EA048365B1 - REGULATORY ELEMENTS OF PLANTS AND THEIR USE - Google Patents

Description

Link to related applications

This application claims the benefit of U.S. Provisional Application No. 62/448,019, filed January 19, 2017, which is incorporated herein by reference in its entirety.

Enabling a sequence list

The machine-readable form of the sequence listing, which is contained in a file named MONS436WO-sequence_listing.txt, has a size of 59,917 bytes (as measured by Microsoft Windows®), was generated on January 12, 2018, and is filed electronically concurrently with this application and is incorporated herein by reference in its entirety.

Field of technology to which the invention relates

The invention relates to the field of plant molecular biology and plant genetic engineering. More specifically, the invention relates to DNA molecules that can be used to modulate gene expression in plants.

State of the art

Regulatory elements are genetic elements that regulate gene activity by modulating the transcription of a functionally related transcribed DNA molecule. Such elements may include promoters, leaders, introns, and 3' untranslated regions and may be useful in the fields of plant molecular biology and plant genetic engineering.

Brief summary of the invention

The invention provides new synthetic gene regulatory elements for use in plants. The invention also relates to recombinant DNA molecules and constructs containing the regulatory elements. The present invention also relates to transgenic plant cells, plants and seeds containing the synthetic regulatory elements. In one embodiment, the synthetic regulatory elements are operably linked to a heterologous transcribed DNA molecule. The present invention also provides methods of using the synthetic regulatory elements and methods of producing and using recombinant DNA molecules containing the synthetic regulatory elements and transgenic plant cells, plants and seeds containing the synthetic regulatory elements operably linked to a transcribed DNA molecule.

Thus, in one aspect, the invention relates to a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: (a) a sequence at least 85% identical 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, wherein the fragment has gene regulatory activity; wherein the sequence is operably linked to a heterologous transcribed DNA molecule. By heterologous transcribed DNA molecule is meant that the transcribed DNA molecule is heterologous with respect to the polynucleotide sequence to which it is operably linked. In particular embodiments, the recombinant DNA molecule comprises a DNA sequence that is at least about 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the DNA sequences of SEQ ID NOS: 1-29 and 43-45. In particular embodiments, the DNA sequence comprises a regulatory element. In some embodiments, the regulatory element comprises a promoter. In yet other embodiments, the heterologous transcribed DNA molecule comprises a gene of agronomic interest, such as a gene capable of conferring herbicide resistance in plants or a gene capable of conferring plant pest resistance in plants. In yet other embodiments, the invention relates to a construct comprising a recombinant DNA molecule as provided herein.

In another aspect, provided herein are transgenic plant cells comprising a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: (a) a sequence at least about 85 percent identical to any one of SEQ ID NOs: 1-29 and 43-45; (b) a sequence comprising any one of SEQ ID NOs: 1-29 and 43-45; and (c) a fragment of any one of SEQ ID NOs: 1-29 and 43-45, wherein the fragment has gene regulatory activity; wherein the DNA sequence is operably linked to a heterologous transcribed DNA molecule. In certain embodiments, the transgenic plant cell is a monocot plant cell. In other embodiments, the transgenic plant cell is a dicot plant cell.

In yet another aspect, the present document further provides a transgenic plant or part thereof comprising a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: a) a sequence at least 85 percent identical to any one of SEQ ID NOs: 1-29 and 43-45; b) a sequence comprising any one of SEQ ID NOs: 1-29 and 43-45, and c) a fragment of any one of SEQ ID NOs: 1-29 and 43-45, wherein the

- 1 048365 fragment has gene regulatory activity; wherein the DNA sequence is operably linked to a heterologous transcribed DNA molecule. In specific embodiments, the transgenic plant is a progeny plant of any generation that contains a recombinant DNA molecule. This document also provides a transgenic seed containing a recombinant DNA molecule, which upon germination grows into such a transgenic plant.

In another aspect, the invention relates to a method for producing a commercial product comprising obtaining a transgenic plant or part thereof containing a recombinant DNA molecule according to the invention and obtaining a commercial product therefrom. In one embodiment, the commercial product is processed seeds, grains, plant parts, vegetable oils and cereals.

In another aspect, the invention relates to a method for producing a transgenic plant containing a recombinant DNA molecule according to the invention, comprising transforming a plant cell using a recombinant DNA molecule according to the invention to produce a transformed plant cell and regenerating a transgenic plant from the transformed plant cell.

Brief description of sequences

SEQ ID NO: 1 is the DNA sequence of a group of synthetic expression regulatory elements (EXP), EXP-At.GSP442.nno+At.Cyco:3, containing a synthetic promoter (PAt.GSP442.nno:2) operably linked 5' to a synthetic leader (L-At.GSP442.nno:1) operably linked 5' to an intron (1-A1.Cyco:2).

SEQ ID NO: 2 is the synthetic promoter sequence, PAt.GSP442.nno:2.

SEQ ID NO: 3 is the synthetic leader sequence, LAt.GSP442.nno:1.

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).

SEQ ID NO: 5 is the synthetic promoter sequence, PAt.GSP571.nno:5.

SEQ ID NO: 6 is the synthetic leader sequence, LAt.GSP571.nno:1.

SEQ ID NO: 7 is the DNA sequence of a group of synthetic expression regulatory elements (EXP), EXP-At.GSP571.nno+At.Cyco:2, containing a synthetic promoter (PAt.GSP571.nno:5) operably linked 5' to a synthetic leader (L-At.GSP571.nno:1) operably linked 5' to an intron ([-A1.Cyco:2).

SEQ ID NO: 8 is the DNA sequence of a group of synthetic expression regulatory elements (EXP), EXP-At.GSP571.nno+At.GSI21.nno:10, containing 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).

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

SEQ ID NO: 10 is the DNA sequence of a synthetic EXP, EXPAt.GSP571.nno+At.GSI102.nno:1, comprising 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).

SEQ ID NO: 11 is a synthetic intron sequence, IAt.GSI102.nno:1.

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

SEQ ID NO: 13 is the synthetic promoter sequence, PAt.GSP564.nno:3.

SEQ ID NO: 14 is the synthetic leader sequence, LAt.GSP564.nno:1.

SEQ ID NO: 15 is the DNA sequence of a synthetic EXP, EXPAt.GSP564.nno+At.Cyco:2, containing 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 (IA1Cyco:2).

SEQ ID NO: 16 is the DNA sequence of a synthetic EXP, EXPAt.GSP564.nno+At.GSI17.nno:2, comprising 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).

SEQ ID NO: 17 is a synthetic intron sequence, I- 2 048365

AtGSI17.rno:1.

SEQ ID NO:18 is the DNA sequence of a synthetic EXP, EXPAt.GSP564.nno+At.GSI102.nno:1, comprising 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).

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

SEQ ID NO:20 is the synthetic promoter sequence, PAt.GSP579.nno:2.

SEQ ID NO:21 is the synthetic leader sequence, LAt.GSP579.nno:1.

SEQ ID NO: 22 is the DNA sequence of a synthetic EXP, EXPAt.GSP579.nno+At.GSI102.nno:3, comprising 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).

SEQ ID NO: 23 is the DNA sequence of a synthetic EXP, EXPAt.GSP571.nno+At.GSP442.nno+At.Cyco:1, comprising a synthetic chimeric promoter (PAt.GSP571/442, which consists of a synthetic enhancer (E-At.GSP571.nno:1) operably linked 5' to a 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).

SEQ ID NO:24 is a synthetic enhancer sequence, E-At.GSP571.nno:1.

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

SEQ ID NO: 26 is the DNA sequence of a synthetic EXP, EXPAt.GSP576.nno+At.GSI17.nno:3, comprising a synthetic promoter (P-At.GSP576.nno:4) operably linked 5' to a synthetic leader (L-At.GSP57 6.nno:2) operably linked 5' to a synthetic intron (I-At.GSI17.nno:1).

SEQ ID NO:27 is the synthetic promoter sequence, PAt.GSP576.nno:4.

SEQ ID NO: 28 is the synthetic leader sequence, LAt.GSP576.nno:2.

SEQ ID NO:29 is the synthetic 3' untranslated region, T-Zm.GST59.nno:1.

SEQ ID NO: 30 is the DNA sequence of a synthetic EXP, EXPAt.GSP221+At.Cyco:3, comprising a synthetic promoter (P-At.GSP221:3) operably linked 5' to a synthetic leader (L-At.GSP221:1) operably linked 5' to an intron (T-LTCyco^).

SEQ ID NO:31 is the synthetic promoter sequence, PAt.GSP221:3.

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

SEQ ID NO:33 is the intron sequence I-Lt.Cyco:2 derived from the Arabidopsis cytochrome c oxidase subunit VIa gene.

SEQ ID NO: 34 is the sequence of the 3'-untranslated region, T-Mt.Sali3-21:2:1, derived from the Sali3 gene of Medicago truncatula.

SEQ ID NO: 35 is the 3' untranslated region sequence, T-Mt.Oxr1:2:1, derived from the putative oxidoreductase protein (OXR) gene from Medicago truncatula.

SEQ ID NO: 36 is the 3' untranslated region sequence, T-Gb.FbL2:1, derived from the FbLate-2 gene of Gossypium barbadense.

SEQ ID NO: 37 is the 3' untranslated region sequence, T-Mt.RD221:2:1, derived from the dehydration sensitive protein gene RD22 of Medicago truncatula.

SEQ ID NO: 38 is the EXP DNA sequence obtained from the Arabidopsis cytochrome c oxidase subunit VIa gene, EXP-At.Cyco:1:1, containing 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 (IAt.Cyco-1:1:1).

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

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

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

SEQ ID NO: 42 is the coding sequence for β-glucuronidase (GUS) with

- 3 048365 modifiable intron derived from the light-inducible tissue-specific potato ST-LS1 gene (Genbank Accession: X04753).

SEQ ID NO: 43 is an EXP DNA sequence, EXP-At.GSP442+LI-At.Cyco, containing a 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-A1.Cyco-1:1:2, operably linked 5' to an intron, 1-AbCyco:2.

SEQ ID NO: 44 is the DNA sequence of the synthetic 3' untranslated region, T-Zm.GST7.nno:2.

SEQ ID NO: 45 is an EXP DNA sequence, EXP-At.GSP576.nno+At.Cyco:1, containing a synthetic promoter, P-At.GSP564.nno:3, operably linked 5' to a synthetic leader, L-At.GSP5 64.nno:1, which is operably linked 5' to an intron, 1-AbCyco:2.

SEQ ID NO: 46 is the DNA sequence of EXP, EXP-CaMV.35S, including the 35S promoter and leader derived from cauliflower mosaic virus.

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

SEQ ID NO: 48 is the DNA sequence of the 3' untranslated region, TOs.LTP: 1, derived from the lipid transfer protein (LTP)-like protein gene from Oryza sativa.

