in light of this disclosure. This is within the skill of the art and is included within the scope of the present invention.

[0062] 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, which may be obtained by comparing the starting DNA This can vary depending on the modifications made to the molecule and the purpose of the modifications.

[0063] construct As used herein, the term "construct" refers to a construct derived from any source and containing genomic information. capable of synthesizing or self-replicating and in which at least one DNA molecule is functionally linked to another DNA molecule Any DNA molecule containing a DNA molecule operably linked to a Recombinant DNA molecules, such as plasmids, cosmids, viruses, phages, or linear As used herein, "vector" refers to a DNA or RNA molecule that is circular or circular. The term "transformation" refers to the introduction of heterologous DNA or RNA into a host cell. Constructs typically refer to any construct that can be used to As used herein, "expression cassette" refers to one or more operably linked to the regulatory elements, typically at least the promoter and 3'UTR refers to a DNA molecule that contains at least one transcribable DNA molecule.

[0064] As used herein, the term "operably linked" refers to a first DNA molecule binds to a second DNA molecule, and this first DNA molecule affects the function of the second DNA molecule. The first and second DNA molecules are arranged so that they affect each other. The molecules may or may not be part of a single, continuous DNA molecule and may be adjacent For example, a promoter may or may not be involved in the transcription of a transcribable DNA of interest within a cell. The promoter is operably linked to a transcribable DNA molecule if it regulates transcription of the molecule. For example, a leader may affect the transcription or translation of a DNA sequence. operably linked to the NA sequence.

[0065] In one embodiment, the constructs of the present invention are derived from A. tumefaciens cells. T-DNA, together with a transfer molecule, allows the T-DNA to integrate into the genome of the plant cell Tipla isolated from Agrobacterium tumefaciens, including The right border (RB or AGRtu.RB) and the left border (LB or AGRtu.RB) of the smid It can be provided as a dual tumor-inducing (Ti) plasmid border construct with a .LB) region ( (See, e.g., U.S. Patent No. 6,603,061.) The constructs may also be used in bacterial cells. A plasmid backbone DNA segment that provides replication functions and antibiotic selection, e.g., ori Escherichia coli replication origin, such as 322, oriV or oriRi broad-host-range origin of replication, such as β-actin, and the ability to bind to spectinomycin or streptomycin. Tn7 aminoglycoside adenyltransferase (aadA) confers resistance to Selectable markers such as Spec / Strp or gentamicin (Gm, Gen t) It may also contain a coding region for a selectable marker gene. , the host bacterial strain is often A. tumefaciens ABI, C58, or LB A4404, but other strains known to those skilled in the art of plant transformation may also be used herein. It can work in the invention.

[0066] 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 well known in the art. For the practice of the invention, reference is made to methods for preparing and using the constructs and host cells. Conventional compositions and methods are well known to those skilled in the art. Exemplary vectors for use are well known in the art and include Agrobacterium Vectors derived from the Ti plasmid of C. tumefaciens and pCaMVC N transposition control vectors are included.

[0067] A variety of regulatory elements, including any of the regulatory elements provided herein, can be incorporated into the construct. Any such regulatory element may be included in combination with other regulatory elements. Such combinations can be designed or modified to provide the desired regulatory function. In one embodiment, the construct of the present invention is operably linked to a 3'UTR. at least one regulatory element operably linked to the transcribable DNA molecule include.

[0068] The constructs of the present invention can be prepared using any of the constructs provided herein or known in the art. For example, the promoters of the present invention may be incorporated into heterologous Operable with untranslated 5' leaders (e.g., from heat shock protein genes) Alternatively, the leaders of the present invention may be linked to a heterologous promoter, e.g., It may be operably linked to the ripe flower mosaic virus 35S transcript promoter.

[0069] Expression cassettes also can be used to express operably linked proteins in various organisms, particularly chloroplasts, leucoplasts, or or other plastid organelles, mitochondria, peroxisomes, vacuoles, or extracellular Transport peptides encoding peptides useful for subcellular targeting to a location. Many chloroplast-localized proteins are derived from nuclear genes as precursors and It is expressed as a chloroplast-transit peptide (CTP) and targeted to the chloroplast. Examples of suitable isolated chloroplast proteins include, but are not limited to, ribulose- 1,5-bisphosphate carboxylase small subunit (SSU), ferredoxin, eredoxin oxidoreductase, light-harvesting complex protein I and protein II, thio Redoxin F and enolpyruvylshikimate phosphate synthase (EPSPS) Chloroplast transit peptides include those described in, for example, U.S. Patent No. 7,193,133. Non-chloroplast proteins are introduced by introducing genes encoding non-chloroplast proteins. By expressing a heterologous CTP operably linked to a gene, it can be targeted to the chloroplast. It has been proven that this is the case.

