Plant regulatory elements and uses thereof

Abstract

To provide DNA molecules useful for modulating gene expression 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] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 448,019, filed January 19, 2017, which is incorporated by reference in its entirety.

[0002] Inclusion of sequence listing The computer readable form of the sequence listing contained in the file titled "MONS436WO-sequence_listing.txt" is 59,917 bytes (as measured in Microsoft Windows®), was created on January 12, 2018, was filed electronically concurrently with the present application, and is hereby incorporated by reference in its entirety.

[0003] FIELD OF THEINVENTION 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 regulating gene expression in plants. [Background technology]

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

[0005] The present invention provides novel synthetic genetic regulatory elements for use in plants. The present invention further provides recombinant DNA molecules and constructs comprising the regulatory elements. The present invention further provides transgenic plant cells, plants and seeds comprising the synthetic regulatory elements. In one embodiment, the synthetic regulatory elements are operably linked to a heterologous transcribable DNA molecule. The present invention further provides methods of using the synthetic regulatory elements, as well as methods of making and using recombinant DNA molecules comprising the synthetic regulatory elements, and transgenic plant cells, plants and seeds comprising the synthetic regulatory elements operably linked to a transcribable DNA molecule.

[0006] Thus, in one aspect, the present invention provides a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: (a) a sequence having at least 85 percent sequence identity with any of SEQ ID NOs: 1-29 and 43-45; (b) a sequence comprising any of SEQ ID NOs: 1-29 and 43-45; and (c) a fragment of any of SEQ ID NOs: 1-29 and 43-45 having gene regulatory activity, wherein the sequence is operably linked to a heterologous transcribable DNA molecule. By "heterologous transcribable DNA molecule" it is meant that the transcribable DNA molecule is heterologous with respect to the polynucleotide sequence operably linked to it. In specific embodiments, the recombinant DNA molecule comprises a DNA sequence having at least about 90 percent, at least 91 percent, at least 92 percent, at least 93 percent, at least 94 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, or at least 99 percent sequence identity with any of SEQ ID NOs: 1-29 and 43-45. In specific embodiments, the DNA sequence comprises a regulatory element. In some embodiments, the regulatory element comprises a promoter. In yet other embodiments, the heterologous transcribable DNA molecule comprises a gene of agricultural scientific interest, such as a gene that can provide herbicide resistance in plants, or a gene that can provide plant pest resistance in plants. In yet other embodiments, the present invention provides a construct that comprises the 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: 1-29 and 43-45, (b) a sequence comprising any of SEQ ID NOs: 1-29 and 43-45, and (c) a fragment of any of SEQ ID NOs: 1-29 and 43-45, said fragment having gene regulatory activity, wherein the DNA sequence is operably linked to a heterologous transcribable DNA molecule. In certain embodiments, the transgenic plant cell is a monocotyledonous plant cell. In other embodiments, the transgenic plant cell is a dicotyledonous plant cell.

[0008] In yet another aspect, provided herein is a transgenic plant or portion thereof comprising a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: a) a sequence having at least 85 percent sequence identity with any of SEQ ID NOs: 1-29 and 43-45; b) a sequence comprising any of SEQ ID NOs: 1-29 and 43-45; and c) a fragment of any of SEQ ID NOs: 1-29 and 43-45, said fragment having gene regulatory activity, wherein said sequence is operably linked to a heterologous transcribable DNA molecule. In a specific embodiment, the transgenic plant is a progeny plant of any generation comprising the recombinant DNA molecule. Also provided herein is a transgenic seed comprising the recombinant DNA molecule that when grown produces such a transgenic plant.

[0009] In another aspect, the present invention provides a method for producing a commodity product comprising obtaining a transgenic plant or part thereof containing a recombinant DNA molecule of the present invention and producing the commodity product therefrom, in one embodiment, the commodity products are processed seeds, grains, plant parts, oils and meal.

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

[0011] A simple explanation of the sequence SEQ ID NO:1 is the DNA sequence of a synthetic regulatory expression element group (EXP), EXP-At.GSP442.nno+At.Cyco:3, which comprises a synthetic promoter (P-At.GSP442.nno:2) operably linked 5' to a synthetic leader (L-At.GSP442.nno:1) operably linked 5' to an intron (I-At.Cyco:2).

[0012] SEQ ID NO:2 is the synthetic promoter sequence, P-At.GSP442.nno:2.

[0013] SEQ ID NO:3 is the synthetic leader sequence, L-At.GSP442.nno:1.

[0014] SEQ ID NO:4 is the DNA sequence of a synthetic EXP, EXP-At.GSP571, containing a synthetic promoter (P-At.GSP571.nno:5) operably linked 5' to a synthetic leader (L-At.GSP571.nno:1).

[0015] SEQ ID NO:5 is the synthetic promoter sequence, P-At.GSP571.nno:5.

[0016] SEQ ID NO:6 is the synthetic leader sequence, L-At.GSP571.nno:1.

[0017] SEQ ID NO: 7 is the DNA sequence of a synthetic regulatory expression element group (EXP), EXP-At.GSP571.nno+At.Cyco:2, which comprises a synthetic promoter (P-At.GSP571.nno:5) operably linked 5' to a synthetic leader (L-At.GSP571.nno:1) operably linked 5' to an intron (I-At.Cyco:2).

[0018] SEQ ID NO:8 is the DNA sequence of a synthetic regulatory expression element group (EXP), EXP-At.GSP571.nno+At.GSI21.nno:10, which comprises a synthetic promoter (P-At.GSP571.nno:5) operably linked 5' to a synthetic leader (L-At.GSP571.nno:1) operably linked 5' to a synthetic intron (I-At.GSI21.nno:2).

[0019] SEQ ID NO:9 is the synthetic intron sequence, I-At.GSI21.nno:2.

[0020] SEQ ID NO: 10 is the DNA sequence of a synthetic EXP, EXP-At.GSP571.nno+At.GSI102.nno:1, which comprises a synthetic promoter (P-At.GSP571.nno:5) operably linked 5' to a synthetic leader (L-At.GSP571.nno:1) operably linked 5' to a synthetic intron (I-At.GSI102.nno:1).

[0021] SEQ ID NO:11 is the synthetic intron sequence, I-At.GSI102.nno:1.

[0022] SEQ ID NO:12 is the DNA sequence of a synthetic EXP, EXP-At.GSP564, containing a synthetic promoter (P-At.GSP564.nno:3) operably linked 5' to a synthetic leader (L-At.GSP564.nno:1).

[0023] SEQ ID NO: 13 is the synthetic promoter sequence, P-At.GSP564.nno:3.

[0024] SEQ ID NO:14 is the synthetic leader sequence, L-At.GSP564.nno:1.

[0025] SEQ ID NO: 15 is the DNA sequence of a synthetic EXP, EXP-At.GSP564.nno+At.Cyco:2, which comprises a synthetic promoter (P-At.GSP564.nno:3) operably linked 5' to a synthetic leader (L-At.GSP564.nno:1) operably linked 5' to an intron (I-At.Cyco:2).

[0026] SEQ ID NO: 16 is the DNA sequence of a synthetic EXP, EXP-At.GSP564.nno+At.GSI17.nno:2, which comprises a synthetic promoter (P-At.GSP564.nno:3) operably linked 5' to a synthetic leader (L-At.GSP564.nno:1) operably linked 5' to a synthetic intron (I-At.GSI17.nno:1).

[0027] SEQ ID NO: 17 is the synthetic intron sequence, I-At.GSI17.nno:1.

[0028] SEQ ID NO: 18 is the DNA sequence of a synthetic EXP, EXP-At.GSP564.nno+At.GSI102.nno:1, which comprises a synthetic promoter (P-At.GSP564.nno:3) operably linked 5' to a synthetic leader (L-At.GSP564.nno:1) operably linked 5' to a synthetic intron (I-At.GSI102.nno:1).

[0029] SEQ ID NO:19 is the DNA sequence of a synthetic EXP, EXP-At.GSP579, containing a synthetic promoter (P-At.GSP579.nno:2) operably linked 5' to a synthetic leader (L-At.GSP579.nno:1).

[0030] SEQ ID NO:20 is the synthetic promoter sequence, P-At.GSP579.nno:2.

[0031] SEQ ID NO:21 is the synthetic leader sequence, L-At.GSP579.nno:1.

[0032] SEQ ID NO: 22 is the DNA sequence of a synthetic EXP, EXP-At.GSP579.nno+At.GSI102.nno:3, which comprises a synthetic promoter (P-At.GSP579.nno:2) operably linked 5' to a synthetic leader (L-At.GSP579.nno:1) operably linked 5' to a synthetic intron (I-At.GSI102.nno:1).

[0033] SEQ ID NO: 23 is the DNA sequence of a synthetic EXP, EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1, comprising a synthetic chimeric promoter (P-At.GSP571 / 442, which consists of a synthetic enhancer (E-At.GSP571.nno:1) operably linked 5' to the synthetic promoter (P-At.GSP442.nno:2)) operably linked 5' to a synthetic leader (L-At.GSP442.nno:1) operably linked 5' to a leader (L-At.Cyco-1:1:2) operably linked 5' to an intron (I-At.Cyco:2).

[0034] SEQ ID NO:24 is the synthetic enhancer sequence, E-At.GSP571.nno:1.

[0035] SEQ ID NO: 25 is the DNA sequence of a synthetic chimeric promoter, P-At.GSP571 / 442, consisting of a synthetic enhancer (E-At.GSP571.nno:1) operably linked to the 5' side of a synthetic promoter (P-At.GSP442.nno:2).

[0036] SEQ ID NO: 26 is the DNA sequence of a synthetic EXP, EXP-At.GSP576.nno+At.GSI17.nno:3, which comprises a synthetic promoter (P-At.GSP576.nno:4) operably linked 5' to a synthetic leader (L-At.GSP576.nno:2) operably linked 5' to a synthetic intron (I-At.GSI17.nno:1).

[0037] SEQ ID NO:27 is the synthetic promoter sequence, P-At.GSP576.nno:4.

[0038] SEQ ID NO:28 is the synthetic leader sequence, L-At.GSP576.nno:2.

[0039] SEQ ID NO:29 is the synthetic 3'UTR, T-Zm.GST59.nno:1.

[0040] SEQ ID NO:30 is the DNA sequence of a synthetic EXP, EXP-At.GSP221+At.Cyco:3, which comprises a synthetic promoter (P-At.GSP221:3) operably linked 5' to a synthetic leader (L-At.GSP221:1) operably linked 5' to an intron (I-At.Cyco:2).

[0041] SEQ ID NO:31 is the synthetic promoter sequence, P-At.GSP221:3.

[0042] SEQ ID NO:32 is the synthetic leader sequence L-At.GSP221:1.

[0043] SEQ ID NO:33 is an intron sequence, I-At.Cyco:2, derived from the cytochrome c oxidase subunit VIa gene from Arabidopsis.

[0044] SEQ ID NO:34 is the 3'UTR sequence from the Sali3 gene of Medicago truncatula, T-Mt.Sali3-2-1:2:1.

[0045] SEQ ID NO:35 is the 3'UTR sequence from a putative oxidoreductase (OXR) protein gene from Medicago truncatula, T-Mt.Oxr-1:2:1.

[0046] SEQ ID NO: 36 is the 3'UTR sequence from the Gossypium barbadense FbLate-2 gene, T-Gb.FbL2:1.

[0047] SEQ ID NO:37 is the 3'UTR sequence from the dehydration response protein RD22 gene from Medicago truncatula, T-Mt.RD22-1:2:1.

[0048] SEQ ID NO:38 is the DNA sequence of EXP, EXP-At.Cyco:1:1, from the cytochrome c oxidase subunit VIa gene from Arabidopsis, comprising a promoter (P-At.Cyco-1:1:2) operably linked 5' to a leader (L-At.Cyco-1:1:2) operably linked 5' to an intron (I-At.Cyco-1:1:1).

[0049] SEQ ID NO:39 is the promoter sequence derived from the cytochrome c oxidase subunit VIa gene from Arabidopsis, P-At.Cyco-1:1:2.

[0050] SEQ ID NO:40 is the leader sequence derived from the cytochrome c oxidase subunit VIa gene from Arabidopsis, L-At.Cyco-1:1:2.

[0051] SEQ ID NO:41 is an intron sequence, I-At.Cyco-1:1:1, derived from the cytochrome c oxidase subunit VIa gene from Arabidopsis.

[0052] SEQ ID NO: 42 is the coding sequence for β-glucuronidase (GUS) with a processable intron derived from the potato light-inducible tissue-specific ST-LS1 gene (Genbank accession: X04753).

[0053] SEQ ID NO: 43 is the DNA sequence of EXP, EXP-At.GSP442+LI-At.Cyco, which contains a synthetic promoter, P-At.GSP442.nno:2, operably linked to the 5' side of a synthetic leader, L-At.GSP442.nno:1, which is operably linked to the 5' side of a leader, L-At.Cyco-1:1:2, which is operably linked to the 5' side of an intron, I-At.Cyco:2.

[0054] SEQ ID NO:44 is the DNA sequence of the synthetic 3'UTR, T-Zm.GST7.nno:2.

[0055] SEQ ID NO: 45 is the DNA sequence of EXP, EXP-At.GSP576.nno+At.Cyco:1, which contains a synthetic promoter, P-At.GSP564.nno:3, operably linked 5' to a synthetic leader, L-At.GSP564.nno:1, which is operably linked 5' to an intron, I-At.Cyco:2.

[0056] SEQ ID NO:46 is the DNA sequence of EXP containing the 35S promoter and leader from Cauliflower Mosaic Virus, EXP-CaMV.35S.

[0057] SEQ ID NO:47 is the DNA sequence of an intron, I-Zm.DnaK:1, from the heat shock protein 70 (Hsp70) gene (DnaK) of Zea mays.

[0058] SEQ ID NO:48 is the DNA sequence of the 3'UTR, T-Os.LTP:1, derived from a lipid transfer protein-like gene (LTP) from Oryza sativa.