SEQ ID NO: 49 is the coding sequence for the NanoLuc® (Promega, Madison, WI 53711) luciferase fluorescent protein, Nluc, which was generated by directed evolution from deep-sea shrimp (Oplophorus gacilirostris) luciferase.

SEQ ID NO: 50 is the EXP DNA sequence, EXP-At.Bglu21+At.Cyco:2, containing the promoter and leader of the Arabidopsis thaliana beta-glucuronidase 21 gene, operably linked 5' to the intron, I-A1Cyco-1:1:1.

SEQ ID NO: 51 is the DNA sequence of EXP, EXP-CaMV.35SEnh+Ph.DnaK:1:3, containing the 35S promoter of the enhanced cauliflower mosaic virus operably linked 5' to the gene of the leader of the heat shock protein 70 (HSP70) gene from petunia hybrids.

SEQ ID NO: 52 is the DNA sequence of EXP, EXP-Gm.Sphas1:1:1, containing the promoter and leader of the soybean 7S alpha primary gene.

SEQ ID NO: 53 is the EXP DNA sequence, EXP-CaMV.35SEnh+Zm.DnaK:1:1, containing the cauliflower mosaic virus 35S promoter operably linked 5' to the intron, I-Zm.DnaK:1.

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

SEQ ID NO: 55 is the DNA sequence of the 3' untranslated region, TAGRtu.nos-1:1:13, derived from the nopaline synthase gene of Agrobacterium tumefaciens.

SEQ ID NO: 56 is the DNA sequence of EXP, EXP-CaMV.35S-Enh-Lhcb1, containing the enhanced 35S promoter of cauliflower mosaic virus operably linked 5' to the leader of the chlorophyll a/b binding gene of the light harvesting complex of Triticum aestivum (wheat).

SEQ ID NO: 57 is the DNA sequence encoding luciferase protein (CRRen.hRenilla Lucife-0:0:1) derived from Renilla reniformis.

Detailed description of the invention

The invention provides synthetic regulatory elements having gene regulatory activity in plants. The nucleotide sequences of these synthetic regulatory elements are presented as SEQ ID NO: 1-32 and SEQ ID NO: 43-45. Such synthetic regulatory elements are capable of influencing the expression of an operatively linked transcribed DNA molecule in plant tissues and, therefore, regulating the expression of genes of an operatively linked transgene in transgenic plants. The invention also provides methods for modifying, producing and using recombinant DNA molecules that contain the provided synthetic regulatory elements. The invention also provides compositions that include transgenic plant cells, plants, plant parts and seeds containing recombinant DNA molecules according to the invention, and methods for producing and using them.

The following explanations of terms and methods are provided to better describe the present compounds, compositions and methods and to instruct those skilled in the art in practicing the present disclosure. Unless otherwise indicated, terms should be understood in accordance with standard usage by those skilled in the art.

DNA molecules

As used herein, the term DNA or DNA molecule refers to a double-stranded DNA molecule of genomic or synthetic origin, that is, a polymer of deoxyribonucleotide bases or a DNA molecule read from the 5' end (upstream) to the 3' end (downstream). As used herein, the term DNA sequence refers to the nucleotide sequence of a DNA molecule. The nomenclature used herein is consistent with that of 37 U.S.C.F.R. 1.822 and is set forth in

- 4 048365 tables in WIPO ST.25 (1998), Appendix 2, Tables 1 and 3.

As used herein, the term recombinant DNA molecule is a DNA molecule that contains a combination of DNA molecules that would not naturally occur in such a combination without human intervention. For example, a recombinant DNA molecule may be a DNA molecule that consists of at least two DNA molecules that are heterologous to each other, a DNA molecule that contains a DNA sequence that differs from DNA sequences that exist in nature, a DNA molecule that contains a synthetic DNA sequence, or a DNA molecule that has been incorporated into the DNA of a host cell by genetic transformation or gene editing.

In the context of this document, the term synthetic nucleotide sequence or synthetic polynucleotide sequence represents a nucleotide sequence that is not known to occur in nature, that does not occur in nature, or that does not occur without human intervention. The gene regulatory elements of the present invention include synthetic nucleotide sequences. Preferably, the synthetic nucleotide sequences have little or no extended homology to natural sequences. Extended homology in this context typically refers to 100% sequence identity extending beyond about 25 nucleotides of contiguous sequence.

Reference in this application to an isolated DNA molecule or an equivalent term or phrase is intended to mean that the DNA molecule is a molecule that is present alone or in combination with other compositions, but not in its natural environment. For example, nucleic acid elements such as a coding sequence, an intron sequence, an untranslated leader sequence, a promoter sequence, a transcription termination sequence, and the like that are naturally found in the DNA of an organism's genome are not considered isolated as long as the element is in the genome of the organism and at the location within the genome in which it naturally occurs. However, each of these elements and portions thereof will be isolated within the scope of this disclosure if the element is outside the genome of the organism and the location within the genome in which it naturally occurs. In one embodiment, the term isolated refers to a DNA molecule that is at least partially separated from certain nucleic acids that normally flank the DNA molecule in its native or natural state. Thus, DNA molecules condensed with regulatory or coding sequences to which they are not normally associated, such as as a result of recombinant technologies, are considered isolated herein. Such molecules are considered isolated when they are integrated into a chromosome of a host cell or are present in a nucleic acid solution with other DNA molecules, since they are not in their natural state. For the purposes of the present disclosure, any transgenic nucleotide sequence, i.e., a DNA nucleotide sequence inserted into the genome of plant or bacterial cells or present in an extrachromosomal vector, shall be considered an isolated nucleotide sequence, regardless of whether it is present in a plasmid or similar structure used to transform cells, in the genome of a plant or bacterium, or is present in detectable amounts in tissues, progeny, biological samples, or commercial products obtained from a plant or bacterium.

As used herein, the term sequence identity refers to the degree to which two optimally aligned polynucleotide sequences or two optimally aligned polypeptide sequences are identical. An optimal sequence alignment is created by manually aligning two sequences, such as a reference sequence and another sequence, in a manner that maximizes the number of nucleotide matches in the sequence alignment with appropriate internal nucleotide insertions, deletions, or gaps. As used herein, the term reference sequence refers to the DNA sequence represented by SEQ ID NOs: 1-32 and SEQ ID NOs: 43-45.

As used herein, the term percent sequence identity or percent identity or % identity is the proportion of identity multiplied by 100. The proportion of identity for a sequence optimally aligned to a reference sequence is the number of nucleotide matches in the optimal alignment divided by the total number of nucleotides in the reference sequence, such as the total number of nucleotides in the full length of the entire reference sequence. Thus, one embodiment of the invention relates to a DNA molecule comprising a sequence that, when optimally aligned to a reference sequence provided herein as any of SEQ ID NOs: 1-32 and SEQ ID NOs: 43-45, is at least 85% identical to the reference sequence, at least 86% identical to the reference sequence, at least 87% identical to the reference sequence, at least

- 5 048365

88% identical to the reference sequence by at least 89%, identical to the reference sequence by at least 90%, identical to the reference sequence by at least 91%, identical to the reference sequence by at least 92%, identical to the reference sequence by at least 93%, identical to the reference sequence by at least 94%, identical to the reference sequence by at least 95%, identical to the reference sequence by at least 96%, identical to the reference sequence by at least 97%, identical to the reference sequence by at least 98%, identical to the reference sequence by at least 99% or identical to the reference sequence by at least 100%. In yet other specific embodiments, a sequence having percent identity to any of SEQ ID NOs: 1-32 and SEQ ID NOs: 43-45 may be defined as exhibiting the promoter activity that the original sequence from which it is derived has. A sequence having percent identity to any of SEQ ID NOs: 1-32 and SEQ ID NO: 4345 may further comprise a minimal promoter that provides a basic level of transcription and consists of a TATA box or equivalent sequence for recognizing and binding the RNA polymerase II complex to initiate transcription.

Regulatory elements

Regulatory elements such as promoters, leaders (also known as 5'-untranslated regions), enhancers, introns, and transcription termination regions (or 3'-untranslated regions) play an integral role in the overall expression of genes in living cells. As used herein, the term regulatory element refers to a DNA molecule that has gene regulatory activity. The term gene regulatory activity, as used herein, refers to the ability to influence the expression of a functionally linked transcribed DNA molecule, such as by affecting the transcription and/or translation of a functionally linked transcribed DNA molecule. Regulatory elements such as promoters, leaders, enhancers, introns, and 3'-untranslated regions that function in plants are useful for modifying plant phenotypes through genetic engineering.

As used herein, the term expression regulatory element group or EXP may refer to a group of operably linked regulatory elements such as enhancers, promoters, leaders, and introns. For example, a group of expression regulatory elements may consist of, for example, a promoter operably linked 5' to a leader sequence. EXP useful for practicing the present invention include SEQ ID NOs: 1, 4, 7, 8, 10, 12, 15, 16, 18, 19, 22, 23, 26, 30, 43, and 45.

Regulatory elements may be characterized by an inherent gene expression pattern, such as positive and/or negative effects such as constitutive expression or temporal, spatial, developmental, tissue, environmental, physiological, pathological, cell cycle-related and/or chemically responsive expression, and any combination thereof, as well as quantitative or qualitative indicators. As used herein, the term gene expression pattern is any pattern of transcription of an operatively linked DNA molecule into a transcribed RNA molecule. The transcribed RNA molecule may be translated to produce 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), and the like.

As used herein, protein expression is any pattern of translation of a transcribed RNA molecule into a protein molecule. Protein expression may be characterized by its temporal, spatial, developmental, or morphological qualities, as well as by quantitative or qualitative measures.

A promoter is useful as a regulatory element for modulating the expression of an operably linked transcribed DNA molecule. As used herein, the term promoter generally refers to a DNA molecule that is involved in recognizing and binding RNA polymerase II and other proteins, such as trans-acting transcription factors, to initiate transcription. A promoter may be originally isolated from the 5'-untranslated region (5'-UTR) of a genomic copy of a gene. Alternatively, promoters may be synthetically produced or modified DNA molecules. Promoters may also be chimeric. Chimeric promoters are produced by fusing two or more heterologous DNA molecules. Promoters useful for practicing the present invention include promoter elements contained in any of SEQ ID NOs: 2, 5, 13, 20, 25, 27, 31, and 39, or fragments or variants thereof. In particular embodiments, the claimed DNA molecules and any variants or derivatives thereof as described herein are further defined as comprising promoter activity, i.e., they are capable of acting as a promoter in a host cell, such as a transgenic plant cell. In yet other particular embodiments, the fragment may be defined as exhibiting the promoter activity possessed by the original promoter molecule from which it is derived, or the fragment may comprise a minimal promoter that provides a basal level of trans

- 6 048365 transcription and consists of a TATA box or equivalent DNA sequence for recognition and binding of the RNA polymerase II complex to initiate transcription.