[0070] transcribable DNA molecule As used herein, the term "transcribeable DNA molecule" refers to a molecule capable of being transcribed into an RNA molecule. refers to any DNA molecule capable of being transcribed into a protein, including but not limited to: Produce DNA molecules with coding sequences and RNA molecules with sequences useful for gene silencing Examples of types of DNA molecules include, but are not limited to, , DNA molecules from the same plant, DNA molecules from different plants, DNA molecules from different organisms or synthetic DNA molecules, e.g., DNA molecules containing antisense messages of genes. or an artificial, synthetic, or otherwise modified version of the transgene. Exemplary transcribable DNA molecules for incorporation into the constructs of the present invention include: Suitable DNA molecules include, for example, DNA molecules derived from a species other than the species into which the DNA molecule is to be incorporated. or genes, or originating from or existing in the same species but not developed using classical breeding techniques. Examples include genes that are inserted into recipient cells by genetic engineering rather than by gene transfer. do.

[0071] 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 Refers to a transcribable DNA molecule that has been artificially integrated into the genome of a host cell.

[0072] Regulatory elements, such as promoters, enhancers, introns or 3's of the present invention The UTR is operably linked to a transcribable DNA molecule that is heterologous to the regulatory element. As used herein, the term "heterologous" refers to two or more DNA molecules. A combination of offspring when such a combination is not normally found in nature For example, the two DNA molecules may be from different species and / or the two DNA molecules may be from different species. The NA molecules may be derived from different genes (e.g., different genes from the same species, or (The same gene from different species). Thus, regulatory elements are For replicable DNA molecules, if such combinations are not normally found in nature, That is, the transcribable DNA molecule does not naturally occur in operably linked with regulatory elements. In some cases, it is heterogeneous.

[0073] A transcribable DNA molecule is generally any DNA molecule from which expression of a transcript is desired. Expression of such transcripts results in translation of the resulting mRNA molecules and thus Alternatively, for example, a transcribable DNA molecule can be transcribed to result in protein expression. Specifically, they can be designed to cause the downregulation of specific genes or proteins. In one embodiment, this uses a transcribable DNA molecule oriented in the antisense direction. Those skilled in the art are familiar with the use of such antisense technology. Any gene can be negatively regulated in this manner, and in one embodiment, transcriptionally A specific DNA molecule can be expressed through the expression of dsRNA, siRNA, or miRNA molecules. It can be designed to suppress a gene.

[0074] Thus, one embodiment of the present invention is a method for producing a gene encoding a gene for a transgenic plant cell, comprising: When integrated into a system, transcription of a transcribable DNA molecule can be controlled at a desired level or in a desired pattern. operably linked to a heterologous transcribable DNA molecule in a manner that regulates transcription, Regulatory elements of the invention, such as those provided as 2-8, 12-14 and 16-19 In one embodiment, the transcribable DNA molecule is a recombinant DNA molecule comprising In another embodiment, the transcribable DNA molecule comprises a gene encoding a protein. The antisense region of the gene is included.

[0075] 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 desirable trait. The product of this study is related to plant morphology, physiology, growth, development, yield, grain composition, nutritional profile, disease resistance, and or cause effects on pest resistance and / or environmental or chemical tolerance. They may act within the plant to protect it from insects, or they may act as insecticides by providing food for pests that feed on the plant. In one embodiment of the present invention, the regulatory element of the present invention The element is operably linked to a transcribable DNA molecule that is a gene of agricultural interest. In transgenic plants containing such constructs, Thus, expression of genes of agronomic interest can confer beneficial agronomic traits. These include, but are not limited to, herbicide tolerance, insect control, altered yield, Disease resistance, pathogen resistance, modified plant growth and development, modified starch content, modified Altered oil content, altered fatty acid content, altered protein content, altered fruit development Ripening, animal and human nutritional enhancement, biopolymer production, environmental stress resistance, pharmaceutical peptides, Improved processing quality, improved flavor, usefulness in hybrid seed production, improved fiber production, and Biofuel production may be desirable.

