Plant regulatory elements and their use

Abstract

To provide novel synthetic gene regulatory elements for use in plants.SOLUTION: The invention provides recombinant DNA molecules and constructs, as well as their nucleotide sequences, useful for modulating gene expression in plants. The invention also provides: transgenic plants, plant cells, plant parts and seeds comprising the recombinant DNA molecules operably linked to heterologous transcribable DNA molecules; and methods of their use.SELECTED DRAWING: None

Description

Technical Field

[0001] Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 714,228, filed Aug. 3, 2018 and the entire disclosure of that application is incorporated herein by reference.

[0002] Incorporation of Sequence Listing The sequence listing contained in the file named “38-21-62691-0001_Seqlist_ST25.txt” is 31,060 bytes (measured in MS-Windows®) and was created on Jul. 2, 2019. This sequence listing is submitted electronically with this application and is incorporated herein by reference.

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

Background Art

[0004] Regulatory elements are genetic elements that regulate gene activity by modulating the transcription of an operably linked transcribable DNA molecule. Such elements include promoters, leaders, introns, and 3′ untranslated regions, which are useful in the fields of plant molecular biology and plant genetic engineering.

Summary of the Invention

[0005] The present invention provides novel synthetic gene regulatory elements for use in plants. The present invention also provides recombinant DNA molecule constructs containing the regulatory elements. The present invention provides regulatory Transgenic plant cells, plants, and seeds containing the element are also provided. In one embodiment form, the regulatory element is operably linked to a transcribable DNA molecule. In a certain embodiment, the transcribable DNA molecule may be heterologous to the regulatory sequence. Thus, the regulatory element sequences provided by the present invention can be defined as being operably linked to a heterologous transcribable DNA molecule in certain embodiments. The present invention also provides methods of making and using regulatory elements, recombinant DNA molecules containing the regulatory elements, and transgenic plant cells, plants, and seeds containing a regulatory element operably linked to a transcribable DNA molecule.

[0006] Thus, in one aspect, the present invention provides a recombinant DNA molecule comprising (a) a sequence having at least about 85% sequence identity to any of SEQ ID NOs: 1-19 and SEQ ID NO: 26, (b) a sequence comprising any of SEQ ID NOs: 1-19 and SEQ ID NO: 26, and (c) a DNA sequence selected from the group consisting of fragments of any of SEQ ID NOs: 1-19 and SEQ ID NO: 26 having gene regulatory activity, wherein the sequence is operably linked to a heterologous transcribable DNA molecule. "Heterologous transcribable DNA molecule" means that the transcribable DNA molecule is heterologous to the polynucleotide sequence to which it is operably linked. In certain embodiments, the recombinant DNA molecule has at least about 85%, at least about 86%, at least about 87%, at least about 88% sequence identity to any of the DNA sequences of SEQ ID NOs: 1-19 and SEQ ID NO: 26. percent, at least about 86 percent, at least about 87 percent, at least about 88 percent, at least about 89 percent, at least about 90 percent, at least 9 1 percent, at least 92 percent, at least 93 percent, at least 94 percent, at least 95 percent, at least 96 percent, at least 97 per cent, at least 98 percent, or at least 99 percent sequence identity and comprising a DNA sequence. In certain embodiments, the DNA sequence comprises regulatory elements . In some embodiments, the regulatory element comprises a promoter. In still other embodiments , the regulatory element comprises an intron. In still other embodiments, the regulatory element comprises a 3’UTR. In still other embodiments, the heterologous transcribable DNA molecule comprises a gene for an agricultural purpose, for example, a gene capable of providing herbicide resistance in a plant, or a gene capable of providing plant pest resistance in a plant. In still other embodiments , the heterologous transcribable DNA molecule comprises a sequence encoding a small molecule RNA (e.g., dsRNA, miRNA, or siRNA). In still other embodiments, the present invention provides a construct comprising a recombinant DNA molecule provided herein.

[0007] In another aspect, provided herein is a transgenic plant cell comprising a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of (a) a sequence having at least about 85 percent sequence identity to any of SEQ ID NOs: 1-19 and SEQ ID NO: 26, (b) a sequence comprising any of SEQ ID NOs: 1-19 and SEQ ID NO: 26, and (c) a fragment of any of SEQ ID NOs: 1-19 and SEQ ID NO: 26 having gene regulatory activity and said Provided are transgenic plant cells in which a DNA sequence is operably linked to a heterologous transcribable DNA molecule. In certain embodiments, the transgenic plant cells are monocotyledonous plant cells. In other embodiments, the transgenic plant cells are dicotyledonous plant cells.

[0008] In yet another aspect, further provided herein is a transgenic plant or a part thereof that comprises a DNA sequence selected from the group consisting of: a) a sequence having at least 85% sequence identity to any of SEQ ID NOs: 1-19 and SEQ ID NO: 26; b) a sequence comprising any of SEQ ID NOs: 1-19 and SEQ ID NO: 26; and c) a fragment of any of SEQ ID NOs: 1-19 and SEQ ID NO: 26 that has gene regulatory activity, wherein the sequence is operably linked to a heterologous transcribable DNA molecule. In certain embodiments, the transgenic plant is a progeny plant of any generation that comprises the recombinant DNA molecule. Also provided herein are transgenic seeds that comprise a recombinant DNA molecule that produces such a transgenic plant when grown.

[0009] In another aspect, the invention provides a method for producing a commercial product, the method comprising obtaining a transgenic plant or a part thereof that comprises the recombinant DNA molecule of the invention, and producing a commercial product therefrom. In one embodiment, the commercial product is seeds, processed seeds, protein concentrates, protein isolates, starch, grains, plant parts, seed oils, biomass, flours, and grits.

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

[0011] Brief Description of the Sequences SEQ ID NO: 1 is the DNA sequence of a synthetic regulatory expression element group (EXP) EXP-Zm.GSP850, which comprises a synthetic leader (L-Zm.GSP850.nno:3) and a synthetic promoter (P-Zm.GSP850.nno:4) operably linked at the 5'-side. SEQ ID NO: 1 is the DNA sequence of a synthetic regulatory expression element group (EXP) EXP-Zm.GSP850, which comprises a synthetic leader (L-Zm.GSP850.nno:3) and a synthetic promoter (P-Zm.GSP850.nno:4) operably linked at the 5'-side. SEQ ID NO: 1 is the DNA sequence of a synthetic regulatory expression element group (EXP) EXP-Zm.GSP850, which comprises a synthetic leader (L-Zm.GSP850.nno:3) and a synthetic promoter (P-Zm.GSP850.nno:4) operably linked at the 5'-side.

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

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

[0014] SEQ ID NO: 4 is the DNA sequence of a synthetic EXP, EXP-Zm.GSP850.nno+Zm.GSI153.nno:2, which comprises a synthetic intron (I-Zm.GSI153.nno:1) operably linked at the 5'-side, a synthetic leader (L-Zm.GSP850.nno:3) operably linked at the 5'-side, and a synthetic promoter (P-Zm.GSP850.nno:4) operably linked at the 5'-side. SEQ ID NO: 4 is the DNA sequence of a synthetic EXP, EXP-Zm.GSP850.nno+Zm.GSI153.nno:2, which comprises a synthetic intron (I-Zm.GSI153.nno:1) operably linked at the 5'-side, a synthetic leader (L-Zm.GSP850.nno:3) operably linked at the 5'-side, and a synthetic promoter (P-Zm.GSP850.nno:4) operably linked at the 5'-side. SEQ ID NO: 4 is the DNA sequence of a synthetic EXP, EXP-Zm.GSP850.nno+Zm.GSI153.nno:2, which comprises a synthetic intron (I-Zm.GSI153.nno:1) operably linked at the 5'-side, a synthetic leader (L-Zm.GSP850.nno:3) operably linked at the 5'-side, and a synthetic promoter (P-Zm.GSP850.nno:4) operably linked at the 5'-side. SEQ ID NO: 4 is the DNA sequence of a synthetic EXP, EXP-Zm.GSP850.nno+Zm.GSI153.nno:2, which comprises a synthetic intron (I-Zm.GSI153.nno:1) operably linked at the 5'-side, a synthetic leader (L-Zm.GSP850.nno:3) operably linked at the 5'-side, and a synthetic promoter (P-Zm.GSP850.nno:4) operably linked at the 5'-side. SEQ ID NO: 4 is the DNA sequence of a synthetic EXP, EXP-Zm.GSP850.nno+Zm.GSI153.nno:2, which comprises a synthetic intron (I-Zm.GSI153.nno:1) operably linked at the 5'-side, a synthetic leader (L-Zm.GSP850.nno:3) operably linked at the 5'-side, and a synthetic promoter (P-Zm.GSP850.nno:4) operably linked at the 5'-side.

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

[0016] SEQ ID NO: 6 is a synthetic EXP, EXP-Zm.GSP990 DNA sequence that contains a synthetic leader (L-Zm.GSP990.nno:1) operably linked 5' to a synthetic promoter (P-Zm.GSP990.nno:2).

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

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

[0019] SEQ ID NO: 9 is a synthetic EXP, EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 DNA sequence that contains a synthetic intron (I-Zm.GSI197.nno:1) operably linked 5' to a synthetic leader (L-Zm.GSP990.nno:1) which is operably linked 5' to a synthetic promoter (P-Zm.GSP990.nno:2).

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

[0021] SEQ ID NO: 11 is a synthetic EXP, EXP-Zm.GSP850.nno+Zm.GSI140.nno:1 DNA sequence that contains a synthetic intron (I-Zm.GSI140.nno:1) operably linked 5' to a synthetic leader (L-Zm.GSP850.nno:3) which is operably linked 5' to a synthetic promoter (P-Zm.GSP850.nno:4).

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

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

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

[0025] SEQ ID NO: 15 is the DNA sequence of synthetic EXP, EXP-Zm.GSP850.nno+Zm.DnaK:1, which is operably linked on the 5’ side to an intron (I-Zm.DnaK:1) and operably linked on the 5’ side to a synthetic leader (L-Zm.GSP850.nno:3) and contains a synthetic promoter (P-Zm.GSP850.nno:4). There is. There is. There is.

[0026] SEQ ID NO: 16 is the DNA sequence of synthetic EXP, EXP-Zm.GSP990.nno+Zm.DnaK:1, which is operably linked on the 5’ side to an intron (I-Zm.DnaK:1) and operably linked on the 5’ side to a synthetic leader (L-Zm.GSP990.nno:1) and contains a synthetic promoter (P-Zm.GSP990.nno:2). There is. There is. There is.

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

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

[0029] SEQ ID NO: 19 is the 3’UTR derived from the NLTP4 (non-specific lipid transfer protein 4) gene of Sorghum bicolor, the DNA sequence of T-Sb.Nltp4-1:1:2.

[0030] SEQ ID NO: 20 is a synthetic coding sequence optimized for plant expression of β-glucuronidase (GUS) with a processable intron derived from the potato light-inducible tissue-specific ST-LS1 gene (Genbank accession: X04753).

[0031] SEQ ID NO: 21 is the DNA sequence of EXP, EXP-CaMV.35S, containing the 35S promoter and leader derived from cauliflower mosaic virus.

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

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

[0034] SEQ ID NO: 24 is the coding sequence of β-glucuronidase (GUS) with a processable intron derived from the potato light-inducible tissue-specific ST-LS1 gene (Genbank accession: X04753).

[0035] SEQ ID NO: 25 is the coding sequence of NanoLuc® luciferase fluorescent protein (Promega, Madison, WI 53711). Nluc is a deep-sea ​​​​​​​​​​Directed evolution of luciferase from shrimp (Oplophorus gacilirostris) which has been engineered by

[0036] SEQ ID NO: 26 is the DNA sequence of the synthetic 3’UTR, T-Zm.GST43.nno:1 there is

BEST MODE FOR CARRYING OUT THE INVENTION

[0037] The present invention provides a synthetic regulatory element having gene regulatory activity in plants. The nucleotide sequence of such a synthetic regulatory element is provided as SEQ ID NOs: 1 to 18 and SEQ ID NO: 26. Such a synthetic regulatory element can affect the expression of a transcriptionally active DNA molecule operably linked therein, and thus can regulate the gene expression of a transgene operably linked in a transgenic plant. Further, the present invention also provides a novel endogenous regulatory element having gene regulatory activity in plants and provided as SEQ ID NO: 19. The present invention also provides methods for modifying, producing, and using recombinant DNA molecules containing the provided synthetic and endogenous regulatory elements. The present invention also provides transgenic plant cells, plants, plant parts, and compositions containing seeds, as well as methods for preparing and using these, which contain the recombinant DNA molecules of the present invention. as such. Such a synthetic regulatory element can affect the expression of a transcriptionally active DNA molecule operably linked therein, and thus can regulate the gene expression of a transgene operably linked in a transgenic plant. Further, the present invention also provides a novel endogenous regulatory element having gene regulatory activity in plants and provided as SEQ ID NO: 19. The present invention also provides methods for modifying, producing, and using recombinant DNA molecules containing the provided synthetic and endogenous regulatory elements. The present invention also provides transgenic plant cells, plants, plant parts, and compositions containing seeds, as well as methods for preparing and using these, which contain the recombinant DNA molecules of the present invention. in plant tissues and can affect the expression of a transcriptionally active DNA molecule operably linked therein, and thus can regulate the gene expression of a transgene operably linked in a transgenic plant. Further, the present invention also provides a novel endogenous regulatory element having gene regulatory activity in plants and provided as SEQ ID NO: 19. The present invention also provides methods for modifying, producing, and using recombinant DNA molecules containing the provided synthetic and endogenous regulatory elements. The present invention also provides transgenic plant cells, plants, plant parts, and compositions containing seeds, as well as methods for preparing and using these, which contain the recombinant DNA molecules of the present invention. in plant tissues and can affect the expression of a transcriptionally active DNA molecule operably linked therein, and thus can regulate the gene expression of a transgene operably linked in a transgenic plant. Further, the present invention also provides a novel endogenous regulatory element having gene regulatory activity in plants and provided as SEQ ID NO: 19. The present invention also provides methods for modifying, producing, and using recombinant DNA molecules containing the provided synthetic and endogenous regulatory elements. The present invention also provides transgenic plant cells, plants, plant parts, and compositions containing seeds, as well as methods for preparing and using these, which contain the recombinant DNA molecules of the present invention. in plant tissues and can affect the expression of a transcriptionally active DNA molecule operably linked therein, and thus can regulate the gene expression of a transgene operably linked in a transgenic plant. Further, the present invention also provides a novel endogenous regulatory element having gene regulatory activity in plants and provided as SEQ ID NO: 19. The present invention also provides methods for modifying, producing, and using recombinant DNA molecules containing the provided synthetic and endogenous regulatory elements. The present invention also provides transgenic plant cells, plants, plant parts, and compositions containing seeds, as well as methods for preparing and using these, which contain the recombinant DNA molecules of the present invention. The present invention also provides a novel endogenous regulatory element having gene regulatory activity in plants and provided as SEQ ID NO: 19. The present invention also provides methods for modifying, producing, and using recombinant DNA molecules containing the provided synthetic and endogenous regulatory elements. The present invention also provides transgenic plant cells, plants, plant parts, and compositions containing seeds, as well as methods for preparing and using these, which contain the recombinant DNA molecules of the present invention. The present invention also provides a novel endogenous regulatory element having gene regulatory activity in plants and provided as SEQ ID NO: 19. The present invention also provides methods for modifying, producing, and using recombinant DNA molecules containing the provided synthetic and endogenous regulatory elements. The present invention also provides transgenic plant cells, plants, plant parts, and compositions containing seeds, as well as methods for preparing and using these, which contain the recombinant DNA molecules of the present invention. The present invention also provides methods for modifying, producing, and using recombinant DNA molecules containing the provided synthetic and endogenous regulatory elements. The present invention also provides transgenic plant cells, plants, plant parts, and compositions containing seeds, as well as methods for preparing and using these, which contain the recombinant DNA molecules of the present invention. The present invention also provides transgenic plant cells, plants, plant parts, and compositions containing seeds, as well as methods for preparing and using these, which contain the recombinant DNA molecules of the present invention. and compositions containing seeds, as well as methods for preparing and using these, which contain the recombinant DNA molecules of the present invention.

[0038] The following definitions and methods are provided to better define the present invention and to provide guidance to those skilled in the art for practicing the present invention. Unless otherwise specified, terms are to be understood according to their conventional usage by those skilled in the relevant art. The following definitions and methods are provided to better define the present invention and to provide guidance to those skilled in the art for practicing the present invention. Unless otherwise specified, terms are to be understood according to their conventional usage by those skilled in the relevant art. The following definitions and methods are provided to better define the present invention and to provide guidance to those skilled in the art for practicing the present invention. Unless otherwise specified, terms are to be understood according to their conventional usage by those skilled in the relevant art.