In one embodiment, fragments of a promoter sequence disclosed herein are provided. The promoter fragments may comprise promoter activity as described above, and may be useful alone or in combination with other promoters and promoter fragments, such as in the construction of chimeric promoters, or in combination with other expression elements and expression element fragments. In particular embodiments, fragments of a promoter are provided that comprise at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900, or at least about 1000 contiguous nucleotides or more of a DNA molecule having promoter activity as described herein. In certain embodiments, the invention provides fragments of a promoter as provided herein that have the activity of a full-length sequence. Methods for obtaining such fragments from the original promoter molecule are well known in the art.

Compositions derived from any of the promoter elements contained in any of SEQ ID NOs: 2, 5, 13, 20, 25, 27, 31 and 39, such as, for example, internal or 5' deletions, can be made using methods known in the art to improve or alter expression, including by removing elements that have a positive or negative effect on expression; redundant elements that have a positive or negative effect on expression and/or duplicating or removing elements that have a tissue-specific or cell-specific effect on expression. Compositions derived from any of the promoter elements contained in any of SEQ ID NOs: 2, 5, 13, 20, 25, 27, 31 and 39, consisting of 3' deletions in which the TATA box element or its equivalent sequence and the subsequent downstream sequence are removed can be used, for example, to create enhancer elements. Further deletions can be made to remove any elements that have positive or negative; tissue-specific, cell-specific or time-specific (e.g., but not limited to, circadian rhythm) effects on expression. Any of the promoter elements contained in any of SEQ ID NOs: 2, 5, 13, 20, 25, 27, 31 and 39 and fragments or enhancers derived therefrom can be used to produce compositions of chimeric transcriptional regulatory elements.

According to the invention, a promoter or promoter fragment can be analyzed for the presence of known promoter elements, i.e., DNA sequence characteristics such as the TATA box 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 an expression pattern similar to the original promoter.

As used herein, the term leader refers to a DNA molecule isolated from the 5' untranslated region (5'-UTR) of a gene and is typically defined as the nucleotide segment between the transcription start site (TSS) and the start site of the protein coding sequence. Alternatively, leaders may be synthetically produced or modified DNA molecules. A leader may be used as a 5' regulatory element to modulate the expression of an operably linked transcribed DNA molecule. Leader molecules may be used with a heterologous promoter or with their native promoter. Leaders useful for practicing the present invention include 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, or fragments or variants thereof. In particular embodiments, such DNA sequences can be determined to be 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 containing leader activity.

Leader sequences (also referred to as 5' UTRs) represented by 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 that may influence the transcription or translation of an operably linked transcribed DNA molecule. Leader sequences represented by 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 can be used in accordance with the invention to create chimeric regulatory elements that affect the transcription or translation of an operably linked transcribed DNA molecule.

As used in this document, the term intron refers to a DNA molecule that can

- 7 048365 be isolated or identified from a gene and can be defined, in general, as a region removed by splicing during processing of messenger RNA (mRNA) prior to translation. Alternatively, an intron can be a synthetically produced or modified DNA element. An intron can contain enhancer elements that affect the transcription of functionally related genes. An intron can be used as a regulatory element to modulate the expression of a functionally related transcribed DNA molecule. The construct can contain an intron, and the intron may or may not be heterologous to the transcribed DNA molecule. Examples of introns known in the art include the rice actin intron and the maize HSP70 intron.

In plants, inclusion of certain introns in gene constructs results in increased mRNA and protein accumulation compared to constructs lacking the intron. This effect has been termed intron-mediated enhancement (IME) of gene expression. Introns known to promote expression in plants have been identified in maize genes (e.g. tubA1, Adh1, Sh1, and Ubi1), in rice genes (e.g. tpi), and in genes from dicotyledonous plants such as petunia (e.g. rbcS), potato (e.g. st-ls1), and from Arabidopsis thaliana (e.g. ubq3 and pat1). Deletions or mutations in intron splice sites have been shown to reduce gene expression, suggesting that splicing may be required for IME. However, IME in dicotyledonous plants has been shown by point mutations in the splice sites of the pat1 gene from A. thaliana. It has been shown that multiple use of the same intron in one plant has disadvantages. In these cases, it is necessary to have a set of basic control elements for constructing the corresponding recombinant DNA elements. Representative introns useful for practicing the present invention are provided as SEQ ID NOs: 9, 11, 17, 33, and 41.

As used herein, the terms 3' transcription termination molecule, 3' untranslated region, or 3'-untranslated region refer to a DNA molecule that is used during transcription into the untranslated region of the 3' portion of an mRNA molecule. The 3'-untranslated region of an mRNA molecule may be created by specific cleavage and 3' polyadenylation, also known as a polyA tail. The 3'-untranslated region may be located downstream of and operably linked to the DNA molecule being transcribed, and may include a polyadenylation signal and other regulatory signals that can affect transcription, mRNA processing, or gene expression. PolyA tails are believed to be involved in mRNA stability and translation initiation. Examples of 3' transcription termination molecules in this region include the 3' region of nopaline synthase; the 3' region of wheat hsp17, the 3' region of pea RubisCo small subunits, the 3' region of cotton Eb and the 3' untranslated region of koisin.

The 3'-untranslated regions generally find useful application for recombinant expression of specific DNA molecules. A weak 3'-untranslated region has the potential to generate read-through, which may affect the expression of DNA molecules located in adjacent expression cassettes. Appropriate control of transcription termination can prevent read-through of DNA sequences (e.g., other expression cassettes) located upstream of transcription and may additionally allow efficient recycling of RNA polymerase to improve gene expression. Efficient transcriptional termination (release of RNA polymerase II from DNA) is a prerequisite for transcription reinitiation and thus directly affects the overall transcription level. After transcription termination, mature mRNA is released from the site of synthesis and is transported into the cytoplasm. Eukaryotic mRNAs accumulate in vivo as poly(A) forms, making detection of transcription termination sites difficult by conventional methods. However, predicting the location of functional and effective 3'-UTRs using bioinformatics methods is hampered by the lack of conserved DNA sequences that would allow easy prediction of the presence of an effective 3'-UTR.

From a practical standpoint, it is generally advantageous for the 3' untranslated region used in an expression cassette to have the following characteristics. First, the 3' untranslated region should be able to effectively and efficiently terminate transcription of the transgene and prevent readthrough of the transcript into any adjacent DNA sequence, which may consist of another expression cassette, such as in the case of multiple expression cassettes present on a single transfer DNA (T-DNA) or adjacent chromosomal DNA into which the T-DNA is inserted. Second, the 3' untranslated region should not cause a reduction in the transcriptional activity conveyed by the promoter, leader, enhancers, and introns that are used to stimulate expression of the DNA molecule. Finally, in plant biotechnology, the 3'-untranslated region is often used to initiate amplification reactions of reverse transcribed RNA isolated from a transformed plant and used to: (1) assess the transcriptional activity or expression of an expression cassette after integration into the plant chromosome; (2) assess the copy number of inserts in the plant DNA; and (3) assess the zygosity of the resulting seeds after multiplication. The 3'-untranslated region

- 8 048365 region is also used in amplification reactions of DNA isolated from a transformed plant to assess the integrity of the inserted cassette. The 3'-untranslated region useful for practicing the present invention is shown as SEQ ID NO: 29, 34, 35, 36, 37 and 44.

As used herein, the term enhancer or enhancer element refers to a cis-acting regulatory element, a so-called cis-element, that confers an aspect of a general expression pattern but is usually insufficient to stimulate transcription of a functionally related transcribed DNA molecule. Unlike promoters, enhancer elements typically do not include 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 transcription of a functionally related DNA sequence. An enhancer element may also be fused to a promoter to produce a chimeric cis-element promoter that confers an aspect of general modulation of gene expression. An example of an enhancer element derived from the synthetic promoter, P-At.GSP571.nno:5 (SEQ ID NO: 5), is shown as SEQ ID NO: 24 (E-At.GSP571.nno: 1).

Many promoter enhancer elements are thought to bind DNA-binding proteins and/or affect DNA topology by creating local conformations that selectively permit or restrict access of RNA polymerase to the DNA template or that promote selective opening of the double helix at the transcription initiation site. An enhancer element may mediate the binding of transcription factors that regulate transcription. Some enhancer elements bind more than one transcription factor, and transcription factors may interact with different affinities with more than one enhancer domain. Enhancer elements can be identified by a number of techniques including deletion analysis, i.e., removal of one or more nucleotides from the 5' end or from the internal portion of the promoter; DNA binding assay using DNase I; methylation interference; electrophoretic mobility shift assay; in vivo genomic footprinting by ligation-mediated polymerase chain reaction (PCR) and other conventional assays or DNA sequence similarity assays using known cis-element motifs or enhancer elements as the target sequence or target motif using conventional DNA sequence comparison methods such as BLAST. The fine structure of the enhancer domain can be further studied by mutagenesis (or substitution) of one or more nucleotides or other conventional methods known in the art. Enhancer elements can be produced by chemical synthesis or isolation from regulatory elements that include such elements, and they can be synthesized with additional flanking nucleotides that contain useful restriction enzyme sites to facilitate subsequence manipulation. Thus, the present invention encompasses the development, design, and use of enhancer elements in accordance with the methods disclosed herein to modulate the expression of operably linked transcribed DNA molecules. An exemplary enhancer useful in practicing the invention is set forth as SEQ ID NO: 24.

As used herein, the term "chimeric" refers to a single DNA molecule produced by fusing a first DNA molecule to a second DNA molecule, wherein neither the first nor the second DNA molecules are normally found in this configuration, i.e., fused to each other. Thus, a chimeric DNA molecule is a new DNA molecule that is not normally found in nature. As used herein, the term "chimeric promoter" refers to a promoter produced by such manipulations of DNA molecules. A chimeric promoter may combine two or more DNA fragments, such as fusing a promoter to an enhancer element. Thus, the invention encompasses the development, construction, and use of chimeric promoters according to the methods disclosed herein to modulate the expression of operably related transcribed DNA molecules. An exemplary chimeric promoter is provided herein as SEQ ID NO: 25 (P-ALGSP571/442).

Chimeric regulatory elements can be designed to include different constituent elements that can be operably linked by various methods known in the art, such as restriction enzyme digestion and ligation, ligation-independent cloning, modular assembly of PCR products during amplification, or direct chemical synthesis of the regulatory element, as well as other methods known in the art. The resulting different chimeric regulatory elements can be composed of the same or variants of the same constituent elements, but differ in the DNA sequence or DNA sequences that contain the DNA binding sequence or sequences that allow the constituent parts to be operably linked. In the invention, the DNA sequences shown as SEQ ID NOs: 1-32 and SEQ ID NOs: 43-45 can provide reference sequences of regulatory elements, wherein the constituent elements that make up the reference sequence can be linked by methods known in the art and can contain substitutions, deletions and/or insertions of one or more nucleotides or

- 9,048,365 mutations that occur naturally during the transformation of bacterial and plant cells.