[0076] Examples of genes of agronomic interest known in the art include genes for herbicide resistance (U.S. Pat. No. 6,449,299; U.S. Pat. No. 6,449,299). ,803,501;No.6,448,476;No.6,248,876;No.6,225 ,114;No.6,107,549;No.5,866,775;No.5,804,425 Nos. 5,633,435 and 5,463,175), yield increase (U.S. Pat. Nos. U .... 5,633,435 SRE No. 38,446; No. 6,716,474; No. 6,663,906; No. 6,47 No. 6,295; No. 6,441,277; No. 6,423,828; No. 6,399,33 No. 0; 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; 6,71 No. 3,063; No. 6,686,452; No. 6,657,046; No. 6,645,49 No. 7; No. 6,642,030; No. 6,639,054; No. 6,620,988; No. No. 6,593,293; No. 6,555,655; No. 6,538,109; No. 6,53 No. 7,756; No. 6,521,442; No. 6,501,009; No. 6,468,52 No. 3; No. 6,326,351; No. 6,313,378; No. 6,284,949; No. No. 6,281,016; No. 6,248,536; No. 6,242,241; No. 6,22 No. 1,649; No. 6,177,615; No. 6,156,573; No. 6,153,81 No. 4; No. 6,110,464; No. 6,093,695; No. 6,063,756; No. No. 6,063,597; No. 6,023,013; No. 5,959,091; No. 5,94 No. 2,664; No. 5,942,658; No. 5,880,275; No. 5,763,245 Nos. 6,653,280; and 5,763,241), fungal disease resistance (U.S. Pat. No. 6,653,280 ; No. 6,573,361; No. 6,506,962; No. 6,316,407; No. 6, No. 215,048; No. 5,516,671; No. 5,773,696; No. 6,121, Nos. 436; 6,316,407; and 6,506,962), virus resistance ( U.S. Patent Nos. 6,617,496; 6,608,241; 6,015,940; Nos. 6,013,864; 5,850,023; and 5,304,730), wire Insect resistance (U.S. Patent No. 6,228,992), bacterial disease resistance (U.S. Patent No. 5,516 ,671), plant growth and development (U.S. Patent Nos. 6,723,897 and 6,518,4 No. 88), starch production (U.S. Patent Nos. 6,538,181; 6,538,179; Nos. 6,538,178; 5,750,876; and 6,476,295), amended Altered oil production (U.S. Patent Nos. 6,444,876; 6,426,447; 6,3 80,462), high oil production (U.S. Patent Nos. 6,495,739; 5,608,14 No. 9; No. 6,483,008; No. 6,476,295), modified fatty acid content (rice National 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. 6,58 No. 9,767; No. 6,537,750; No. 6,489,461; No. 6,459,01 No. 8), 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. Pat. No. 6,723,837 ; No. 6,653,530; No. 6,5412,59; No. 5,985,605; No. 6, 171,640), biopolymers (U.S. Patent Nos. USRE37,543; 6,228, 623; 5,958,745, and 6,946,588), environmental stress resistance (U.S. Patent No. 6,072,103), medicinal peptides and secretory peptides (U.S. Patent No. Nos. 6,812,379; 6,774,283; 6,140,075; and 6, 080,560), improved processing traits (U.S. Patent No. 6,476,295), Improved polymerization rate (U.S. Patent No. 6,531,648), low raffinose (U.S. Patent No. 6,1 66,292), industrial enzyme production (U.S. Patent No. 5,543,576), flavor improvement ( U.S. Patent No. 6,011,199), nitrogen fixation (U.S. Patent No. 5,229,114), Hybrid seed production (U.S. Patent No. 5,689,041), fiber production (U.S. Patent No. 6,5 Nos. 76,818; 6,271,443; 5,981,834; and 5,869 ,720), and biofuel production (U.S. Pat. No. 5,998,700). do.

[0077] 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., U.S. Pat. App. Pub. No. 2005 / 0109994), Nos. 2006 / 0200878 and 2008 / 0066206, and U.S. Patent Application Nos. As described in No. 11 / 974,469, miRNA, siRNA, trans This includes regulation of gene expression by siRNA- and phase-sensitive sRNA-mediated mechanisms. affect the plant traits or phenotypes mentioned above by co-suppression or by co-suppression mediated mechanisms. RNA can also be engineered to cleave the desired endogenous mRNA product. Catalytic RNA molecules (e.g., ribozymes or riboswitches; see, e.g., U.S. Pat. App. Pub. No. 2005 / 0109994). 2006 / 0200878). The construct is then constructed and delivered to the cell in such a way that it is transcribed into a molecule that can cause gene silencing. Methods for introduction are known in the art.

[0078] 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 refers to any transcribable DNA molecule that can be cored. Selectable marker genes for the purpose of cloning and their associated selection and screening techniques are also described in the present application. Various enzymes known in the art include, but are not limited to, β-glucuronidase ( GUS), green fluorescent protein (GFP), a protein that confers antibiotic resistance, and Examples include transcribable DNA molecules that encode proteins that confer herbicide resistance. An example of a marker transgene is provided as SEQ ID NO:11.

[0079] 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.

[0080] The term "transformation" refers to the introduction of a DNA molecule into a recipient host. As used herein, the term "host" means a bacterium, fungus, or plant, which It includes any cell, tissue, organ, or progeny of a bacterium, fungus, or plant. The target plant tissues and cells include protoplasts, callus, roots, tubers, seeds, stems, etc. , leaves, seedlings, embryos, and pollen.

[0081] As used herein, the term "transformed" refers to the introduction of a foreign DNA molecule (e.g., refers to a cell, tissue, organ, or organism into which an introduced DNA molecule (e.g., construct) has been introduced. into the genomic DNA of the recipient cell, tissue, organ, or organism, The DNA molecule may be passed on to subsequent generations. A "transformed" cell or organism includes the progeny of the cell or organism, and any subsequent matings. produced from breeding programs using transgenic parents such as Progeny that exhibit phenotypic alterations resulting from the presence of the introduced NA molecule may also be included. The DNA molecule is transiently introduced into the recipient cell, and the introduced DNA molecule is transmitted to the next generation The term "transgenic" refers to one or more Refers to bacteria, fungi, or plants that contain heterologous DNA molecules.