[0039] DNA molecule As used herein, the terms "DNA" or "DNA molecule" refer to double-stranded DNA molecules of genomic or synthetic origin read from the 5' (upstream ) end to the 3' (downstream) end (i.e., polymers of deoxyribonucleotide bases or DNA molecules). As used herein, the term "DNA sequence" refers to the nucleotide sequence of a DNA molecule. The nomenclature used herein corresponds to the nomenclature of 37 CFR 1.822 and is set forth in the tables of WIPO Standard ST.25 (1998), Appendices 2, Tables 1 and 3.

[0040] As used herein, a "recombinant DNA molecule" is a DNA molecule that contains a combination of DNA molecules that are not naturally found together without human intervention. For example, a recombinant DNA molecule can be a DNA molecule composed of at least two DNA molecules that are heterologous to each other, a DNA molecule containing a DNA sequence that deviates from a DNA sequence that exists in nature, a DNA molecule containing a synthetic DNA sequence, or a DNA molecule that has been incorporated into the DNA of a host cell by genetic transformation or gene editing.

[0041] As used herein, a "synthetic nucleotide sequence" or "artificial nucleotide sequence" is a nucleotide sequence that is not known to occur in nature or does not occur naturally. The gene regulatory elements of the present invention include synthetic nucleotide sequences. Preferably, the synthetic nucleotide sequence shares little or no extended homology with natural sequences. Extended homology in this context generally means 100% sequence identity that extends more than about 25 nucleotides of adjacent sequences.

[0042] ​​​​​​​​​​​​​​ References to "isolated DNA molecule" or equivalent terms or expressions in this application are intended to mean that the DNA molecule exists alone or in combination with other compositions, but not in its natural environment. For example, nucleic acid elements such as coding sequences, intron sequences, untranslated leader sequences, promoter sequences, transcription termination sequences, etc., naturally found within the DNA of an organism's genome are not considered "isolated" as long as the element is within the organism's genome and in the position within the genome where the element is naturally found. However, each of these elements, and subparts of these elements, are considered "isolated" within the scope of this disclosure as long as the element is not within the organism's genome and not in the position within the genome where the element is naturally found. Similarly, a nucleotide sequence encoding an insecticidal protein or any naturally occurring insecticidal variant of that protein is considered an isolated nucleotide sequence as long as the nucleotide sequence is not within the DNA of the bacterium in which the sequence encoding the protein naturally occurs. A synthetic nucleotide sequence encoding the amino acid sequence of a naturally occurring insecticidal protein is considered isolated for the purposes of this disclosure. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA inserted into the genome of a plant or bacterial cell or present within an extrachromosomal vector, is considered isolated whether it is present within a plasmid or similar construct used for cell transformation, within the genome of a plant or bacterium, or derived from a plant or bacterium. not within the organism's genome and not in the position within the genome where the element is naturally found. is considered "isolated" within the scope of this disclosure. Similarly, a nucleotide sequence encoding an insecticidal protein or any naturally occurring insecticidal variant of that protein is considered an isolated nucleotide sequence as long as the nucleotide sequence is not within the DNA of the bacterium in which the sequence encoding the protein naturally occurs. A synthetic nucleotide sequence encoding the amino acid sequence of a naturally occurring insecticidal protein is considered isolated for the purposes of this disclosure. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA inserted into the genome of a plant or bacterial cell or present within an extrachromosomal vector, is considered isolated whether it is present within a plasmid or similar construct used for cell transformation, within the genome of a plant or bacterium, or derived from a plant or bacterium. of that protein is considered an isolated nucleotide sequence as long as the nucleotide sequence is not within the DNA of the bacterium in which the sequence encoding the protein naturally occurs. A synthetic nucleotide sequence encoding the amino acid sequence of a naturally occurring insecticidal protein is considered isolated for the purposes of this disclosure. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA inserted into the genome of a plant or bacterial cell or present within an extrachromosomal vector, is considered isolated whether it is present within a plasmid or similar construct used for cell transformation, within the genome of a plant or bacterium, or derived from a plant or bacterium. naturally occurs. A synthetic nucleotide sequence encoding the amino acid sequence of a naturally occurring insecticidal protein is considered isolated for the purposes of this disclosure. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA inserted into the genome of a plant or bacterial cell or present within an extrachromosomal vector, is considered isolated whether it is present within a plasmid or similar construct used for cell transformation, within the genome of a plant or bacterium, or derived from a plant or bacterium. A synthetic nucleotide sequence encoding the amino acid sequence of a naturally occurring insecticidal protein is considered isolated for the purposes of this disclosure. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA inserted into the genome of a plant or bacterial cell or present within an extrachromosomal vector, is considered isolated whether it is present within a plasmid or similar construct used for cell transformation, within the genome of a plant or bacterium, or derived from a plant or bacterium. is considered isolated for the purposes of this disclosure. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA inserted into the genome of a plant or bacterial cell or present within an extrachromosomal vector, is considered isolated whether it is present within a plasmid or similar construct used for cell transformation, within the genome of a plant or bacterium, or derived from a plant or bacterium. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA inserted into the genome of a plant or bacterial cell or present within an extrachromosomal vector, is considered isolated whether it is present within a plasmid or similar construct used for cell transformation, within the genome of a plant or bacterium, or derived from a plant or bacterium. inserted into the genome of a plant or bacterial cell or present within an extrachromosomal vector, is considered isolated whether it is present within a plasmid or similar construct used for cell transformation, within the genome of a plant or bacterium, or derived from a plant or bacterium. within a plasmid or similar construct used for cell transformation, within the genome of a plant or bacterium, or derived from a plant or bacterium. even if it is present within a plasmid or similar construct used for cell transformation, within the genome of a plant or bacterium, or derived from a plant or bacterium. even if present in a detectable amount in an organism, progeny, biological sample, or commercial product, is considered an isolated nucleotide sequence. is regarded as such.

[0043] As used herein, the term "sequence identity" means the degree to which two optimally aligned polynucleotide sequences or two optimally aligned polypeptide sequences are identical. Optimal sequence alignment is created by manually aligning two sequences, e.g., a reference sequence and another sequence, to maximize the number of nucleotide matches within the sequence alignment with appropriate internal nucleotide insertions, deletions, or gaps. As used herein, the term "reference sequence" means the DNA sequences provided as SEQ ID NOs: 1 to 19 and SEQ ID NO: 26. or gaps. As used herein, the term "reference sequence" means the DNA sequences provided as SEQ ID NOs: 1 to 19 and SEQ ID NO: 26. to 19 and SEQ ID NO: 26.

[0044] As used herein, the term "percent sequence identity" or "percent identity" or " % identity" is 100 times the ratio of identity. The "ratio of identity" in a sequence optimally aligned to a reference sequence is the number of nucleotide matches in the optimal alignment divided by the total number of nucleotides in the reference sequence, e.g., the total number of nucleotides in the entire full-length reference sequence. Thus, one embodiment of the present invention is that when optimally aligned to the reference sequences shown as SEQ ID NOs: 1 to 19 and SEQ ID NO: 26 in this specification, it has at least about 85 percent identity, at least about 86 percent identity, at least about 87 percent identity, at least about 88 percent identity, at least about 89 percent identity, at least about 90 percent identity to the reference sequence. identity, at least about 87 percent identity, at least about 88 percent identity, at least about 89 percent identity, at least about 90 percent identity identity, at least about 91 percent identity, at least about 92 percent identity , at least about 93 percent identity, at least about 94 percent identity, at least about 95 percent identity, at least about 96 percent identity, at least about 9 7 percent identity, at least about 98 percent identity, at least about 99 percent identity, or DNA molecules comprising a sequence having at least about 100 percent identity are provided. A DNA molecule having a certain percent sequence identity to a reference molecule may exhibit the activity of the reference sequence.

[0045] Regulatory elements Regulatory elements, such as promoters, leaders (also known as 5'UTRs), enhancers, introns, and transcription termination regions (or 3'UTRs), play essential roles in the overall expression of genes in living cells. As used herein, the term "regulatory element" means a DNA molecule having gene regulatory activity. As used herein, the term "gene regulatory activity" means the ability to affect the expression of an operably linked transcribable DNA molecule, for example, by affecting the transcription and / or translation of the operably linked transcribable DNA molecule. Regulatory elements that function in plants, such as promoters, leaders, enhancers, introns, and 3'UTRs, are useful for modifying the phenotype of plants by genetic engineering. As used herein, the term "regulatory expression element group" or "EXP" sequence refers to a group of operably linked regulatory elements, such as enhancers, promoters, leaders, and As used herein, the term "gene regulatory activity" means the ability to affect the expression of an operably linked transcribable DNA molecule, for example, by affecting the transcription and / or translation of the operably linked transcribable DNA molecule. Regulatory elements that function in plants, such as promoters, leaders, enhancers, introns, and 3'UTRs, are useful for modifying the phenotype of plants by genetic engineering. transcriptionally active DNA molecule, thereby affecting the expression of the operably linked transcribable DNA molecule. Regulatory elements that function in plants, such as promoters, leaders, enhancers, introns, and 3'UTRs, are useful for modifying the phenotype of plants by genetic engineering. As used herein, the term "regulatory element" means a DNA molecule having gene regulatory activity. As used herein, the term "gene regulatory activity" means the ability to affect the expression of an operably linked transcribable DNA molecule, for example, by affecting the transcription and / or translation of the operably linked transcribable DNA molecule. Regulatory elements that function in plants, such as promoters, leaders, enhancers, introns, and 3'UTRs, are useful for modifying the phenotype of plants by genetic engineering. As used herein, the term "regulatory expression element group" or "EXP" sequence refers to a group of operably linked regulatory elements, such as enhancers, promoters, leaders, and 3'UTR are useful for modifying the phenotype of plants by genetic engineering.

[0046] As used herein, the term "regulatory expression element group" or "EXP" sequence refers to a group of operably linked regulatory elements, such as enhancers, promoters, leaders, and introns, that are operably linked, such as enhancers, promoters, leaders, and may mean an intron. For example, the regulatory expression element group may be composed of, for example, a leader sequence and a promoter operably linked on the 5'-side. Useful EXPs for practicing the present invention include SEQ ID NOs: 1, 4, 6, 9, 11, 15, and 16.

[0047] Regulatory elements can be characterized by their gene expression patterns. For example, positive and / or negative effects, such as constitutive expression or temporal, spatial, developmental, tissue, environmental, physiological, pathological, cell cycle, and / or chemical-responsive expression, and any combination thereof, can be characterized by additional quantitative or qualitative indicators. As used herein, the term "gene expression pattern" refers to any pattern in which an operably linked DNA molecule is transcribed into a transcribed RNA molecule. The transcribed RNA molecule may be translated into a protein molecule, or may result in an antisense or other regulatory RNA molecule, such as double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), small interfering RNA (siRNA), etc. There are also cases.

[0048]

[0049] As used herein, the term "protein expression" refers to any pattern in which a transcribed RNA molecule is translated into a protein molecule. Protein expression can be further characterized by its temporal, spatial, developmental, or morphological properties, as well as by quantitative or qualitative indicators.

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

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

[0051] In further embodiments, fragments of the enhancer or intron sequences disclosed herein are provided. Enhancer or intron fragments can include the activity of the base molecule from which they are derived and can be useful alone or in combination with other regulatory elements (promoters, leaders, other enhancers, other introns, or fragments thereof). 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 ​​​​​​​​​​​at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900, or at least about 1000 consecutive nucleotides, or more, of a DNA molecule having enhancer or intron activity as disclosed herein is provided. Fragments of the enhancer or intron are provided. Methods for producing such fragments from starting molecules are well known in the art.

[0052] In other embodiments, fragments of the 3'UTR sequences disclosed herein are provided. The 3'UTR fragments can include the activity of the base 3'UTR molecules from which they are derived and can be useful alone or in combination with other regulatory elements (promoters, leaders, introns, or fragments thereof). In certain embodiments, at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900, or at least about 1000 consecutive nucleotides, or more, of a DNA molecule having 3'UTR activity as disclosed herein are provided. Methods for producing such fragments from starting 3'UTR molecules are well known in the

[0053] Provided as SEQ ID NO: 2 and 7, or contained in any of SEQ ID NO: 1, 4, 6, 9, 11, 15, and 16 (e.g., internal or 5' deletion) promoter elements Compositions derived from any of these can be produced by methods known in the art for improving or modifying expression, e.g., by removing elements that have either a positive or negative effect on expression, duplicating elements that have a positive or negative effect on expression, and / or duplicating or removing elements that have a tissue-specific or cell-specific effect on expression. Compositions derived from any of the promoter elements provided as SEQ ID NO: 2 and 7, or contained in any of SEQ ID NO: 1, 4, 6, 9, 11, 15, and 16, which are composed of a TATA box element or its equivalent sequence and a 3' deletion in which the downstream sequence has been removed, can be used to produce, for example, enhancer elements. Further deletions can be made to remove elements that have a positive or negative tissue-specific, cell-specific, or time-specific (e.g., circadian rhythm, but not limited to) effect on expression. Using the promoter elements provided as SEQ ID NO: 2 and 7, or contained in any of SEQ ID NO: 1, 4, 6, 9, 11, 15, and 16, as well as fragments or enhancers derived from these, chimeric transcriptional regulatory element compositions can be produced.

[0054] In the present invention, a promoter or promoter fragment can be analyzed for the presence of known promoter element DNA sequence characteristics, i.e., a TATA box and other known transcription factor binding site motifs. Such known promoter ele The identification information of the menthol can be used by those skilled in the art to design a promoter variant having an expression pattern similar to that of the original promoter.

[0055] As used herein, the term "leader" refers to a DNA molecule isolated from the untranslated 5' region (5'UTR) of a gene and generally defined as the nucleotide segment between the transcription start site (TSS) and the start site of the protein coding sequence. Alternatively, the leader may be a synthetically produced or engineered DNA element. The leader can be used as a 5' regulatory element to regulate the expression of a transcribable DNA molecule to which it is operably linked. The leader molecule can be used with a heterologous promoter or its native promoter. Leaders useful in practicing the present invention include any of the leader elements contained in SEQ ID NO: 3 and 8, or any of SEQ ID NO: 1, 4, 6, 9, 11, 15, and 16, or fragments or variants thereof. In certain embodiments, such DNA sequences can be defined as capable of acting as a leader in a host cell (e.g., including transgenic plant cells). In one embodiment, such sequences are decoded as containing leader activity.

[0056] The leader sequences presented as SEQ ID NO: 3 and 8 (also referred to as 5'UTR), or any of the leader elements contained in any of SEQ ID NO: 1, 4, 6, 9, 11, 15, and 16 may in some cases be composed of regulatory elements or may be operably linked transcription ​​​​​​​​​​​​​​​It may also adopt a secondary structure that can affect the transcription or translation of a possible DNA molecule. The leader sequences presented as SEQ ID NOs: 3 and 8, or any of the leader elements contained in any of SEQ ID NOs: 1, 4, 6, 9, 11 , 15, and 16 are used according to the present invention to produce a chimeric regulatory element that affects the transcription or translation of a transcriptionally operably linked DNA molecule.

[0057] As used herein, the term "intron" refers to a DNA molecule that can be isolated or identified from a gene and can generally be defined as a region that is spliced out during messenger RNA (mRNA) processing before translation. Alternatively, the intron may be a synthetically generated or engineered DNA element. The intron can contain enhancer elements that result in the transcription of an operably linked gene. The intron can be used as a regulatory element to regulate the expression of an operably linked transcriptionally active DNA molecule. A construct can contain an intron, and the intron may or may not be heterologous to the transcriptionally active DNA molecule. Examples of introns in the art include the rice actin intron and the maize HSP70 intron.