[0059] SEQ ID NO:49 is the coding sequence for Nluc, a NanoLuc® luciferase fluorescent protein engineered by directed evolution from deep sea shrimp (Oplophorus gacilirostris) luciferase (Promega, Madison, WI 53711).

[0060] SEQ ID NO:50 is the DNA sequence of EXP, EXP-At.Bglu21+At.Cyco:2, which contains the promoter and leader of the beta-glucuronidase 21 gene from Arabidopsis thaliana operably linked 5' to the intron I-At.Cyco-1:1:1.

[0061] SEQ ID NO:51 is the DNA sequence of EXP containing the enhanced cauliflower mosaic virus 35S promoter operably linked 5' to the heat shock protein 70 (HSP70) leader from Petunia x hybrid, EXP-CaMV.35S-enh+Ph.DnaK:1:3.

[0062] SEQ ID NO:52 is the DNA sequence of EXP, EXP-Gm.Sphas1:1:1, which contains the promoter and leader of the soybean 7S alpha prime gene.

[0063] SEQ ID NO:53 is the DNA sequence of EXP, EXP-CaMV.35S-enh+Zm.DnaK:1:1, which contains an enhanced cauliflower mosaic virus 35S promoter operably linked 5' to the intron I-Zm.DnaK:1.

[0064] SEQ ID NO:54 is the DNA sequence encoding the luciferase protein from Photinus pyralis (firefly) (LUCIFERASE:1:3).

[0065] SEQ ID NO:55 is the DNA sequence of the 3'UTR from the Agrobacterium tumefaciens nopaline synthase gene, T-AGRtu.nos-1:1:13.

[0066] SEQ ID NO:56 is the DNA sequence of EXP-CaMV.35S-enh-Lhcb1, an EXP containing an enhanced Cauliflower Mosaic Virus 35S promoter operably linked 5' to the leader of a chlorophyll a / b-binding gene of the light-harvesting complex of Triticum aestivum (wheat).

[0067] SEQ ID NO:57 is the DNA sequence encoding the luciferase protein from Renilla reniformis (CR-Ren.hRenilla Lucife-0:0:1). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] The present invention provides synthetic regulatory elements having gene regulation activity in plants. The nucleotide sequences of these synthetic regulatory elements are provided as SEQ ID NOs: 1-32 and 43-45. These synthetic regulatory elements are capable of affecting expression of an operably linked transcribable DNA molecule in plant tissue and therefore capable of regulating gene expression of an operably linked transgene in a transgenic plant. The present invention further provides methods of modifying, making and using recombinant DNA molecules containing the provided synthetic regulatory elements. The present invention further provides compositions comprising transgenic plant cells, plants, plant parts and seeds containing the recombinant DNA molecules of the invention, as well as methods of preparing and using the same.

[0069] The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the art.

[0070] dna molecule As used herein, the term "DNA" or "DNA molecule" refers to a double-stranded DNA molecule of genomic or synthetic origin, i.e., a polymer of deoxynucleotide bases, or a DNA molecule read from the 5' (upstream) end to the 3' (downstream) end. As used herein, the term "DNA sequence" refers to the nucleotide sequence of a DNA molecule. The nomenclature used herein corresponds to that of Title 37 of the Code of Federal Regulations §1.822 and as set forth in Tables 1 and 3 of Appendix 2 of WIPO Standard ST.25 (1998).

[0071] As used herein, a "recombinant DNA molecule" is a DNA molecule that contains a combination of DNA molecules that do not occur together in nature without human intervention. For example, a recombinant DNA molecule may be a DNA molecule that is composed of at least two DNA molecules that are heterologous to each other, a DNA molecule that contains a DNA sequence that deviates from a naturally occurring DNA sequence, a DNA molecule that contains a synthetic DNA sequence, or a DNA molecule that is incorporated into the DNA of a host cell by genetic transformation or gene editing.

[0072] As used herein, "synthetic nucleotide sequence" or "artificial nucleotide sequence" is a nucleotide sequence that is not known to occur in nature, does not occur in nature, or does not occur without human intervention. The gene regulatory element of the present invention comprises a synthetic nucleotide sequence. Preferably, the synthetic nucleotide sequence shares little or no extensive homology with natural sequences. In this context, extensive homology generally refers to 100% sequence identity that extends beyond about 25 nucleotides of a continuous sequence.

[0073] Reference in this application to an "isolated DNA molecule" or an equivalent term or phrase is intended to mean that the DNA molecule is present alone or in combination with other components but not in its natural environment. For example, nucleic acid elements naturally found in the DNA of the genome of an organism, such as coding sequences, intron sequences, non-translated leader sequences, promoter sequences, transcription termination sequences, etc., are not considered "isolated" as long as the elements are in the genome of the organism and in the location in the genome in which they are naturally found. On the other hand, each of these elements and small portions of these elements will be "isolated" within the scope of this disclosure as long as the elements are not in the genome of the organism and in the location in which they are naturally found. In one embodiment, the term "isolated" refers to a DNA molecule that is at least partially separated from some of the nucleic acids that are normally present on both sides of it in its original or natural state. Thus, a DNA molecule that is fused to regulatory or coding sequences that are not normally present together, for example as a result of recombinant techniques, is considered to be isolated herein. Such molecules are considered to be isolated in that they are not in their original state when integrated into the chromosome of a host cell or present in a nucleic acid solution with other DNA molecules. For 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 in an extrachromosomal vector, will be considered to be an isolated nucleotide sequence, whether it is present in a plasmid or similar construct used to transform the cell, in the genome of the plant or bacteria, or present in detectable amounts in tissue, progeny, biological sample, or commercial product derived from the plant or bacteria.

[0074] As used herein, the term "sequence identity" refers to the degree to which two optimally aligned polynucleotide sequences or two optimally aligned polypeptides are identical. An optimal sequence alignment is created by manually aligning two sequences, such as a reference sequence and another sequence, to maximize the number of nucleotide matches in the sequence alignment using appropriate internal nucleotide insertions, deletions, or gaps. As used herein, the term "reference sequence" refers to the DNA sequences provided as SEQ ID NOs: 1-32 and 43-45.

[0075] As used herein, the term "percent sequence identity" or "percent identity" or "% identity" refers to the percentage of identity multiplied by 100. The "percent identity" of a sequence optimally aligned with 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 full length of the entire reference sequence. Thus, one embodiment of the present invention provides a DNA molecule comprising a sequence that, when optimally aligned with a reference sequence provided herein as any of SEQ ID NOs: 1-32 and 43-45, 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, 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 97 percent identity, at least about 98 percent identity, at least about 99 percent identity, or at least about 100 percent identity to the reference sequence. In yet another specific embodiment, a sequence having a percent identity with any of SEQ ID NOs: 1-32 and 43-45 may be defined as exhibiting the promoter activity possessed by the starting sequence from which it was derived. A sequence having a percent identity to any of SEQ ID NOs:1-32 and 43-45 may further include a "minimal promoter" that provides a basal level of transcription, as well as consisting of a TATA box or equivalent sequence for recognition and binding of the RNA polymerase II complex for initiation of transcription.

[0076] 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. The term "regulatory element", as used herein, refers to a DNA molecule that has gene regulatory activity. The term "gene regulatory activity", as used herein, refers to 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, such as promoters, leaders, enhancers, introns and 3'UTRs that function in plants, are useful in modifying plant phenotypes by genetic engineering.

[0077] As used herein, a "regulatory expression element" or "EXP" sequence can refer to a group of operably linked regulatory elements, such as an enhancer, promoter, leader, and intron. For example, a regulatory expression element can consist of a promoter operably linked 5' to a leader sequence. EXPs useful in practicing the invention include SEQ ID NOs: 1, 4, 7, 8, 10, 12, 15, 16, 18, 19, 22, 23, 26, 30, 43, and 45.

[0078] Regulatory elements can be characterized by their gene expression patterns, such as positive and / or negative effects, such as constitutive expression or temporal, spatial, developmental, tissue, environmental, physiological, pathological, cell cycle, and / or chemical response expression, and any combination thereof, as well as by quantitative or qualitative adaptation. As used herein, a "gene expression pattern" is any pattern of transcription from an operably linked DNA molecule to a transcribed RNA molecule. The transcribed RNA molecule may be translated into a protein molecule or may provide an antisense or other regulatory RNA molecule, such as double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), etc.

[0079] As used herein, the term "protein expression" refers to any manner of translation of a transcribed RNA molecule into a protein molecule. Protein expression can be characterized not only by its temporal, spatial, developmental, or morphological qualities, but also by its quantitative or qualitative adaptation.

[0080] Promoters are useful as regulatory elements for regulating the expression of operably linked transcribable DNA molecules. As used herein, the term "promoter" generally refers to a DNA molecule that is involved in the recognition and binding of RNA polymerase II and other proteins, such as trans-acting transcription factors, to initiate transcription. A promoter may be initially isolated from the 5' untranslated region (5'UTR) of a genomic copy of a gene. Alternatively, a promoter may be a synthetically produced or engineered DNA molecule. A promoter may also be chimeric. A chimeric promoter is produced by the fusion of two or more heterologous DNA molecules. Promoters useful in carrying out the present invention include promoter elements contained in SEQ ID NOs: 2, 5, 13, 20, 25, 27, 31 and 39, or any of their fragments or variants. In a specific embodiment of the present invention, the claimed DNA molecules and any variants or derivatives thereof described herein are further defined as those that comprise promoter activity, i.e., capable of acting as a promoter in a host cell, such as a transgenic plant. In yet other specific embodiments, a fragment may be defined as one that exhibits the promoter activity possessed by the starting promoter molecule from which it is derived, or a fragment may contain a "minimal promoter" that provides a basal level of transcription as well as consisting of a TATA box or equivalent DNA sequence for recognition and binding of the RNA polymerase II complex for initiation of transcription.

[0081] In one embodiment, a fragment of the promoter sequence disclosed herein is provided. The promoter fragment may contain promoter activity as described above and may be useful alone or in combination with other promoters and promoter fragments, for example when constructing chimeric promoters, or in combination with other expression elements and expression element fragments. In a specific embodiment, a fragment of a promoter is provided that comprises 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, at least about 1000 or more consecutive nucleotides of a DNA molecule having promoter activity disclosed herein. In a specific embodiment, the present invention provides a fragment of a promoter provided herein that has the activity of a full-length sequence. Methods for producing such fragments from a starting promoter molecule are well known in the art.

[0082] Constructs derived from any of the promoter elements contained within any of SEQ ID NOs: 2, 5, 13, 20, 25, 27, 31 and 39 can be made using methods known in the art to improve or alter expression, such as by internal or 5' deletions, for example 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. Constructs derived from any of the promoter elements contained within any of SEQ ID NOs: 2, 5, 13, 20, 25, 27, 31 and 39, consisting of a 3' deletion that removes the TATA box element or its equivalent and downstream sequences, can be used, for example, to create enhancer elements. Further deletions can be made to remove any elements that have a positive or negative; tissue-specific; cell-specific; or timing-specific (for example, but not limited to, circadian rhythm) effect on expression. Any of the promoter elements contained within any of SEQ ID NOs: 2, 5, 13, 20, 25, 27, 31 and 39 and fragments or enhancers derived therefrom can be used to make chimeric transcriptional regulatory element constructs.

[0083] According to the present invention, a promoter or promoter fragment can be analyzed for the presence of known promoter elements, i.e., DNA sequence features such as TATA boxes and other known transcription factor binding site motifs. Identification of such known promoter elements can be used by one skilled in the art to design promoter variants having expression patterns similar to the original promoter.

[0084] The term "leader" as used herein refers to a DNA molecule generally defined as a nucleotide segment between the transcription start site (TSS) and the protein coding sequence start site, isolated from the untranslated 5' region (5'UTR) of a gene. Alternatively, a leader may be a synthetically produced or engineered DNA element. A leader can be used as a 5' regulatory element to regulate expression of an operably linked transcribable DNA molecule. A leader molecule may 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 NOs: 3, 6, 14, 21, 28, 32, and 40, or SEQ ID NOs: 1, 4, 7, 8, 10, 12, 15, 16, 18, 19, 22, 23, 26, 30, 43, and 45, or any of their fragments or variants. In specific embodiments, such DNA sequences may be defined as being capable of acting as a leader in a host cell, including, for example, a transgenic plant cell. In one embodiment, such sequences are decoded as comprising leader activity.

[0085] The leader sequences (also referred to as 5'UTRs) shown as any of SEQ ID NOs: 3, 6, 14, 21, 28, 32 and 40 or any of the leader elements contained in any of SEQ ID NOs: 1, 4, 7, 8, 10, 12, 15, 16, 18, 19, 22, 23, 26, 30 and 43 may consist of regulatory elements or may adopt secondary structures capable of influencing the transcription or translation of an operably linked transcribable DNA molecule. The leader sequences shown as any of SEQ ID NOs: 3, 6, 14, 21, 28, 32 and 40 or any of the leader elements contained in any of SEQ ID NOs: 1, 4, 7, 8, 10, 12, 15, 16, 18, 19, 22, 23, 26, 30, 43 and 45 may be used in accordance with the present invention to create chimeric regulatory elements that influence the transcription or translation of an operably linked transcribable DNA molecule.

[0086] As used herein, the term "intron" refers to a DNA molecule that can be isolated or identified from a gene and generally defined as a region that is excised during messenger RNA (mRNA) processing prior to translation. Alternatively, an intron may be a synthetically produced or engineered DNA element. An intron may contain an enhancer element that affects the transcription of an operably linked gene. An intron may be used as a regulatory element to regulate the expression of an operably linked transcribable DNA molecule. A construct may contain an intron, which may or may not be heterologous with respect to the transcribable DNA molecule. Examples of introns known in the art include the rice actin intron and the maize HSP70 intron.