[0082] There are numerous 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 transfecting the host cell with a vector. and obtaining the transformed host cell. Method for transforming plant cells by introducing a plant construct into the plant genome The methods and materials may include any well-known and proven method. Examples include, but are not limited to, bacterial infections (e.g., Agrobacterium m), binary BAC vectors, direct delivery of DNA (e.g., PEG-mediated transformation, Desiccation / inhibition-mediated DNA uptake, electroporation, agitation with silicon carbide fibers, and acceleration of DNA-coated particles), gene editing (e.g., CRISPR-Cas systems) Examples include:

[0083] 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 fungal, bacterial, or insect cell. The transformed cells may include cells from crop plants.

[0084] Transgenic plants are then regenerated from the transgenic plant cells of the invention. Seeds can be produced from this transgenic plant using conventional breeding techniques or self-pollination. Such seeds and the resulting progeny plants grown from such seeds may be produced. contains a recombinant DNA molecule of the present invention and is therefore transgenic.

[0085] 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. The present invention provides seeds of homozygous transgenic plants of the present invention (i.e., plants of the same or similar species), or non-transgenic plants of the present invention. Transgenic plants or crossed with other transgenic plants to produce recombinant DNA and (b) providing seeds of heterozygous transgenic plants of the present invention (heterozygous for the A molecule). Both such homozygous and heterozygous transgenic plants can be used in this invention. These are referred to herein as "progeny plants." Progeny plants are plants derived from the original transgenic plant. The transgenic plant of the present invention contains a recombinant DNA molecule. Seeds produced using the genetic plants are harvested and grown to contain the constructs of the invention and for agronomic purposes. Transgenic plants expressing the gene, i.e., progeny plants of the invention, are grown. A description of breeding methods commonly used for various crops. can be found in one of several references, e.g., Allard, Pri ciples of Plant Breeding,John Wiley & S. ons,NY,U.of CA,Davis,CA,50-98(1960);Simm onds,Principles of Crop Improvement,Long man, Inc., NY, 369-399 (1979); Sneep and Hend riksen,Plant breeding Perspectives,Wagen ingen(ed),Center for Agricultural Publis hing and Documentation(1979);Fehr,Soybea ns:Improvement,Production and Uses,2nd E dition, Monograph, 16:249(1987); Fehr, Princ iples of Variety Development,Theory and Technique,(Vol.1)and Crop Species Soybea n(Vol.2), Iowa State Univ., Macmillan Pub. Co., NY, 360-376 (1987).

[0086] Transformed plants are characterized by the presence of the gene(s) of interest, as well as the presence of the regulatory elements of the present invention. The expression level and / or profile of the resulting gene can be analyzed. We recognize the many methods available for analyzing recombinant plants. For example, methods for plant analysis include: , including but not limited to Southern blot or Northern blot, PCR-based Approaches, biochemical analyses, phenotypic screening methods, field evaluations, and immunodiagnostic assays Expression of transcribable DNA molecules can be performed using TaqMan® (Appli Reagents and manufacturers are Ed Biosystems (Foster City, CA). Using the methods described, as well as the TaqMan® Testing Matrix Alternatively, PCR cycle times can be measured using Invader (registered trademark). Trademark)(Third Wave Technologies(Madison,WI)) Transgene expression may be assessed using reagents and methods described by the manufacturer.

[0087] The present invention also provides parts of the plants of the present invention. Plants of the present invention include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovules, and pollen. Parts of an object can be viable, non-viable, regenerable, and / or non-regenerable. The present invention also includes and provides transformed plant cells containing the DNA molecules of the present invention. The clearly transformed or transgenic plant cells may be regenerable and / or non-regenerable. Possible plant cells are included.

[0088] 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 gene products of the present invention are also provided. A detectable amount of a DNA sequence selected from the group consisting of 2 to 14 and 16 to 19. As used herein, "commercial product" refers to a recombinant DNA molecule of the present invention. Material derived from transgenic plants, seeds, plant cells, or plant parts, including offspring Commercial products include, but are not limited to, any composition or product consisting of: The present invention also provides a method for producing a commercial product of a plant, including but not limited to processed seeds, grains, plant parts, and meal. The product will contain a detectable amount of DNA corresponding to a recombinant DNA molecule of the present invention. To determine the content or source of a commercial product, one or more of these DNA fragments in a sample may be detected. Detection may be used any standard DNA detection method, including those disclosed herein. Molecular detection methods may also be used.