[0058] In plants, including several introns in a gene construct increases the accumulation of mRNA and protein compared to a construct without introns. This effect is referred to as "intron-mediated enhancement" (IME) of gene expression. Stimulating expression within a plant Introns that are known to exist are found in maize genes (e.g., tubA1, Ad h1, Sh1, and Ubi1), rice genes (e.g., tpi), and petunia ( e.g., rbcS), potato (e.g., st-ls1), and dicotyledonous plant genes such as Arabidopsi s thaliana (e.g., ubq3 and pat1). Deletion or mutation within the splice site of an intron has been shown to reduce gene expression, indicating that splicing may be required for IME. However, IME in dicotyledonous plants has been shown by point mutations within the splice site of the pat1 gene of A. thaliana. Multiple uses of the same intron in one plant have been shown to have disadvantages. In such cases, it is necessary to have a set of basic regulatory elements for constructing appropriate recombinant DNA elements. Exemplary introns useful in practicing the present invention are presented as SEQ ID NOs: 5, 10, and 12. As used herein, the terms "3' transcription termination molecule," "3' untranslated region," or "3' UTR" mean a DNA molecule that is used during transcription into the untranslated region of the 3' portion of an mRNA molecule. The 3' untranslated region of an mRNA molecule can be generated by specific cleavage and 3' polyadenylation (also known as the polyA tail). The 3'UTR can be operably linked to a transcribable DNA molecule and located downstream thereof, and can contain a polyadenylation signal and other regulatory signals that can affect transcription, mRNA processing, or gene expression. The polyA tail is an mRNA feature. Multiple uses of the same intron in one plant have been shown to have disadvantages. In such cases, it is necessary to have a set of basic regulatory elements for constructing appropriate recombinant DNA elements. Exemplary introns useful in practicing the present invention are presented as SEQ ID NOs: 5, 10, and 12. As used herein, the terms "3' transcription termination molecule," "3' untranslated region," or "3' UTR" mean a DNA molecule that is used during transcription into the untranslated region of the 3' portion of an mRNA molecule. The 3' untranslated region of an mRNA molecule can be generated by specific cleavage and 3' polyadenylation (also known as the polyA tail). The 3'UTR can be operably linked to a transcribable DNA molecule and located downstream thereof, and can contain a polyadenylation signal and other regulatory signals that can affect transcription, mRNA processing, or gene expression. The polyA tail is an mRNA

[0059] As used herein, the terms "3' transcription termination molecule," "3' untranslated region," or "3' UTR" mean a DNA molecule that is used during transcription into the untranslated region of the 3' portion of an mRNA molecule. The 3' untranslated region of an mRNA molecule can be generated by specific cleavage and 3' polyadenylation (also known as the polyA tail). The 3'UTR can be operably linked to a transcribable DNA molecule and located downstream thereof, and can contain a polyadenylation signal and other regulatory signals that can affect transcription, mRNA processing, or gene expression. The polyA tail is an mRNA feature. The 3' untranslated region of an mRNA molecule can be generated by specific cleavage and 3' polyadenylation (also known as the polyA tail). The 3'UTR can be operably linked to a transcribable DNA molecule and located downstream thereof, and can contain a polyadenylation signal and other regulatory signals that can affect transcription, mRNA processing, or gene expression. The polyA tail is an mRNA feature. The 3'UTR can be operably linked to a transcribable DNA molecule and located downstream thereof, and can contain a polyadenylation signal and other regulatory signals that can affect transcription, mRNA processing, or gene expression. The polyA tail is an mRNA feature. is thought to function in stability and translation initiation. Examples of 3' transcriptional termination elements in the art include the nopaline synthase 3' region, the wheat hsp17 3' region, the pea rubisco small subunit 3' region, the cotton E6 3' region, and the coixin 3' UTR.

[0060] 3' UTRs typically find useful applications in the recombinant expression of specific DNA molecules. Weak 3' UTRs have the potential to cause readthrough, which may affect the expression of DNA molecules within adjacent expression cassettes. By appropriately controlling transcriptional termination, readthrough to downstream DNA sequences (e.g., other expression cassettes) can be prevented, and efficient recycling of RNA polymerase for improving gene expression can be achieved. Efficient transcriptional termination (release of RNA polymerase II from DNA) is a prerequisite for transcriptional restart, which directly affects the overall transcriptional level. Following transcriptional termination, mature mRNA is released from the synthesis site and the template is transported to the cytoplasm. Since eukaryotic mRNAs accumulate in a poly(A) form in vivo, it is difficult to detect transcriptional termination sites by conventional methods. However, predicting 3' UTRs functionally and efficiently by bioinformatics methods is difficult because there are no conserved DNA sequences that allow for easy prediction of effective 3' UTRs.

[0061] From a practical perspective, it is typically beneficial for 3' UTRs used in expression cassettes to have the following characteristics. First, the 3' UTR efficiently and effectively should be terminable and any adjacent DNA sequences that can be composed of another expression cassette, such as in the case of multiple expression cassettes present within one transfer DNA (T-DNA), or should be able to prevent read-through of transcripts into adjacent chromosomal DNA into which the T-DNA has been inserted. Next, the 3’UTR should not cause a reduction in transcriptional activity conferred by promoters, leaders, enhancers, and introns used to promote the expression of the DNA molecule. Finally, in plant biotechnology, the 3’UTR is often used for priming the amplification reaction of reverse transcribed RNA extracted from transgenic plants, ( 1) evaluation of the transcriptional activity or expression of an expression cassette once integrated into the plant chromosome, (2) evaluation of the copy number of the insert within the plant DNA, and (3) evaluation of the zygosity of the seeds obtained after breeding is used. Also, the 3’UTR is also used for the amplification reaction of DNA extracted from transgenic plants to characterize the integrity of the inserted cassette. The 3’UTRs useful in practicing the present invention are presented as SEQ ID NOs: 13, 14, 19, and 26. When used herein, the term “enhancer” or “enhancer element” means a cis-acting regulatory element (also known as a cis element). This results in one aspect of the overall expression pattern of a transcribable DNA molecule bound in an operative manner, but usually is insufficient to drive transcription alone. Unlike a promoter, enhancer elements usually do not contain a transcription start site (TSS) or TATA box, or equivalent DNA sequences. A promoter or promoter fragment is naturally in an operative configuration, and the 3’UTRs useful in practicing the present invention are presented as SEQ ID NOs: 13, 14, 19, and 26.

[0062] As used herein, the term “enhancer” or “enhancer element” means a cis-acting regulatory element (also known as a cis element). This results in one aspect of the overall expression pattern of a transcribable DNA molecule bound in an operative manner, but usually is insufficient to drive transcription alone. Unlike a promoter, enhancer elements usually do not contain a transcription start site (TSS) or TATA box, or equivalent DNA sequences. A promoter or promoter fragment is naturally in an operative configuration, and usually enhancer elements do not contain a transcription start site (TSS) or TATA box, or equivalent DNA sequences. A promoter or promoter fragment is naturally in an operative configuration, Comprising one or more enhancer elements that affect the transcription of a DNA sequence linked thereto It is possible. Also, the enhancer element can be fused with a promoter to produce a chimeric pro moter element, which results in an overall regulation of gene expression in one aspect.

[0063] Many promoter enhancer elements bind to DNA-binding proteins and / or affect DNA topology, resulting in a local three-dimensional structure that selectively permits or restricts the access of RNA polymerase to the DNA template, or promotes the selective opening of the double helix at the transcription start site. It is thought that enhancer elements can function to bind to transcription factors that regulate transcription. Some enhancer elements bind to two or more transcription factors, and the transcription factors can interact with two or more enhancer domains with different affinities. The identification of enhancer elements can be carried out by a plurality of techniques including deletion analysis (i.e., deleting one or more nucleotides from the 5'-end or internally with respect to the promoter), DNA-binding protein analysis using DNase I footprinting, methylation interference, electrophoretic mobility shift assay, in vivo genomic footprinting by ligation-mediated polymerase chain reaction (PCR), and other conventional assays, or by DNA sequence similarity analysis using a known cis element motif or enhancer element as a target sequence or target motif together with a conventional DNA sequence comparison method such as BLAST. The fine structure of the enhancer domain can be determined by mutagenesis of one or more nucleotides of the local three-dimensional structure that selectively permits or restricts the access of RNA polymerase to the DNA template, or promotes the selective opening of the double helix at the transcription start site. It is thought that enhancer elements can function to bind to transcription factors that regulate transcription. Some enhancer elements bind to two or more transcription factors, and the transcription factors can interact with two or more enhancer domains with different affinities. The identification of enhancer elements can be carried out by a plurality of techniques including deletion analysis (i.e., deleting one or more nucleotides from the 5'-end or internally with respect to the promoter), DNA-binding protein analysis using DNase I footprinting, methylation interference, electrophoretic mobility shift assay, in vivo genomic footprinting by ligation-mediated polymerase chain reaction (PCR), and other conventional assays, or by DNA sequence similarity analysis using a known cis element motif or enhancer element as a target sequence or target motif together with a conventional DNA sequence comparison method such as BLAST. The fine structure of the enhancer domain can be determined by mutagenesis of one or more nucleotides of the local three-dimensional structure that selectively permits or restricts the access of RNA polymerase to the DNA template, or promotes the selective opening of the double helix at the transcription start site. It is thought that enhancer elements can function to bind to transcription factors that regulate transcription. Some enhancer elements bind to two or more transcription factors, and the transcription factors can interact with two or more enhancer domains with different affinities. The identification of enhancer elements can be carried out by a plurality of techniques including deletion analysis (i.e., deleting one or more nucleotides from the 5'-end or internally with respect to the promoter), DNA-binding protein analysis using DNase I footprinting, methylation interference, electrophoretic mobility shift assay, in vivo genomic footprinting by ligation-mediated polymerase chain reaction (PCR), and other conventional assays, or by DNA sequence similarity analysis using a known cis element motif or enhancer element as a target sequence or target motif together with a conventional DNA sequence comparison method such as BLAST. The fine structure of the enhancer domain can be determined by mutagenesis of one or more nucleotides of the local three-dimensional structure that selectively permits or restricts the access of RNA polymerase to the DNA template, or promotes the selective opening of the double helix at the transcription start site. It is thought that enhancer elements can function to bind to transcription factors that regulate transcription. Some enhancer elements bind to two or more transcription factors, and the transcription factors can interact with two or more enhancer domains with different affinities. The identification of enhancer elements can be carried out by a plurality of techniques including deletion analysis (i.e., deleting one or more nucleotides from the 5'-end or internally with respect to the promoter), DNA-binding protein analysis using DNase I footprinting, methylation interference, electrophoretic mobility shift assay, in vivo genomic footprinting by ligation-mediated polymerase chain reaction (PCR), and other conventional assays, or by DNA sequence similarity analysis using a known cis element motif or enhancer element as a target sequence or target motif together with a conventional DNA sequence comparison method such as BLAST. The fine structure of the enhancer domain can be determined by mutagenesis of one or more nucleotides of the local three-dimensional structure that selectively permits or restricts the access of RNA polymerase to the DNA template, or promotes the selective opening of the double helix at the transcription start site. It is thought that enhancer elements can function to bind to transcription factors that regulate transcription. Some enhancer elements bind to two or more transcription factors, and the transcription factors can interact with two or more enhancer domains with different affinities. The identification of enhancer elements can be carried out by a plurality of techniques including deletion analysis (i.e., deleting one or more nucleotides from the 5'-end or internally with respect to the promoter), DNA-binding protein analysis using DNase I footprinting, methylation interference, electrophoretic mobility shift assay, in vivo genomic footprinting by ligation-mediated polymerase chain reaction (PCR), and other conventional assays, or by DNA sequence similarity analysis using a known cis element motif or enhancer element as a target sequence or target motif together with a conventional DNA sequence comparison method such as BLAST. The fine structure of the enhancer domain can be determined by mutagenesis of one or more nucleotides of the local three-dimensional structure that selectively permits or restricts the access of RNA polymerase to the DNA template, or promotes the selective opening of the double helix at the transcription start site. It is thought that enhancer elements can function to bind to transcription factors that regulate transcription. Some enhancer elements bind to two or more transcription factors, and the transcription factors can interact with two or more enhancer domains with different affinities. The identification of enhancer elements can be carried out by a plurality of techniques including deletion analysis (i.e., deleting one or more nucleotides from the 5'-end or internally with respect to the promoter), DNA-binding protein analysis using DNase I footprinting, methylation interference, electrophoretic mobility shift assay, in vivo genomic footprinting by ligation-mediated polymerase chain reaction (PCR), and other conventional assays, or by DNA sequence similarity analysis using a known cis element motif or enhancer element as a target sequence or target motif together with a conventional DNA sequence comparison method such as BLAST. The fine structure of the enhancer domain can be determined by mutagenesis of one or more nucleotides of the local three-dimensional structure that selectively permits or restricts the access of RNA polymerase to the DNA template, or promotes the selective opening of the double helix at the transcription start site. It is thought that enhancer elements can function to bind to transcription factors that regulate transcription. Some enhancer elements bind to two or more transcription factors, and the transcription factors can interact with two or more enhancer domains with different affinities. The identification of enhancer elements can be carried out by a plurality of techniques including deletion analysis (i.e., deleting one or more nucleotides from the 5'-end or internally with respect to the promoter), DNA-binding protein analysis using DNase I footprinting, methylation interference, electrophoretic mobility shift assay, in vivo genomic footprinting by ligation-mediated polymerase chain reaction (PCR), and other conventional assays, or by DNA sequence similarity analysis using a known cis element motif or enhancer element as a target sequence or target motif together with a conventional DNA sequence comparison method such as BLAST. The fine structure of the enhancer domain can be determined by mutagenesis of one or more nucleotides of the local three-dimensional structure that selectively permits or restricts the access of RNA polymerase to the DNA template, or promotes the selective opening of the double helix at the transcription start site. It is thought that enhancer elements can function to bind to transcription factors that regulate transcription. Some enhancer elements bind to two or more transcription factors, and the transcription factors can interact with two or more enhancer domains with different affinities. The identification of enhancer elements can be carried out by a plurality of techniques including deletion analysis (i.e., deleting one or more nucleotides from the 5'-end or internally with respect to the promoter), DNA-binding protein analysis using DNase I footprinting, methylation interference, electrophoretic mobility shift assay, in vivo genomic footprinting by ligation-mediated polymerase chain reaction (PCR), by substitution, or by other conventional methods known in the art can be further studied. Enhancer elements can be obtained by chemical synthesis or by isolation from regulatory elements containing elements such as and can be synthesized together with additional adjacent nucleotides containing useful restriction enzyme sites for facilitating the manipulation of the subsequences. Thus, the design, construction, and use of enhancer elements according to the methods disclosed herein for modulating the expression of operably linked transcribable DNA molecules are encompassed by the present invention. Exemplary enhancers useful in practicing the present invention are presented as SEQ ID NOs: 17 and 18.

[0064] As used herein, the term "chimeric" means a single DNA molecule produced by fusing a first DNA molecule with a second DNA molecule, where neither the first DNA molecule nor the second DNA molecule is normally found in this configuration (i.e., fused with the other). Thus, a chimeric DNA molecule is a novel DNA molecule not normally found in nature by other means. As used herein, the term "chimeric promoter" means a promoter produced by the manipulation of such a DNA molecule. A chimeric promoter can combine two or more DNA fragments (e.g., fuse a promoter with an enhancer element). Thus, the design, construction, and use of chimeric promoters according to the methods disclosed herein for modulating the expression of operably linked transcribable DNA molecules are encompassed by the present invention.

[0065] Chimeric regulatory elements can be operably linked by various methods known in the art, such as restriction enzyme digestion and ligation, ligation-independent cloning, modular assembly of PCR products during amplification, or direct chemical synthesis of regulatory elements, as well as other methods known in the art, and can be designed to include various constituent elements that can be operably linked. The various chimeric regulatory elements obtained may be composed of the same constituent elements or variants of the same constituent elements, but the DNA sequence(s) containing the binding DNA sequence(s) that enable the constituents to be operably linked are different. In the present invention, the DNA sequences provided as SEQ ID NOs: 1 to 19 and SEQ ID NO: 26 may provide regulatory element reference sequences, in which case the constituent elements constituting the reference sequences can be ligated by methods known in the art and can also include substitutions, deletions, and / or insertions, or mutations of one or more nucleotides that occur naturally in the transformation of bacterial and plant cells. and ligation, ligation-independent cloning, modular assembly of PCR products during amplification, or direct chemical synthesis of regulatory elements, as well as other methods known in the art, and can be designed to include various constituent elements that can be operably linked. The various chimeric regulatory elements obtained may be composed of the same constituent elements or variants of the same constituent elements, but the DNA sequence(s) containing the binding DNA sequence(s) that enable the constituents to be operably linked are different. In the present invention, the DNA sequences provided as SEQ ID NOs: 1 to 19 and SEQ ID NO: 26 may provide regulatory element reference sequences, in which case the constituent elements constituting the reference sequences can be ligated by methods known in the art and can also include substitutions, deletions, and / or insertions, or mutations of one or more nucleotides that occur naturally in the transformation of bacterial and plant cells. The various chimeric regulatory elements obtained may be composed of the same constituent elements or variants of the same constituent elements, but the DNA sequence(s) containing the binding DNA sequence(s) that enable the constituents to be operably linked are different. In the present invention, the DNA sequences provided as SEQ ID NOs: 1 to 19 and SEQ ID NO: 26 may provide regulatory element reference sequences, in which case the constituent elements constituting the reference sequences can be ligated by methods known in the art and can also include substitutions, deletions, and / or insertions, or mutations of one or more nucleotides that occur naturally in the transformation of bacterial and plant cells. In the present invention, the DNA sequences provided as SEQ ID NOs: 1 to 19 and SEQ ID NO: 26 may provide regulatory element reference sequences, in which case the constituent elements constituting the reference sequences can be ligated by methods known in the art and can also include substitutions, deletions, and / or insertions, or mutations of one or more nucleotides that occur naturally in the transformation of bacterial and plant cells. In the present invention, the DNA sequences provided as SEQ ID NOs: 1 to 19 and SEQ ID NO: 26 may provide regulatory element reference sequences, in which case the constituent elements constituting the reference sequences can be ligated by methods known in the art and can also include substitutions, deletions, and / or insertions, or mutations of one or more nucleotides that occur naturally in the transformation of bacterial and plant cells. In the present invention, the DNA sequences provided as SEQ ID NOs: 1 to 19 and SEQ ID NO: 26 may provide regulatory element reference sequences, in which case the constituent elements constituting the reference sequences can be ligated by methods known in the art and can also include substitutions, deletions, and / or insertions, or mutations of one or more nucleotides that occur naturally in the transformation of bacterial and plant cells. In the present invention, the DNA sequences provided as SEQ ID NOs: 1 to 19 and SEQ ID NO: 26 may provide regulatory element reference sequences, in which case the constituent elements constituting the reference sequences can be ligated by methods known in the art and can also include substitutions, deletions, and / or insertions, or mutations of one or more nucleotides that occur naturally in the transformation of bacterial and plant cells. In the present invention, the DNA sequences provided as SEQ ID NOs: 1 to 19 and SEQ ID NO: 26 may provide regulatory element reference sequences, in which case the constituent elements constituting the reference sequences can be ligated by methods known in the art and can also include substitutions, deletions, and / or insertions, or mutations of one or more nucleotides that occur naturally in the transformation of bacterial and plant cells. In the present invention, the DNA sequences provided as SEQ ID NOs: 1 to 19 and SEQ ID NO: 26 may provide regulatory element reference sequences, in which case the constituent elements constituting the reference sequences can be ligated by methods known in the art and can also include substitutions, deletions, and / or insertions, or mutations of one or more nucleotides that occur naturally in the transformation of bacterial and plant cells.