[0087] In plants, the incorporation of some introns into gene constructs leads to increased accumulation of mRNA and protein compared to constructs lacking the intron. This effect has been called "intron-mediated enhancement" (IME) of gene expression. Introns known to stimulate expression in plants have been identified in maize genes (e.g., tubA1, Adh1, Sh1, and Ubi1), rice genes (e.g., tpi), and dicotyledonous genes, such as genes from petunia (e.g., rbcS), potato (e.g., st-ls1), and Arabidopsis thaliana (e.g., ubq3 and pat1). Deletions or mutations within the splicing sites of introns have been shown to reduce gene expression, indicating that splicing may be required for IME. However, a point mutation within the splicing site of the pat1 gene from A. thaliana demonstrated IME in dicotyledonous plants. It has been shown that the use of multiple identical introns in one plant can lead to disadvantages. In that case, a collection of basic control elements is required for the construction of a suitable recombinant DNA element. Exemplary introns useful in practicing the present invention are shown as SEQ ID NOs: 9, 11, 17, 33 and 41.

[0088] As used herein, the term "3' transcription termination molecule", "3' untranslated region" or "3'UTR" refers to a DNA molecule used during transcription of the 3' portion of an mRNA molecule into the untranslated region. The 3' untranslated region of an mRNA molecule can be generated by specific cleavage and 3' polyadenylation, also known as polyA tail. The 3'UTR can be operably linked and positioned downstream of a transcribable DNA molecule and can include polyadenylation signals and other regulatory signals that can affect transcription, mRNA processing or gene expression. The polyA tail is believed to function in mRNA stability and translation initiation. In the art, examples of 3' transcription termination molecules are nopaline synthase 3' region, wheat hsp17 3' region, pearubisco small subunit 3' region, cotton E6 3' region, and coixin 3'UTR.

[0089] 3'UTRs typically have beneficial applications for recombinant expression of specific DNA molecules. Weak 3'UTRs have the potential to cause read-through, which may affect the expression of DNA molecules placed in adjacent expression cassettes. Proper control of transcription termination can prevent read-through into downstream located DNA sequences (e.g., other expression cassettes) and even allow efficient recycling of RNA polymerase to improve gene expression. Efficient transcription termination (release of RNA polymerase II from DNA) is a prerequisite for transcription resumption, thereby affecting the overall transcription level. Following transcription termination, mature mRNA is released from the site of synthesis and template and transported to the cytoplasm. Eukaryotic mRNA accumulates in vivo in the form of poly(A), making transcription termination sites difficult to detect by traditional methods. However, bioinformatics prediction of functional and efficient 3'UTRs is difficult in that there is no conserved DNA sequence that would allow for easy prediction of effective 3'UTRs.

[0090] From a practical standpoint, it is typically beneficial for the 3'UTR used in an expression cassette to have the following attributes: First, the 3'UTR must be able to efficiently and effectively terminate transcription of the transgene and prevent read-through of the transcript into any adjacent DNA sequences that may consist of another expression cassette, such as when multiple expression cassettes are present in one transfer DNA (T-DNA) or in adjacent chromosomal DNA into which the T-DNA is inserted. Second, the 3'UTR must not result in attenuation of the transcriptional activity conferred by the promoters, leaders, enhancers, and introns used to drive the expression of the DNA molecule. Finally, in plant biotechnology, 3'UTRs are often used to prime amplification reactions of reverse transcribed RNA extracted from transformed plants for use in (1) assessing the transcriptional activity or expression of expression cassettes once integrated into plant chromosomes, (2) assessing the copy number of the insert in the plant DNA, and (3) assessing the zygosity of the seeds obtained after breeding. The 3'UTRs are also used in amplification reactions of DNA extracted from transformed plants to assess the integrity of the inserted cassette. 3'UTRs useful in carrying out the present invention are set forth as SEQ ID NOs: 29, 34, 35, 36, 37 and 44.

[0091] As used herein, the term "enhancer" or "enhancer element" refers to a cis-acting regulatory element, also known as a cis-element, which confers an aspect of the overall expression pattern, but is usually insufficient by itself to promote transcription of an operably linked transcribable DNA molecule. Unlike a promoter, an enhancer element usually does not contain a transcription start site (TSS) or a TATA box or equivalent DNA sequence. A promoter or promoter fragment may naturally contain one or more enhancer elements that affect the transcription of an operably linked DNA sequence. Enhancer elements may be fused to promoters to generate chimeric promoter cis-elements that confer an aspect of global regulation of gene expression. An example of an enhancer element derived from the synthetic promoter P-At.GSP571.nno:5 (SEQ ID NO:5) is provided as SEQ ID NO:24 (E-At.GSP571.nno:1).

[0092] Many promoter enhancer elements are believed to bind DNA-binding proteins and / or affect DNA topology, thereby selectively allowing or restricting RNA polymerase to contact the DNA template or facilitating the selective opening of the double helix at the transcription start site. Enhancer elements may function to bind transcription factors that regulate transcription. Some enhancer elements bind more than one transcription factor, and a transcription factor may interact with more than one enhancer domain with different affinities. Enhancer elements can be identified by many techniques, including deletion analysis, i.e., deleting one or more nucleotides from the 5' end or inside of 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 conventional DNA sequence comparison methods such as BLAST, using known cis-element motifs or enhancer elements as target sequences or target motifs. The fine structure of the enhancer domain can be further investigated by mutagenesis (or substitution) of one or more nucleotides, or other conventional methods known in the art. Enhancer elements can be obtained by chemical synthesis, or by isolation from a regulatory element containing such an element, and may also contain additional flanking sequences containing restriction enzyme sites useful for facilitating manipulation of the resulting sequence. It can be synthesized with nucleotides. Thus, the design, construction and use of enhancer elements according to the methods disclosed herein to regulate the expression of an operably linked transcribable DNA molecule are encompassed by the present invention. An exemplary enhancer useful in carrying out the present invention is shown as SEQ ID NO:24.

[0093] As used herein, the term "chimeric" refers to a single DNA molecule generated by fusing a first DNA molecule with a second DNA molecule, where neither the first nor the second DNA molecule is normally found in a configuration, i.e., fused to the other. Thus, a chimeric DNA molecule is a novel DNA molecule that is not normally found elsewhere in nature. As used herein, the term "chimeric promoter" refers to a promoter generated by such manipulation of DNA molecules. A chimeric promoter can be a combination of two or more DNA fragments, for example, a promoter fused to an enhancer element. Thus, the design, construction and use of chimeric promoters according to the methods disclosed herein to regulate the expression of a transcribable DNA molecule operably linked to them are encompassed by the present invention. An exemplary chimeric promoter is set forth herein as SEQ ID NO: 25 (P-At.GSP571 / 442).

[0094] Chimeric regulatory elements can be designed to contain various components that can be operably linked by various methods known in the art, such as restriction enzyme digestion and ligation, ligation-free cloning, modular assembly of PCR products during amplification, or direct chemical synthesis of regulatory elements, as well as other methods known in the art. The resulting various chimeric regulatory elements can consist of the same components or variants thereof, but can differ in one or more DNA sequences, including one or more linking DNA sequences that allow the components to be operably linked. In the present invention, the DNA sequences provided as SEQ ID NOs: 1-32 and 43-45 can provide regulatory element reference sequences, where the components that make up the reference sequences may be spliced ​​together by methods known in the art and may include one or more nucleotide substitutions, deletions and / or insertions, or mutations that occur naturally in bacterial and plant cell transformation.

[0095] As used herein, the term "variant" refers to a second DNA molecule, such as a regulatory element, that is similar but not identical in composition to a first DNA molecule, yet still maintains the approximate functionality, i.e., the same or similar expression pattern, for example, by a somewhat equivalent transcriptional activity of the first DNA molecule. A variant may be a shorter or truncated form of the first DNA molecule, or may have a modified form of the sequence of the first DNA molecule, e.g., a different restriction enzyme site and / or internal deletion, substitution or insertion. "Variant" may also include a derivative regulatory element, which has a nucleotide sequence that includes a substitution, deletion or insertion of one or more nucleotides of the reference sequence, and has a somewhat equivalent transcriptional or translational activity compared to the corresponding parent regulatory molecule. In the present invention, the polynucleotide sequences provided as SEQ ID NOs: 1-32 and 43-45 may be used to generate variants that are similar but not identical in composition to the DNA sequence of the original regulatory element, while still maintaining the approximate functionality, i.e., the same or similar expression pattern as the original regulatory element. The production of such variants of the present invention is well within the skill of those in the art in light of the instant disclosure and is within the scope of the present invention.

[0096] The effectiveness of the modifications, duplications or deletions described herein on the desired expression pattern of a particular transgene can be empirically tested to verify the results in stable and transient plant assays as described in the working examples, but the results may vary depending on the modifications made in the starting DNA molecule and the purpose of the modifications.

[0097] Constructs As used herein, the term "construct" refers to any recombinant DNA molecule, such as a plasmid, cosmid, virus, phage, or linear or circular DNA or RNA molecule, from any source, capable of genomic integration or autonomous replication, including at least one DNA molecule functionally linked, i.e. operably linked, to another DNA molecule. As used herein, the term "vector" refers to any construct that can be used for the purpose of transformation, i.e., the introduction of heterologous DNA or RNA into a host cell. A construct typically contains one or more expression cassettes. As used herein, "expression cassette" refers to a DNA molecule that contains at least a transcribable DNA molecule operably linked to one or more regulatory elements, typically at least a promoter and a 3'UTR.

[0098] As used herein, the term "operably linked" refers to a first DNA molecule spliced ​​to a second DNA molecule, the first and second DNA molecules being 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 if it controls the transcription of the transcribable DNA molecule of interest in a cell. For example, a leader is operably linked to a DNA sequence if it can affect the transcription or translation of the DNA sequence.

[0099] The constructs of the invention may, in one embodiment, be provided as Ti plasmid border constructs having the right border (RB or AGRtu.RB) and left border (LB or AGRtu.LB) regions of a dual tumor-inducing (Ti) plasmid isolated from A. tumefaciens containing T-DNA, which allows the T-DNA to be integrated into the genome of a plant cell together with a transfer molecule provided by an Agrobacterium tumefaciens cell (see, e.g., U.S. Pat. No. 6,603,061). The constructs may further contain a plasmid backbone DNA segment providing replication functions and antibiotic selection in bacterial cells, e.g., an Escherichia coli origin of replication such as ori322, a broad host range origin of replication such as oriV or oriRi, and a selection marker such as Spec / Strp encoding the Tn7 aminoglycoside adenyltransferase (aadA) conferring resistance to spectinomycin or streptomycin, or the coding region of a gentamicin (Gm, Gent) selection marker gene. For plant transformation, the host bacterial strain is often A. tumefaciens ABI, C58, or LBA4404, although other strains known to those skilled in the art of plant transformation may work in the present invention.

[0100] Methods for assembling and introducing constructs into cells to transcribe transcribable DNA molecules into functional mRNA molecules that are translated and expressed as proteins are known to those of skill in the art. Conventional compositions and methods for making and using constructs and host cells in the practice of the present invention are well known to those of skill in the art. Exemplary vectors useful for the expression of nucleic acids in higher plants are well known in the art and include vectors derived from the Ti plasmid of Agrobacterium tumefaciens and the pCaMVCN transfer control vector.

[0101] A variety of regulatory elements, including any of those 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 desired regulatory properties. In one embodiment, the construct of the present invention comprises at least one regulatory element operably linked to a transcribable DNA molecule operably linked to a 3'UTR.

[0102] The constructs of the invention can include any promoter or leader provided herein or known in the art. For example, a promoter of the invention can be operably linked to a heterologous untranslated 5' leader, such as one derived from a heat shock protein gene. Alternatively, a leader of the invention can be operably linked to a heterologous promoter, such as the cauliflower mosaic virus 35S transcription promoter.

[0103] The expression cassette may further comprise a transit peptide coding sequence encoding a peptide useful for directing an operably linked protein into a cell, specifically to a chloroplast, leucoplast or other plastid organelle; mitochondria; peroxisome; vacuole; or extracellular location. Many chloroplast-localized proteins are expressed as precursors from nuclear genes and are directed to the chloroplast by a chloroplast transit peptide (CTP). Examples of such isolated chloroplast proteins include, but are not limited to, those related to the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase, ferredoxin, ferredoxin oxidoreductase, light-harvesting complex protein I and protein II, thioredoxin F, and enolpyruvylshikimate phosphate synthase (EPSPS). Chloroplast transit peptides are described, for example, in U.S. Pat. No. 7,193,133. It has been demonstrated that non-chloroplast proteins can be directed to the chloroplast by expression of a heterologous CTP operably linked to a transgene encoding a non-chloroplast protein.

[0104] Transcribable DNA molecules As used herein, the term "transcribeable DNA molecule" refers to any DNA molecule that can be transcribed into an RNA molecule, including, but not limited to, those having protein coding sequences and those that produce RNA molecules having sequences useful for gene silencing. The types of DNA molecules can include, but are not limited to, DNA molecules from the same plant, from another plant, from a different organism, or synthetic DNA molecules, such as DNA molecules containing an antisense message of a gene or a DNA molecule encoding an artificial, synthetic, or modified form of a transgene. Exemplary transcribable DNA molecules for incorporation into the constructs of the invention include, for example, DNA molecules or genes from a species other than the species into which the DNA molecule is to be incorporated, or genes originating from or present in the same species but incorporated into the recipient cell by genetic engineering methods rather than classical breeding techniques.

[0105] "Transgene" refers to a transcribable DNA molecule that is heterologous to a 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 of the present or any prior generation of the cell.

[0106] The regulatory elements of the present invention, such as synthetic promoters, can be operably linked to a heterologous transcribable DNA molecule. As used herein, the term "heterologous" refers to a combination of two or more DNA molecules, where such a combination is not normally found in nature. For example, the two DNA molecules can be from different species, and / or the two DNA molecules can be from different genes, such as different genes from the same species, or the same gene from different species, or one of the DNA molecules is not naturally found but synthetic. A regulatory element is heterologous with respect to the transcribable DNA molecule to which it is operably linked, when such a combination is not normally found in nature, i.e., when the transcribable DNA molecule does not naturally exist in a state where it is operably linked to the regulatory element.