As used herein, the term "variant" refers to a second DNA molecule, such as a regulatory element, which is similar in composition to, but not identical to, the first DNA molecule, and wherein the second DNA molecule still retains the overall functionality, i.e., the same or similar expression pattern, such as through more or less equivalent transcriptional activity of the first DNA molecule. A variant may be a shorter or truncated version of the first DNA molecule, or an altered version of the sequence of the first DNA molecule, such as a version with different restriction enzyme sites and/or internal deletions, substitutions or insertions. A variant may also include a regulatory element having a nucleotide sequence comprising a substitution, deletion or insertion of one or more nucleotides of a reference sequence, wherein the derived regulatory element has greater or lesser or equivalent transcriptional or translational activity compared to the corresponding parent regulatory molecule. In the present invention, the polynucleotide sequence presented as SEQ ID NO: 1-32 and SEQ ID NO: 43-45 can be used to create variants that are similar in composition, but not identical to the DNA sequence of the original regulatory element, while maintaining the overall functionality, i.e., the same expression pattern as the original regulatory element or similar. The production of such variants of the invention is within the competence of one of ordinary skill in the art in light of this disclosure and is within the scope of the invention.

The effectiveness of the modifications, duplications, or deletions described herein on desired aspects of expression of a particular transgene can be tested empirically by performing stable and transient expression assays in plants, such as those described in the working examples, to confirm results that may vary depending on the changes made and the purpose of the modification of the original DNA molecule.

Constructions

As used herein, the term construct means any recombinant DNA molecule, such as a plasmid, cosmid, virus, phage, or a linear or circular DNA or RNA molecule obtained from any source capable of genomic integration or autonomous replication, containing a DNA molecule, wherein at least one DNA molecule has been linked to another DNA molecule in a functionally effective manner, i.e., operably linked. As used herein, the term vector means any construct that can be used for the purpose of transformation, i.e., introducing heterologous DNA or RNA into a host cell. The construct typically includes one or more expression cassettes. As used herein, the term expression cassette refers to a DNA molecule comprising at least a transcribed DNA molecule operably linked to one or more regulatory elements, typically at least a promoter and a 3' untranslated region.

As used herein, the term operably linked refers to a connection of a first DNA molecule with a second DNA molecule, wherein the first and second DNA molecules are arranged such that the first DNA molecule affects the function of the second DNA molecule. The two DNA molecules may or may not be part of a single contiguous DNA molecule and may or may not be contiguous. For example, a promoter is operably linked to a transcribed DNA molecule if the promoter modulates the transcription of the transcribed DNA molecule of interest in a cell. A leader, for example, is operably linked to a DNA sequence when it is capable of affecting the transcription or translation of the DNA sequence.

The constructs according to the invention can be presented in one embodiment in the form of double plasmid border constructs, tumor inducing (Ti), which have a right border (RB or AGRtu.RB) and a left border (LB or AGRtu.LB) region of the Ti plasmid isolated from Agrobacterium tumefaciens, including T-DNA, which, together with the transfer molecules provided by the A. tumefaciens cells, allows the T-DNA to be integrated into the genome of the plant cell (see, for example, US Patent 6,603,061). The constructs may also comprise plasmid backbone DNA segments that provide replication function and antibiotic selection in bacterial cells, such as an Escherichia coli origin of replication such as ori322, broad host range origins of replication such as oriV or oriRi, and a coding region for a selectable marker such as Spec/Strp, which encodes the aminoglycoside adenyltransferase Tn7 (aadA) that confers resistance to spectinomycin or streptomycin, or the gentamicin selectable marker gene (Gm, Gent). For plant transformation, the bacterial host strain is often A. tumefaciens ABI, C58 or LBA4404, but other strains known to those skilled in the art of plant transformation may also be used in the invention.

In this technical field, methods are known for assembling and introducing constructs into a cell in such a way that the transcribed DNA molecule is transcribed into a functional mRNA molecule, which is translated and expressed as a protein. For practical application of the invention, a typical

- 10 048365 compositions and methods for producing and using the constructs and host cells are well known to those skilled in the art. Typical vectors used for expressing nucleic acids in higher plants are well known in the art and include vectors derived from the Ti plasmid of Agrobacterium tumefaciens and the transfer control vector pCaMVCN.

Various constructs may be included in the construct, including any of those described herein. Any such regulatory elements may be provided in combination with other regulatory elements. Such combinations may be designed or modified to provide the desired regulatory features. In one embodiment, the constructs of the invention comprise at least one regulatory element operably linked to a transcribed DNA molecule operably linked to a 3' untranslated region.

Constructs according to the invention may include any promoter or leader provided herein or known in the art. For example, a promoter according to the invention may be operably linked to a heterologous untranslated 5' leader, such as a gene derived from a heat shock protein gene. Alternatively, a leader according to the invention may be operably linked to a heterologous promoter, such as the cauliflower mosaic virus 35S transcript promoter.

The expression cassettes may also include a transit peptide coding sequence that encodes a peptide useful for subcellular targeting of the functionally related protein, particularly to a chloroplast, leukoplast, or other plastid organelle; a mitochondrion; a peroxis; a vacuole; or an extracellular location. Many chloroplast-localized proteins are expressed from nuclear genes as precursors and are targeted to the chloroplast by the chloroplast transit peptide (CTP). Examples of such isolated chloroplast proteins include, but are not limited to, ribulose-1,5 bisphosphate carboxylase small subunit (SSU)-related proteins, 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. Patent No. 7,193,133. It has been demonstrated that non-chloroplast proteins can be targeted to the chloroplast by expression of a heterologous CTP operably linked to a transgene encoding non-chloroplast proteins.

Transcribed DNA molecules

As used herein, the term transcribable DNA molecule refers to any DNA molecule capable of being transcribed into an RNA molecule, including, but not limited to, molecules having protein encoding sequences and molecules that produce RNA molecules having sequences useful for gene silencing. The type of DNA molecule may include, but is not limited to, a DNA molecule from the same plant, a DNA molecule from a different plant, a DNA molecule from a different organism, or a synthetic DNA molecule, such as a DNA molecule containing an antisense message of a gene, or a DNA molecule encoding an artificial, synthetic, or otherwise modified version of a transgene. Typical transcribed DNA molecules for inclusion in constructs according to the invention include, for example, DNA molecules or genes from a species different from the species into which the DNA molecule is included, or genes that originate or are present in the same species but are included in the recipient cells by genetic engineering methods rather than by classical selection methods.

A transgene refers to a transcribed DNA molecule that is heterologous to a host cell, at least with respect to its location in the host cell genome and/or a transcribed DNA molecule that has been artificially introduced into the host cell genome in the current or any previous generation of cells.

A regulatory element, such as a synthetic promoter according to the present invention, may be operably linked to a heterologous transcribed DNA molecule. As used herein, the term heterologous refers to a combination of two or more DNA molecules in cases where such a combination does not normally occur in nature. For example, the two DNA molecules may be derived from different species, and/or the two DNA molecules may be derived from different genes, such as different genes of the same species or the same genes of different species, or one of the DNA molecules may be synthetic and not naturally occurring. A regulatory element is heterologous with respect to an operably linked transcribed DNA molecule if such a combination does not normally occur in nature, i.e., under natural conditions, the transcribed DNA molecule is not operably linked to the regulatory element.

The transcribed DNA molecule may generally be any DNA molecule for which transcript expression is desired. Such transcript expression may result in translation of the resulting mRNA molecule and thus protein expression. Alternatively, for example, the transcribed DNA molecule may be engineered to ultimately cause a reduction in the expression of a particular gene or protein. In one embodiment, this may be achieved by using a transcribed DNA molecule that is oriented in antisense to

- 11 048365 board. The person skilled in the art is familiar with the use of such antisense technology. Thus, any gene can be subjected to negative regulation, and in one embodiment, the transcribed DNA molecule can be designed to suppress a specific gene by expressing a dsRNA, siRNA or microRNA molecule.

Thus, one embodiment of the invention is a recombinant DNA molecule comprising a regulatory element of the invention, such as those represented by SEQ ID NOs: 1-32 and SEQ ID NOs: 43-45, operably linked to a heterologous transcribed DNA molecule in a manner that modulates transcription of the transcribed 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 transcribed DNA molecule comprises a protein-coding region of a gene, and in another embodiment, the transcribed DNA molecule comprises an antisense region of a gene.

Genes of agronomic interest

The transcribed DNA molecule may be a gene of agronomic interest. As used herein, the term gene of agronomic interest refers to a transcribed DNA molecule that, when expressed in a particular plant tissue, cell, or cell type, confers a desired characteristic. The substance produced by the gene of agronomic interest may act in the plant to influence the plant's morphology, physiology, growth, development, yield, grain composition, nutritional profile, disease or pest resistance, and/or environmental or chemical resistance, or may act as a pesticidal agent in the diet of a pest that feeds on the plant. In one embodiment, the regulatory element of the invention is included in the construct such that the regulatory element is operably linked to a transcribed DNA molecule that is a gene of agronomic interest. In a transgenic plant containing such a construct, expression of a gene of agronomic interest may impart a beneficial agronomic trait. The beneficial agronomic trait may include, for example, but not limited to: herbicide resistance, insect control, yield modification, disease resistance, pathogen resistance, plant growth and development modification, starch content modification, oil content modification, fatty acid content modification, protein content modification, fruit ripening modification, increased nutritional value for animals and humans, biopolymer production, environmental stress resistance, pharmaceutical peptides, improved processing qualities, improved flavor, usefulness for hybrid seed production, improved fiber production, and desirable biofuel production.