[0066] As used herein, the term "variant" refers to a second DNA molecule whose composition is similar but not identical to that of a first DNA molecule, for example, a regulatory element, where the second DNA molecule still maintains the general functionality of the first DNA molecule, i.e., the same or similar expression pattern, for example, by approximately equivalent transcriptional activity. A variant may be a shorter or truncated version of the first DNA molecule, or a modified version of the sequence of the first DNA molecule, for example, having different restriction enzyme sites and / or internal deletions. As used herein, the term "variant" refers to a second DNA molecule whose composition is similar but not identical to that of a first DNA molecule, for example, a regulatory element, where the second DNA molecule still maintains the general functionality of the first DNA molecule, i.e., the same or similar expression pattern, for example, by approximately equivalent transcriptional activity. As used herein, the term "variant" refers to a second DNA molecule whose composition is similar but not identical to that of a first DNA molecule, for example, a regulatory element, where the second DNA molecule still maintains the general functionality of the first DNA molecule, i.e., the same or similar expression pattern, for example, by approximately equivalent transcriptional activity. As used herein, the term "variant" refers to a second DNA molecule whose composition is similar but not identical to that of a first DNA molecule, for example, a regulatory element, where the second DNA molecule still maintains the general functionality of the first DNA molecule, i.e., the same or similar expression pattern, for example, by approximately equivalent transcriptional activity. As used herein, the term "variant" refers to a second DNA molecule whose composition is similar but not identical to that of a first DNA molecule, for example, a regulatory element, where the second DNA molecule still maintains the general functionality of the first DNA molecule, i.e., the same or similar expression pattern, for example, by approximately equivalent transcriptional activity. As used herein, the term "variant" refers to a second DNA molecule whose composition is similar but not identical to that of a first DNA molecule, for example, a regulatory element, where the second DNA molecule still maintains the general functionality of the first DNA molecule, i.e., the same or similar expression pattern, for example, by approximately equivalent transcriptional activity. 、a version having substitutions, or insertions. Also, a "variant" has a nucleotide sequence that includes substitutions, deletions, or insertions of one or more nucleotides of a reference sequence, and may also include regulatory elements, where the derivative regulatory element has transcriptional or translational activity that is greater than, less than, or equivalent to that of the corresponding parental regulatory molecule. Also, a regulatory element "variant" includes variants that arise from mutations that occur naturally in the transformation of bacteria and plant cells. In the present invention, using the polynucleotide sequences provided as SEQ ID NOs: 1-19 and SEQ ID NO: 26, variants can be created that have a composition similar but not identical to the DNA sequence of the original regulatory element, while still maintaining the general functionality of the original regulatory element, i.e., the same or a similar expression pattern. The production of such variants of the present invention is within the ordinary skill of the art in light of the present disclosure and is encompassed within the scope of the present invention. a nucleotide sequence that includes substitutions, deletions, or insertions of one or more nucleotides of a reference sequence Regulatory elements can also be included, where the derivative regulatory element has transcriptional or translational activity that is greater than, less than, or equivalent to that of the corresponding parental regulatory molecule. Also, a regulatory element "variant" includes variants that arise from mutations that occur naturally in the transformation of bacteria and plant cells. In the present invention, using the polynucleotide sequences provided as SEQ ID NOs: 1-19 and SEQ ID NO: 26, variants can be created that have a composition similar but not identical to the DNA sequence of the original regulatory element, while still maintaining the general functionality of the original regulatory element, i.e., the same or a similar expression pattern. The production of such variants of the present invention is within the ordinary skill of the art in light of the present disclosure and is encompassed within the scope of the present invention. Using the polynucleotide sequences provided as SEQ ID NOs: 1-19 and SEQ ID NO: 26 in the present invention, a variant can be created that has a composition similar but not identical to the DNA sequence of the original regulatory element, while still maintaining the general functionality of the original regulatory element, i.e., the same or a similar expression pattern. The production of such variants of the present invention is within the ordinary skill of the art in light of the present disclosure and is encompassed within the scope of the present invention. The effectiveness of a particular transgene in the modifications, duplications, or deletions described herein with respect to a desired expression pattern can be empirically tested and verified in a stable transient plant assay, such as the assays described in the working examples herein. The results will vary depending on the changes made to the input DNA molecule and the purpose of the changes. The production of such variants of the present invention is within the ordinary skill of the art in light of the present disclosure and is encompassed within the scope of the present invention. The production of such variants of the present invention is within the ordinary skill of the art in light of the present disclosure and is encompassed within the scope of the present invention.

[0067] The effectiveness of a particular transgene in the modifications, duplications, or deletions described herein with respect to a desired expression pattern can be empirically tested and verified in a stable transient plant assay, such as the assays described in the working examples herein. The results will vary depending on the changes made to the input DNA molecule and the purpose of the changes. The effectiveness of a particular transgene in the modifications, duplications, or deletions described herein with respect to a desired expression pattern can be empirically tested and verified in a stable transient plant assay, such as the assays described in the working examples herein. The results will vary depending on the changes made to the input DNA molecule and the purpose of the changes. The results can be verified by empirical testing in a stable transient plant assay, such as the assays described in the working examples herein. The results will vary depending on the changes made to the input DNA molecule and the purpose of the changes. The results will vary depending on the changes made to the input DNA molecule and the purpose of the changes.

[0068] Construct As used herein, the term "construct" refers to a DNA molecule that is derived from any source, is capable of genomic integration or self-replication, and includes at least one DNA molecule that is functionally operably linked, i.e., operably linked, to another DNA molecule. a DNA molecule that is derived from any source, is capable of genomic integration or self-replication, and includes at least one DNA molecule that is functionally operably linked, i.e., operably linked, to another DNA molecule a DNA molecule that is derived from any source, is capable of genomic integration or self-replication, and includes at least one DNA molecule that is functionally operably linked, i.e., operably linked, to another DNA molecule Recombinant DNA molecules, for example, plasmids, cosmids, viruses, phages, or linear or circular DNA or RNA molecules. As used herein, the term "vector" refers to any construct that can be used for the purpose of transformation, i.e., introducing heterologous DNA or RNA into a host cell. A construct typically contains one or more expression cassettes. As used herein, an "expression cassette" refers to a DNA molecule containing at least one transcribable DNA molecule operably linked to one or more regulatory elements, typically at least a promoter and a 3'UTR.

[0069] As used herein, the term "operably linked" means that a first DNA molecule is linked to a second DNA molecule and the first and second DNA molecules are arranged such that the first DNA molecule affects the function of the second DNA molecule. The two DNA molecules may or may not be part of a single continuous DNA molecule and may or may not be adjacent. For example, a promoter is operably linked to a transcribable DNA molecule when the promoter regulates the transcription of the transcribable DNA molecule of interest in a cell. For example, a leader is operably linked to a DNA sequence when it can affect the transcription or translation of the DNA sequence.

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

[0071] A transcribable DNA molecule is transformed into a functional mRNA molecule that is translated and expressed as a protein. Methods for assembling and introducing constructs into cells in a transfected manner are described in the art. The practice of the invention involves the preparation and use of constructs and host cells. Conventional compositions and methods for use are well known to those skilled in the art. Exemplary vectors useful for expressing nucleic acids in vectors are well known in the art and include, The vector is derived from the Ti plasmid of Agrobacterium tumefaciens. Vectors include the pCaMVCN transfer control vector.

[0072] Various regulatory elements, including any regulatory elements provided herein, can be included in the construct. Any such regulatory element can be provided 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 transcribable DNA molecule operably linked to a 3'UTR and includes at least one regulatory element. The construct of the present invention can include any promoter or leader provided herein or known in the art. For example, the promoter of the present invention can be operably linked to a heterologous untranslated 5' leader (e.g., one derived from a heat shock protein gene). Alternatively, the leader of the present invention can be operably linked to a heterologous promoter (e.g., the cauliflower mosaic virus 35S transcript promoter). The expression cassette can also include a transit peptide coding sequence that encodes a peptide useful for targeting the operably linked protein at the subcellular level, particularly to chloroplasts, leucoplasts, or other plastid cell organelles, mitochondria, peroxisomes, vacuoles, or extracellular locations. Many chloroplast-localized proteins are expressed as precursors from nuclear genes and targeted to the chloroplast by a chloroplast transit peptide (CTP). Examples of such isolated chloroplast proteins include, but are not limited to, ribulose-1, and can be combined with other regulatory elements to provide the desired regulatory function. In one embodiment, the construct of the present invention is operably linked to a transcribable DNA molecule operably linked to a 3'UTR and includes at least one regulatory element. The construct of the present invention can include any promoter or leader provided herein or known in the art. For example, the promoter of the present invention can be operably linked to a heterologous untranslated 5' leader (e.g., one derived from a heat shock protein gene). Alternatively, the leader of the present invention can be operably linked to a heterologous promoter (e.g., the cauliflower mosaic virus 35S transcript promoter). including at least one regulatory element.

[0073] The construct of the present invention can be provided herein or can include any promoter or leader known in the art. For example, the promoter of the present invention can be operably linked to a heterologous untranslated 5' leader (e.g., one derived from a heat shock protein gene). Alternatively, the leader of the present invention can be operably linked to a heterologous promoter (e.g., the cauliflower mosaic virus 35S transcript promoter). The expression cassette can also include a transit peptide coding sequence that encodes a peptide useful for targeting the operably linked protein at the subcellular level, particularly to chloroplasts, leucoplasts, or other plastid cell organelles, mitochondria, peroxisomes, vacuoles, or extracellular locations. Many chloroplast-localized proteins are expressed as precursors from nuclear genes and targeted to the chloroplast by a chloroplast transit peptide (CTP). Examples of such isolated chloroplast proteins include, but are not limited to, ribulose-1, The expression cassette can also include a transit peptide coding sequence that encodes a peptide useful for targeting the operably linked protein at the subcellular level, particularly to chloroplasts, leucoplasts, or other plastid cell organelles, mitochondria, peroxisomes, vacuoles, or extracellular locations. Many chloroplast-localized proteins are expressed as precursors from nuclear genes and targeted to the chloroplast by a chloroplast transit peptide (CTP). Examples of such isolated chloroplast proteins include, but are not limited to, ribulose-1, ). Alternatively, the leader of the present invention can be operably linked to a heterologous promoter (e.g., the cauliflower mosaic virus 35S transcript promoter). The expression cassette can also include a transit peptide coding sequence that encodes a peptide useful for targeting the operably linked protein at the subcellular level, particularly to chloroplasts, leucoplasts, or other plastid cell organelles, mitochondria, peroxisomes, vacuoles, or extracellular locations. Many chloroplast-localized proteins are expressed as precursors from nuclear genes and targeted to the chloroplast by a chloroplast transit peptide (CTP). Examples of such isolated chloroplast proteins include, but are not limited to, ribulose-1, The expression cassette can also include a transit peptide coding sequence that encodes a peptide useful for targeting the operably linked protein at the subcellular level, particularly to chloroplasts, leucoplasts, or other plastid cell organelles, mitochondria, peroxisomes, vacuoles, or extracellular locations. Many chloroplast-localized proteins are expressed as precursors from nuclear genes and targeted to the chloroplast by a chloroplast transit peptide (CTP). Examples of such isolated chloroplast proteins include, but are not limited to, ribulose-1,

[0074] The expression cassette can also include a transit peptide coding sequence that encodes a peptide useful for targeting the operably linked protein at the subcellular level, particularly to chloroplasts, leucoplasts, or other plastid cell organelles, mitochondria, peroxisomes, vacuoles, or extracellular locations. Many chloroplast-localized proteins are expressed as precursors from nuclear genes and targeted to the chloroplast by a chloroplast transit peptide (CTP). Examples of such isolated chloroplast proteins include, but are not limited to, ribulose-1, The expression cassette can also include a transit peptide coding sequence that encodes a peptide useful for targeting the operably linked protein at the subcellular level, particularly to chloroplasts, leucoplasts, or other plastid cell organelles, mitochondria, peroxisomes, vacuoles, or extracellular locations. Many chloroplast-localized proteins are expressed as precursors from nuclear genes and targeted to the chloroplast by a chloroplast transit peptide (CTP). Examples of such isolated chloroplast proteins include, but are not limited to, ribulose-1, The expression cassette can also include a transit peptide coding sequence that encodes a peptide useful for targeting the operably linked protein at the subcellular level, particularly to chloroplasts, leucoplasts, or other plastid cell organelles, mitochondria, peroxisomes, vacuoles, or extracellular locations. Many chloroplast-localized proteins are expressed as precursors from nuclear genes and targeted to the chloroplast by a chloroplast transit peptide (CTP). Examples of such isolated chloroplast proteins include, but are not limited to, ribulose-1, The expression cassette can also include a transit peptide coding sequence that encodes a peptide useful for targeting the operably linked protein at the subcellular level, particularly to chloroplasts, leucoplasts, or other plastid cell organelles, mitochondria, peroxisomes, vacuoles, or extracellular locations. Many chloroplast-localized proteins are expressed as precursors from nuclear genes and targeted to the chloroplast by a chloroplast transit peptide (CTP). Examples of such isolated chloroplast proteins include, but are not limited to, ribulose-1, The expression cassette can also include a transit peptide coding sequence that encodes a peptide useful for targeting the operably linked protein at the subcellular level, particularly to chloroplasts, leucoplasts, or other plastid cell organelles, mitochondria, peroxisomes, vacuoles, or extracellular locations. Many chloroplast-localized proteins are expressed as precursors from nuclear genes and targeted to the chloroplast by a chloroplast transit peptide (CTP). Examples of such isolated chloroplast proteins include, but are not limited to, ribulose-1, and can be combined with other regulatory elements to provide the desired regulatory function. In one embodiment, the construct of the present invention is operably linked to a transcribable DNA molecule operably linked to a 3'UTR and includes at least one regulatory element. The small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase, ferredoxin, ferred doxin oxidoreductase, light-harvesting complex proteins I and II, thiored oxin F, and enolpyruvylshikimate phosphate synthase (EPSPS) are examples. Chloroplast transit peptides are described, for example, in U.S. Patent No. 7,193,133 Non-chloroplast proteins can be targeted to the chloroplast by expressing a heterologous CTP operably linked to a transgene encoding the non-chloroplast protein This has been demonstrated.

[0075] Transcribable DNA molecule As used herein, the term "transcribable DNA molecule" means any DNA molecule capable of being transcribed into an RNA molecule, including, but not limited to, molecules having protein-coding sequences and molecules that produce RNA molecules useful for gene silencing DNA molecules include, but are not limited to, DNA molecules from the same plant, DNA molecules from another plant, DNA molecules from different organisms, or synthetic DNA molecules, such as DNA molecules containing the antisense message of a gene, or DNA molecules encoding an artificial, synthetic, or otherwise modified transgene version. Exemplary transcribable DNA molecules for incorporation into the constructs of the present invention include, for example, DNA molecules or genes from species other than the species into which the DNA molecule is to be incorporated, or genes that occur in or are present in the same species but are incorporated into recipient cells by genetic engineering methods rather than classical breeding techniques

[0076] ​​​​​​​​A "transgene" means a transcribable DNA molecule that is heterologous to the host cell, at least with respect to its location within the host cell genome, and / or a transcribable DNA molecule that has been artificially integrated into the genome of the host cell in the current or any past generation of the cell. Regulatory elements, such as the promoter of the present invention, may be operably linked to a transcribable DNA molecule that is heterologous to the regulatory element. As used herein, the term "heterologous" means a combination of two or more DNA molecules that is not normally found in nature when such a combination of two or more DNA molecules 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 genes (e.g., different genes from the same species or the same gene from different species).