[0107] The transcribable DNA molecule may generally be any DNA molecule for which expression of a transcription product is desired. Such expression of a transcription product may result in the translation of the resulting mRNA molecule and thus the expression of a protein. Alternatively, the transcribable DNA molecule may be designed to, for example, ultimately cause a reduction in the expression of a particular gene or protein. In one embodiment, this may be accomplished by using a transcribable DNA molecule oriented in an antisense direction. Those skilled in the art are familiar with the use of such antisense technology. Any gene may be negatively regulated in this way, and in one embodiment, the transcribable DNA molecule may be designed to suppress a particular gene by the expression of a dsRNA, siRNA or miRNA molecule.

[0108] Thus, one embodiment of the present invention is a recombinant DNA molecule comprising a regulatory element of the invention, e.g., those set forth as SEQ ID NOs: 1-32 and 43-45, operably linked to a heterologous transcribable DNA molecule such that the regulatory element regulates transcription of the transcribable DNA molecule at a desired level or in a desired manner when the construct is integrated into the genome of a transgenic plant cell. In one embodiment, the transcribable DNA molecule comprises a protein coding region of a gene, and in another embodiment, the transcribable DNA molecule comprises an antisense region of a gene.

[0109] Genes of Agricultural Scientific Interest The transcribable DNA molecule may be a gene of agricultural scientific interest. As used herein, the term "gene of agricultural scientific interest" refers to a transcribable DNA molecule that confers a desired attribute when expressed in a particular plant tissue, cell or cell type. The product of the gene of agricultural scientific interest may act in the plant to affect plant morphology, physiology, growth, development, yield, grain composition, nutritional profile, disease or insect resistance, and / or environmental or chemical resistance, or may act as an insecticide in the diet of a pest that feeds on the plant. In one embodiment of the present invention, the regulatory element of the invention is incorporated into a construct such that the regulatory element is operably linked to the transcribable DNA molecule of agricultural scientific interest. Expression of the gene of agricultural scientific interest in a transgenic plant containing such a construct may confer advantageous agricultural traits. Advantageous agronomic traits may include, for example and without limitation, 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, enhanced animal and human nutrition, biopolymer production, environmental stress tolerance, pharmaceutical peptides, improved processing qualities, improved flavor, hybrid seed production utility, improved fiber production, and desirable biofuel production.

[0110] Examples of genes of agricultural scientific interest known in the art include, but are not limited to, those related to herbicide resistance (U.S. Pat. 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. Pat. Nos. USRE38,446, 6,716,474, 6,663,906, 6,476,295, 6,441,277, 6,423,826, and 6,463,175). 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, 6,593,293, 6,555,655, 6,538,109, 6,537,756, 6,521,442, 6,501,009, 6,468, ,523, No. 6,326,351, No. 6,313,378, No. 6,284,949, No. 6,281,016, No. 6,248,5 No. 36, No. 6,242,241, No. 6,221,649, No. 6,177,615, No. 6,156,573, No. 6,153,814 No. 6,110,464, No. 6,093,695, No. 6,063,756, No. 6,063,597, No. 6,023,013, No. 5,959,091, No. 5,942,664, No. 5,942,658, No. 5,880,275, No. 5,763,245 and No. 5,763,241), fungal disease resistance (U.S. Patent Nos. 6,653,280, 6,573,361, 6,506,962, 6,316,407, 6,215,048, 5,516,671, 5,773,696, 6,121,436, 6,316,407 and 6,506,962), virus resistance (U.S. Patent Nos. 6,617,496, 6,608,241, 6,015,940, 6,013,864, 5,850,023 and 5,304,730), nematode resistance (U.S. Patent Nos. 6,228,No. 992), bacterial disease resistance (U.S. Patent No. 5,516,671), plant growth and development (U.S. Patent Nos. 6,723,897 and 6,518,488), starch production (U.S. Patent Nos. 6,538,181, 6,538,179, 6,538,178, 5,750,876, 6,476,295), modified oil production (U.S. Patent Nos. 6,444,876, 6,426,447 and 6,380,462), high oil yield (U.S. Patent Nos. 6,495,739, 5,608,149, 6,483,008 and and 6,476,295), modified fatty acid content (U.S. Patent Nos. 6,828,475, 6,822,141, 6,770,465, 6,706,950, 6,660,849, 6,596,538, 6,589,767, 6,537,750, 6,489,461 and 6,459,018), high protein yield (U.S. Patent No. 6,380,466), fruit ripening (U.S. Patent No. 5,512,466), enhanced animal and human nutrition (U.S. Patent Nos. 6,723,837, 6, Nos. 6,5412,59, 5,985,605 and 6,171,640), biopolymers (U.S. Patents. USRE37,543, 6,228,623, 5,958,745 and 6,946,588), environmental stress resistance (U.S. Patent No. 6,072,103), pharmaceutical and secretory peptides (U.S. Patents. 6,812,379, 6,774,283, 6,140,075 and 6,080,560), improved processing traits (U.S. Patent No. 6,476,295), modified Improved digestibility (U.S. Patent No. 6,531,648), low raffinose (U.S. Patent No. 6,166,292), industrial enzyme production (U.S. Patent No. 5,543,576), improved flavor (U.S. Patent No. 6,011,199), nitrogen fixation (U.S. Patent No. 5,229,114), hybrid seed production (U.S. Patent No. 5,689,041), fiber production (U.S. Patent Nos. 6,576,818, 6,271,443, 5,981,834, and 5,869,720), and biofuel production (U.S. Patent No. 5,998,700).

[0111] Alternatively, the gene of agricultural scientific interest can encode an RNA molecule that causes directed regulation of gene expression of the endogenous gene, e.g., by antisense (see, e.g., U.S. Pat. No. 5,107,065), inhibitory RNA ("RNAi" including regulation of gene expression by miRNA-, siRNA-, trans-acting siRNA- and phasic sRNA-mediated mechanisms as described, e.g., in Published Applications US2006 / 0200878 and US2008 / 0066206 and U.S. Patent Application No. 11 / 974,469) or co-suppression mediated mechanisms. The RNA may be a catalytic RNA molecule (e.g., a ribozyme or riboswitch, see, e.g., US2006 / 0200878) engineered to cleave a desired endogenous mRNA product. Methods for constructing and introducing constructs into cells such that a transcribable DNA molecule is transcribed into a molecule capable of causing gene repression are known in the art.

[0112] Selection marker Selection marker transgenes may be used with the regulatory elements of the present invention. As used herein, the term "selection marker transgene" refers to any transcribable DNA molecule whose expression or lack thereof in a transgenic plant, tissue or cell can be screened or scored in some manner. Selection marker genes and associated selection and screening techniques used in the practice of the present invention are known in the art and include, but are not limited to, transcribable DNA molecules encoding β-glucuronidase (GUS), green fluorescent protein (GFP), proteins that confer antibiotic resistance, and proteins that confer herbicide resistance. An example of a selection marker transgene is provided as SEQ ID NO: 42.

[0113] Cell transformation The present invention further relates to methods for producing transformed cells and plants comprising one or more regulatory elements operably linked to a transcribable DNA molecule.

[0114] The term "transformation" refers to the introduction of a DNA molecule into a recipient host. As used herein, the term "host" refers to a bacterium, fungus, or plant, including any cell, tissue, organ, or progeny of the bacterium, fungus, or plant. Plant tissues and cells of particular interest include protoplasts, callus, roots, tubers, seeds, stems, leaves, seedlings, embryos, and pollen.

[0115] As used herein, the term "transformed" refers to a cell, tissue, organ, or organism into which a foreign DNA molecule, such as a construct, has been introduced. The introduced DNA molecule may be integrated into the genomic DNA of the recipient cell, tissue, organ, or organism such that the introduced DNA molecule is inherited by subsequent offspring. A "transgenic" or "transformed" cell or organism may include the progeny of the cell or organism, as well as progeny produced from a breeding program employing such transgenic organisms as parents in a cross and exhibiting an altered phenotype resulting from the presence of the foreign DNA molecule. The introduced DNA molecule may also be transiently introduced into the recipient cell such that the introduced DNA molecule is not inherited by subsequent offspring. The term "transgenic" refers to a bacterium, fungus, or plant that contains one or more heterologous DNA molecules.

[0116] There are many methods well known to those skilled in the art for introducing DNA molecules into plant cells. The process generally includes the steps of selecting a suitable host cell, transforming the host cell with a vector, and obtaining the transformed host cell. The methods and materials for transforming plant cells by introducing a plant construct into the plant genome in the practice of the present invention can include any well-known and proven method. Suitable methods include, but are not limited to, bacterial infection (e.g., Agrobacterium), binary BAC vectors, direct delivery of DNA (e.g., by PEG-mediated transformation, desiccation / inhibition-mediated DNA uptake, electroporation, stirring with silicon carbide fibers, and accelerating DNA-coated particles), and gene editing (e.g., CRISPR-Cas system), among others.

[0117] The present disclosure further contemplates that the synthetic expression element of the present disclosure can be engineered in plants by using various gene editing methods known in the art.Such techniques used for genome editing include, but are not limited to, ZFN (zinc finger nuclease), meganuclease, TALEN (transcription activator-like effector nuclease) and CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated) system.These genome editing methods can be used to change the expression element sequence to a different sequence in plant cells.

[0118] The host cell can be any cell or organism, for example, a plant cell, an algal cell, an algae, a fungal cell, a fungus, a bacterial cell, or an insect cell. In particular embodiments, the host cell and the transformed cell can include cells from a crop plant.

[0119] A transgenic plant can then be regenerated from the transgenic plant cell of the invention. Seeds can be produced from the transgenic plant using conventional breeding techniques or self-pollination. Such seeds, and the resulting progeny plants grown from such seeds, will contain a recombinant DNA molecule of the invention and thus be transgenic.

[0120] A transgenic plant of the invention can be self-pollinated to provide seeds of a homozygous transgenic plant (homozygous for the recombinant DNA molecule) of the invention, or can be crossed with a non-transgenic plant or a different transgenic plant to provide seeds of a heterozygous transgenic plant (heterozygous for the recombinant DNA molecule) of the invention. Both such homozygous and heterozygous transgenic plants are referred to herein as "progeny plants." Progeny plants are transgenic plants that are descendants of the original transgenic plant and contain a recombinant DNA molecule of the invention. Seeds produced using the transgenic plants of the invention can be harvested and used to grow generations of transgenic plants of the invention that contain a construct of the invention and express a gene of agricultural scientific interest, i.e., progeny plants. Descriptions of commonly used breeding methods for various crops can be found in one of several reference books, such as Allard, Principles of Plant Breeding, John Wiley & Sons, NY, U. of CA, Davis, CA, 50-98 (1960); Simmonds, Principles of Crop Improvement, Longman, Inc., NY, 369-399 (1979); Sneep and Hendriksen, Plant breeding Perspectives, Wageningen (ed), Center for Agricultural Publishing and Documentation (1979); Fehr, Soybeans: Improvement, Production and Uses, 2nd Edition, Monograph, 16:249 (1987); Fehr, Principles of Variety Development, Theory and Technique, (Vol. 1), and Crop Species Soybean (Vol. 2), Iowa State Univ., Macmillan, 1989; See Pub. Co., NY, 360-376 (1987).

[0121] Transformed plants can be analyzed for the presence of one or more genes of interest and the expression levels and / or profile conferred by the regulatory elements of the present invention. Those skilled in the art are aware of the many methods available for analyzing transformed plants. For example, plant analysis methods include, but are not limited to, Southern or Northern blots, PCR-based techniques, biochemical analysis, phenotypic screening methods, field evaluation, and immunodiagnostic assays. Expression of transcribable DNA molecules can be measured using TaqMan® (Applied Biosystems, Foster City, CA) reagents and methods described by the manufacturer, and PCR cycle times determined using the TaqMan® Testing Matrix. Alternatively, transgene expression can be assessed using Invader® (Third Wave Technologies, Madison, WI) reagents and methods described by the manufacturer.

[0122] The present invention further provides parts of the plants of the present invention. Plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovules and pollen. Plant parts of the present invention can be viable, non-viable, regenerable and / or non-regenerable. The present invention further includes and provides transformed plant cells comprising the DNA molecules of the present invention. Transformed or transgenic plant cells of the present invention include regenerable and / or non-regenerable plant cells.

[0123] The present invention further provides a commodity product produced from a transgenic plant or part thereof containing a recombinant DNA molecule of the present invention. The commodity product of the present invention contains a detectable amount of DNA comprising a DNA sequence selected from the group consisting of SEQ ID NOs: 1-32 and SEQ ID NOs: 43-45. As used herein, "commodity product" refers to any composition or product of material derived from a transgenic plant, seed, plant cell, or plant part containing a recombinant DNA molecule of the present invention. Commercial products include, but are not limited to, processed seeds, grains, plant parts, and meal. The commodity product of the present invention will contain a detectable amount of DNA corresponding to a recombinant DNA molecule of the present invention. Detection of one or more of this DNA in a sample may be used to determine the content or source of the commodity product. Any standard method for detecting DNA molecules may be used, including the detection methods disclosed herein.

[0124] The present invention may be more readily understood by reference to the following examples, which are provided as illustrations and are not intended to limit the present invention unless expressly stated. Those skilled in the art will recognize that the techniques disclosed in the following examples represent techniques discovered by the inventors and which function well in the practice of the present invention. However, in light of this disclosure, those skilled in the art will recognize that many changes can be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention, and therefore, all matters shown or depicted in the accompanying drawings should be interpreted as illustrative and not limiting. EXAMPLES

[0125] Example 1 Design, synthesis and cloning of synthetic regulatory elements The regulatory elements shown in Table 1 are novel synthetic expression elements designed by algorithmic methods. These computer-designed synthetic regulatory elements were chemically synthesized and cloned to create the family of synthetic regulatory expression elements (EXP). Well over 1,000 synthetic regulatory elements were designed and tested in soybean protoplasts and stably transformed soybean plants to identify synthetic regulatory elements that provide desired protein expression levels and characteristics such as expression patterns. The synthetic regulatory elements listed in Table 1 provide various modes of expression that are useful for driving the expression of various coding sequences and interfering RNAs of agricultural scientific interest.