[0089] The present invention may be more readily understood by reference to the following examples, which Unless otherwise specified, the following are provided by way of example only and are not intended to limit the invention. Those skilled in the art will recognize that the techniques disclosed in the following examples are based on the present invention, which was discovered by the inventors. It should be understood that these represent fully functional techniques for practicing the invention. However, those skilled in the art will appreciate that, in light of this disclosure, many modifications can be made to the specific embodiments disclosed. may be used, and still use similar or similar components without departing from the spirit and scope of the present invention. It should be understood that results will be obtained, and therefore all content shown is for illustrative purposes only. The terms and conditions of the present invention shall be construed as meaning the present invention and not as limiting. [Example]

[0090] Example 1 Identification and cloning of regulatory elements Novel transcriptional regulatory elements and expression regulatory elements (EXPs) were identified and several It was cloned from the genomic DNA of monocotyledonous plant species.

[0091] Three novel major untranslated region (3'UTR) sequences were identified from the unique sequences of several monocotyledonous plant species. Table 1 below lists various 3'UTRs and the monocotyledonous plants from which they are derived. The 3'UTR, T-SETit.Ams1:1 (SEQ ID NO: 1), is a fragment of the 3'UTR gene of the present invention. Previously presented as SEQ ID NO: 270 in patent application US20130031672. TIFF2025176124000001.tif83170

[0092] Furthermore, a promoter isolated from Zea mays genomic DNA (P-Zm.L tp-1:1:2) and leader (L-Zm.Ltp-1:1:3), as well as Seta Exp sequences containing introns isolated from ria italic genomic DNA were prepared. EXP, EXP-Zm.LTP-SETit.Act4 (sequence Number 6) is a promoter P-Zm operably linked to the 5' of the 5'UTR Ltp-1:1:2 (SEQ ID NO: 7), an L operably linked to the 5' of the intron -Zm.Ltp-1:1:3 (SEQ ID NO: 8), I-SETit.Act4:2 (SEQ ID NO: 9; previously presented as SEQ ID NO: 627 in U.S. Patent Application US20130031672) It is composed.

[0093] These 3'UTR and EXP-Zm.LTP-SETit.Act4 were obtained by the method of the present invention. The vectors were cloned into binary plant transformation vectors using methods known in the art.

[0094] Example 2 Analysis of the 3'UTR and transgene in stably transformed maize plants their effect on the expression of Maize plants were transfected with vectors, specifically, the β-glucuronidase (GUS) transgene. The transcriptional regulatory elements driving expression of the seven (7) 3'UTs presented in Example 1 The plants were transformed with a plant expression vector containing one of each of the R regulatory elements. To evaluate the effect of the 3'UTR regulatory element on GUS protein expression, The resulting plants were analyzed.

[0095] Maize plants were transformed with a plant GUS expression construct. Using this method, the 3'UTR regulatory elements were cloned into a plant-based expression vector. The obtained plant expression vector was transformed into Agrobacterium tumefaciens. The left border region derived from s and transgenic plants that confer resistance to the herbicide glyphosate The first transgene selection cassette used for cell selection and the activity of the 3'UTR regulatory element A second transgene cassette for assessing sexuality, seven (7) of which are shown in Example 1 operably linked at the 5' side to one of each of the 3'UTR regulatory elements of , potato light-inducible tissue-specific ST-LS1 gene (Genbank accession: β-glucuronidase containing a processible intron derived from Encoding a synthetic coding sequence designed for expression in plant cells (GUS, GOI-Ec. operably linked to the 5' side of uidA+St.LS1.nno:1, SEQ ID NO: 11) 5 for EXP, EXP-Zm.LTP-SETit.Act4 (SEQ ID NO: 6) operably linked to the 'side, in a reverse complementary orientation, The second one contains a motor-derived enhancer (E-CaMV.35S-RC, SEQ ID NO: 10). transgene cassette, followed by Agrobacterium tumefaciens The right border region of origin was included.

[0096] Corn plant cells derived from corn variety 01DKD2 are cultured using methods known in the art. As previously mentioned, these binary forms can be produced by Agrobacterium-mediated transformation. The resulting transformed plant cells were transformed with the transformation vector construct. It was induced to form Kosi plants.

[0097] Histochemical GUS analysis was used for qualitative and quantitative expression analysis of transformed plants. The whole section was stained with GUS staining solution X-Gluc (5-bromo-4-chloro-3-indolyl-b- Incubate with 1 mg / ml of HCl (glucuronide) for the appropriate time and rinse. GUS activity was determined by direct visual inspection or by in vitro transfection of selected plants. Qualitative determinations were made by microscopic examination of organs and tissues.

[0098] For quantitative analysis of GUS expression, whole tissue was extracted from selected tissues of transformed maize plants. Proteins were extracted. One microgram of total protein was extracted from 50 microliters of total reaction. Fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide (MUG) in a reactive volume The reaction product, 4-methylumbelliferone (4-MU), was maximally fluorescent at high pH. The hydroxyl group is ionized when the basic solution of sodium carbonate is Upon addition, the assay is stopped and the pH is adjusted to quantify the fluorescent product. Fluorescence was measured using a FLUOstar® Omega microplate reader at 36 Measurements were made with excitation at 5 nm and emission at 445 nm. Values ​​are given per mg of total protein. The unit is nmol of GUS per hour (nmol GUS / hour / mg). .