[0077] Thus, a regulatory element is heterologous to an operably linked transcribable DNA molecule if such a combination is not normally found in nature, i.e., if the transcribable DNA molecule does not occur naturally operably linked to the regulatory element. A transcribable DNA molecule is generally any DNA molecule for which expression of the transcript is desired. Expression of such a transcript can result in translation of the resulting mRNA molecule and thus protein expression. Alternatively, for example, a transcribable DNA molecule may be designed to ultimately cause a decrease in the expression of a particular gene or protein. In one embodiment, this can be achieved by using a transcribable DNA molecule oriented in the antisense direction. Those skilled in the art are familiar with the use of such antisense techniques. When two or more DNA molecules are not normally found in nature, the term "heterologous" means such a combination of two or more DNA molecules. That is, the transcribable DNA molecule is heterologous if it does not occur naturally operably linked to the regulatory element.

[0078] A transcribable DNA molecule is generally any DNA molecule for which expression of the transcript is desired. Expression of such a transcript can result in translation of the resulting mRNA molecule and thus protein expression. Alternatively, for example, a transcribable DNA molecule may be designed to ultimately cause a decrease in the expression of a particular gene or protein. In one embodiment, this can be achieved by using a transcribable DNA molecule oriented in the antisense direction. Those skilled in the art are familiar with the use of such antisense techniques. ​​is proficient in. Any gene can be negatively regulated by this method. In one embodiment, the transcriptionally active DNA molecule can be designed to suppress a specific gene through the expression of dsRNA, siRNA or miRNA molecules.

[0079] Accordingly, one embodiment of the present invention is a recombinant DNA molecule comprising a regulatory element of the present invention (e.g., those provided as SEQ ID NOs 1 to 19 and SEQ ID NO: 26), which is operably linked to a heterologous transcriptionally active DNA molecule such that when the construct is integrated into the genome of a transgenic plant cell, it regulates the transcription of the transcriptionally active DNA molecule at a desired level or in a desired pattern. In one embodiment, the transcriptionally active DNA molecule comprises the protein-coding region of a gene, and in another embodiment, the transcriptionally active DNA molecule comprises the antisense region of a

[0080] Gene for agricultural purposes The transcriptionally active DNA molecule can be a gene for agricultural purposes. As used herein, the term "gene for agricultural purposes" means a transcriptionally active DNA molecule that confers desirable characteristics when expressed in a particular plant tissue, cell, or cell type. The product of a gene for agricultural purposes can act within a plant to cause effects on plant morphology, physiology, growth, development, yield, grain composition, nutritional profile, disease or pest resistance, and / or environmental or chemical tolerance, or can act as an insecticide in the diet of pests that commonly feed on the plant. In one embodiment of the present invention, the regulatory element of the present invention is such that the regulatory element is Sea urchins are incorporated into the construct. In transgenic plants containing such a construct, the expression of genes for agronomic purposes can confer beneficial agronomic traits. Examples of beneficial agronomic traits include, but are not limited to, herbicide tolerance, insect control, modified yield, disease resistance, pathogen resistance, modified plant growth and development, modified starch content, modified oil content, modified fatty acid content, modified protein content, modified fruit ripening, animal and human nutrition enhancement, biopolymer production, environmental stress resistance, pharmaceutical peptides, improved processing quality, improved flavor, hybrid seed production utility , improved fiber production, and desirable biofuel production.

[0081] Non-limiting examples of genes for agronomic purposes known in the art include herbicide resistance (U.S. Patent Nos. 6,803,501; 6,448,476; 6,248,876 ; 6,225,114; 6,107,549; 5,866,775; 5, 804,425; 5,633,435; and 5,463,175), increased yield (U.S. Patent Nos. USRE38,446; 6,716,474; 6,663,906 ; 6,476,295; 6,441,277; 6,423,828; 6 ,399,330; 6,372,211; 6,235,971; 6,222 ,098; and 5,716,837), insect control (U.S. Patent Nos. 6,809,078 ; 6,713,063; 6,686,452; 6,657,046; 6 ,645,497; 6,642,030; 6,639,054; 6,620 ,988; No. 6,593,293; No. 6,555,655; No. 6,538,109 ; No. 6,537,756; No. 6,521,442; No. 6,501,009; No. 6 ,468,523; No. 6,326,351; No. 6,313,378; No. 6,284 ,949; No. 6,281,016; No. 6,248,536; No. 6,242,241 ; No. 6,221,649; No. 6,177,615; No. 6,156,573; No. 6 ,153,814; No. 6,110,464; No. 6,093,695; No. 6,063 ,756; No. 6,063,597; No. 6,023,013; No. 5,959,091 ; No. 5,942,664; No. 5,942,658; 5,880,275; No. 5, 763,245; and No. 5,763,241), fungal disease resistance (U.S. Patent No. 6,6 53,280; No. 6,573,361; No. 6,506,962; No. 6,316,4 07; No. 6,215,048; No. 5,516,671; No. 5,773,696; No. 6,121,436; No. 6,316,407; and No. 6,506,962), virus resistance (U.S. Patent No. 6,617,496; No. 6,608,241; No. 6,01 5,940; No. 6,013,864; No. 5,850,023; and No. 5,304, 730), nematode resistance (U.S. Patent No. 6,228,992), bacterial disease resistance (U.S. Pat ent No. 5,516,671), plant growth and development (U.S. Patent Nos. 6,723,897 and 6,518,488), starch production (U.S. Patent Nos. 6,538,181; No. 6,53 8,179; No. 6,538,178; No. 5,750,876; No. 6,476,29 5), modified oil production (U.S. Patent Nos. 6,444,876; No. 6,426,447 ; U.S. Patent No. 6,380,462), high oil production (U.S. Patent No. 6,495,739; No. 5,6 08,149; No. 6,483,008; and No. 6,476,295), modified fatty acid content (U.S. Patent No. 6,828,475; No. 6,822,141; No. 6,770 ,465; No. 6,706,950; No. 6,660,849; No. 6,596,538 ; No. 6,589,767; No. 6,537,750; No. 6,489,461; No. 6 ,459,018), high protein production (U.S. Patent No. 6,380,466), fruit ripening (U.S. Patent No. 5,512,466), animal and human nutrition enhancement (U.S. Patent No. 6,7 23,837; No. 6,653,530; No. 6,5412,59; No. 5,985,6 05; No. 6,171,640), biopolymers (U.S. Patent No. USRE37,543; No. 6,228,623; and No. 5,958,745, and No. 6,946,588) , environmental stress resistance (U.S. Patent No. 6,072,103), pharmaceutical peptides and secretory peptides (U.S. Patent No. 6,812,379; No. 6,774,283; No. 6,140,0 75; and No. 6,080,560), improvement of processing traits (U.S. Patent No. 6,47 6,295), improvement of digestibility (U.S. Patent No. 6,531,648), low raffinose (U.S. Patent No. 6,166,292), industrial enzyme production (U.S. Patent No. 5,543,576 ), improvement of flavor (U.S. Patent No. 6,011,199), nitrogen fixation (U.S. Patent No. 5,22 9,114), hybrid seed production (U.S. Patent No. 5,689,041), fiber production (U.S. Patent No. 6,576,818; No. 6,271,443; No. 5,981,834 ; and U.S. Patent No. 5,869,720, as well as biofuel production (U.S. Patent No. 5,998,7 00).

[0082] Alternatively, genes for agronomic purposes can, for example, by encoding RNA molecules that trigger targeted regulation of the gene expression of endogenous genes, antisense (see, e.g., U.S. Patent No. 5,107,065), inhibitory RNA ("RNAi"; e.g., published applications U.S. 2006 / 0200878 and U.S. 2008 / 0066206, as well as U.S. Patent Application No. 11 / 974,469, as described, miRNA, siRNA, trans acting siRNA, and regulation of gene expression by mechanisms mediated by phased sRNA are included), or by mechanisms mediated by co-suppression, affect the above plant characteristics or phenotypes. Further, the RNA can be an engineered catalytic RNA molecule (e.g., ribozyme or riboswitch; e.g., see U.S. 200 6 / 0200878) that cleaves the desired endogenous mRNA product. Methods for constructing constructs and introducing them into cells such that the transcribed DNA molecule is transcribed into a molecule capable of causing gene silencing are known in the art.

[0083] Selectable Marker A selectable marker transgene may be used in conjunction with the regulatory elements of the present invention. As used herein, the term "selectable marker transgene" means any transcribable DNA molecule whose expression, or lack of expression, in transgenic plants, tissues, or cells can be screened or scored in some way. ​​​​​​​​​A selectable marker gene for use thereon, and related selection and screening techniques are known in the art and include, but are not limited to, β-glucuronidase (GUS), green fluorescent protein (GFP), proteins conferring antibiotic resistance, and transcribable DNA molecules encoding proteins conferring herbicide tolerance. Examples of selectable marker transgenes are provided as SEQ ID NOs: 20 and 24.

[0084] Cell transformation The present invention also relates to a method for producing transformed cells and plants, which method comprises one or more regulatory elements operably linked to a transcribable

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

[0086] As used herein, the term "transformed" means a cell, tissue, organ, or organism into which a foreign DNA molecule (e.g., a construct) has been introduced. The introduced DNA molecule can be integrated into the genomic DNA of the recipient cell, tissue, organ, or organism such that the introduced DNA molecule is inherited by subsequent progeny. "Transgenic" or "transformed" cells or organisms include the progeny of the Produced from breeding programs that use such transgenic organisms as parents in mating , progeny showing phenotypic modifications caused by the presence of foreign DNA molecules may also be included. Also, the introduced DNA molecule can be transiently introduced into the recipient cell so that the introduced DNA molecule is not inherited by the next generation. The term "transgenic" means a bacterium, fungus, or plant containing one or more heterologous DNA molecules.

[0087] There are numerous methods for introducing DNA molecules into plant cells and are well known to those skilled in the art . This process generally includes the steps of selecting a suitable host cell, transforming the host cell with a vector , and obtaining the transformed host cell. In practicing the present invention, methods and materials for transforming plant cells by introducing a plant construct into the plant genome may include any well-known and proven method. Suitable methods include, but are not limited to, among others, bacterial infection (e.g., Agrobacteri um), 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 stem). stem).

[0088] The host cell can be any cell or organism, such as a plant cell, an algal cell, an alga, a fungal cell, a fungus, a bacterial cell, or an insect cell. In certain embodiments, the host cell and the transformed cell may include cells from crop plants.

[0089] The transgenic plants can then be regenerated from the transgenic plant cells of the present invention. Seeds can be produced from such transgenic plants using conventional breeding techniques or self-pollination. Such seeds, and the resulting progeny plants grown from such seeds, contain the recombinant DNA molecule of the present invention and are therefore transgenic. The transgenic plants of the present invention can self-pollinate to provide seeds of the homozygous transgenic plants of the present invention (homozygous for the recombinant DNA molecule), or can be crossed with non-transgenic plants or different transgenic plants to provide seeds of the heterozygous transgenic plants of the present invention (heterozygous for the recombinant DNA molecule). Such homozygous and heterozygous transgenic plants are both referred to herein as "progeny plants". Progeny plants are transgenic plants derived from the original transgenic plants and containing the recombinant DNA molecule of the present invention. Seeds produced using the transgenic plants of the present invention can be harvested and used to grow a new generation of transgenic plants that contain the construct of the present invention and express genes for agricultural purposes, i.e., the progeny plants of the present invention. Descriptions of breeding methods commonly used for various crops can be found in one of several references. For example, Allard, Principles of Plant Breeding, John Wiley & Sons, NY, U. of CA, Davis, CA, 50-98 (1960); Simmonds, Principles of Crop Impr

[0090] ​​​​​​​​​​​​​​​​​ovement, Longman, Inc., NY, 369 - 399(1979); Sn eep and Hendriksen, Plant breeding Perspe ctives, Wageningen(ed), Center for Agricul tural Publishing and Documentation(1979) ; Fehr, Soybeans: Improvement, Production a nd Uses, 2nd Edition, Monograph, 16:249(198 7); Fehr, Principles of Variety Developmen t, Theory and Technique, (Vol.1) and Crop S pecies Soybean(Vol.2), Iowa State Univ., M acmillan Pub.Co., NY, 360 - 376(1987). See

[0091] Transgenic plants can be analyzed for the presence of the gene(s) of interest, as well as the expression level and / or profile conferred by the regulatory ele ments of the present invention. Those skilled in the art are aware of numerous methods available for the analysis of transgenic plants. For example, methods of plant analysis include, but are not limited to, Southern or Northern blotting, PCR -based approaches, biochemical analysis, phenotypic screening methods, field evaluation, and immunoassay assays. The expression of a transcribable DNA molecule can be determined by the reagents and methods described by the manufacturer of TaqMan® (A pplied Biosystems (Foster City, CA)), as well as the TaqMan® Testing Matri x described by the manufacturer, and It can be measured using the PCR cycle time determined using x. Alternatively, In vader® (Third Wave Technologies (Madi son, WI)) reagents and the methods described by the manufacturer can be used to evaluate the expression of the transgene. It may be evaluated.

[0092] The present invention also provides parts of the plants of the present invention. Examples of plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovules, and pollen. The parts of the plants of the present invention 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 transformed or transgenic plant cells of the present invention include regenerable and / or non-regenerable plant cells. Examples of plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovules, and pollen. The parts of the plants of the present invention 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 transformed or transgenic plant cells of the present invention include regenerable and / or non-regenerable plant cells. The present invention also includes and provides transformed plant cells containing the DNA molecules of the present invention. The transformed or transgenic plant cells of the present invention include regenerable and / or non-regenerable plant cells. The transformed or transgenic plant cells of the present invention include regenerable and / or non-regenerable plant cells. The transformed or transgenic plant cells of the present invention include regenerable and / or non-regenerable plant cells.

[0093] The present invention also provides transgenic plants containing the recombinant DNA molecules of the present invention or commodity products produced from parts thereof. The commodity products of the present invention contain a detectable amount of DNA comprising a DNA sequence selected from the group consisting of SEQ ID NOs: 1 to 19 and SEQ ID NO: 26. As used herein, "commodity product" means any composition or product composed of materials derived from transgenic plants, seeds, plant cells, or parts of plants containing the recombinant DNA molecules of the present invention. Examples of commodity products include, but are not limited to, processed seeds, grains, plant parts, and meal. The commodity products of the present invention will contain a detectable amount of DNA corresponding to the recombinant DNA molecules of the present invention. To determine the content or source of the commodity product, detection of one or more of this DNA in a sample The present invention also provides transgenic plants containing the recombinant DNA molecules of the present invention or commodity products produced from parts thereof. The commodity products of the present invention contain a detectable amount of DNA comprising a DNA sequence selected from the group consisting of SEQ ID NOs: 1 to 19 and SEQ ID NO: 26. The present invention also provides transgenic plants containing the recombinant DNA molecules of the present invention or commodity products produced from parts thereof. The commodity products of the present invention contain a detectable amount of DNA comprising a DNA sequence selected from the group consisting of SEQ ID NOs: 1 to 19 and SEQ ID NO: 26. The present invention also provides transgenic plants containing the recombinant DNA molecules of the present invention or commodity products produced from parts thereof. The commodity products of the present invention contain a detectable amount of DNA comprising a DNA sequence selected from the group consisting of SEQ ID NOs: 1 to 19 and SEQ ID NO: 26. The present invention also provides transgenic plants containing the recombinant DNA molecules of the present invention or commodity products produced from parts thereof. The commodity products of the present invention contain a detectable amount of DNA comprising a DNA sequence selected from the group consisting of SEQ ID NOs: 1 to 19 and SEQ ID NO: 26. The present invention also provides transgenic plants containing the recombinant DNA molecules of the present invention or commodity products produced from parts thereof. The commodity products of the present invention contain a detectable amount of DNA comprising a DNA sequence selected from the group consisting of SEQ ID NOs: 1 to 19 and SEQ ID NO: 26. The present invention also provides transgenic plants containing the recombinant DNA molecules of the present invention or commodity products produced from parts thereof. The commodity products of the present invention contain a detectable amount of DNA comprising a DNA sequence selected from the group consisting of SEQ ID NOs: 1 to 19 and SEQ ID NO: 26. The present invention also provides transgenic plants containing the recombinant DNA molecules of the present invention or commodity products produced from parts thereof. The commodity products of the present invention contain a detectable amount of DNA comprising a DNA sequence selected from the group consisting of SEQ ID NOs: 1 to 19 and SEQ ID NO: 26. The present invention also provides transgenic plants containing the recombinant DNA molecules of the present invention or commodity products produced from parts thereof. The commodity products of the present invention contain a detectable amount of DNA comprising a DNA sequence selected from the group consisting of SEQ ID NOs: 1 to 19 and SEQ ID NO: 26. can be used. Any standard DN, including the detection methods disclosed in this specification A molecular detection method can be used.