[0126] The computer-designed synthetic regulatory elements do not have extensive homology to any known naturally occurring nucleic acid sequences. The synthetic EXPs and corresponding promoters, leaders, introns and 3'UTRs are shown in Table 1. Using methods known in the art, the synthetic EXPs were cloned into binary plant transformation vectors operably linked to a β-glucuronidase (GUS) coding sequence, and the vectors were used to evaluate the level and pattern of expression provided by the synthetic EXPs in stably transformed soybean, cotton and corn plants.

[0127] Analysis of the transcription start site (TSS) and intron / exon splice junctions of the synthetic regulatory elements can be performed using transformed plant tissue. Briefly, plants are transformed with a plant expression vector containing a cloned DNA fragment operably linked to a heterologous transcribable DNA molecule. The TSS and intron / exon splice junctions of the synthetic regulatory elements are then confirmed by analyzing the DNA sequence of the resulting mRNA transcripts using the 5'RACE system for rapid amplification of cDNA ends, Version 2.0 (Invitrogen, Carlsbad, California 92008). [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0128] Example 2 Analysis of synthetic EXPs, EXP-At.GSP442.nno+At.Cyco:3 and EXP-At.GSP221+At.Cyco:3, driving GUS expression in stably transformed soybean plants Soybean plants were transformed with vectors, specifically plant expression vectors, containing regulatory elements driving the expression of a β-glucuronidase (GUS) transgene, and the resulting plants were analyzed for GUS protein expression to assess the effect of the selected regulatory elements on expression.

[0129] Soybean plants were transformed with plant GUS expression constructs containing the endogenous EXP, EXP-At.Cyco:1:1 (SEQ ID NO:38), and two synthetic EXPs, EXP-At.GSP442.nno+At.Cyco:3 (SEQ ID NO:1) and EXP-At.GSP221+At.Cyco:3 (SEQ ID NO:30). EXP-At.Cyco:1:1 (SEQ ID NO:38) is derived from the cytochrome c oxidase subunit VIa gene from Arabidopsis and consists of a promoter, P-At.Cyco-1:1:2 (SEQ ID NO:39), operably linked 5' to a leader, L-At.Cyco-1:1:2 (SEQ ID NO:40), operably linked 5' to an intron, I-At.Cyco-1:1:1 (SEQ ID NO:41). EXP-At.GSP442.nno+At.Cyco:3 (SEQ ID NO:1) and EXP-At.GSP221+At.Cyco:3 (SEQ ID NO:30) each contained a synthetic promoter and leader operably linked to the 5' side of an intron, I-At.Cyco:2 (SEQ ID NO:33). The sequence of I-At.Cyco:2 (SEQ ID NO:33) is identical to that of I-At.Cyco-1:1:1 (SEQ ID NO:41), except that the sequence of I-At.Cyco-1:1:1 contains two nucleotides after the intron splice site. Both I-At.Cyco introns splice in the same way.

[0130] Regulatory elements were cloned into a basic plant expression vector using standard methods known in the art. The resulting plant expression vector contained the right border from Agrobacterium tumefaciens (B-AGRtu.right border), a first transgene selection cassette used to select transformed plant cells conferring resistance to the antibiotic spectinomycin, and a second transgene cassette for evaluating the activity of a regulatory element comprising an EXP sequence operably linked 5' to the coding sequence for β-glucuronidase (GUS, GOI-Ec.uidA+St.LS1:1:1, SEQ ID NO: 42) containing a processable intron from the potato light-inducible tissue-specific ST-LS1 gene (Genbank accession: X04753) operably linked 5' to the 3'UTR from the Gossypium barbadense FbLate-2 gene (T-Gb.FbL2:1, SEQ ID NO: 36), and the left border from Agrobacterium tumefaciens (B-AGRtu.left border).

[0131] These binary transformation vector constructs were used to transform soybean plant cells by Agrobacterium-mediated transformation as is well known in the art, and the resulting transformed plant cells were induced to form whole soybean plants.

[0132] Histochemical GUS assays were used for qualitative and quantitative expression analysis of transformed plants. Whole tissue sections were incubated with GUS staining solution X-Gluc (5-bromo-4-chloro-3-indolyl-b-glucuronide) (1 milligram / milliliter) for an appropriate length of time, rinsed, and visually examined for blue coloration. Selected plant organs and tissues were used to qualitatively determine GUS activity by direct visual or microscopic examination.

[0133] For quantitative analysis of GUS expression, total protein was extracted from selected tissues of transformed soybean plants. One microgram of total protein was used 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 where the hydroxyl group is ionized. Addition of a basic solution of sodium carbonate stops the assay and the pH is adjusted for quantification of the fluorescent product. Fluorescence was measured in a Micromax reader using a Fluoromax-3 with excitation at 365 nm and emission at 445 nm, with slit widths set at 2 nm excitation and 3 nm emission. Values ​​are provided in nmol GUS / hour / mg total protein.

[0134] The following tissues were sampled for GUS expression in the R0 generation: roots, leaves-sinus at V5 stage R1, root, leaf-petiole, leaf-source, and flower at stage R2, seed-immature and pod at stage R3, seed-cotyledon at stage R5, and seed-embryo and seed-cotyledon at stage R8. Table 2 shows the average quantitative GUS expression of each sampled tissue driven by the EXP regulatory elements tested, with "ND" in the table indicating that expression in a particular tissue was not determined. Table 2. Average quantitative GUS expression in stably transformed soybean plants driven by synthetic regulatory elements and the endogenous EXP, EXP-At.Cyco:1:1. [Table 2]

[0135] As can be seen from Table 2, each of the synthetic regulatory elements has a unique expression pattern compared to the endogenous EXP in the tissues sampled. For example, the synthetic At.GSP442 promoter P-At.GSP442.nno:2 (SEQ ID NO:2) and leader L-At.GSP442.nno:1 (SEQ ID NO:3) of EXP-At.GSP442.nno+At.Cyco:3 (SEQ ID NO:1) results in higher levels of GUS expression in all organs assayed compared to the endogenous EXP-At.Cyco:1:1 (SEQ ID NO:38), which contains the same intron sequence. Analysis of the TSS demonstrated a consistent TSS. In the resulting mRNA, the intron was properly removed as expected. Furthermore, the synthetic At.GSP221 promoter P-At.GSP221:3 (SEQ ID NO: 31) and leader L-At.GSP221:1 (SEQ ID NO: 32) of EXP-At.GSP221+At.Cyco:3 (SEQ ID NO: 30) also resulted in higher levels of constitutive expression in most organs assayed compared to the endogenous EXP-At.Cyco:1:1, demonstrating a consistent TSS. However, the TSS of EXP-At.GSP221+At.Cyco:3 was not located in the predicted location - multiple TATA elements could be present. This raises potential concerns about multiple transcripts that could result in multiple coding sequences. For this reason, EXP-At.GSP221+At.Cyco:3 was not deemed acceptable to be used to drive transgene expression in stably transformed dicotyledonous plants. This demonstrates one of the complexities in designing synthetic expression elements. In the development and identification of synthetic expression elements, many synthetic elements were assayed, but only a small subset conferred the desired traits and regulatory activity, illustrating the complexities in designing effective synthetic transcriptional regulatory elements.

[0136] As can be seen from Table 2, the synthetic promoters P-At.GSP442.nno:2 (SEQ ID NO:2) and L-At.GSP442.nno:1 (SEQ ID NO:3) contained in EXP-At.GSP442.nno+At.Cyco:3 (SEQ ID NO:1) can drive constitutive transgene expression of operably linked transgenes in stably transformed soybean plants.

[0137] Example 3 Analysis of the synthetic At.GSP571 promoter and leader and the synthetic At.GSI21 and At.GSI102 introns driving GUS expression in stably transformed soybean plants Soybean plants were transformed with vectors, specifically plant expression vectors, containing regulatory elements driving the expression of a β-glucuronidase (GUS) transgene, and the resulting plants were analyzed for GUS protein expression to assess the effect of the selected regulatory elements on expression.

[0138] Soybean plants were transformed with plant GUS expression constructs including the synthetic EXPs EXP-At.GSP571 (SEQ ID NO: 4), EXP-At.GSP571.nno+At.Cyco:2 (SEQ ID NO: 7), EXP-At.GSP571.nno+At.GSI21.nno:10 (SEQ ID NO: 8) and EXP-At.GSP571.nno+At.GSI102.nno:1 (SEQ ID NO: 10). Each of the synthetic EXPs contained the synthetic At.GSP571 promoter (SEQ ID NO: 5) and leader (SEQ ID NO: 6). EXP-At.GSP571.nno+At.Cyco:2 contained the endogenous Arabidopsis intron I-At.Cyco:2 (SEQ ID NO: 33). EXP-At.GSP571.nno+At.GSI21.nno:10 and EXP-At.GSP571.nno+At.GSI102.nno:1 contained the synthetic introns I-At.GSI21.nno:2 (SEQ ID NO: 9) and I-At.GSI102.nno:1 (SEQ ID NO: 11), respectively. The binary plant transformation vectors were similar to those described in Example 2, except that each of the At.GSP571EXP vectors contained the 3'UTR, T-Mt.Sali3-2-1:2:1 (SEQ ID NO: 34), derived from the Sali3 gene of Medicago truncatula.

[0139] Quantitative and qualitative GUS expression analysis was performed as described in Example 2. The tissue samples used for analysis were the same as those described in Example 2. Table 3 shows the average quantitative GUS expression of each sampled tissue driven by the synthetic EXP regulatory elements tested, with "ND" in the table indicating that expression in a particular tissue was not determined. [Table 3]

[0140] As can be seen from Table 3, the synthetic At.GSP571 promoter and leader resulted in constitutive expression in all organs assayed. Expression was highest in leaves and seeds. Analysis of the TSS demonstrated consistent TSS. Operative linking of intron sequences altered expression in many organs, providing a means to "fine tune" constitutive expression. Differences in expression were observed when the synthetic introns I-At.GSI21.nno:2 (SEQ ID NO: 9) and I-At.GSI102.nno:1 (SEQ ID NO: 11) were operably linked. The synthetic introns enhanced expression in several tissues, but the level of enhancement varied from organ to organ. For example, enhancement in pods of R3 using synthetic intron I-At.GSI21.nno:2 was stronger than that seen with synthetic intron I-At.GSI102.nno:1, and with endogenous intron I-At.Cyco:2 was stronger than that seen with EXP-At.GSP571. In petioles of R1, expression was only slightly enhanced by the three operably linked introns. In flowers of R1, I-At.GSI21.nno:2 and I-At.Cyco:2 enhanced expression, with I-At.GSI21.nno:2 providing a high level of enhanced expression and I-At.Cyco:2 providing a moderate level of enhanced expression. Interestingly, I-At.GSI102.nno:1 reduced expression in flowers of R1.

[0141] Analysis of the resulting mRNAs demonstrated proper and consistent processing of the intron elements.

[0142] The synthetic promoter P-At.GSP571.nno:5 (SEQ ID NO:5) and leader L-At.GSP571.nno:1 (SEQ ID NO:6) contained within EXP-At.GSP571 (SEQ ID NO:4) confer constitutive expression of an operably linked transgene in stably transformed soybean plants. EXP-At.GSP571.nno+At.Cyco:2 (SEQ ID NO: 7), which contains the Arabidopsis intron I-At.Cyco:2 (SEQ ID NO: 33), and EXP-At.GSP571.nno+At.GSI21.nno:10 (SEQ ID NO: 8) and EXP-At.GSP571.nno+At.GSI102.nno:1 (SEQ ID NO: 10), which contain the synthetic introns I-At.GSI21.nno:2 (SEQ ID NO: 9) and I-At.GSI102.nno:1 (SEQ ID NO: 11), respectively, are synthetic EXPs but confer a unique constitutive expression pattern in stably transformed soybean plants. The synthetic introns I-At.GSI21.nno:2 (SEQ ID NO: 9) and I-At.GSI102.nno:1 (SEQ ID NO: 11), when operably linked to EXP-At.GSP571 (SEQ ID NO: 4), result in enhanced or regulated expression in many of the plant organs. These unique expression patterns can be used to direct specific transgenes to which one of the four At.GSP571EXPs has the most desirable intrinsic expression pattern.

[0143] Example 4 Analysis of the synthetic At.GSP564 promoter and leader and the synthetic At.GSI17 and At.GSI102 introns driving GUS expression in stably transformed soybean plants Soybean plants were transformed with vectors, specifically plant expression vectors, containing regulatory elements driving the expression of a β-glucuronidase (GUS) transgene, and the resulting plants were analyzed for GUS protein expression to assess the effect of the selected regulatory elements on expression.

[0144] Soybean plants were transformed with plant GUS expression constructs including the synthetic EXPs EXP-At.GSP564 (SEQ ID NO: 12), EXP-At.GSP564.nno+At.Cyco:2 (SEQ ID NO: 15), EXP-At.GSP564.nno+At.GSI17.nno:2 (SEQ ID NO: 16) and EXP-At.GSP564.nno+At.GSI102.nno:1 (SEQ ID NO: 18). Each of the synthetic EXPs contained the synthetic P-At.GSP564.nno:3 promoter (SEQ ID NO: 13) and the synthetic L-At.GSP564.nno.1 leader (SEQ ID NO: 14). EXP-At.GSP564.nno+At.Cyco:2 contained the Arabidopsis intron I-At.Cyco:2 (SEQ ID NO: 33). EXP-At.GSP564.nno+At.GSI17.nno:2 and EXP-At.GSP564.nno+At.GSI102.nno:1 contained the synthetic introns I-At.GSI17.nno:1 (SEQ ID NO: 17) and I-At.GSI102.nno:1 (SEQ ID NO: 11), respectively. The binary plant transformation vectors were similar to those described in Example 2, except that each of the At.GSP564EXP vectors contained the 3'UTR, T-Mt.Oxr-1:2:1 (SEQ ID NO: 35), derived from a putative oxidoreductase (OXR) protein gene from Medicago truncatula.