[0099] The following tissues were analyzed using the 3'UTR regulatory elements shown in Example 1 in the R0 generation: Five (5) samples were taken for GUS expression: leaves and roots at V4 stage; leaves and roots at V7 stage; Roots; flowers / anthers, leaves, and roots at VT stage; cobs / silk at R1 stage; 21 days after pollination Seed embryos and seed endosperms at the R3 stage after 1 day of maturity (DAP). Tables 2 and 3 below show the relationship between GUS expression and The influence of each of the 3'UTR regulatory elements on the expression of ribosomal RNA is shown. TIFF2025176124000002.tif85170TIFF2025176124000003.tif85170

[0100] As can be seen in Tables 2 and 3, each 3'UTR regulatory element is related to the same enhancer / promoter. When operably linked to a combination promoter / intron construct, The promoter P-Zm.Ltp-1: 1:2 has been experimentally confirmed to be preferentially expressed in roots. Addition of MV.35S-RC adds a constitutive expression component. Each of the components is driven by a combination of enhancer / promoter / intron constructs. For example, the 3'UTR, T-SETit.Ams1:1, Compared with T-ERAte.Hsp17.9:1 and T-ANDge.Hsp / Sb.Hsp Expression in leaves using T-SETit.Ams1:1 was also enhanced. The expression level of T-Cl.Hsp16.9_2:1 in roots was much lower than that in roots. Expression in maize plants containing T-SETit.Ams1:1 was as high as in maize plants containing T-SETit.Ams1:1. , leaf expression compared to maize plants containing T-SETit.Ams1:1. The expression of T-Cl.Hsp16.9_2:1 in roots was higher in plants containing T-Cl.Hsp16.9_2:1 at the V4 stage. The results were lower in maize plants containing ERIra.Hsp16.9_3:1, but not in V7 to V The number of VT flowers and anthers, R1 cobs and hairs, and 21 DAP embryos gradually increased until the T stage. Regarding T-Cl.Hsp16.9, compared with the other four 3'UTR regulatory elements, Expression was highest in maize plants containing _2:1. Expression in the 21 DAP endosperm was Compared with four other 3'UTR regulatory elements, including T-SETit.Ams1:1 It was highest in maize plants.

[0101] Maize plants also contain two of the 3'UTR regulatory elements presented in Example 1. T-SACra.Hsp16.9:2 (SEQ ID NO: 12) and T-Cl.Hsp16. The plants were transformed with a plant GUS expression construct as described above containing GUS 9:2 (SEQ ID NO: 13). US expression was determined as described above. Only leaves and roots at the V2 stage were sampled. Table 4 shows the results of the experiments using stably transformed strains of tallow containing two 3'UTR regulatory elements. Average GUS expression in sorghum plants is shown. TIFF2025176124000004.tif42170

[0102] As seen in Table 4 above, T-SACra.Hsp16.9:2 (SEQ ID NO: 12) showed overall growth in roots and leaves when compared to T-Cl.Hsp16.9:2 (SEQ ID NO: 13). The expression of T-SACra.Hsp16.9:2 was high. The level of T-Cl.Hsp16.9:2 was approximately 6.3 times higher in roots than in leaves. The root-to-leaf expression ratio of T-SACra.Hsp16.9:2 was approximately 4.2 times higher than that of T-SACra.Hsp16.9:2. It was low. Each of the seven (7) 3'UTR regulatory elements controls the expression of the GUS transgene. These unique patterns of gene expression in various tissues can be regulated. The current pattern can be used to fine-tune the expression of specific transgenes of interest to target specific preferred combinations. It provides an expression profile in the target tissue while reducing expression in other less favorable tissues. Each of the seven 3'UTR regulatory elements may This allows for great flexibility to drive specific traits in plants.

[0103] Example 3 EXP- for transgene expression in stably transformed maize plants Analysis of Zm.LTP-SETit.Act4 Maize plants were transfected with vectors, specifically the β-glucuronidase (GUS) gene. The plants were transformed with a plant expression vector containing a transcriptional regulatory element that drives gene expression. The plants were analyzed for GUS protein expression and analyzed for EXP, EXP-Zm.LTP The expression characteristics of -SETit.Act4 (SEQ ID NO: 6) were evaluated.

[0104] Maize plants were transformed with a plant GUS expression construct using methods known in the art. EXP, EXP-Zm.LTP-SETit.Act4 (SEQ ID NO: 6) was synthesized using the method The resulting plant expression vector was cloned into an Agro-based plant expression vector. The left border region from bacterium tumefaciens and the herbicide glyphosate A first transgene selection method is used to select transformed plant cells that confers resistance to thiol. The cassette and the 5' side of the 3'UTR regulatory element T-SETit.Ams1:1 for expression in plant cells encoding a β-glucuronidase operably linked to The designed synthetic coding sequence (GUS, GOI-Ec.uidA+St.LS1.nno: 1) and EXP, EXP-Zm.LTP-SETit.Ac operably linked to the 5' side A second study to assess the activity of EXP-Zm.LTP-SETit.Act4 containing t4 The transgene cassette and subsequently the Agrobacterium tumefaciens-derived The right border region of the thoracic vein was included.