Examples of genes of agronomic interest known in the art include, but are not limited to, herbicide resistance genes (U.S. Patent Nos. 6,803,501; 6,448,476; 6,248,876; 6,225,114; 6,107,549; 5,866,775; 5,804,425; 5,633,435; and 5,463,175), increased yield (U.S. Patent Nos. US RE 38,446; 6,716,474; 6,663,906; 6,476,295; 6,441,277; 6,423,828; 6,399,330; 6,372,211; 6,235,971; 6,222,098; and 5,716,837), with insects (US Patents Nos. 6,809,078; 6,713,063; 6,686,452; 6,657,046; 6,645,497;

6642030; 6639054; 6620988; 6593293; 6555655; 6538109; 6537756; 6521442; 6501009; 6468523; 6326351;

6313378; 6284949; 6281016; 6248536; 6242241; 6221649; 6177615; 6156573; 6153814; 6110464; 6093695;

6063756; 6063597; 6023013; 5959091; 5942664; 5942658, 5880275; 5,763,245 and 5,763,241), resistance to fungal diseases (US Patents 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), resistance to viruses (US Patents Nos. 6,617,496; 6,608,241; 6,015,940 ; 6,013,864; 5,850,023 and 5,304,730), resistance to nematodes (US Patent Nos. 6,228,992), resistance to bacterial infections (US Patent Nos. 5,516,671), plant growth and development (U.S. Pat. Nos. 6,723,897 and 6,518,488), starch production (U.S. Pat. Nos. 6,538,181; 6,538,179; 6,538,178; 5,750,876; 6,476,295), altered oil production (U.S. Pat. Nos. 6,444,876; 6,426,447 and 6,380,462 ), high oil production (US Patents 6,495,739; 5,608,149; 6,483,008 and 6,476,295), altered fatty acid content (US Patents 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 production (US Patent Nos. 6,380,466), fruit ripening (US Patent Nos. 5,512,466), increased nutritional value for animals and humans (US Patent Nos. 6,723,837 ; 6653530; 6541259; 5985605 and 6171640), biopolymers (US Patents Nos. USRE 37543; 6,228,623 and 5,958,745 and 6,946,588), environmental stress tolerance (U.S. Patent Nos. 6,072,103), pharmaceutical peptides and secreted peptides (U.S. Patent Nos. 6,812,379; 6,774,283; 6,140,075 and 6,080,560), improved processing characteristics (U.S. Patent Nos. 6,476,295), improved digestibility (US Patent No. 6,531,648), low raffinose content (US Patent No. 6,166,292), production of industrial enzymes (US Patent No. 5,543,576), improved taste (US 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).

Alternatively, a gene of agronomic interest may influence the above-mentioned plant traits or phenotypes by encoding an RNA molecule that induces a target

- 12 048 365 modulation of gene expression of an endogenous gene, such as by antisense (see, for example, US Patent 5,107,065) using inhibitory RNA (RNAi, including modulation of gene expression by miRNA-, siRNA-, trans-acting siRNA- and stepwise sRNA-mediated mechanisms, such as described in published applications US 2006/0200878 and US 2008/0066206 and in US Patent Application 11/974469) or co-suppression-mediated mechanisms. The RNA may also be a catalytic RNA molecule (e.g., a ribozyme or riboswitch; see, for example, US 2006/0200878) designed to cleave a desired endogenous mRNA product. Methods are known in the art for constructing and introducing constructs into a cell in such a way that the transcribed DNA molecule is transcribed into a molecule capable of causing gene suppression.

Selective markers

Selectable marker transgenes may also be used with the regulatory elements of the invention. As used herein, the term selectable marker transgene refers to any transcribed DNA molecule whose expression in a transgenic plant, tissue, or cell, or the lack thereof, can be screened or assessed in some manner. Selectable marker genes and associated selection and screening methods for use in the practice of the invention are known in the art and include, but are not limited to, transcribed DNA molecules encoding B-glucuronidase (GUS), green fluorescent protein (GFP), proteins that confer antibiotic resistance, and proteins that confer herbicide resistance. An example of a selectable marker transgene is set forth as SEQ ID NO: 42.

Cell transformation

The invention also relates to a method for producing transformed cells and plants that contain one or more regulatory elements functionally linked to a transcribed DNA molecule.

The term transformation refers to the introduction of a DNA molecule into a recipient host. As used herein, the term host refers to bacteria, fungi, or plants, including any cells, tissues, organs, or progeny of bacteria, fungi, or plants. Plant tissues and cells of particular interest include protoplasts, calli, roots, tubers, seeds, stems, leaves, seedlings, embryos, and pollen.

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 a 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 also include the progeny of the cell or organism and the offspring resulting from a breeding program that uses such a transgenic organism as a parent in a cross and that exhibit an altered phenotype resulting from the presence of the foreign DNA molecule. The introduced DNA molecule may also be transiently introduced into a 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.

There are many methods well known to those skilled in the art for introducing DNA molecules into plant cells. The process typically includes the steps of selecting a suitable host cell, transforming the host cell with a vector, and obtaining a transformed host cell. Methods and materials for transforming plant cells by introducing a plant construct into the plant genome in the practice of the present invention may include any of the well-known and demonstrated methods. Suitable methods include, but are not limited to, bacterial infection (e.g., Agrobacterium), binary BAC vectors, direct DNA delivery (e.g., via PEG-mediated transformation, dehydration/inhibition-mediated DNA uptake, electroporation, silicon carbide fiber excitation, and DNA-coated particle acceleration), and gene editing (e.g., CRISPR-Cas systems), among others.

This disclosure further contemplates that the disclosed synthetic expression elements can be created in planta using various gene editing techniques known in the art. Such technologies used for genome editing include, but are not limited to, ZFN (zinc finger nuclease), meganucleases, TALEN (transcription activator-like effector nucleases), and CRISPR (clustered regularly intersected short palindromic repeats)/Cas (CRISPR-associated) systems. Such genome editing methods can be used to change the sequence of an expression element in a plant cell to a different sequence.

The host cells may be any cells or organisms, such as plant cells, algal cells, algae, fungal cells, fungi, bacterial cells, or insect cells. In particular embodiments, the host cells and transformed cells may include cells from

- 13,048,365 agricultural plants.

The transgenic plant can subsequently be regenerated from the transgenic plant cell according to the invention. Seeds can be obtained from such a transgenic plant using standard propagation or self-pollination methods. Such a seed and the resulting progeny plant grown from such a seed will contain a recombinant DNA molecule according to the invention and will therefore be transgenic.

The transgenic plants according to the invention can be self-pollinated to produce seeds in the case of homozygous transgenic plants according to the invention (homozygous for the recombinant DNA molecule) or crossed with non-transgenic plants or different transgenic plants to produce seeds in the case of heterozygous transgenic plants according to the invention (heterozygous for the recombinant DNA molecule). Both such homozygous and heterozygous transgenic plants are referred to herein as progeny plants. Progeny plants are transgenic plants derived from the original transgenic plant and containing the recombinant DNA molecule according to the invention. Seeds obtained using the transgenic plant according to the invention can be collected and used to grow generations of transgenic plants, i.e., progeny plants according to the invention containing the construct according to the present invention and expressing a gene of agronomic interest. Descriptions of breeding methods commonly used for different crops can be found in one of several handbooks, see, for example, 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, 369399 (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 Pub. Co., NY, 360-376 (1987).

The transformed plants can be analyzed for the presence of the gene or genes of interest and the expression levels and/or profile provided by the regulatory elements of the invention. Those skilled in the art are familiar with numerous methods available for analyzing transformed plants. For example, plant analysis methods include, but are not limited to, Southern or Northern blots, PCR-based approaches, biochemical assays, phenotypic screening methods, field assessments, and immunodiagnostic assays. Expression of the transcribed DNA molecule can be measured using TaqMan® reagents and methods (Applied Biosystems, Foster City, Calif.) as described by the manufacturer, the number of PCR cycles is determined using a TaqMan® assay array. In addition, Invader® reagents and methods (Third Wave Technologies, Madison, Wis.) as described by the manufacturer can be used to assess transgene expression.

The invention also provides plant parts according to the invention. Plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovule and pollen. Plant parts according to the invention may be viable, non-viable, regenerable and/or non-regenerable. The invention also includes and provides transformed plant cells comprising a DNA molecule according to the invention. Transformed or transgenic plant cells according to the invention include regenerable and/or non-regenerable plant cells.

The invention also provides a commercial product that is obtained from a transgenic plant or part thereof comprising a recombinant DNA molecule according to the invention. Commercial products according to the invention comprise a detectable amount of DNA comprising a DNA sequence selected from the group consisting of SEQ ID NO: 1-32 and SEQ ID NO: 43-45. As used herein, the term commercial product refers to any composition or product that consists of material obtained from a transgenic plant, seed, plant cell or plant part comprising a recombinant DNA molecule according to the invention. Commercial products include, but are not limited to, processed seeds, grains, plant parts and cereals. A commercial product according to the invention will comprise a detectable amount of DNA corresponding to a recombinant DNA molecule according to the invention. Detection of one or more of this DNA in a sample can be used to determine the content or source of the commercial product. Any standard method for detecting DNA molecules may be used, including the detection methods disclosed herein.

The invention may be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention unless otherwise indicated. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventor that function well in the practice of the present invention. However, those skilled in the art, in light of the present disclosure, will appreciate that in the specific embodiments disclosed

- 14 048365 Many changes may be made in the embodiments and still obtain the same or similar result without departing from the spirit and scope of the invention, therefore all matter set forth or shown in the accompanying drawings are to be interpreted as illustrative and not in a limiting sense.

Examples

Example 1. Design, synthesis and cloning of synthetic regulatory elements

The regulatory elements presented in Table 1 are novel synthetic expression elements designed using algorithmic methods. These artificially designed synthetic regulatory elements were chemically synthesized and cloned to generate arrays of synthetic expression regulatory elements (EXRs). More than 1000 synthetic regulatory elements were constructed and analyzed in soybean protoplasts and stably transformed soybean plants to find synthetic regulatory elements that provide desired characteristics such as protein expression levels and expression patterns. The synthetic regulatory elements described in Table 1 provide diverse expression patterns useful for driving the expression of many different coding sequences and interfering RNAs of agronomic interest.

The synthetic regulatory elements designed using appropriate computer programs do not have extended homology to any known nucleic acid sequences that exist in nature. The synthetic EXPs and the corresponding promoters, leaders, introns, and 3'-untranslated regions are presented in Table 1. The synthetic EXPs were cloned using methods known in the art into binary plant transformation vectors operably linked to the coding sequence for β-glucuronidase (GUS), and the vectors were used to evaluate the expression levels and patterns provided by the synthetic EXPs in stably transformed soybean, cotton, and maize plants.

Analysis of the transcriptional site of the synthetic regulatory element (TSS) and the intron-exon splice junction can be accomplished using transformed plant tissue. Briefly, plants are transformed with plant expression vectors containing cloned DNA fragments operably linked to a heterologous transcribed DNA molecule. The 5' RACE system for rapid amplification of cDNA ends, version 2.0 (Invitrogen, Carlsbad, California 92008) is then used to confirm the TSS of the synthetic regulatory element and the intron-exon splice junction by DNA sequence analysis of the resulting mRNA transcripts.