[0145] Quantitative and qualitative GUS expression analysis was performed as described in Example 2. The tissue samples used for analysis were the same as those described in Example 2. Table 4 shows the average quantitative GUS expression of each sampled tissue driven by the synthetic EXP regulatory elements tested, with "ND" in the table indicating that expression in a particular tissue was not determined. [Table 4]

[0146] As can be seen from Table 4, the synthetic At.GSP564 promoter and leader resulted in constitutive expression in all organs assayed. Expression was highest in leaves and seeds. Analysis of the TSS demonstrated consistent TSS. Operable linking of intron sequences altered expression in many organs, providing a means to "fine tune" constitutive expression. Differences in expression were observed when the synthetic introns I-At.GSI17.nno:1 (SEQ ID NO: 17) and I-At.GSI102.nno:1 (SEQ ID NO: 11) were operably linked. The synthetic introns enhanced expression compared to EXP-At.GSP564 in some tissues, but the level of enhancement varied from organ to organ. For example, enhancement in source leaves of V5 using the synthetic intron I-At.GSI102.nno:1 was stronger than that seen using the synthetic intron I-At.GSI17.nno:1. In roots of R1, the enhancement with the synthetic intron I-At.GSI17.nno:1 was stronger than that conferred by the synthetic intron I-At.GSI102.nno:1. Both synthetic introns led to a greater enhancement of expression in source leaves of R1 compared to the endogenous intron I-At.Cyco:2.

[0147] Analysis of the resulting mRNAs demonstrated proper and consistent processing of the intron elements.

[0148] The synthetic At.GSP564 promoter, P-At.GSP564.nno.3 (SEQ ID NO: 13) and leader, L-At.GSP564.nno:1 (SEQ ID NO: 14), which constitute EXP-At.GSP564 (SEQ ID NO: 12), confer constitutive expression of an operably linked transgene in stably transformed soybean plants. EXP-At.GSP564.nno+At.Cyco:2 (SEQ ID NO: 15), which contains the Arabidopsis intron I-At.Cyco:2 (SEQ ID NO: 33), and EXP-At.GSP564.nno+At.GSI17.nno:2 (SEQ ID NO: 16) and EXP-At.GSP564.nno+At.GSI102.nno:1 (SEQ ID NO: 18), which contain the synthetic introns I-At.GSI17.nno:1 (SEQ ID NO: 17) and I-At.GSI102.nno:1 (SEQ ID NO: 11), respectively, are synthetic EXPs but confer a unique constitutive expression pattern in stably transformed soybean plants. The synthetic introns I-At.GSI17.nno:1 (SEQ ID NO: 17) and I-At.GSI102.nno:1 (SEQ ID NO: 11) when operably linked to EXP-At.GSP564 (SEQ ID NO: 12) result in enhanced or regulated expression in many of the plant organs. These unique expression patterns can be used to direct a particular transgene to one of the four At.GS564EXPs, which has the most desirable native expression pattern.

[0149] Example 5 Analysis of EXP-At.GSP579.nno+At.GSI102.nno:3, a synthetic EXP that drives GUS expression in stably transformed soybean plants Soybean plants were transformed with vectors, specifically plant expression vectors, containing synthetic regulatory elements driving the expression of a β-glucuronidase (GUS) transgene. The resulting plants were analyzed for GUS protein expression to assess the effect of selected synthetic regulatory elements on expression.

[0150] Soybean plants were transformed with a plant GUS expression construct containing the synthetic EXP, EXP-At.GSP579.nno+At.GSI102.nno:3 (SEQ ID NO:22). EXP-At.GSP579.nno+At.GSI102.nno:3 contains EXP-At.GSP579 (SEQ ID NO:19), which consists of the At.GSP promoter and leader (SEQ ID NO:20 and 21, respectively) operably linked to the 5' side of the synthetic intron, I-At.GSI102.nno:1 (SEQ ID NO:11). The GUS transgene cassette further contains the 3'UTR sequence, T-Mt.RD22-1:2:1 (SEQ ID NO:37), derived from the dehydration response protein RD22 gene from Medicago truncatula.

[0151] Quantitative and qualitative GUS expression analysis was performed as described in Example 2. The tissue samples used for analysis were the same as those described in Example 2. Table 5 shows the average quantitative GUS expression of each sampled tissue stimulated by the synthetic EXP, EXP-At.GSP579.nno+At.GSI102.nno:3, where "ND" indicates that expression in a particular tissue was not determined. Table 5. Average quantitative GUS expression in stably transformed soybean plants driven by EXP-At.GSP579.nno+At.GSI102.nno:3. [Table 5]

[0152] As can be seen from Table 5, EXP-At.GSP579.nno+At.GSI102.nno:3 (SEQ ID NO: 22) results in constitutive expression in stably transformed soybean plants. The synthetic promoter P-At.GSP579.nno:2 (SEQ ID NO: 20) and leader L-At.GSP579.nno:1 (SEQ ID NO: 21) contained in EXP-At.GSP579 (SEQ ID NO: 19) drive constitutive expression of the operably linked transgene. From the previous examples in which the synthetic intron I-At.GSI102.nno:1 (SEQ ID NO: 11) was operably linked to another constitutive synthetic promoter, it can be inferred that I-At.GSI102.nno:1 enhanced or regulated the constitutive expression conferred by EXP-At.GSP579 in at least some of the sampled organs.

[0153] Example 6 Analysis of EXP-At.GSP571.nno+At.Cyco:2, a synthetic EXP that drives GUS expression in stably transformed cotton plants Cotton plants were transformed with vectors, specifically plant expression vectors, containing synthetic regulatory elements that drive expression of a β-glucuronidase (GUS) transgene, and the resulting plants were analyzed for GUS protein expression to assess the effect of the synthetic regulatory elements on expression.

[0154] Cotton plants were stably transformed using a plant binary vector containing the synthetic EXP, EXP-At.GSP571.nno+At.Cyco:2 (SEQ ID NO: 7), similar to that described in Example 3. The GUS transgene cassette contained EXP-At.GSP571.nno+At.Cyco:2 operably linked 5' to the coding sequence of β-glucuronidase (GUS, GOI-Ec.uidA+St.LS1:1:1, SEQ ID NO: 42) containing a processable intron from the potato light-inducible tissue-specific ST-LS1 gene (Genbank accession: X04753) operably linked 5' to the 3'UTR from the Gossypium barbadense FbLate-2 gene (T-Gb.FbL2:1, SEQ ID NO: 36). The resulting transformed cotton events were grown and tissue samples from the 4th node leaf; the 8th node petiole, sink and source leaves; the bracts and buds of pre-pollination buds; the anthers and floral ovules at anthesis; and the pod wall at 8 days after pollination (DAP) were sampled and assayed for qualitative and quantitative GUS expression.

[0155] Table 6 shows the average quantitative GUS expression of each sampled tissue stimulated by the synthetic EXP, EXP-At.GSP571.nno+At.Cyco:2. Table 6. Average quantitative GUS expression in stably transformed cotton plants driven by EXP-At.GSP571.nno+At.Cyco:2. [Table 6]

[0156] As can be seen from Table 6, EXP-At.GSP571.nno+At.Cyco:2 was expressed in all tissues sampled. Expression was highest in the leaves at the 4th node and lowest in the anthers at the anthesis stage. Expression in the sink and source leaves at the 8th node was relatively the same, about half of that in the leaves at the 4th node. Expression was also high in the pod wall. Table 6 demonstrates that the promoter P-At.GSP571.nno:5 (SEQ ID NO:5) can also promote constitutive expression in stably transformed cotton plants. The intron I-At.Cyco:2 (SEQ ID NO:33) in EXP-At.GSP571.nno+At.Cyco:2 enhanced the expression of the P-At.GSP571.nno:5 promoter in stably transformed soybean plants, as shown in Example 3.

[0157] Example 7 Analysis of the synthetic chimeric promoter P-At.GSP571 / 442 driving GUS expression in stably transformed soybean plants Soybean plants were transformed with vectors, specifically plant expression vectors, containing regulatory elements driving the expression of a β-glucuronidase (GUS) transgene, and the resulting plants were analyzed for GUS protein expression to assess the effect of selected synthetic regulatory elements on expression.

[0158] Soybean plants were transformed with a plant binary vector comprising a synthetic EXP, EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1 (SEQ ID NO: 23), consisting of a synthetic chimeric promoter P-At.GSP571 / 442 (SEQ ID NO: 25) operably linked to the 5' side of the synthetic promoter P-At.GSP442.nno:2 (SEQ ID NO: 2), operably linked to the 5' side of the leader L-At.Cyco-1:1:2 (SEQ ID NO: 40), which is operably linked to the 5' side of the intron I-At.Cyco:2 (SEQ ID NO: 33), and comprising a synthetic enhancer E-At.GSP571.nno:1 (SEQ ID NO: 24) derived from the synthetic promoter P-At.GSP571.nno:5 (SEQ ID NO: 5) operably linked to the 5' side of the synthetic leader L-At.GSP442.nno:1 (SEQ ID NO: 3). The GUS transgene cassette comprised EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1 operably linked 5' to the coding sequence for β-glucuronidase (GUS, GOI-Ec.uidA+St.LS1:1:1, SEQ ID NO:42) containing a processable intron derived from the potato light-inducible tissue-specific ST-LS1 gene (Genbank accession: X04753) operably linked 5' to the synthetic 3'UTR, T-Zm.GST59.nno:1 (SEQ ID NO:29).

[0159] A plant binary vector was also constructed to be used to compare the activity of the chimeric promoters. The vector contained an EXP, EXP-At.GSP442+LI-At.Cyco (SEQ ID NO: 43), which consists of a synthetic promoter, P-At.GSP442.nno:2 (SEQ ID NO: 2), operably linked 5' to a synthetic leader, L-At.GSP442.nno:1 (SEQ ID NO: 3), operably linked 5' to a leader, L-At.Cyco-1:1:2 (SEQ ID NO: 40), operably linked 5' to an intron, I-At.Cyco:2 (SEQ ID NO: 33). The binary vector is similar to that described in Examples 2-6, except that each GUS transgene cassette has a synthetic 3'UTR, T-Zm.GST59.nno:1 (SEQ ID NO: 29), operably linked 3' to the GUS coding sequence.

[0160] Soybean plants were transformed with the two binary vectors. Tissue samples of selected organs were taken at specific developmental stages and assayed for qualitative and quantitative GUS expression. Table 7 shows the average quantitative GUS expression of each sampled tissue stimulated by the synthetic EXPs EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1 and EXP-At.GSP442+LI-At.Cyco. Table 7. Average quantitative GUS expression in stably transformed soybean plants driven by EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1 and EXP-At.GSP442+LI-At.Cyco. [Table 7]

[0161] As can be seen from Table 7, the addition of the synthetic enhancer E-At.GSP571.nno:1 enhanced expression in many of the tissues sampled. Both EXPs resulted in constitutive expression in stably transformed soybean plants. The synthetic 3'UTR, T-Zm.GST59.nno:1, functioned similarly to the natural 3'UTR in providing proper termination and polyadenylation of the transcript.

[0162] Example 8 Analysis of the synthetic chimeric promoter P-At.GSP571 / 442 driving GUS expression in stably transformed cotton plants Cotton plants were transformed with vectors, specifically plant expression vectors, containing synthetic regulatory elements that drive expression of a β-glucuronidase (GUS) transgene, and the resulting plants were analyzed for GUS protein expression to assess the effect of selected synthetic regulatory elements on expression.

[0163] Cotton plants were transformed with a plant binary vector comprising a synthetic EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1 (SEQ ID NO: 23), which is composed of a synthetic chimeric promoter P-At.GSP571 / 442 (SEQ ID NO: 25) comprising a synthetic enhancer E-At.GSP571.nno:1 (SEQ ID NO: 24) derived from the synthetic promoter P-At.GSP571.nno:5 (SEQ ID NO: 5) operably linked to the 5' side of the synthetic promoter P-At.GSP442.nno:2 (SEQ ID NO: 2) operably linked to the 5' side of the synthetic leader L-At.GSP442.nno:1 (SEQ ID NO: 3), which is operably linked to the 5' side of the leader L-At.Cyco-1:1:2 (SEQ ID NO: 40), which is operably linked to the 5' side of the intron I-At.Cyco:2 (SEQ ID NO: 33). The GUS transgene cassette comprised EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1 operably linked 5' to the coding sequence for β-glucuronidase (GUS, GOI-Ec.uidA+St.LS1:1:1, SEQ ID NO:42) containing a processable intron derived from the potato light-inducible tissue-specific ST-LS1 gene (Genbank accession: X04753) operably linked 5' to the synthetic 3'UTR, T-Zm.GST59.nno:1 (SEQ ID NO:29). The resulting transformed cotton events were grown and tissue samples from the 4th node leaf; the 8th node petiole, sink and source leaves; the bracts and buds of pre-pollination buds; the anthers and floral ovules at anthesis; and the pod wall at 8 days after pollination (DAP) were sampled and assayed for qualitative and quantitative GUS expression.

[0164] Table 8 shows the average quantitative GUS expression of each sampled tissue stimulated by the synthetic EXP, EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1, where "bdl" means below the detection limit. Table 8. Average quantitative GUS expression in stably transformed cotton plants driven by EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1. [Table 8]

[0165] As can be seen from Table 8, EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1 (SEQ ID NO: 23) was able to drive constitutive GUS expression in sampled tissues. Expression in petioles was determined to be below the detection limit. Expression was highest in the source leaf at the 8th node. Expression was relatively equal in anthers and floral ovules at the anthesis stage. Furthermore, the synthetic 3'UTR, T-Zm.GST59.nno:1 (SEQ ID NO: 29), functioned similarly to the natural 3'UTR in providing proper termination and polyadenylation of the transcript.