[0105] Corn plant cells derived from corn variety 01DKD2 are cultured using methods known in the art. As shown, Agrobacterium-mediated transformation can be used to generate the above binary traits. The transformation vector construct was used to transform the resulting transformed plant cells. Quantitative and qualitative measurements of GUS expression were performed at the V2 stage. The leaves and roots were used to determine the phenotype as described in the previous example. Table 5 shows the phenotype of stably transformed plants. The average GUS expression in the leaves and roots of V2 plants and the ratio of GUS expression in the roots compared to the leaves are shown. do. TIFF2025176124000005.tif44170

[0106] As can be seen in Table 5, the GUS expression is much higher in roots than in leaves. Root expression is 35% of that measured in leaves. (35)-fold increase in expression. This expression pattern favors root expression over aboveground expression. It may be advantageous to drive a particular transgene.

[0107] Example 4 The orientation of the enhancer allows for the differentiation of genes between the roots and leaves of stably transformed maize plants. Affects the current ratio This example demonstrates the construction of a promoter containing the Dahlia mosaic virus enhancer in different orientations. Illustrates the effect of operably linking and altering the relative expression of transgenes in leaves and roots. Maize plants have been shown to express enhancer sequences in both the forward and reverse directions for native promoters. The plants were transformed with a plant expression vector containing EXP with an enhancer in either the reverse orientation. The plants were analyzed to identify the enhancer sequences for GUS protein expression in roots and leaves. The influence of the direction was determined.

[0108] Maize plants were transformed with plant GUS expression constructs. -DaMV.H-Flt+Zm.Ltp+SETit.Act4:1 (SEQ ID NO: 16), EXP-DaMV.H-Flt+Zm.Ltp+Zm.Ltp+SETit.Act4: 4 (SEQ ID NO: 17), EXP-DaMV.FLT+Td.RCc3_1+SETit.1 4-3-3C-5:1 (SEQ ID NO: 18), and EXP-DaMV.FLT+Td.RCc 3_1+Td.RCc3_1+SETit.14-3-3C-5:5 (SEQ ID NO: 19) , and cloned into a plant-based expression vector using methods known in the art. The resulting plant expression vectors were used to express transformed plants that confer tolerance to the herbicide glyphosate. The first transgene selection cassette used for selection of Agrobacterium cells. Left border derived from Agrobacterium tumefaciens The boundary region and the 3'UTR regulatory element, T-SETit.Ams1:1, are operable 5' to A synthetic compound designed for expression in plant cells encoding β-glucuronidase linked to The coding sequence (GUS, GOI-Ec.uidA+St.LS1.nno:1) is located 5' to a second transgene cassette for evaluating the activity of the EXP, which is operably linked to the second transgene cassette; The right border of Agrobacterium tumefaciens followed by This includes the area.

[0109] Each EXP contained an enhancer derived from the dahlia mosaic virus promoter. The enhancer is located in the forward orientation (within the native promoter of the Dahlia mosaic virus genome). enhancer direction) or reverse (native promoter of the Dahlia mosaic virus genome) Each EXP promoter is either in the opposite direction to the enhancer in the promoter EXP-DaMV.H-Flt was operably linked to the 5' end of the EXP. +Zm.Ltp+SETit.Act4:1 (SEQ ID NO: 16) and EXP-DaMV.H -Flt+Zm.Ltp+Zm.Ltp+SETit.Act4:4 (SEQ ID NO: 17) , a chimeric reconstituted enhancer derived from multiple published dahlia mosaic virus promoters The first fragment contained E-DaMV.H-Flt:1 (SEQ ID NO: 14). The promoter of the DaMV-Holland (DaMV-H) strain, Genbank accession number Originated from session EU090957, nucleotides 1177-1494. This flag The second fragment, nucleotide 1, is derived from the DaMV-H promoter. The native DaMV promoter was operably linked to the nucleotides 1003-1176. In the construction, the second fragment precedes the first fragment. , compared to SEQ ID NO: 14, nucleotides 287-288 and nucleotides 319-3 22 is a DaMV promoter homologue in Genbank accession JX272320. The EXP-DaMV.FLT+Td.RC arrangement has been changed to a similar position. c3_1+SETit.14-3-3C-5:1 (SEQ ID NO: 18) and EXP-DaMV .FLT+Td.RCc3_1+Td.RCc3_1+SETit.14-3-3C-5 :5 (SEQ ID NO: 19) is the Dahlia mosaic virus promoter, Genbank Accession No. session EF513491, enhancer derived from nucleotides 1–322, E-Da It contained MV.FLT:2 (SEQ ID NO: 15).