[0094] The present invention can be more easily understood by referring to the following examples. This example is presented by way of illustration only and is not intended to limit the present invention unless otherwise specified. Those skilled in the art should understand that the techniques disclosed in the following examples correspond to the techniques sufficient to function in practicing the present invention discovered by the inventor. However, those skilled in the art should be able to make many changes to the specific embodiments disclosed in this disclosure and still obtain similar or identical results without departing from the spirit and scope of the present invention. Therefore, all the content described or shown in the accompanying drawings should be construed as illustrative only and not in a limiting sense should not be construed.

Examples

[0095] The novel synthetic transcriptional regulatory elements are synthetic expression elements designed by an algorithm method. These computationally designed regulatory elements were chemically synthesized and cloned to generate a group of synthetic regulatory expression elements (EXP). More than 1,000 synthetic regulatory elements were designed and assayed in maize protoplasts and stably transformed maize plants to identify synthetic regulatory elements that confer desired properties (e.g., protein expression level and expression pattern). The synthetic elements of the present invention have many ​​​​​​​Diverse patterns that drive the expression of interfering RNAs with different coding sequences and for agricultural purposes result in constitutive expression.

[0096] The designed synthetic transcriptional regulatory elements do not extend homology with any known nucleic acid sequences existing in nature, yet still affect the transcription of operably linked coding sequences in the same manner as naturally occurring promoters, leaders, introns, and 3’UTRs. Synthetic EXPs and their corresponding synthetic promoters, leaders, introns, and synthetic 3’UTRs are presented in Table 1. Synthetic EXPs were cloned in a binary plant transformation vector operably linked to a β-glucuronidase (GUS) coding sequence and the expression levels and patterns were evaluated in stably transformed maize plants using methods known in the art.

[0097] Analysis of regulatory elements TSS and intron / exon splice junctions can be performed using transformed plant tissues. Briefly, a plant expression vector containing a cloned DNA fragment operably linked to a heterologous transcribable DNA molecule was used to transform plants. Next, the regulatory element TSS and intron / exon splice junctions were identified by analyzing the DNA sequences of the produced mRNA transcripts using the 5’ RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (Invitrogen (Carlsbad, California 92008)). The synthetic 3’UTR was characterized for its effect on gene expression and proper termination of transcripts.

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

[0099] Example 2 Analysis of Synthetic Regulatory Elements Driving GUS in Maize Leaf Protoplasts Maize leaf protoplasts were transformed with a vector, specifically, an expression vector containing a test regulatory element that drives the expression of the β-glucuronidase (G US) transgene. The resulting transformed maize leaf protoplasts were analyzed for GUS protein expression to evaluate the effect of the selected regulatory element on expression.

[0100] Maize protoplasts derived from leaf tissue were transformed with an expression vector containing synthetic expression elements. The levels and patterns of expression of these synthetic expression element vectors in maize protoplasts were compared to the levels and patterns of expression of expression elements known in the art. Separate experiments were conducted for EXP with EXP-Zm .GSP850 (SEQ ID NO: 1) and EXP-Zm.GSP990 (SEQ ID NO: 6), for intron with I-Zm.GSI153.nno:1 (SEQ ID NO: 5) and I-Zm.GSI 197.nno:1 (SEQ ID NO: 10), and for 3’UTR with T-Zm.GST9.nno ​​​​​:2 (Accession No. 13) and T-Zm.GST18.nno:2 (Accession No. 14) were evaluated. The expression elements were cloned within an expression vector and linked operably to a GUS coding sequence containing a processable intro n and GOI-Ec.uidA+St.LS1:1:1 (Accession No. 24). The control expression vector contained known expression elements of different configurations, which varied according to the type of element being evaluated (EXP, intron, or 3’UTR). Plasmids for use in co-transformation of protoplasts and normalization of data were also constructed using methods known in the art. This plasmid contained a transgene cassette composed of EXP, EXP-CaMV .35S (Accession No. 21) operably linked on the 5’ side to a coding sequence encoding NanoLuc® luciferase fluorescent protein (Promega (

[0101] Madison, WI 53711)) (referred to herein as Nluc ( Accession No. 25)) and operably linked on the 5’ side to T-Os.LTP:1 (Accession No. 23). Maize leaf protoplasts were transformed using the PEG-based transformation method as is known in the 5 art. Protoplast cells were transformed in 96-well format. 12 micrograms of test vector DNA or control vector DNA and 6 The expression of luciferase and GUS was measured using the lysate. To lyse the cells, the cells in the plate were pelleted by centrifugation, washed, resuspended in a small volume, and transferred to a strip well tube. The tube was centrifuged again, and the supernatant was aspirated, leaving the protoplast cell pellet. The cell pellet was resuspended in QB buffer (100 mM KPO, pH 7.8; 1 mM EDTA; 1% Triton X-100; 10% glycerol; 1 mM DTT). The cells were vigorously pipetted several times, the tube was vortexed, and the tube was incubated on ice for 5 minutes to lyse the cells. The lysate was then centrifuged to pellet the cell debris. The resulting lysate was then transferred to a clean plate. The cells in the plate were pelleted by centrifugation, washed, resuspended in a small volume, and transferred to a strip well tube. The tube was centrifuged again, and the supernatant was aspirated, leaving the protoplast cell pellet. The cell pellet was resuspended in QB buffer (100 mM KPO 4 , pH 7.8 ; 1 mM EDTA; 1% Triton X-100; 10% glycerol; 1 mM DTT) The cells were vigorously pipetted several times, the tube was vortexed, and the tube was incubated on ice for 5 minutes to lyse the cells. The lysate was then centrifuged to pellet the cell debris. The resulting lysate was then transferred to a clean plate.

[0102] Luciferase activity was assayed with Nano-Glo® Luciferase Assay Substrate (Promega (Madison, WI 53711)) in QB buffer. Briefly, a small volume of lysate, QB buffer, and Nano-Glo® Luciferase Assay Substrate / QB solution were mixed together in a white 96-well plate. Fluorescence was then measured using a PHERAstar® plate reader (BMG LABTECH Inc. (Cary, NC 27513)). Luciferase activity was assayed with Nano-Glo® Luciferase Assay Substrate (Promega (Madison, WI 53711)) in QB buffer. Briefly, a small volume of lysate, QB buffer, and Nano-Glo® Luciferase Assay Substrate / QB solution were mixed together in a white 96-well plate. Luciferase activity was assayed with Nano-Glo® Luciferase Assay Substrate / QB solution. solution were mixed together in a white 96-well plate. Fluorescence was then measured using a PHERAstar® plate reader (BMG LABTECH Inc. (Cary, NC 27513)).

[0103] GUS activity was assayed with the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide (MUG) in a total reaction volume of 50 microliters. The reaction product 4-methylumbelliferone (4-MU) is maximally fluorescent at high pH, at which time the hydroxyl group GUS activity was assayed with the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide (MUG) in a total reaction volume of 50 microliters. 4-methylumbelliferone (4-MU) is maximally fluorescent at high pH, at which time the hydroxyl group is ionized. Addition of a basic solution of sodium carbonate stops the assay and simultaneously adjusts the pH for quantifying the fluorescent product. An aliquot of the lysate was mixed with an aliquot of MUG dissolved in QB buffer - and incubated at 37°C. Small aliquots of the lysate / MUG G reaction mixture were taken at three different time points: (1) immediately after mixing the lysate / MUG reactant as "time zero minutes", (2) at 20 minutes, and (3) at 60 minutes and added to the stop buffer. Fluorescence was measured at 355 nm excitation using a PHERAstar® plate reader (BMG LABTECH Inc. (Cary, NC 27513)) and 460 nm emission. The level of expression was represented as "MUG hydrolysis nM" derived from the in-plate standard curve.

[0104] For each plate, each construct was transformed in 4 - 8 wells. Aliquots were taken from each transformation for the MUG assay and "MUG hydrolysis nM" was derived from the in-plate standard curve. Also, aliquots were taken from each transformation for NanoLuc® reading (NanoLuc® RLU). The average MUG hydrolysis nM / NanoLuc® RLU for each construct was normalized to the EXP-CaMV.35S / I-Zm.DnaK:1 / T-Os.LTP:1 construct set at 100%.

[0105] Analysis of GUS expression in maize leaf protoplasts driven by synthetic EXP, EXP-Zm.GSP850. An expression element driving GUS expression constructed using methods known in the art The maize leaf protoplast cells were transformed with an expression vector containing the transgene cassette containing the synthetic EXP, EXP-Zm.GSP850 (SEQ ID NO: 1). The synthetic EXP-Zm.GSP850 was composed of a synthetic promoter P-Zm.GSP850.nno:4 (SEQ ID NO: 2) operably linked at the 5'-side to a synthetic leader L-Zm.GSP850.nno:3 (SEQ ID NO: 3). The first test vector contained EXP-Zm.GSP850 operably linked at the 5'-side to a coding sequence encoding GUS (SEQ ID NO: 24) containing a processable intron operably linked at the 5'-side to 3'UTR, T-Os.LTP:1 (SEQ ID NO: 23). The second transgene cassette contained EXP-Zm.GSP850 operably linked at the 5'-side to a GUS coding sequence operably linked at the 5'-side to 3'UTR, T-Os.LTP:1 and further operably linked at the 5'-side to intron I-Zm.DnaK:1 (SEQ ID NO: 22).

[0106] Three control expression vectors were also constructed and used for the transformation of maize leaf protoplasts. The first control expression vector contained a transgene cassette without a promoter and was composed of 3'UTR, T-Os.LTP:1 operably linked at the 5'-side to a GUS coding sequence and further operably linked at the 5'-side to intron I-Zm.DnaK:1. The second control vector contained a transgene cassette without an intron and was composed of 3'UTR, T-Os.LTP:1 operably linked at the 5'-side to a GUS coding sequence and further operably linked at the 5'-side to The control vector was G operably linked on the 5' side to a 3'UTR, T-Os.LTP:1 and on the 5' side operably linked to an intron I-Zm.DnaK:1 and on the 5' side operably linked to a US coding sequence and contained a transgene cassette containing an EXP, EXP-CaMV.35S operably linked on the 5' side to it.

[0107] Maize leaf protoplasts were transformed with all five vectors. Transformation and lysis of protoplast cells were carried out as described herein. Luciferase and GUS expression were assayed as described herein. Table 2 shows the average GUS expression assayed and is represented as a percentage of expression compared to a third control expression vector containing EXP-CaMV.35S and I -Zm.DnaK:1 driving GUS.

Table 2

[0108] As can be seen from Table 2 above, EXP-Zm.GSP850 (SEQ ID NO: 1) was able to drive GUS transgene expression in maize leaf protoplasts compared to maize leaf protoplast cells transformed with a construct without a promoter .

[0109] Analysis of GUS expression in maize leaf protoplasts driven by synthetic EXP, EXP-Zm.GSP990 . Maize leaf protoplast cells were transformed with an expression vector constructed to contain an expression element driving GUS expression. The test expression vector was a coding sequence encoding GUS (SEQ ID NO: 20) operably linked on the 5' side to a 3'UTR, T-Os.LTP:1 and on the 5' side operably linked to it.​​ and intron I-Zm.DnaK:1 (SEQ ID NO: 22) operably linked on the 5'-side, and a synthetic EXP, EXP-Zm.GSP990 (SEQ ID NO: 6) operably linked on the 5'-side The transgenic cassette contained. Synthetic EXP-Zm.GSP990 (SEQ ID NO: 6 ) consists of a synthetic leader L-Zm.GSP990.nno:1 (SEQ ID NO: 8) and a synthetic promoter P-Zm.GSP990.nno:2 (SEQ ID NO: 7) operably linked on the 5'-side from which it is composed. Three control expression vectors were also transformed into maize leaf protoplasts and constructed as described above. Table 3 shows the average expression percentage compared to the third control expression vector containing EXP-CaMV.35S driving GUS and I-Zm.DnaK:1 as shown.

Table 3

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

[0111] Analysis of Enhancement of GUS Expression by Synthetic Intron I-Zm.GSI153.nno:1 Maize leaf protoplast cells were transformed with an expression vector constructed to contain an expression element driving GUS expression. Using the test expression vector, the enhancement of GUS expression from synthetic intron I-Zm.GSI153.nno:1 (SEQ ID NO: 5) driven by EXP-CaMV. 35 was assayed. The transgenic cassette contained a 3'UTR, T- ​​The coding sequence encoding GUS (sequence No. 24) operably linked on the 5'-side, and the synthetic intron I-Zm.GSI153.n no:1 (SEQ ID NO: 5) operably linked on the 5'-side, and EXP, EXP-CaMV.35 were included. Two control expression vectors were also constructed and used for the transformation of maize leaf protoplasts. The first control expression vector contained a transgene cassette without an intron, including a 3'UTR, the GUS coding sequence operably linked on the 5'-side to T-Os.LTP:1, and EXP, EXP-CaMV.35S operably linked on the 5'-side. The second control vector contained a transgene cassette including a 3'UTR, the GUS coding sequence operably linked on the 5'-side to T-Os.LTP:1, and EXP, EXP-CaMV.35S operably linked on the 5'-side to the intron I-Zm.Dna K:1. Table 4 shows the average expression percentages compared to the second control expression vector containing both EXP-CaMV.35S and I-Zm.DnaK:1 that drive GUS. As can be seen from Table 4, the synthetic intron I-Zm.GSI153.nno:1 (SEQ ID NO: 5) enhanced the expression of the GUS transgene in maize leaf protoplasts driven by EXP-CaMV.35S compared to the control expression vector without an intron. Analysis of the enhancement of GUS expression by the synthetic intron I-Zm.GSI197.nno:1

Table 4

[0112] As shown in Table 4, the synthetic intron I-Zm.GSI153.nno:1 (SEQ ID NO: 5) enhanced the expression of the GUS transgene in maize leaf protoplasts driven by EXP-CaMV.35S compared to the control expression vector without an intron.

[0113] Analysis of the enhancement of GUS expression by the synthetic intron I-Zm.GSI197.nno:1 ​​​Maize leaf protoplast cells were transformed with an expression vector constructed to contain an expression element that drives GUS expression. Using a test expression vector, enhancement of GUS expression from synthetic intron I-Zm.GSI197.nno:1 (SEQ ID NO: 10) driven by EXP-CaMV.35 was assayed. The transgene cassette included a coding sequence encoding GUS (SEQ ID NO: 24) operably linked 5' to 3'UTR, T-Os.LTP:1 and operably linked 5' to a synthetic intron I-Zm.GSI197. nno:1 and operably linked 5' to EXP, EXP-CaMV.35. Three control expression vectors were also constructed and used for transformation of maize leaf protoplasts. The first control expression vector included a transgene cassette without a promoter and was composed of a GUS coding sequence operably linked 5' to 3'UTR, T-Os.LTP:1 and operably linked 5' to intron I-Zm.DnaK:1. The second control vector included a transgene cassette without an intron and was composed of a GUS coding sequence linked 5' to 3'UTR, T-Os.LTP:1 and linked 5' to EXP, EXP-CaMV.35S. The third control vector included a transgene cassette containing 3'UTR, T-Os.LTP:1 operably linked 5' to a GUS coding sequence and operably linked 5' to intron I-Zm.DnaK:1 and operably linked 5' to EXP, EXP-CaMV.35S. Table 5 shows the average percent expression compared to the third control expression vector containing both EXP-CaMV.35S and I-Zm.DnaK:1 that drive GUS. ​​​​​​​​​​

Table 5

[0114] As can be seen from Table 5, the synthetic intron I-Zm.GSI197.nno:1 (SEQ ID NO: 10) enhanced the expression of the introduced GUS gene in maize leaf protoplasts driven by EXP-CaMV.35S compared to the control expression vector without intron. The enhancement of expression was greater than that conferred by intron I-Zm.DnaK:1 when compared to a third control expression vector containing EXP-CaMV.35S operably linked at the 5' side to the E XP. Analysis of the enhancement of GUS expression by the synthetic 3'UTR, T-Zm.GST9.nno:2, and T-Zm.GST18.nno: 2.

[0115] Maize leaf protoplast cells were transformed with an expression vector constructed to contain an expression element driving GUS expression. The two test vectors contained the introduced gene cassette used for the analysis of the enhancement of GUS expression conferred by the 3'UTR, T-Zm.G ST9.nno:2 (SEQ ID NO: 13) and T-Zm.GST18.nno:2 (SEQ ID NO: 14), and were composed of a coding sequence encoding GUS (SEQ ID NO: 24) operably linked at the 5' side to an intron I-Zm.DnaK:1 operably linked at the 5' side to EXP-CaMV.35S, with either the 3'UTR, T-Zm.G ST9.nno:2 (SEQ ID NO: 13) or the 3'UTR, T-Zm.G ST18.nno:2 (SEQ ID NO: 14) operably linked at the 5' side. As described above, three control expression vectors were also constructed and used to transform maize leaf protoplasts ​​​​​​It was used for the transformation of plastids. Table 6 shows the average expression percentage compared to a third control expression vector containing both EXP-CaMV.35S driving GUS and I-Zm.DnaK:1. As can be seen from Table 6, the 3'UTR, T-Zm.GST9.nno:2 (SEQ ID NO: 13) and T-Zm.GST18.nno:2 (SEQ ID NO: 14) enhanced GUS expression compared to the control of maize leaf protoplasts.