[0166] Example 9 Analysis of EXP-At.GSP576.nno+At.Cyco:1, a synthetic EXP that drives GUS expression in stably transformed soybean plants Soybean plants were transformed with vectors, specifically plant expression vectors, containing synthetic regulatory elements driving the expression of a β-glucuronidase (GUS) transgene. The resulting plants were analyzed for GUS protein expression to assess the effect of selected synthetic regulatory elements on expression.

[0167] Soybean plants were transformed with a plant binary vector containing the synthetic EXP, EXP-At.GSP576.nno+At.Cyco:1 (SEQ ID NO: 45). The GUS transgene cassette also contained the 3'UTR from the FbLate-2 gene of Gossypium barbadense (T-Gb.FbL2:1, SEQ ID NO: 36) operably linked 3' to the GUS coding sequence. The resulting transformed soybean events were grown and tissue samples of selected organs from several developmental stages were sampled and assayed for qualitative and quantitative GUS expression. The expression of GUS in stably transformed soybean plants driven by EXP-At.GSP576.nno+At.Cyco:1 is shown in Table 9. Table 9. Average quantitative GUS expression in stably transformed soybean plants driven by EXP-At.GSP576.nno+At.Cyco:1. [Table 9]

[0168] As can be seen from Table 9, EXP-At.GSP576.nno+At.Cyco:1 (SEQ ID NO: 45) resulted in constitutive expression in stably transformed soybean plants. The synthetic promoter P-At.GSP576.nno:4 (SEQ ID NO: 27) and leader L-At.GSP576.nno:2 (SEQ ID NO: 28) drive the constitutive expression of the operably linked transgene. From the previous examples where the intron I-At.Cyco:2 (SEQ ID NO: 33) was operably linked to another constitutive synthetic promoter, it can be inferred that I-At.Cyco:2 enhanced or regulated the constitutive expression conferred by P-At.GSP576.nno:4 in at least some of the sampled organs.

[0169] Example 10 Analysis of EXP-At.GSP576.nno+At.GSI17.nno:3, a synthetic EXP that drives GUS expression in stably transformed soybean plants Contains a group of regulatory elements that drive expression of the β-glucuronidase (GUS) transgene The vectors, specifically plant expression vectors, were transformed into soybean plants, and the resulting plants were analyzed for GUS protein expression to assess the effect of the selected regulatory elements on expression.

[0170] Soybean plants are transformed with plant binary vectors containing either the synthetic EXP EXP-At.GSP576.nno+At.GSI17.nno:3 (SEQ ID NO:26) or the EXP EXP-At.Cyco:1:1 (SEQ ID NO:38). The GUS transgene cassette further contains the 3'UTR from the FbLate-2 gene of Gossypium barbadense (T-Gb.FbL2:1, SEQ ID NO:36) operably linked 3' to the GUS coding sequence. The resulting transformed soybean events are grown and tissue samples of selected organs from several developmental stages are sampled and assayed for qualitative and quantitative GUS expression. Expression of GUS in stably transformed soybean plants driven by EXP-At.GSP576.nno+At.GSI17.nno:3 is compared to that driven by EXP-At.Cyco:1:1. Expression of GUS in stably transformed soybean plants driven by EXP-At.GSP576.nno+At.GSI17.nno:3 demonstrates the ability of the synthetic promoter P-At.GSP576.nno:4 (SEQ ID NO:27) and leader L-At.GSP576.nno:2 (SEQ ID NO:28) to drive constitutive expression of an operably linked transgene.

[0171] As demonstrated in Examples 9 and 11, the synthetic promoter P-At.GSP576.nno:4 (SEQ ID NO:27) and leader L-At.GSP576.nno:2 (SEQ ID NO:28) drive constitutive expression of operably linked transgenes. As demonstrated in Example 4, the synthetic intron I-At.GSI17.nno:1 (SEQ ID NO:17) enhanced or regulated transgene expression in many plant organs when operably linked to EXP-At.GSP564 (SEQ ID NO:12). Similarly, it can be reasonably expected that the expression of the synthetic promoter P-At.GSP576.nno:4 and leader L-At.GSP576.nno:2 will be enhanced or regulated in the same way.

[0172] Example 11 Analysis of EXP-At.GSP576.nno+At.GSI17.nno:3, a synthetic EXP that drives GUS expression in stably transformed cotton plants Cotton plants were transformed with vectors, specifically plant expression vectors, containing synthetic regulatory elements that drive expression of a β-glucuronidase (GUS) transgene, and the resulting plants were analyzed for GUS protein expression to assess the effect of selected synthetic regulatory elements on expression.

[0173] Cotton plants were transformed with a binary vector containing the synthetic EXP, EXP-At.GSP576.nno+At.GSI17.nno:3 (SEQ ID NO:26), as described above in Example 10. The GUS transgene cassette also contained the 3'UTR from the Gossypium barbadense FbLate-2 gene (T-Gb.FbL2:1, SEQ ID NO:36) operably linked 3' to the GUS coding sequence. The resulting transformed cotton events were grown and tissue samples from the 4th node leaf; the 8th node petiole, sink and source leaves; the bracts and buds of the buds before pollination; the anthers and floral ovules at anthesis; and the pod wall at 8 days after pollination (DAP) were sampled and assayed for qualitative and quantitative GUS expression.

[0174] Table 10 shows the average quantitative GUS expression in each sampled tissue driven by the synthetic EXP-At.GSP576.nno+At.GSI17.nno:3. Table 10. Average quantitative GUS expression in stably transformed cotton plants driven by EXP-At.GSP576.nno+At.GSI17.nno:3. [Table 10]

[0175] As can be seen from Table 10, EXP-At.GSP576.nno+At.GSI17.nno:3 (SEQ ID NO: 26) promoted constitutive expression of GUS transgene in stably transformed cotton plants. Expression was highest in the 4th node leaf, the 8th node source leaf, and the pod wall at 8 DAP. The synthetic promoter P-At.GSP576.nno:4 (SEQ ID NO: 27) and leader L-At.GSP576.nno:2 (SEQ ID NO: 28) can promote constitutive expression of operably linked transgene in stably transformed cotton plants. As demonstrated in Example 4, the synthetic intron I-At.GSI17.nno:1 (SEQ ID NO: 17) enhanced or regulated transgene expression in many plant organs when operably linked to EXP-At.GSP564 (SEQ ID NO: 12). Similarly, it can be reasonably expected that expression of the synthetic promoter P-At.GSP576.nno:4 and leader L-At.GSP576.nno:2 in stably transformed cotton plants will be similarly enhanced or regulated.

[0176] Example 12 Enhancer elements derived from regulatory elements The enhancer is derived from the promoter elements shown as SEQ ID NO: 2, 5, 13, 20, 25, 27, 31 and 39. The enhancer element may consist of one or more cis-regulatory elements that can enhance or regulate the expression level of a transcribable DNA molecule when operably linked 5' or 3' to the promoter element or to additional enhancer elements operably linked to the promoter, or can bring about the expression of a transcribable DNA molecule in a particular cell type or plant organ or at a particular time point of development or circadian rhythm. The enhancer is made by removing the TATA box or functionally similar element and any downstream sequence from the promoter that initiates transcription from the promoters shown as SEQ ID NO: 2, 5, 13, 20, 25, 27, 31 and 39 or fragments thereof. For example, the synthetic enhancer E-At.GSP571.nno:1 (SEQ ID NO: 24) was derived from the synthetic promoter P-At.GSP571.nno:5 (SEQ ID NO: 5) and consists of nucleotides 1 to 422 of P-At.GSP571.nno:5, excluding the 3' downstream sequence containing the TATA box of the synthetic promoter.

[0177] Further refinement of the enhancer element may be required and is empirically established. In addition, the position of the enhancer element relative to other elements in the group of chimeric regulatory elements is also determined empirically, since the order of each element in the group of chimeric regulatory elements may have different effects depending on the relative position of each element. Some promoter elements will have multiple TATA boxes or TATA box-like elements and potentially multiple transcription start sites. In such a situation, it may be necessary to first identify where the first TSS is located and then start designing the enhancer using the first TSS to prevent cryptic transcription initiation in the putative enhancer element.

[0178] Enhancer elements derived from the synthetic promoter elements set forth as SEQ ID NOs: 2, 5, 13, 20, 25, 27, 31 and 39 can be cloned using methods known in the art and operably linked to the 5' or 3' side of the promoter element or to additional enhancer elements operably linked to the promoter. Alternatively, enhancer elements can be cloned using methods known in the art to provide larger enhancer elements consisting of two or more copies of the enhancer, and can be cloned using methods known in the art and operably linked to the 5' or 3' side of the promoter element or to additional enhancer elements operably linked to the promoter to generate chimeric transcriptional regulatory elements. Enhancer elements can also be cloned using methods known in the art and operably linked 5' to promoter elements from organisms of different genus, or operably linked 5' or 3' to additional enhancer elements from organisms of other genus that are operably linked to promoters from organisms of either the same or different genus, resulting in a chimeric regulatory element.The GUS-expressing plant transformation vector comprises a right border region from Agrobacterium tumefaciens (B-AGRtu.right border), a first transgene selection cassette used to select transformed plant cells that confer resistance to the antibiotic spectinomycin, and a 5' promoter element operably linked to a coding sequence for β-glucuronidase (GUS, GOI-Ec.uidA+St.LS1:1:1, SEQ ID NO: 42) containing a processable intron derived from the potato light-inducible tissue-specific ST-LS1 gene (Genbank accession: X04753) operably linked to the 3' termination region. Alternatively, a second transgene cassette for testing enhancer elements, consisting of an enhancer element operably linked 5' or 3' to a promoter operably linked 5' or 3' to an additional enhancer element operably linked to a promoter operably linked 5' to a leader element, and the left border region (B-AGRtu.left border) from A. tumefaciens, may be constructed using methods known in the art similar to the construct described in Example 2 resulting in a plant expression vector containing the left border region (B-AGRtu.left border) from A. tumefaciens. The resulting plasmid is used to transform soybean plants or plants of other genera by the methods described in the Examples. Alternatively, protoplast cells derived from soybean or other plant genera are transformed using methods known in the art to perform transient assays.

[0179] GUS expression driven by the regulatory element containing one or more enhancers is evaluated in a stable or transient plant assay to determine the effect of the enhancer element on the expression of the transcribable DNA molecule. Modifications to one or more enhancer elements or duplication of one or more enhancer elements can be performed based on empirical experiments and the resulting gene expression regulation observed with each regulatory element composition. The resulting change in the relative position of one or more enhancers in the regulatory or chimeric regulatory element may affect the transcriptional activity or specificity of the regulatory or chimeric regulatory element, and is empirically determined to identify the best enhancer for the desired transgene expression profile in soybean plants or plants of multiple genera.

[0180] Example 13 Analysis of the effect of the synthetic 3'UTR, T-Zm.GST7.nno:2, on GUS expression in stably transformed soybean plants Soybean plants were transformed with vectors, specifically plant expression vectors, containing regulatory elements driving the expression of a β-glucuronidase (GUS) transgene, and the resulting plants were analyzed for GUS protein expression to assess the effect of the selected regulatory elements on expression.

[0181] Soybean plants were transformed with two binary vectors containing EXP-At.GSP571 (SEQ ID NO: 4) driving GUS expression. The GUS transgene cassette also contained either the endogenous 3'UTR T-Mt.Sali3-2-1:2:1 (SEQ ID NO: 34) or the synthetic 3'UTR T-Zm.GST7.nno:2 (SEQ ID NO: 44). GUS protein expression was quantitatively measured in organs of stably transformed soybean plants transformed with the two constructs. GUS expression was compared between the constructs. Table 11 below shows the average GUS expression regulated by the synthetic 3'UTR T-Zm.GST7.nno:2 compared to the endogenous 3'UTR T-Mt.Sali3-2-1:2:1, where "nd" means not determined and "bdl" means below the detection limit. Table 11. Average quantitative GUS expression in stably transformed soybean plants. [Table 11]

[0182] As can be seen from Table 11, the synthetic 3'UTR T-Zm.GST7.nno:2 attenuated expression in all tissues assayed compared to the 3'UTR T-Mt.Sali3-2-1:2:1. The degree of attenuation varied from tissue to tissue, from 1.5-fold in R1 roots to 7.4-fold in V5 sink leaves. The use of 3'UTRs to attenuate expression in stably transformed plants is very useful. For example, 3'UTRs can be used in combination with other regulatory elements, such as promoters, leaders, and introns, to fine-tune the expression of transgenes, especially transgenes whose high expression may lead to phenotypic deviation effects that are detrimental to the transformed plant. Analysis of the resulting GUS transcripts confirmed that the synthetic 3'UTR T-Zm.GST7.nno:2 conferred proper termination of the transcript. The synthetic 3'UTR, T-Zm.GST7.nno:2, is capable of regulating expression and properly terminating transcription in stably transformed soybean plants.

[0183] Example 14 Analysis of synthetic 3'UTRs T-Zm.GST7.nno:2 and T-Zm.GST59.nno:1 for GUS expression in maize protoplast cells Maize leaf protoplasts were transformed with vectors, specifically expression vectors, containing the test regulatory elements driving expression of a β-glucuronidase (GUS) transgene. The resulting transformed maize leaf protoplasts were analyzed for GUS protein expression to assess the effect of selected regulatory elements on expression.