Table 1. Groups of synthetic transcriptional expression regulatory elements, promoters, leaders, introns and 3'-untranslated regions

Abstract SEQ ID NO: Size (p. H.) Description and/or regulatory elements of EXP linked in direction 5 3’ (SEQ ID NO): EXP- At.GSP442.nno+At. Suso:3 1 855 EXP: P-At.GSP442.nno:2 (SEQ ID NO:2), L- At.GSP442.nno:1 (SEQ ID NO:3), I-At.Cyco:2 (SEQ ID NO:33) P-At.GSP442.nno:2 2 480 Promoter L-At.GSP442.nno:1 3 20 Leader EXP-At.GSP571 4 500 EXP: P-At.GSP571.nno:5 (SEQ ID NO:5), L- At.GSP571.nno:1 (SEQ ID NO: 6) P-At.GSP571.nno:5 5 451 Promoter

- 15 048365

L-AP.GSP571.nno:1 6 49 Leader EXP- At.GSP571.nno+AP. Cyco:2 7 855 EXP: P-At.GSP571.nno:5 (SEQ ID NO:5), Ε- ΑΡ. GSP57 1. nno: 1 (SEQ ID NO:6), I-At.Cyco:2 (SEQ ID NO:33) EXP- AP.GSP571.nno+At. GSI21.nno:10 8 816 EXP: P-At.GSP571.nno:5 (SEQ ID NO:5), Ε- ΑΡ. GSP57 1. nno: 1 (SEQ ID NO:6), I-AP .GS121.nno:2 (SEQ ID NO:9) I-At.GSI21.nno:2 9 309 Intron EXP- AP.GSP571.nno+At. GSI102.nno:1 10 810 EXP: P-AP.GSP571.nno:5 (SEQ ID NO:5), Ε- ΑΡ.GSP571.nno:1 (SEQ ID NO:6), I-AP.GSI102.nno:1 (SEQ ID NO:11) I-At.GSI102.nno:1 11 310 Intron EXP-AP.GSP564 12 500 EXP: P-AP.GSP564.nno:3 (SEQ ID NO:13), Ε- ΑΡ.GSP564.nno:1 (SEQ ID NO:14) P-AP.GSP564.nno:3 13 461 Promoter L-At.GSP564.nno:1 14 39 Leader EXP- At .GSP564.nno+At. Cyco:2 15 855 EXP: P-AP.GSP564.nno:3 (SEQ ID NO:13), Ε- ΑΡ.GSP564.nno:1 (SEQ ID NO:14), I-AP.Cyco:2 (SEQ ID NO:33) EXP- At .GSP564.nno+At. GSI17.nno:2 16 807 EXP: P-AP.GSP564.nno:3 (SEQ ID NO:13), Ε- ΑΡ.GSP564.nno:1 (SEQ ID NO:14), I-AP.GSI17.nno:1 (SEQ ID NO:17) I-At.GSI17.nno:1 17 300 Intron

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EXP- At.GSP564.nno+At. GSI102.nno:1 18 810 EXP: P-At.GSP564.nno:3 (SEQ ID NO:13), Ε- ΑΡ. GSP5 64. nno: 1 (SEQ ID NO:14), I-At.GSI102.nno:1 (SEQ ID NO:11) EXP-At.GSP579 19 500 EXP: P-At.GSP579.nno:2 (SEQ ID NQ:20), L- At.GSP579.nno:1 (SEQ ID NO:21) P-At.GSP579.nno:2 20 449 Promoter L-At .GSP579.nno:1 21 51 Leader EXP- At .GSP579.nno+At. GSI102.nno:3 22 810 EXP: P-At.GSP579.nno:2 (SEQ ID NO:20), L- At.GSP579.nno:1 (SEQ ID NO:21), I-At.GSI102.nno:1 (SEQ ID NO:11) EXP- At .GSP571.nno+At. GSP442.nno+At.Cyc o: 1 23 1350 EXP: E-At.GSP571.nno:1 (SEQ ID NO:24), P- At.GSP442.nno:2 (SEQ ID NO:2), L-At.GSP442.nno:1 (SEQ ID NO:3), L-At.Cyco1:1:2 (SEQ ID NO:40), I- At.Cyco:2 (SEQ ID NO:33) E-At .GSP571.nno:1 24 422 Enhancer P-At.GSP571/442 25 902 Chimeric promoter E- At.GSP571.nno:1 (SEQ ID NO:24), P-At.GSP442.nno:2 (SEQ ID NO:2) EXP- At .GSP576.nno+At. GSI17.nno:3 26 800 EXP: P-At.GSP57 6.nno:4 (SEQ ID NO:27), L- At.GSP576.nno:2 (SEQ ID NO:28), I-At.GSI17.nno:1 (SEQ ID NO:17) P-At.GSP576.nno:4 27 458 Promoter L-At.GSP576.nno:2 28 42 Leader

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T-Zm.GST59.nno:1 29 400 3’ UTR EXP- At .GSP221+At.Suso : 3 30 947 EXP: P-At.GSP221 : 3 (SEQ ID NO:31), L-At.GSP221 : 1 (SEQ ID NO:32), I-At.Cyco:2 (SEQ ID NO:33) P-At.GSP221:3 31 370 Promoter L-At.GSP221:1 32 229 Leader EXP-At.GSP442+L- I-At.Suso 43 928 EXP: P-At.GSP442.nno:2 (SEQ ID NO:2), L- At.GSP442.nno:1 (SEQ ID NO:3), L-At.Cyco-1:1:2 (SEQ ID NQ:40), I- At.Cyco:2 (SEQ ID NO:33) T-Zm.GST7.nno:2 44 300 3’ UTR EXP- At .GSP576.nno+At. Suso:1 45 855 EXP: P-At.GSP576.nno:4 (SEQ ID NO:27), L- At.GSP576.nno:2 (SEQ ID NO:28), I-At.Cyco:2 (SEQ ID NO:33)

Example 2. Analysis of synthetic EXPs: EXP-At.GSP442.nno+At.Cyco:3 and EXP-At.GSP221+At.Cyco:3, stimulating GUS expression in stably transformed soybean plants

Soybean plants were transformed with vectors, specifically plant expression vectors, containing groups of regulatory elements that stimulate expression of the β-glucuronidase (GUS) transgene. The resulting plants were analyzed for GUS protein expression to assess the effect of selected groups of regulatory elements on expression.

Soybean plants were transformed with GUS expressing plant constructs containing an 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 Arabidopsis cytochrome c oxidase subunit VIa gene and consists of the L-At.Cyco-1:1:2 promoter (SEQ ID NO: 39) operably linked 5' to a leader, L-At.Cyco-1:1:2 (SEQ ID NO: 40), which is operably linked 5' to an intron, I-At.Cyco:1:1:1 (SEQ ID NO: 41). EXP-At.Cyco:3 (SEQ ID NO: 1) and EXP-At.Cyco:3 (SEQ ID NO: 30) each contain a synthetic promoter and leader operably linked 5' to 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 LA1Cyco-1:1:1 (SEQ ID NO: 41), except that it contains two nucleotides after the intron splice site included in the sequence LA1Cyco-1:1:1. Both introns of LA1Cyco are spliced in the same way.

The regulatory elements were cloned into basic plant expression vectors using standard techniques known in the art. The resulting plant expression vectors contained the right border region from Agrobacterium tumefaciens (B-AGRtu.right border), the first transgene selection cassette used to select transformed plant cells that confer resistance to the antibiotic spectinomycin; a second transgene cassette for assessing the activity of a regulatory element that contains an EXP sequence operably linked 5' to the coding sequence for β-glucuronidase (GUS, GOI-Ec.uidA+St.LSI:1:1, SEQ ID NO: 42) containing a modifiable intron derived from the potato ST-LS1 tissue-specific photosensitive gene (Genbank Accession: X04753), operably linked 5' to the 3' untranslated region of the Gossypium barbadense FbLate-2 gene (T-Gb.FbL2:1, SEQ ID NO: 36) and the left border region of Agrobacterium tumefaciens (B-AGRtu.left border).

Soybean plant cells were transformed by Agrobacterium-mediated transformation using these binary transformation vector constructs as is well known in the art. The resulting transformed plant cells were induced to form whole soybean plants.

For qualitative and quantitative analysis of the expression of transformed plants, histochemical GUS analysis was used. Whole tissue sections were incubated with the GUS staining solution X-Gluc (5-6-rom-4-chloro-3-indolyl-L-glucuronide) (1 mg/ml) for the appropriate time, washed, and visually inspected for blue coloration. GUS activity was qualitatively determined by direct visual inspection or microscopic examination using selected plant organs and tissues.

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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-b-D-glucuronide (MUG) in a total reaction volume of 50 μL. The reaction product, 4-methylumbelliferone (4-MU), is maximally fluorescent at high pH, where the hydroxyl group is ionized. The addition of a stock sodium carbonate solution both stops the assay and adjusts the pH for quantification of the fluorescent product. Fluorescence was measured with excitation at 365 nm and emission at 445 nm using a Fluoromax-3 with a Micromax reader, with the slit width set at 2 nm excitation and 3 nm emission. Values are reported as nmol GUS/hr/mg total protein.

The following tissues were selected for GUS expression in the R0 generation: V5 root, leaf (storage tissue), and leaf (source); R1 root, leaf (petiole), leaf (source), and flowers; R3 seed (immature seed and silique), R5 seed (cotyledon), and R8 seed (embryo and cotyledon). Table 2 shows the mean quantitative GUS expression for each of the selected tissues stimulated by the tested EXP regulatory element groups, where H/O indicates that expression in a particular tissue was not determined.

Table 2. Average quantitative expression of GUS in stably transformed soybean plants due to synthetic groups of regulatory elements and endogenous EXP, 1 '' XP-Ai.Cvco: 1: 1

Stage of development Organ EXP- At.Suso:1: 1 (SEQ ID NO:38) EXP- At.GSP442.nno +At.Suso: 3 (SEQ ID NO:1) EXP- At .GSP221+At. Suso:3 (SEQ ID NO:30) V5 Root 151 399 928 Sheet (storage fabric) 39 65 59 Sheet (source) 52 109 100 R1 Root BUT 616 1893 Leaf (petiole) 97 470 136 Sheet (source) 46 177 240 Flowers 71 277 140 R3 Immature seeds 64 477 H/O Pod 84 575 702 R5 Seed (cotyledon) 91 564 58 R8 Seed (embryo) 57 149 301 Seed (cotyledon) 100 1118 414

As can be seen from Table 2, each of the groups of synthetic regulatory elements has a unique expression pattern in tissue samples compared to endogenous EXP. For example, the synthetic At.GSP442 promoter, P-At.GSP442.nno:2 (SEQ ID NO: 2) and the leader L-At.GSP442.nno:1 (SEQ ID NO: 3), EXPAt.GSP442.nno+At.Cyco:3 (SEQ ID NO: 1) provide higher levels of GUS expression in all analyzed organs compared to endogenous EXP-At.Cyco:1:1 (SEQ ID NO: 38), which contains an identical intron sequence. TSS analysis demonstrated TSS consistency. The intron was properly excised in the resulting mRNA, as expected. Furthermore, the synthetic At.GSP221 promoter, P-At.GSP221:3 (SEQ ID NO: 31) and leader, L-AtGSP221:1 (SEQ ID NO: 32), EXPAt.GSP221+At.Cyco:3 (SEQ ID NO: 30) also provided higher levels of constitutive expression in most organs analyzed compared to endogenous EXP-At.Cyco:1:1 and displayed a consistent TSS. However, the TSS of EXP-At.GSP221+At.Cyco:3 was not located in the predicted

- 19 048365 multiple potential TATA elements were present. This creates potential problems for multiple transcripts that may result in multiple coding sequences. Therefore, EXP-At.GSP221+At.Cyco:3 was not considered suitable for use in driving transgene expression in stably transformed dicotyledonous plants. This demonstrates one of the challenges in designing synthetic expression elements. In the design and identification of synthetic expression elements, many synthetic elements have been analyzed, but only a small subset exhibited the desired characteristics and regulatory activity, illustrating the difficulty in designing effective synthetic transcriptional regulatory elements.