Table 6

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

[0117] Example 3 Analysis of GUS expression driven by synthetic EXP, EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 and EXP-Zm.GSP850.nno+Zm.GSI140.nno:1 in stably transformed LH244 variety maize plants .GSP850.nno+Zm.GSI153.nno:2 and EXP-Zm.GSP8 50.nno+Zm.GSI140.nno:1 in stably transformed LH244 variety maize plants Maize plants were transformed with a vector, specifically a plant expression vector containing a test regulatory element that drives the expression of the β-glucuronidase (GUS) transgene. The resulting plants were analyzed for GUS protein expression to evaluate the effect of the selected regulatory element on expression. Maize plants were transformed with a plant GUS expression construct. Using standard methods known in the art, the regulatory element was cloned into the base plant expression vector. The resulting plant expression vector contained the left border region (B-AGRtu.left border) from Agrobacterium tumefaciens and a herbicide Maize plants were transformed with a plant GUS expression construct. Using standard methods known in the art, the regulatory element was cloned into the base plant expression vector. The resulting plant expression vector contained the left border region (B-AGRtu.left border) from Agrobacterium tumefaciens and a herbicide Maize plants were transformed with a plant GUS expression construct. Using standard methods known in the art, the regulatory element was cloned into the base plant expression vector. The resulting plant expression vector contained the left border region (B-AGRtu.left border) from Agrobacterium tumefaciens and a herbicide

[0118] Maize plants were transformed with a plant GUS expression construct. Using standard methods known in the art, the regulatory element was cloned into the base plant expression vector. The resulting plant expression vector contained the left border region (B-AGRtu.left border) from Agrobacterium tumefaciens and a herbicide Maize plants were transformed with a plant GUS expression construct. Using standard methods known in the art, the regulatory element was cloned into the base plant expression vector. The resulting plant expression vector contained the left border region (B-AGRtu.left border) from Agrobacterium tumefaciens and a herbicide Maize plants were transformed with a plant GUS expression construct. Using standard methods known in the art, the regulatory element was cloned into the base plant expression vector. The resulting plant expression vector contained the left border region (B-AGRtu.left border) from Agrobacterium tumefaciens and a herbicide ns from the left border region (B-AGRtu.left border) and a herbicide A first introduced gene selection cassette used for the selection of transgenic plant cells conferring resistance to glyphosate, and a second introduced gene cassette for evaluating the activity of synthetic regulatory elements, wherein the second introduced gene cassette comprises a 3' termination region, T-Sb.Nltp4-1:1:2 (SEQ ID NO: 19) and a processing intron derived from the potato light-inducible tissue-specific ST-LS1 gene (GenBank accession: X04753) operably linked on the 5' side, a synthetic coding sequence (GUS, GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO: 20) designed for expression in plant cells encoding β-glucuronidase, and a synthetic EXP, EXP-Zm.GSP850.nno+Zm.G SI153.nno:2 (SEQ ID NO: 4) or EXP-Zm.GSP850.nno+Z m.GSI140.nno:1 (SEQ ID NO: 11) operably linked on the 5' side, and a right border region (B-AGRtu.right border) from Agrobacterium tumefaciens. The synthetic EXP, EXP -Zm.GSP850.nno+Zm.GSI153.nno:2 (SEQ ID NO: 4) consists of a synthetic leader L-Zm.GSP850.nno:3 (SEQ ID NO: 3) operably linked on the 5' side to a synthetic intron I-Zm.GSI153.nno:1 (SEQ ID NO: 5) and a synthetic promoter P-Zm.GSP850.nno:4 (SEQ ID NO: 2) operably linked on the 5' side. The synthetic EXP, EXP-Zm.GSP850.nno+Zm.G SI140.nno:1 (SEQ ID NO: 11) consists of a synthetic leader L-Zm.G operably linked on the 5' side to a synthetic intron I-Zm.GSI140. nno:1 (SEQ ID NO: 12) and 5' side, a second introduced gene set containing either one of the above, and was included. The synthetic EXP, EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 (SEQ ID NO: 4) was operably linked on the 5' side to a synthetic intron I-Zm.GSI153.nno:1 (SEQ ID NO: 5), a synthetic leader L-Zm.GSP850.nno:3 (SEQ ID NO: 3) operably linked on the 5' side to a synthetic promoter P-Zm.GSP850.nno:4 (SEQ ID NO: 2). The synthetic EXP, EXP-Zm.GSP850.nno+Zm.G SI140.nno:1 (SEQ ID NO: 11) was operably linked on the 5' side to a synthetic intron I-Zm.GSI140. nno:1 (SEQ ID NO: 12) and 5' A synthetic promoter operably linked 5' - side to SP850.nno:3 (SEQ ID NO: 3) - and consisted of P - Zm.GSP850.nno:4 (SEQ ID NO: 2).

[0119] Maize variety LH244 plant cells were transformed using the above binary transformation vector construct by Agrobacterium - mediated transformation as is well - known in the art. The obtained transformed plant cells were induced to form whole maize plants.

[0120] Qualitative and quantitative GUS assays were used to evaluate the activity of the expression elements in selected plant organs and tissues of the transformed plants. For qualitative analysis of GUS expression by histochemical staining, whole mounts or sections of tissues were incubated with GUS staining solution containing 1 mg / mL of X - Gluc (5 - bromo - 4 - chloro - 3 - indolyl - β - glucuronide) at 37 °C for 5 hours and then decolorized with 35% EtOH and 50% acetic acid. The expression of GUS was qualitatively determined by visually inspecting the blue staining of the selected plant organs or tissues under a dissecting microscope or compound microscope.

[0121] For quantitative analysis of GUS expression by enzyme assay, total proteins were extracted from selected tissues of the transformed maize plants. One to two micrograms of total protein were incubated with 50 microliters of a fluorogenic substrate 4 - methylumbelliferyl - β - D - glucuronide (MUG) at a concentration of 1 mM in a total reaction volume. After incubation at 37 °C for 1 hour, 350 microliters of 200 mM sodium bicarbonate solution were added. ​​​​​​​As a result, the reaction was stopped. The reaction product 4-methylumbelliferone (4-MU) becomes maximally fluorescent at high pH and at this time the hydroxyl group is ionized. When a basic sodium carbonate solution is added, the assay is stopped and at the same time the pH for quantifying the fluorescent product 4-MU is adjusted. The amount of 4-MU formed was estimated by measuring its fluorescence using a FLUOstar Omega micro plate reader (BMG LABTECH) (excitation at 355 nm, emission at 460 nm). The GUS activity value is provided in nanomoles of 4-MU / hour / mg total protein.

[0122] The following tissues were sampled for GUS expression in the R 0 generation: leaves and roots at the V4 stage; leaves and roots at the V7 stage; leaves, roots, flowers / anthers at the VT stage; rachis / hairs at the R1 stage; seed embryos and seed endosperms at the R3 stage 21 days after pollination. Table 7 shows the average quantitative GUS expression values for each synthetic EXP.

Table 7

[0123] As can be seen in Table 7, the synthetic GSP850 promoter and leader (P-Zm.GSP 850.nno:4 (SEQ ID NO: 2) and L-Zm.GSP850.nno:3 (SEQ ID NO: 3)) drove constitutive expression of GUS in stably transformed LH244 variety maize plants. Molecular analysis of the transcription start site showed a consistent TSS for the GSP850 promoter and leader . The synthetic introns I-Zm.GSI153.nno:1 (SEQ ID NO: 5) and I-Zm.GSI140.nno:1 (SEQ ID NO: 12) were sampled ​​​​It had different effects on expression in different tissues. From the molecular analysis of intron splice sites showed consistent processing of the synthetic intron. The overall enhancement of GUS expression was higher in most tissue samples from plants containing I-Zm.GSI153.nno:1 (SEQ ID NO: 5), but the V4-stage leaves, V7-stage roots, and R3 seed embryos were exceptions where the GUS expression levels were relatively similar. The enhancement of expression conferred by I-Zm.GSI153.nno:1 (SEQ ID NO: 5) was approximately 7.5-fold higher in V4 roots, 7.7-fold higher in VT leaves, 6.0-fold higher in VT roots, 4.3-fold higher in VT flowers / anthers, 3.2-fold higher in R1 ear axes / hairs, and 2.3-fold higher in R3 seed endosperm compared to I-Zm.GSI140.nno:1 (SEQ ID NO: 12).

[0124] Example 4 Analysis of GUS expression driven by synthetic EXP, EXP-Zm .GSP850.nno+Zm.DnaK:1, EXP-Zm.GSP850.nno+ Zm.GSI153.nno:2, and EXP-Zm.GSP850.nno+Zm.G SI140.nno:1 in stably transformed 01DKD2 variety maize plants Maize plants were transformed with a vector, specifically a plant expression vector containing a test regulatory element that drives the expression of the β-glucuronidase (GUS) transgene. The resulting plants were analyzed for GUS protein expression to evaluate the effect of the selected regulatory element on expression.

[0125] Maize plants were transformed with a plant GUS expression construct. Using standard methods known in the art, the regulatory element was cloned into a base plant expression vector. Thus obtained. The resulting plant expression vector was from Agrobacterium tumefacie The left border region (B-AGRtu.left border) from ns and the herbicide The first introduced gene selection cassette used for the selection of transformed plant cells conferring resistance to glyphosate And the second introduced gene cassette for evaluating the activity of regulatory elements Comprising a synthetic coding sequence (G US, GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO: 20) designed for expression in plant cells encoding β-glucuronidase containing a processable intron derived from the potato light-inducible tissue-specific ST-LS1 gene (Genba nk accession: X04753) and operably linked at the 5'-side And a synthetic EXP, EXP-Zm.GSP850.nno+Zm.Dna K:1 (SEQ ID NO: 15), EXP-Zm.GSP850.nno+Zm.GSI153. nno:2 (SEQ ID NO: 4), or EXP-Zm.GSP850.nno+Zm.GSI 140.nno:1 (SEQ ID NO: 11), and the second introduced gene cassette, and The right border region (B-A GRtu.right border) from Agrobacterium tumefaciens. The synthetic EXP, EXP-Zm.G SP850.nno+Zm.DnaK:1 (SEQ ID NO: 15) was composed of a synthetic leader L-Zm.G SP850.nno:3 (SEQ ID NO: 3) operably linked at the 5'-side and a synthetic promoter - P-Zm.GSP850.nno:4 (SEQ ID NO: 2) operably linked at the 5'-side and an intron I-Zm.D naK:1 (SEQ ID NO: 22). The synthetic EXP Composed of a synthetic promoter, P-Zm.GSP850.nno:4 (SEQ ID NO: 2), operably linked at the 5'-side to a synthetic leader L-Zm.G SP850.nno:3 (SEQ ID NO: 3) and at the 5'-side to an intron I-Zm.D P, EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 (SEQ ID NO 4) and EXP-Zm.GSP850.nno+Zm.GSI140.nno:1 (SEQ ID NO 11) are described in Example 3.

[0126] Maize variety 01DKD2 plant cells were transformed with the above binary transformation vector con struct by Agrobacterium-mediated transformation as is well known in the art. The resulting transformed plant cells were induced to form whole maize plants. Qualitative and quantitative GUS expression was assayed as described in Example 3. Table 8 shows the average quantitative GUS expression values for each synthetic EXP.

Table 8

[0127] As can be seen in Table 8, the synthetic GSP850 promoter and leader (P-Zm.GSP 850.nno:4 (SEQ ID NO 2) and L-Zm.GSP850.nno:3 (SEQ ID NO 3)) drove constitutive expression of GUS in stably transformed 01DKD2 variety maize plants. The synthetic introns I-Zm.GSI153.nno:1 (SEQ ID NO 5) and I-Zm.GSI140.nno:1 (SEQ ID NO 12) enhanced expression in all tissues assayed compared to intron I-Zm.Dn aK:1 (SEQ ID NO 22).

[0128] Example 5 In stably transformed 01DKD2 variety maize plants, the synthetic EXP, EXP-Z m.GSP990.nno+Zm.DnaK:1 and EXP-Zm.GSP990.nn Analysis of GUS expression driven by o+Zm.GSI197.nno:2 Maize plants were transformed with a vector, specifically a plant expression vector containing a test regulatory element that drives the expression of the β-glucuronidase (GUS) transgene. The resulting plants were analyzed for GUS protein expression to evaluate the effect of the selected regulatory element on expression. Maize plants were transformed with a plant GUS expression construct. Using standard methods known in the art, the regulatory element was cloned into a base plant expression vector. The resulting plant expression vector contained the left border region (B-AGRtu.left border) from Agrobacterium tumefaciens, a first transgene selection cassette used for the selection of transformed plant cells conferring resistance to the herbicide glyphosate, and a second transgene cassette for evaluating the activity of the regulatory element.

[0129] The second transgene cassette contained a synthetic coding sequence (GUS, GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO: 20) encoding β-glucuronidase, designed for expression in plant cells, containing a processable intron derived from the potato light-inducible tissue-specific ST-LS1 gene (GenBank accession: X04753), operably linked at the 5' side to the 3' termination region, T-Sb.Nltp4-1:1:2 (SEQ ID NO: 19), and at the 5' side, either EXP-Zm.GSP990.nno+Zm.DnaK:1 (SEQ ID NO: 16) or EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 (SEQ ID NO: 9). The resulting plant expression vector also contained a second transgene cassette containing either EXP-Zm.GSP990.nno+Zm.DnaK:1 (SEQ ID NO: 16) or EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 (SEQ ID NO: 9), and an Agr The resulting plant expression vector contained the left border region (B-AGRtu.left border) from Agrobacterium tumefaciens, a first transgene selection cassette used for the selection of transformed plant cells conferring resistance to the herbicide glyphosate, and a second transgene cassette for evaluating the activity of the regulatory element. The second transgene cassette contained a synthetic coding sequence (GUS, GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO: 20) encoding β-glucuronidase, designed for expression in plant cells, containing a processable intron derived from the potato light-inducible tissue-specific ST-LS1 gene (GenBank accession: X04753), operably linked at the 5' side to the 3' termination region, T-Sb.Nltp4-1:1:2 (SEQ ID NO: 19), and at the 5' side, either EXP-Zm.GSP990.nno+Zm.DnaK:1 (SEQ ID NO: 16) or EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 (SEQ ID NO: 9). The resulting plant expression vector also contained a second transgene cassette containing either EXP-Zm.GSP990.nno+Zm.DnaK:1 (SEQ ID NO: 16) or EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 (SEQ ID NO: 9), and an Agr The resulting plant expression vector contained the left border region (B-AGRtu.left border) from Agrobacterium tumefaciens, a first transgene selection cassette used for the selection of transformed plant cells conferring resistance to the herbicide glyphosate, and a second transgene cassette for evaluating the activity of the regulatory element. The second transgene cassette contained a synthetic coding sequence (GUS, GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO: 20) encoding β-glucuronidase, designed for expression in plant cells, containing a processable intron derived from the potato light-inducible tissue-specific ST-LS1 gene (GenBank accession: X04753), operably linked at the 5' side to the 3' termination region, T-Sb.Nltp4-1:1:2 (SEQ ID NO: 19), and at the 5' side, either EXP-Zm.GSP990.nno+Zm.DnaK:1 (SEQ ID NO: 16) or EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 (SEQ ID NO: 9). The resulting plant expression vector also contained a second transgene cassette containing either EXP-Zm.GSP990.nno+Zm.DnaK:1 (SEQ ID NO: 16) or EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 (SEQ ID NO: 9), and an Agr The second transgene cassette contained a synthetic coding sequence (GUS, GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO: 20) encoding β-glucuronidase, designed for expression in plant cells, containing a processable intron derived from the potato light-inducible tissue-specific ST-LS1 gene (GenBank accession: X04753), operably linked at the 5' side to the 3' termination region, T-Sb.Nltp4-1:1:2 (SEQ ID NO: 19), and at the 5' side, either EXP-Zm.GSP990.nno+Zm.DnaK:1 (SEQ ID NO: 16) or EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 (SEQ ID NO: 9). The resulting plant expression vector also contained a second transgene cassette containing either EXP-Zm.GSP990.nno+Zm.DnaK:1 (SEQ ID NO: 16) or EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 (SEQ ID NO: 9), and an Agr The second transgene cassette contained a synthetic coding sequence (GUS, GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO: 20) encoding β-glucuronidase, designed for expression in plant cells, containing a processable intron derived from the potato light-inducible tissue-specific ST-LS1 gene (GenBank accession: X04753), operably linked at the 5' side to the 3' termination region, T-Sb.Nltp4-1:1:2 (SEQ ID NO: 19), and at the 5' side, either EXP-Zm.GSP990.nno+Zm.DnaK:1 (SEQ ID NO: 16) or EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 (SEQ ID NO: 9). The resulting plant expression vector also contained a second transgene cassette containing either EXP-Zm.GSP990.nno+Zm.DnaK:1 (SEQ ID NO: 16) or EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 (SEQ ID NO: 9), and an Agr The second transgene cassette contained a synthetic coding sequence (GUS, GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO: 20) encoding β-glucuronidase, designed for expression in plant cells, containing a processable intron derived from the potato light-inducible tissue-specific ST-LS1 gene (GenBank accession: X04753), operably linked at the 5' side to the 3' termination region, T-Sb.Nltp4-1:1:2 (SEQ ID NO: 19), and at the 5' side, either EXP-Zm.GSP990.nno+Zm.DnaK:1 (SEQ ID NO: 16) or EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 (SEQ ID NO: 9). The resulting plant expression vector also contained a second transgene cassette containing either EXP-Zm.GSP990.nno+Zm.DnaK:1 (SEQ ID NO: 16) or EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 (SEQ ID NO: 9), and an Agr The right border region from Agrobacterium tumefaciens (B-AGRt u.right border) was included. Synthetic EXP, EXP-Zm.GSP9 90.nno+Zm.GSI197.nno:2 (SEQ ID NO: 9) was a synthetic intron I- Zm.GSI197.nno:1 (SEQ ID NO: 10) operably linked at the 5'-side, and a synthetic leader L-Zm.GSP990.nno:1 (SEQ ID NO: 8) operably linked at the 5'-side to form a synthetic promoter, P-Zm.GSP990.nno:2 (SEQ ID NO: 7). Synthetic EXP, EXP-Zm.GSP990.nno+Zm.DnaK:1( SEQ ID NO: 16) was composed of an intron I-Zm.DnaK:1 (SEQ ID NO: 22) operably linked at the 5'-side and a synthetic leader L-Zm.GSP990.nno:1 (SEQ ID NO: 8) operably linked at the 5 '-side to a synthetic promoter P-Zm.GSP990.nno:2 (SEQ ID NO: 7). Maize variety 01DKD2 plant cells were transformed using the above binary transformation vector construct