[0184] Maize protoplasts derived from leaf tissue were transformed with expression vectors containing synthetic expression elements and compared to expression elements known in the art. Two expression vectors were constructed to evaluate the activity of synthetic 3'UTRs T-Zm.GST7.nno:2 (SEQ ID NO:44) and T-Zm.GST59.nno:1 (SEQ ID NO:29), and two construct expression vectors were also constructed. Each of the four constructs contained a transgene cassette containing a constitutive promoter and leader EXP-CaMV.35S (SEQ ID NO:46) operably linked 5' to intron I-Zm.DnaK:1 (SEQ ID NO:47) operably linked 5' to GUS coding sequence GOI-Ec.uidA+St.LS1:1:1 (SEQ ID NO:42). Expression vectors used to evaluate the synthetic 3'UTRs contained either T-Zm.GST7.nno:2 or T-Zm.GST59.nno:1 operably linked 3' to the GUS coding sequence. One control vector contained the 3'UTR T-Os.LTP:1 (SEQ ID NO:48) operably linked 3' to the GUS coding sequence. The other control vector lacked the 3'UTR.

[0185] The plasmid used for co-transformation of protoplasts and normalization of data was also constructed using methods known in the art and contained a transgene cassette consisting of EXP-CaMV.35S (SEQ ID NO: 46) operably linked 5' to the coding sequence encoding NanoLuc® luciferase fluorescent protein (Promega, Madison, WI 53711) Nluc (SEQ ID NO: 49) operably linked 5' to the 3'UTR, T-Os.LTP:1 (SEQ ID NO: 48).

[0186] Corn leaf protoplasts were transformed using a PEG-based transformation method similar to that known in the art. Protoplast cells were transformed in a 96-well format. 3.2 x 10 cells were transformed per well using 12 micrograms of test or control vector DNA and 6 micrograms of NanoLuc® vector DNA. 5Protoplasts were transformed. After transformation, the protoplasts were incubated at 25°C in the dark for 16-20 hours. After incubation, the protoplasts were lysed and the lysate was used for measuring luciferase and GUS expression. To lyse the cells, the cells in the plate were pelleted by centrifugation, washed, resuspended in a smaller volume, and transferred to a strip-well tube. The tube was centrifuged again and the supernatant was aspirated, leaving behind the protoplast cell pellet. The cell pellet was resuspended in QB buffer (100 mM KPO4, pH 7.8, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 1 mM DTT). The cells were lysed by pipetting the cells vigorously several times, vortexing the tube, and leaving the tube to incubate on ice for 5 minutes. The lysate was then centrifuged to pellet the cell debris. The resulting lysate was then transferred to a clean plate.

[0187] Luciferase activity was assessed using Nano-Glo® Luciferase Assay Substrate (Promega, Madison, WI 53711) in QB buffer. Briefly, small volumes of lysate, QB buffer and Nano-Glo® Luciferase Assay Substrate / QB solution were mixed in a white 96-well plate, and fluorescence was then measured using a PHERAstar® plate reader (BMG LABTECH Inc., Cary, NC27513).

[0188] GUS activity was assessed using 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 where the hydroxyl groups are ionized. Addition of a basic solution of sodium carbonate stops the assay and simultaneously adjusts the pH for quantification of 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 reaction mixture were removed and added to stop buffer at three different time points: (1) immediately after mixing the lysate / MUG reaction as "time zero minutes", (2) at 20 minutes, and (3) at 60 minutes. Fluorescence was measured using a PHERAstar® plate reader (BMG LABTECH Inc., Cary, NC27513) with excitation at 355 nm and emission at 460 nm.

[0189] At least two plates were used for transformation, with 4-8 transformations per plate for each expression vector. Each construct was transformed in 4-8 wells per plate. An aliquot was removed from each transformation for MUG assay and the "nM MUG hydrolyzed" was derived from the standard curve on the plate. An additional aliquot was removed from each transformation for NanoLuc® reading (NanoLuc® RLU). For each expression vector, the average nM MUG hydrolyzed / NanoLuc® RLU was normalized with respect to the EXP-CaMV.35S / I-Zm.DnaK:1 / T-Os.LTP:1 expression vector, defined as 100%. Table 12 shows the average of the average values ​​of all plates used for transformation for each expression vector, including the synthetic 3'UTRs T-Zm.GST7.nno:2 and T-Zm.GST59.nno:1, as well as the controls. Table 12. Mean nM values ​​of hydrolyzed MUG / NanoLuc® RLU for each expression vector. [Table 12]

[0190] As can be seen from Table 12, the expression vector without 3'UTR showed lower expression than the T-Os.LTP:1 control. Compared to the T-Os.LTP:1 control, expression was enhanced by the synthetic 3'UTRs T-Zm.GST7.nno:2 and T-Zm.GST59.nno:1. Analysis of the transcripts demonstrated proper termination conferred by the synthetic 3'UTRs T-Zm.GST7.nno:2 and T-Zm.GST59.nno:1. The synthetic 3'UTRs T-Zm.GST7.nno:2 and T-Zm.GST59.nno:1 can regulate expression and properly terminate transcription in transformed maize leaf protoplast cells.

[0191] Example 15 Analysis of regulatory elements promoting GUS in cotton leaf protoplasts. Cotton leaf protoplasts were transformed with vectors, specifically expression vectors, containing regulatory elements driving the expression of a β-glucuronidase (GUS) transgene. The resulting transformed cotton leaf protoplasts were analyzed for GUS protein expression to assess the effect of the selected regulatory elements on expression.

[0192] The expression vectors containing the synthetic expression elements were transformed into cotton protoplasts derived from leaf tissue and compared to expression elements known in the art. Separate experiments were performed to evaluate the activity of EXPs EXP-At.GSP571 (SEQ ID NO: 4), EXP-At.GSP571.nno+At.GSI21.nno:10 (SEQ ID NO: 8), EXP-At.GSP571.nno+At.GSI102.nno:1 (SEQ ID NO: 10), EXP-At.GSP564.nno+At.GSI17.nno:2 (SEQ ID NO: 16) and EXP-At.GSP579.nno+At.GSI102.nno:3 (SEQ ID NO: 22). The expression elements were cloned into the expression vector and operably linked to the GUS coding sequence GOI-Ec.uidA+St.LS1:1:1 (SEQ ID NO: 42) containing a processible intron. The control expression vectors contained known expression elements in different configurations.

[0193] Two plasmids were also constructed for use in co-transformation and data normalization using methods known in the art. Each plasmid contained a specific luciferase coding sequence driven by a constitutive EXP sequence. The plant vector pFLUC contained a transgene cassette with a constitutive promoter operably linked 5' to an intron (EXP-CaMV.35S-enh+Zm.DnaK:1:1, SEQ ID NO:53) operably linked 5' to a firefly (Photinus pyralis) luciferase coding sequence (LUCIFERASE:1:3, SEQ ID NO:54) operably linked 5' to a 3'UTR from the Agrobacterium tumefaciens nopaline synthase gene (T-AGRtu.nos-1:1:13, SEQ ID NO:55). The plant vector pRLUC contained a transgene cassette with the constitutive EXP sequence (EXP-CaMV.35S-enh-Lhcb1, SEQ ID NO:56) operably linked 5' to the Renilla reniformis luciferase coding sequence (CR-Ren.hRenilla Lucife-0:0:1, SEQ ID NO:57) operably linked 5' to the 3'UTR from the Agrobacterium tumefaciens nopaline synthase gene (T-AGRtu.nos-1:1:13, SEQ ID NO:55).

[0194] Cotton leaf protoplasts were transformed using PEG-based transformation methods known in the art. Protoplast cells were transformed with plasmids, pFLUC and pRLUC, and equimolar amounts of the EXP expression vector. Both GUS and luciferase measurements were performed by placing aliquots of lysate preparations of cells transformed as described above into two different small well trays. One tray was used for GUS measurements and the other tray was used to perform dual luciferase assays using the Dual Luciferase Reporter Assay System (Promega Corp., Madison, WI; see, e.g., Promega Notes Magazine, No: 57, 1996, p. 02). Sample measurements were based on multiple transformations similar to those shown in Example 14. Mean GUS / FLUC values ​​were calculated as in Example 14 but were not normalized to the control EXP vector.

[0195] EXPs EXP-At.GSP571 (SEQ ID NO: 4), EXP-At.GSP571.nno+At.GSI21.nno:10 (SEQ ID NO: 8) and EXP-At.GSP571.nno+At.GSI102.nno:1 (SEQ ID NO: 10) were cloned into plant expression vectors operably linked to the 5'-side of the GUS coding sequence (SEQ ID NO: 42) operably linked to the 5'-side of the 3'UTR T-Mt.Sali3-2-1:2:1 (SEQ ID NO: 34). Two control plant expression vectors were constructed with EXP-At.Bglu21+At.Cyco:2 (SEQ ID NO: 50), an EXP known to express poorly in cotton leaf protoplasts, and EXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO: 51), an EXP known to express well in cotton leaf protoplasts. Control EXP was operably linked to the same GUS and 3'UTR sequences. Additionally, a plant expression vector containing a GUS transgene cassette containing EXP-At.GSP571 (SEQ ID NO: 4), operably linked to GUS, contained a synthetic 3'UTR, T-Zm.GST7.nno:2 (SEQ ID NO: 44), to assess the activity of the synthetic 3'UTR. The average GUS / FLUC values ​​of multiple transformations are shown in Table 13. Table 13. Average GUS / FLUC values ​​from transformed cotton leaf protoplasts. [Table 13]

[0196] As can be seen from Table 13, EXPs EXP-At.GSP571 (SEQ ID NO: 4), EXP-At.GSP571.nno+At.GSI21.nno:2 (SEQ ID NO: 8) and EXP-At.GSP571.nno+At.GSI102.nno:1 (SEQ ID NO: 10) demonstrated expression in cotton leaf protoplast cells. Synthetic 3'UTR T-Zm.GST7.nno:10 (SEQ ID NO: 44) functioned similarly to endogenous 3'UTR T-Mt.Sali3-2-1:2:1.

[0197] EXP, EXP-At.GSP564.nno+At.GSI17.nno:2 (SEQ ID NO:16), was cloned into a plant expression vector operably linked 5' to the GUS coding sequence (SEQ ID NO:42) operably linked 5' to the endogenous 3'UTR, T-Mt.Oxr-1:2:1 (SEQ ID NO:35). Two control plant expression vectors were constructed with EXP-Gm.Sphas1:1:1 (SEQ ID NO:52), an EXP known to express poorly in cotton leaf protoplasts, and EXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO:51), an EXP known to express well in cotton leaf protoplasts. The control EXP was operably linked to the same GUS and 3'UTR sequences. The average GUS / FLUC values ​​of multiple transformations are shown in Table 14. Table 14. Average GUS / FLUC values ​​from transformed cotton leaf protoplasts. [Table 14]

[0198] As can be seen from Table 14, the synthetic EXP, EXP-At.GSP564.nno+At.GSI17.nno:1 (SEQ ID NO: 16), demonstrated expression in cotton leaf cell protoplasts.

[0199] EXP, EXP-At.GSP579.nno+At.GSI102.nno:3 (SEQ ID NO:22), was cloned into a plant expression vector operably linked to the 5' end of the GUS coding sequence (SEQ ID NO:42) operably linked to the 5' end of the endogenous 3'UTR, T-Mt.RD22-1:2:1 (SEQ ID NO:37). Two control plant expression vectors were cloned with EXP-Gm.Sphas1:1:1 (SEQ ID NO:52), an EXP known to express poorly in cotton leaf protoplasts, and EXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO:51), an EXP known to express well in cotton leaf protoplasts. A control EXP was operably linked to the same GUS and 3'UTR sequences. The average GUS / FLUC values ​​of multiple transformations are shown in Table 15. Table 15. Average GUS / FLUC values ​​from transformed cotton leaf protoplasts. [Table 15]

[0200] As can be seen from Table 15, the synthetic EXP, EXP-At.GSP579.nno+At.GSI102.nno:3 (SEQ ID NO: 22), demonstrated expression in cotton leaf cell protoplasts.

[0201] Having illustrated and described the principles of the present invention, it will be apparent to those skilled in the art that modifications in arrangement and detail may be made without departing from such principles. The inventors claim all modifications that come within the spirit and scope of the claims. All publications and published patents cited in this specification 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) a sequence having at least 90 percent sequence identity to SEQ ID NO: 16 or 13 and having gene regulatory activity; and (b) a sequence comprising SEQ ID NO: 16 or 13 A recombinant DNA molecule comprising a DNA sequence selected from the group consisting of:

2. 2. The recombinant DNA molecule of claim 1, wherein said DNA sequence is operably linked to a heterologous transcribable DNA molecule.

3. 2. The recombinant DNA molecule of claim 1, wherein the DNA sequence has at least 95 percent sequence identity with the DNA sequence of SEQ ID NO: 16 or 13 and has gene regulatory activity.

4. 2. The recombinant DNA molecule of claim 1, wherein the DNA sequence has at least 97 percent sequence identity with the DNA sequence of SEQ ID NO: 16 or 13 and has gene regulatory activity.

5. 3. The recombinant DNA molecule of claim 2, wherein said heterologous transcribable DNA molecule comprises a gene of agricultural scientific interest.

6. 6. The recombinant DNA molecule of claim 5, wherein said gene of agricultural scientific interest confers herbicide resistance in plants.

7. 6. The recombinant DNA molecule of claim 5, wherein said gene of agricultural scientific interest confers pest resistance in plants.

8. A transgenic plant cell comprising the recombinant DNA molecule of claim 1.

9. 9. The transgenic plant cell of claim 8, wherein said DNA sequence is operably linked to a heterologous transcribable DNA molecule.

10. The transgenic plant cell of claim 8 , wherein the transgenic plant cell is a monocotyledonous plant cell.

11. 9. The transgenic plant cell of claim 8, wherein the transgenic plant cell is a dicotyledonous plant cell.

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

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

14. 13. A transgenic seed, said seed comprising the recombinant DNA molecule of claim 1.

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

16. 16. The method of claim 15, wherein the commodity product is selected from the group consisting of protein concentrates, protein isolates, grains, starches, seeds, meal, flour, biomass, and seed oils.

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

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