As can be seen from Table 2, the synthetic promoter P-At.GSP442.nno:2 (SEQ ID NO: 2) and LAt.GSP442.nno:1 (SEQ ID NO: 3), contained in EXP-At.GSP442.nno+At.Cyco:3 (SEQ ID NO: 1), is able to stimulate the expression of the constitutive transgene of the functionally linked transgene in a stably transformed soybean plant.

Example 3. Analysis of the synthetic promoter and leader At.GSP571 and the synthetic introns At.GSI21 and At.GSI102 stimulating GUS expression in stably transformed soybean plants

Soybean plants were transformed with vectors, specifically plant expression vectors, containing groups of regulatory elements that stimulate expression of the β-glucuronidase (GUS) transgene. The resulting plants were analyzed for GUS protein expression to assess the effect of selected groups of regulatory elements on expression.

Soybean plants were transformed with GUS expression plant constructs comprising the synthetic EXPs, EXP-At.GSP571 (SEQ ID NO:4), EXP-At.GSP571.nno+At.Cyco:2 (SEQ ID NO:7), EXP-AtGSP571.nno+At.GSI21.nno:10 (SEQ ID NO:8), and EXP-At.GSP571.nno+AtGSI102.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 EXPAt.GSP571.nno+At.GSI102.nno:1 contained the synthetic introns I-At.GSI21.nno:2 (SEQ ID NO: 9) and IAt.GSI102.nno:1 (SEQ ID NO: 11), respectively. The plant binary transformation vectors were similar to those described in Example 2, except that each of the At.GSP571 EXP vectors contained the 3' untranslated region of T-Mt.Sali3-2-1:2:1 (SEQ ID NO: 34), derived from the Medicago truncatula Sali3 gene.

Quantitative and qualitative analysis of GUS expression was performed as described in Example 2. The tissue samples used for the analysis were the same as described in Example 2. Table 3 shows the average quantitative GUS expression for each of the selected tissues stimulated by the tested EXP synthetic regulatory elements, where H/O means that the expression in the specific tissue was not determined.

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Table 3. Average quantitative expression of GUS in stably transformed soybean plants stimulated by synthetic regulatory elements

Stage of development Organ EXP- At. GSP 571 (SEQ ID NO: 4) EXP- At.GSP571 .nno+At.C yco: 2 (SEQ ID NO: 7) EXP- At .GSP571 .nno+At.G SI21.nno: 10 (SEQ ID NO:8) EXP- At .GSP571 .nno+At.G SI102.nno :1 (SEQ ID NO:10) V5 Root 40 57 165 579 Sheet (storage fabric) 650 612 792 1683 Sheet (source) 1379 1090 1475 2128 RI Root 110 H/O 457 645 Leaf (petiole) 951 1091 1267 1167 Sheet (source) 1995 3538 2094 2129 Flowers 703 830 1408 350 R3 Immature seeds 75 609 495 232 Pod 852 2228 4014 1535 R5 Seed (cotyledon) 650 474 540 1433 R8 Seed (embryo) 1153 1004 603 1122 Seed (cotyledon) 2449 4524 2533 2648

As shown in Table 3, the synthetic At.GSP571 promoter and leader mediated constitutive expression in all organs analyzed. The highest expression was observed in leaves and seeds. TSS analysis demonstrated TSS consistency. Functional linkage of the intron sequence altered expression in many organs, providing a means to fine-tune constitutive expression. Differences in expression were observed upon functional linkage of the synthetic introns I-At.GSI21.nno:2 (SEQ ID NO: 9) and I-At.GSI102.nno:1 (SEQ ID NO: 11). The synthetic introns enhanced expression in some tissues, but varied in the level of enhancement for each organ. For example, the enhancement using the synthetic intron I-At.GSI21.nno:2 in the R3 silique was higher than that observed using the synthetic intron I-At.GSI102.nno:1 and the endogenous intron I-At.Cyco:2 relative to EXP-At.GSP571. Expression was only modestly enhanced by the three functionally linked introns in the R1 petiole. In R1 flowers, I-At.GSI21.nno:2 and IAt.Cyco:2 enhanced expression, with I-At.GSI21.nno:2 providing a high level of enhancement and I-At.Cyco:2 providing a moderate level of enhancement. Interestingly, I-At.GSI102.nno:1 reduced expression in R1 flowers.

Analysis of the resulting mRNAs showed proper and consistent processing of intron elements.

The synthetic promoter P-At.GSP571.nno:5 (SEQ ID NO: 5) and leader L-At.GSP571.nno:1 (SEQ ID NO: 6) contained in EXP-At.GSP571 (SEQ ID NO: 4) provide constitutive expression of a functionally linked transgene in stably transformed soybean plants. The synthetic EXP, РХР-А1.С^Р571.нно-А1.Сyко:2 (SEQ ID NO: 7), which includes the Arabidopsis IAt.Cyco:2 intron (SEQ ID NO: 33) and EXP-At. GSP571.nno+At.GSI21.nno:10 (SEQ ID NO: 8) and EXPAt.GSP571.nno+At.GSI102.nno:1 (SEQ ID NO: 10), which contain the synthetic introns IAt.GSI21.nno:2 (SEQ ID NO: 9) and I-At.GSI102.nno:1 (SEQ ID NO: 11), respectively, confer unique constitutive expression patterns 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), confer

- 21 048365 enhanced or modulated expression in multiple plant organs when functionally linked to EXP-At.GSP571 (SEQ ID NO: 4). These unique expression patterns can be used to stimulate specific transgenes in which a particular expression pattern of one of the four At.GSP571 EXPs is most desirable.

Example 4. Analysis of the synthetic promoter and leader At.GSP564 and the synthetic introns At.GSI17 and At.GSI102 stimulating GUS expression in stably transformed soybean plants

Soybean plants were transformed with vectors, specifically plant expression vectors, containing groups of regulatory elements that stimulate expression of the β-glucuronidase (GUS) transgene. The resulting plants were analyzed for GUS protein expression to assess the effect of selected groups of regulatory elements on expression.

Soybean plants were transformed with GUS expressing plant constructs containing 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 promoter P-At.GSP564.nno:3 (SEQ ID NO: 13) and the synthetic leader L-At.GSP564.nno.l (SEQ ID NO: 14). EXPAt.GSP564.nno+At.Cyco:2 contains the Arabidopsis intron, I-At.Cyco:2 (SEQ ID NO: 33). EXPAt.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 plant binary transformation vectors were similar to those described in Example 2, except that each of the At.GSP564 EXP vectors contained the 3'-untranslated region T-Mt.Oxr-1:2:1 (SEQ ID NO: 35), derived from the putative oxidoreductase protein (OXR) gene from Medicago truncatula.

Quantitative and qualitative analysis of GUS expression was performed as described in Example 2. The tissue samples used for the analysis were the same as described in Example 2. Table 4 shows the average quantitative GUS expression for each of the selected tissues stimulated by the tested EXP synthetic regulatory elements, where H/O means that expression in a particular tissue was not determined.

Table 4. Average quantitative expression of GUS in stably transformed soybean plants stimulated by synthetic regulatory elements

Stage of developed FL Organ EXP- At.GSP564 (SEQ ID NO:12) EXP- At.GSP564 .nno+At.0 yco: 2 EXP- At .GSP56 4.nno+At .GSI17.n EXP- At .GSP564 .nno+At.G SI102.nno

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(SEQ ID NO:15) by: 2 (SEQ ID NO:16) : 1 (SEQ ID NO:18) V5 Root 61 108 54 145 Sheet (storage fabric) 38 220 89 259 Sheet (source) 74 421 209 1229 R1 Root 118 165 2348 627 Leaf (petiole) 90 235 273 148 Sheet (source) 140 205 436 917 Flowers 66 91 BUT 305 R3 Immature seeds 26 BUT 101 BUT Pod 40 BUT 749 BUT R5 Seed (cotyledon) 25 88 78 61 R8 Seed (embryo) 38 97 137 70 Seed (cotyledon) 79 288 655 572

As shown in Table 4, the synthetic At.GSP564 promoter and leader mediated constitutive expression in all organs analyzed. Expression was highest in leaves and seeds. TSS analysis demonstrated TSS consistency. Functional linkage of the intron sequence altered expression in many organs, providing a means to fine-tune constitutive expression. Differences in expression were observed upon functional linkage of the synthetic introns I-At.GSI17.nno:1 (SEQ ID NO: 17) and I-At.GSI102.nno:1 (SEQ ID NO: 11). The synthetic introns enhanced expression in some tissues relative to EXP-At.GSP564, but differed in the level of enhancement for each organ. For example, the gain using the synthetic intron I-At.GSI102.nno:1 in the V5 leaf was higher than the gain observed using the synthetic intron I-At.GSI17.nno:1. In the R1 root, the gain using the synthetic intron IAt.GSI17.nno:1 was higher than the gain provided by the synthetic intron I-At.GSI102.nno:1. Both synthetic introns provided greater gain in expression in the R1 leaf than the endogenous intron, I-At.Cyco:2.

Analysis of the resulting mRNAs showed proper and consistent processing of intron elements.