[0110] EXP-DaMV.H-Flt+Zm.Ltp+SETit.Act 4:1 (SEQ ID NO: 16) and EXP-DaMV.H-Flt+Zm.Ltp+Zm.Lt p+SETit.Act4:4 (SEQ ID NO: 17) is a chimeric rearrangement enhancer, promoter E-DaMV.H-Flt:1 (SEQ ID NO: 14) operably linked 5' to the promoter and an intron derived from the actin 4 gene from Setaria italica operably linked at the 5' end to a lipid transfer protein-like protein from Zea mays EXP-DaMV.FLT contains the 5'UTR derived from the protein gene. +Td.RCc3_1+SETit.14-3-3C-5:1 (SEQ ID NO: 18) and EX P-DaMV.FLT+Td.RCc3_1+Td.RCc3_1+SETit.14- 3-3C-5:5 (SEQ ID NO: 19) is an enzyme operably linked to the 5' side of a promoter. enhancer E-DaMV.FLT:2 (SEQ ID NO: 15) and Setaria itali Trypsi operably linked to an intron derived from the 14-3-3C gene from ca It contains the 5'UTR derived from the RCc3 gene from C. cum dactyloides.

[0111] Corn plant cells derived from corn variety 01DKD2 are cultured using methods known in the art. As shown, the above binary forms can be produced by Agrobacterium-mediated transformation. The resulting transformed plant cells were then transformed with the transformation vector construct. Quantitative and qualitative measurements of GUS expression were performed at the V2 stage. The leaves and roots of the four (4) E For each XP, the average GUS expression in leaves and roots of stably transformed V2 is shown. The orientation of the enhancer and the ratio of expression in roots to leaves are also shown in Table 6. TIFF2025176124000006.tif76170

[0112] In Example 3, Table 5, EXP, EXP-Zm.LTP-SETit.Act4 (arrangement Column number 6) shows that roots drive much higher GUS expression than leaves by more than thirty-five (35) times. As shown in Table 6, EXP-DaMV.H-Flt+Zm.Ltp+SETit .Act4:1 (SEQ ID NO: 16) and EXP-DaMV.H-Flt+Zm.Ltp+Z EXP-Zm contained within m.Ltp+SETit.Act4:4 (SEQ ID NO: 17) The chimeric reconstituted enhancer, LTP-SETit, was inserted into the promoter in Act4. E-DaMV.H-Flt:1 (SEQ ID NO: 14) 5' is operably linked to GUS expression was increased in leaves compared to roots. However, the enhancers in these two EXPs Orientation affected the relative levels of GUS expression in roots and leaves. The ratio of root to leaf expression was higher when :1 was oriented in the reverse direction compared to the forward direction. ,EXP-DaMV.FLT+Td.RCc3_1+SETit.14-3-3C-5 In the reverse orientation within E-DaMV.FLT:1 (SEQ ID NO: 18), E-DaMV.FLT:2 (SEQ ID NO: 15) enhancer EXP-DaMV.FLT+Td.RCc 3_1+Td.RCc3_1+SETit.14-3-3C-5:5 (SEQ ID NO: 19) The ratio of GUS expression in roots to leaves was increased when compared with the forward orientation of the two enhancers. By operably linking the enhancer in the reverse orientation, the enhancer is operably linked in the forward orientation. This allows us to obtain an expression profile that results in a higher root-to-leaf expression ratio than when the This feature is useful when you want to use viral enhancers to enhance expression, but want to minimize the risk of infection. Desirable when it is desired to provide a high root to leaf expression ratio.

[0113] 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. Variations in arrangement and detail will be apparent to those skilled in the art. We claim all modifications that come within the spirit and scope of the claims. All publications and published patent documents cited herein are to the extent that each individual publication or patent application is References are hereby incorporated by reference to the same extent as if they were specifically and individually indicated to be incorporated by reference. Incorporated by reference.

Claims

1. 1. A recombinant DNA molecule comprising: a) at least 85% of any of SEQ ID NOs: 2-8, 12-14, and 16-19 a sequence having sequence identity of 100% to 100%; b) a sequence comprising any of SEQ ID NOs: 2 to 8, 12 to 14, and 16 to 19; and c) any of SEQ ID NOS: 2 to 8, 12 to 14, and 16 to 19, which has gene regulatory activity; a fragment of the recombinant D, wherein the sequence is operably linked to a heterologous transcribable DNA molecule. NA molecule.

2. The sequence corresponds to any one of the DNA sequences of SEQ ID NOs: 2 to 8, 12 to 14, and 16 to 19.

2. The recombinant DNA molecule of claim 1, which has at least 90 percent sequence identity with 。

3. The sequence corresponds to any one of the DNA sequences of SEQ ID NOs: 2 to 8, 12 to 14, and 16 to 19.

2. The recombinant DNA molecule of claim 1, which has at least 95 percent sequence identity with 。

4. 2. The recombinant DNA molecule of claim 1, wherein the DNA sequence comprises a 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. A transgenic plant cell comprising the recombinant DNA molecule of claim 1.

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, , grains, plant parts, seed oils, biomass, flours, and meal according to claim 15. method.

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

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