[0130] by Agrobacterium-mediated transformation as is well known in the art. The resulting transformed plant cells were induced to form whole maize plants . Qualitative and quantitative GUS expression was assayed as described previously in Example 3 . Table 9 shows the average quantitative GUS expression values for each synthetic EXP, where "N D" indicates not measured.

Table 9

[0131] As can be seen here, the synthetic GSP990 promoter and leader (P-Zm.GSP 990.nno:2 (SEQ ID NO: 7) and L-Zm.GSP990.nno:1 (SEQ ID NO: 8)) drove GUS expression. Molecular analysis of the transcription start site showed a consistent TSS for the GSP990 promoter and leader. The synthetic intron I-Zm.GSI19 7.nno:1 (SEQ ID NO: 10) weakened expression in some tissues compared to intron I-Zm.DnaK:1 (SEQ ID NO: 22 ) while enhancing expression in other tissues. For example GUS expression was reduced in leaves at the V4, V7, and VT stages. GUS expression was slightly enhanced in roots at V7 and VT compared to I -Zm.DnaK:1. Flower / anther expression was approximately 2.7-fold enhanced by I-Zm.GSI197.nno:1 (SEQ ID NO: :10) compared to I-Zm.DnaK:1. The difference in expression conferred by I-Z m.GSI197.nno:1 (SEQ ID NO: 10) compared to I-Zm.DnaK:1 could be very useful when low leaf expression and high flower / anther expression are desired. Molecular analysis of the intron splice site showed consistent processing of the synthetic intron I-Zm.GSI197.nno:1 (SEQ ID NO: 10).

[0132] Example 6 Analysis of the effects of synthetic 3'UTRs, T-Zm.GST9.nno:2, T-Zm.GST18.nno:2 , and T-Zm.GST43.nno:1, as well as native T-Sb.Ntlp4- 1:1:2 on GUS expression in stably transformed 01DKD2 variety maize plants Maize plants were transformed with vectors, specifically, β-glucuronidase (GUS) transgenes ​Transformed with a plant expression vector containing a test regulatory element that drives gene expression. Obtained The resulting plants were analyzed for GUS protein expression to evaluate the effect of the selected regulatory element on expression .

[0133] Maize plants were transformed with a plant GUS expression construct. Using standard methods known in the art , the regulatory element was cloned into the base plant expression vector . The resulting plant expression vector contained the left border region (B-AGRtu.left border) from Agrobacterium tumefacie ns, a first transgene selection cassette for the selection of transformed plant cells conferring resistance to the herbicide glyphosate, and a second transgene cassette for evaluating the activity of the 3'UTR regulatory element, which was operably linked on the 5' side to a processing-intron-containing β-glucuronidase encoding a synthetic coding sequence (GUS, GOI-Ec.uidA+St.LS 1.nno:1, SEQ ID NO: 20) derived from the potato light-inducible tissue-specific ST-LS1 gene (Genbank accession: X04753), and on the 5' side to an intron I-Zm. DnaK:1 (SEQ ID NO: 22), and on the 5' side to an EXP, EXP-CaM V.35S (SEQ ID NO: 21)-containing second transgene cassette, and the right border region (B-AGRtu.righ t border) from Agrobacterium tumefaciens. The three test expression vectors contained a 3'UTR, T-Zm.GST9.nno:2 (SEQ ID NO: 13), T- operably linked to the GUS coding sequence , and T-Zm.GST9.nno:2 (SEQ ID NO: 13), T- operably linked to the GUS coding sequence , and the right border region (B-AGRtu.righ t border) from Agrobacterium tumefaciens. The three test expression vectors contained a 3'UTR, T-Zm.GST9.nno:2 (SEQ ID NO: 13), T- operably linked to the GUS coding sequence, T-Zm.GST9.nno:2 (SEQ ID NO: 13), T- Zm.GST18.nno:2 (SEQ ID NO: 14), or T-Zm.GST43.nno :1 (SEQ ID NO: 26). Additional test expression vectors were made with a native 3’UTR ligated operably to the GUS coding sequence, T-Sb.Nltp4-1:1:2 (SEQ ID NO: 19), and were used to compare expression between the native and synthetic 3’UTRs.

[0134] Maize variety 01DKD2 plant cells were transformed with the above binary transformation vector constructs by Agrobacterium-mediated transformation as is well known in the art. The resulting transformed plant cells were induced to form whole maize plants.

[0135] Qualitative and quantitative GUS assays were used to assess the expression element activity in the V4 leaves and root tissues of the transformed plants, and were performed as described in Example 3 above. The effect of the synthetic 3’UTR was evaluated by comparison to the effect of expression by 3’UTR, T-Sb.Nltp4-1:1:2 (SEQ ID NO: 19). The resulting transcripts were analyzed to determine if proper termination occurred and if there was no read-through of the transcript. One way to assess for read-through was by using amplification of the transcribed cDNA with an amplification primer corresponding to the portion of the T-DNA border sequence that is 3’ to the 3’UTR. The average GUS expression of plants transformed with four constructs containing T-Zm.GST9.nno:2 (SEQ ID NO: 13), T-Zm.GST18.nno:2 (SEQ ID NO: 14), T-Zm.GST43.nno:1 (SEQ ID NO: 26), and T-Sb.Nltp4-1:1:2 (SEQ ID NO: 19) is shown in Table 10. ​

Table 10

[0136] As can be seen in Table 10, both T-Zm.GST9.nno:2 (SEQ ID NO: 13) and T-Z m.GST18.nno:2 (SEQ ID NO: 14) enhanced GUS expression operably linked to I-Zm.DnaK:1 compared to EXP-CaMV.35S driven by T-Sb.Nltp4-1:1:2. GUS expression in plants containing T-Zm.GST9.nno :2 (SEQ ID NO: 13) was higher than that in plants containing T-Zm.GST18.nno:2 . T-Zm.GST43.nno:1 (SEQ ID NO: 26) enhanced GUS expression in V4 roots compared to T -Sb.Nltp4-1:1:2, but weakened the expression in V4 leaves . Analysis of GUS transcripts from all four constructs showed proper termination of the transcripts and no evidence of read-through in the resulting GUS transcripts . The 3’UTRs, T-Zm.GST9.nno:2 (SEQ ID NO: 13), T-Zm.G ST18.nno:2 (SEQ ID NO: 14), and T-Zm.GST43.nno:1 (SEQ ID NO: 26) functioned in a manner similar to the native 3’UTR and showed regulation of GUS expression compared to the native 3’UTR, T-Sb.Nltp4-1:1:2. All four synthetic 3’UTRs and the additional native 3’UTR, T-Sb.Nltp4-1:1:2 provided a series of expression values useful for fine-tuning expression in stably transformed maize plants .

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

[0138] TATA boxes in plant promoters are not as highly conserved as in some other eukaryotes. Therefore, to define a fragment as an enhancer, first, 'It is necessary to identify the transcription start site (TSS) of the gene where the UTR is first transcribed. The enhancer derived from the synthetic promoter P-Zm.GSP850.nno:4 (SEQ ID NO:2) The enhancer can include nucleotides 1 to 418 of SEQ ID NO:2, and the synthetic enhancer E -Zm.GSP850 (SEQ ID NO: 17) is obtained. The enhancer derived from 990.nno:2 (SEQ ID NO: 7) is and can include synthetic enhancer E-Zm.GSP990 (SEQ ID NO: 18) are obtained. Enhancers derived from these promoters are shown in SEQ ID NO: 17 and fragments of 18, or duplicates of SEQ ID NOs: 17 and 18 or fragments thereof can be included. The effectiveness of synthetic enhancers derived from synthetic promoters is empirically determined by constructing chimeric transcriptional regulatory elements that contain fragments derived from either synthetic promoter P-Zm.GSP850.nno:4 (SEQ ID NO: 2) or P-Zm .GSP990.nno:2 (SEQ ID NO: 7). This element is operably linked to a promoter and a leader and is used to drive the expression of a transcribable

[0139] DNA molecule such as GUS in a stable or transient plant assay. Further refinement of enhancer elements may be required and is empirically verified. In addition, the position of enhancer elements relative to other elements within the chimeric transcriptional regulatory element is also empirically determined, because the order of the elements within the chimeric transcriptional regulatory element can impart different effects depending on the relative position of each element. Some promoter elements have multiple TATA boxes or TATA box-like elements and, in some cases, multiple transcription start sites. Under such circumstances, it may be

[0140] necessary to first identify where the first TSS is located and then use the first TSS to initiate enhancer design to prevent potential transcription operatively coupled, or additional enhancers operably linked to a promoter operatively coupled on the 5' or 3' side with an element, are cloned using methods known in the art Alternatively, the enhancer element may be cloned using methods known in the art to result in a larger enhancer element composed of two or more copies of the enhancer and may also be cloned using methods known in the art to operably couple on the 5' or 3' side with a promoter, or with an additional enhancer element operably linked to a promoter that results in a chimeric transcriptional regulatory element and operably couple on the 5' or 3' side. Enhancer elements derived from promoters derived from genes from organisms of multiple genera can be operably linked to enhancers derived from synthetic promoters Using methods known in the art, a GUS expression plant transformation vector similar to the construct described in Example 3 can be constructed, and the resulting plant expression vector has a left border region from Agrobacterium tumefaciens (B-AGRtu.left border), a first transgene selection cassette used for selection of transformed plant cells conferring resistance to the herbicide glyphosate, and a second transgene cassette for testing enhancer elements comprising the potato light-inducible tissue-specific ST-LS1 gene operably linked to the 3' termination region from the Oryza sativa lipid transfer protein-like gene (T-Os.LTP:1, SEQ ID NO: 23)

[0141] Using methods known in the art, a GUS expression plant transformation vector similar to the construct described in Example 3 can be constructed and the resulting plant expression vector has a left border region from Agrobacterium tumefaciens (B-AGRtu.left border) and a first transgene selection cassette used for selection of transformed plant cells conferring resistance to the herbicide glyphosate and a second transgene cassette for testing enhancer elements comprising the potato light-inducible tissue-specific ST-LS1 gene operably linked to the 3' termination region from the Oryza sativa lipid transfer protein-like gene (T-Os.LTP:1, SEQ ID NO: 23) and operably linked to the 3' termination region ​A processable intro derived from Genbank accession: X04753 coding sequence for β-glucuronidase containing an intron (GOI-Ec.uidA+St.L S1.nno:1, SEQ ID NO: 20), an intron element operably linked to the 5' side, a leader element operably linked to the 5' side, a promoter element operably linked to the 5' or 3' side or operably linked to the promoter and additional enhancer elements operably linked to the 5' or 3' side composed of enhancer elements, a second transgene cassette, and A.tume faciens right border region (B-AGRtu.right border) and is included. The resulting plasmid is used to transform maize plants or other monocotyledonous plants by the above method. Alternatively, protoplast cells derived from maize or other monocotyledonous plants are transformed using methods known in the art to perform a transient assay. using methods known in the art to transform transient assays. Perform a transient assay.

[0142] Evaluate GUS expression driven by regulatory elements containing one or more enhancers in a stable or transient plant assay to determine the effect of enhancer elements on the expression of transcribable DNA molecules. Modification of one or more enhancer elements or duplication of one or more enhancer elements can be carried out based on empirical experiments and the resulting gene expression regulation observed with each regulatory element composition. Altering the relative positions of one or more enhancers within the resulting regulatory or chimeric regulatory element may affect the transcriptional activity or specificity of the regulatory or chimeric regulatory element. or transient plant assays to determine the effect of enhancer elements on the expression of transcribable DNA molecules. One or more enhancer elements or duplication of one or more enhancer elements can be carried out based on empirical experiments and the resulting gene expression regulation observed with each regulatory element composition. using each regulatory element composition. Based on the resulting gene expression regulation observed, it can be carried out. Changing the relative positions of one or more enhancers within the resulting regulatory or chimeric regulatory element may affect the transcriptional activity or specificity of the regulatory or chimeric regulatory element. is present and is empirically determined to identify the best enhancer for a desired transgene expression profile in maize plants or other plants.

[0143] Although the principles of the invention have been illustrated and described, it should be apparent to those skilled in the art that the invention can be modified in arrangement and detail without departing from such principles. The inventors claim all modifications that come within the spirit and scope of the claims. All publications and patent documents cited herein are hereby incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. ​​​​

Claims

1. A recombinant DNA molecule comprising: a) a sequence having at least 90% sequence identity to the DNA sequence of SEQ ID NO: 26 and having transcriptional regulatory activity, and b) a sequence comprising the DNA sequence of SEQ ID NO: 26, wherein the DNA sequence is selected from the group consisting of: said recombinant DNA molecule, wherein said DNA sequence is operably linked to a heterologous transcribable DNA molecule.

2. The recombinant DNA molecule according to claim 1, wherein said sequence has at least 95% sequence identity to the DNA sequence of SEQ ID NO:

26.

3. The recombinant DNA molecule according to claim 1, wherein said sequence has at least 97% sequence identity to the DNA sequence of SEQ ID NO:

26.

4. The recombinant DNA molecule according to claim 1, wherein said DNA sequence has transcriptional regulatory activity.

5. The recombinant DNA molecule according to claim 1, wherein said heterologous transcribable DNA molecule comprises a gene for agricultural purposes.

6. The recombinant DNA molecule according to claim 5, wherein said gene for agricultural purposes confers herbicide tolerance in plants.

7. The recombinant DNA molecule according to claim 5, wherein said gene for agricultural purposes confers pest resistance in plants.

8. The recombinant DNA molecule according to claim 1, wherein said heterologous transcribable DNA molecule encodes dsRNA, miRNA, or siRNA.

9. A transgenic plant cell comprising the recombinant DNA molecule according to claim 1, wherein said DNA sequence is operably linked to a heterologous transcribable DNA molecule.

10. The transgenic plant cell according to claim 9, wherein said transgenic plant cell is a monocotyledonous plant cell.

11. The transgenic plant cell according to claim 9, wherein said transgenic plant cell is a dicotyledonous plant cell.

12. A transgenic plant or a part thereof comprising the recombinant DNA molecule according to claim 1.

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

14. A transgenic seed comprising the recombinant DNA molecule according to claim 1.

15. A method for producing a commodity product, the method comprising obtaining the transgenic plant or a part thereof according to claim 12 and producing the commodity product therefrom.

16. The method according to claim 15, wherein the commodity product is selected from the group consisting of seeds, processed seeds, protein concentrates, protein isolates, starches, grains, plant parts, seed oils, biomass, flours and meals.

17. A method for expressing a transcribable DNA molecule, the method comprising obtaining the transgenic plant according to claim 12 and cultivating the plant, wherein the transcribable DNA is expressed.

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