The synthetic At.GSP564 promoter, P-At.GSP564.nno.3 (SEQ ID NO: 13) and leader, LAt.GSP564.nno:1 (SEQ ID NO: 14), containing EXP-At.GSP564 (SEQ ID NO: 12), provides constitutive expression of a operatively linked transgene in stably transformed soybean plants. The synthetic EXP, EXP-At.GSP564.nno+At.Cyco:2 (SEQ ID NO: 15), which includes the Arabidopsis I-At.Cyco:2 intron (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 synthetic introns, IAt.GSI17.nno:1 (SEQ ID NO: 17) and I-At.GSI102.nno:1 (SEQ ID NO: 11), respectively, provide unique constitutive expression patterns 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) provide enhanced or modulated transgene expression in multiple plant organs when functionally linked to EXP-At.GSP564 (SEQ ID NO: 12). These unique expression patterns can be used to stimulate specific transgenes in which a particular expression pattern of one of the four At.GS564 EXPs is most desirable. Example 5.

Analysis of a synthetic EXP, EXP-At.GSP579.nno+At.GSI102.nno:3, stimulating GUS expression in stably transformed soybean plants

Soybean plants were transformed with vectors, in particular plant expression vectors,

- 23 048365 containing a group of synthetic regulatory elements stimulating the expression of the β-glucuronidase (GUS) transgene. The resulting plants were analyzed for GUS protein expression to assess the effect of the selected group of synthetic regulatory elements on expression.

Soybean plants were transformed with a plant GUS expression construct containing a synthetic EXP, EXP-At.GSP579.nno+At.GSI102.nno:3 (SEQ ID NO: 22). EXPAt.GSP579.nno+At.GSI102.nno:3 contains EXP-At.GSP579 (SEQ ID NO: 19), consisting of the At.GSP promoter and leader (SEQ ID NOs: 20 and 21, respectively), operably linked 5' to a synthetic intron, I-At.GSI102.nno:1 (SEQ ID NO: 11). The GUS transgene cassette also contains a 3' untranslated region, T-Mt.RD22-1:2:1 (SEQ ID NO: 37), derived from the dehydration-sensitive RD22 protein gene from Medicago truncatula.

Quantitative and qualitative analysis of GUS expression was performed as described in Example 2. The tissue samples used for the analysis were the same as described in Example 2. Table 5 shows the average quantitative GUS expression for each of the selected tissues stimulated by the synthetic EXP, EXP-At.GSP579.nno+At.GSI102.nno:3, where H/O means that the expression in the specific tissue was not determined,

Table 5. Average quantitative expression of GUS in stably transformed soybean plants stimulated by EXP-At.GSP57 9.nno+At.GSI102.nno:3

Stage of development Organ EXP- At.GSP579.nno+At.GSI102.nno:3 (SEQ ID NO:22) V5 Root 187 Sheet (storage tissue) 311 Sheet (source) 458 R1 Root 148 Leaf (petiole) 118 Sheet (source) 425 Flowers 130 R3 Immature seeds H/O Pod H/O R5 Seed (cotyledon) H/O R8 Seed (embryo) 127 Seed (cotyledon) 266

As can be seen from Table 5, EXP-At.GSP579.nno+At.GSI102.nno:3 (SEQ ID NO: 22) provides constitutive expression in stably transformed soybean plants. The synthetic promoter PAt.GSP579.nno:2 (SEQ ID NO: 20) and the leader L-At.GSP579.nno:1 (SEQ ID NO: 21) contained in EXPAt.GSP579 (SEQ ID NO: 19) lead to constitutive expression of the operably linked transgene. As could be deduced from the previous examples in which the synthetic intron IAt.GSI102.nno:1 (SEQ ID NO: 11) was operably linked to other constitutive synthetic promoters; I-At.GSI102.nno:1 enhances or modulates constitutive expression mediated by EXP-At.GSP579 in at least some of the selected organs.

Example 6. Analysis of synthetic EXP, EXP-At.GSP571.nno+At.Сусо:2, stimulation of GUS expression in stably transformed cotton plants

Cotton plants were transformed with a vector, specifically a plant expression vector, containing a group of synthetic regulatory elements that stimulate the expression of the glucuronidase (GUS) transgene. The resulting plants were analyzed for GUS protein expression to assess the effect of the group of synthetic regulatory elements on expression.

A plant binary vector containing a synthetic EXP, EXP-At.GSP571.nno+At.Cyco:2 (SEQ ID NO: 7), similar to that described in Example 3, was used to stably transform cotton plants. The GUS transgene cassette contained EXP-At.GSP571.nno+At.Cyco:2 operably linked 5' to the coding sequence for β-glucuronidase (GUS, GOI-Ec.uidA+St.LS1:1:1, SEQ ID NO: 42) containing a recyclable intron derived from the potato ST-LS1 light-sensitive tissue-specific gene (Genbank Accession: X04753) operably linked 5' to the 3' untranslated region from the FbLate-2 gene of Gossypium barbadense (T-Gb.FbL2:1, SEQ ID NO: 36). The resulting transformed cotton events were grown and tissue samples were obtained from 4-node leaf, 8-node petiole, petiole, leaf (source), and leaf (storage tissue); quad

- 24,048,365 antler bracts and square buds before fertilization; anther and ovary during anthesis, and capsule walls 8 days after pollination (DAP) for qualitative and quantitative GUS expression.

Table 6 shows the average quantitative expression of GUS for each of the selected tissues stimulated by the synthetic EXP, EXP-At.GSP571.nno+At.Сусо:2.

Table 6. Average quantitative expression of GUS in stably transformed cotton plants stimulated by EXP-At.GSP571.nno+At.Сусо:2

Stage Organ Average value 4 knot - sheet 1232,57 8 knot - leaf, petiole 223.68 Sheet, storage fabric 612,14 Sheet, source 618.9 Before fertilization - square bracts 381,69 square buds 347.22 Blooming, dust cover 64, 66 Flowering, ovary 210.92 8 DAP Wall of the seed box 835,94

As shown in Table 6, EXP-At.GSP571.nno+At.Cyco:2 was expressed in all tissues sampled. The highest expression was observed in the 4th node leaf and the lowest in the anther during flowering. Expression in the source and storage tissue of the 8th node leaves was relatively equal and was approximately half that in the 4th node leaves. Expression in the boll walls was also high. Table 6 demonstrates that the promoter, P-At.GSP571.nno:5 (SEQ ID NO: 5), is able to drive constitutive expression in stably transformed cotton plants. The I-At.Cyco:2 intron (SEQ ID NO: 33) in EXP-At.GSP571.nno+At.Cyco:2 enhances expression of the P-At.GSP571.nno:5 promoter in stably transformed soybean plants, as shown in Example 3.

Example 7. Analysis of the synthetic chimeric promoter P-At.GSP571/442 stimulating GUS expression in stably transformed soybean plants

Soybean plants were transformed with vectors, specifically plant expression vectors, containing groups of regulatory elements that stimulate expression of the β-glucuronidase (GUS) transgene. The resulting plants were analyzed for GUS protein expression to assess the effect of selected groups of synthetic regulatory elements on expression.

Soybean plants were transformed with a plant binary vector containing a synthetic EXP, EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1 (SEQ ID NO: 23), which consists of the synthetic chimeric promoter P-AEGSP571/442 (SEQ ID NO: 25) containing the synthetic enhancer EAt.GSP571.nno:1 (SEQ ID NO: 24) derived from the synthetic promoter P-At.GSP571.nno:5 (SEQ ID NO: 5), which is operably linked 5' to the synthetic promoter P-At.GSP442.nno:2 (SEQ ID NO: 2) and operably linked 5' to the synthetic leader, L-At.GSP442.nno:1 (SEQ ID NO: 3), functionally linked 5' to the leader, I-At.Cyco-1:1:2 (SEQ ID NO: 40), which is operably linked 5' to the intron, I-At.Cyco:2 (SEQ ID NO: 33). The GUS transgene cassette contained EXPAt.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 recyclable intron derived from the potato ST-LS1 light-sensitive tissue-specific gene (Genbank Accession: X04753) operably linked 5' to the synthetic 3'-untranslated region TZm.GST59.nno:1 (SEQ ID NO: 29).

A plant binary vector used to compare chimeric promoter activity was also constructed. The vector contains an EXP, EXP-At.GSP442+L-I-At.Cyco (SEQ ID NO: 43), consisting of the 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-AECyco1:1:2 (SEQ ID NO: 40), which is operably linked 5' to an intron, I-At.Cyco:2 (SEQ ID NO: 33). The binary vectors are similar to those described in Examples 2-6, except that each GUS transgene cassette has a synthetic 3' untranslated region T-Zm.GST59.nno:1 (SEQ ID NO: 29) operably linked 3' to the GUS coding sequence.

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Soybean plants were transformed with two binary vectors. Tissue samples were taken from selected organs at specific developmental stages and analyzed to assess qualitative and quantitative GUS expression. Table 7 shows the average quantitative GUS expression for each of the selected tissues stimulated with synthetic EXP, EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1 and EXPAt.GSP442+L-I-At.Cyco.

Table 7. Average quantitative expression of GUS in stably transformed soybean plants stimulated by EXP-At.GSP571.nno+At.GSP442.nno+At.Сусо:1 and EXP-At.GSP442+LI-At.Сусо

EXP-At.GSP442+L- I-At.Cyco (SEQ ID NO:43) EXP- At. GSP571.nno+At.GSP4 42.nno+At.Cyco:1 (SEQ ID NO:23) Stage Organ Average value Average value V5 Sheet, storage fabric 69, 61 72,12 Sheet, source 88.22 96, 06 Root 74, 67 102.9 RI Flowers 79.16 62,01 leaf, petiole 77.07 87 Sheet, source 66, 59 114.33 Root 76, 88 123,12 R3 Pod 93, 19 102.54 Seed, immature 71,15 61, 62 R5 Seed, cotyledon 78,72 92.83 R8 Seed, cotyledon 65, 55 72.15 Seed, embryo 129.95 107.66

As shown in Table 7, addition of the synthetic enhancer E-At.GSP571.nno:1 enhanced expression in many tissues selected. Both EXPs provided constitutive expression in stably transformed soybean plants. The synthetic 3'-UTR T-Zm.GST59.nno:1 functioned similarly to the natural 3'-UTR in ensuring proper transcript termination and polyadenylation.

Example 8. Analysis of the synthetic chimeric promoter P-At.GSP571/442, stimulating GUS expression in stably transformed cotton plants

Cotton plants were transformed with a vector, specifically a plant expression vector, containing a group of synthetic regulatory elements stimulating the expression of the β-glucuronidase (GUS) transgene. The resulting plants were analyzed for GUS protein expression to assess the effect of the selected group of synthetic regulatory elements on expression.

Cotton plants were transformed with a plant binary vector containing a synthetic EXP, EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1 (SEQ ID NO: 23), which consists of the synthetic chimeric promoter P-ALGSP571/442 (SEQ ID NO: 25) containing the synthetic enhancer E-At.GSP571.nno:1 (SEQ ID NO: 24) derived from the synthetic promoter PAt.GSP571.nno:5 (SEQ ID NO: 5), which is operably linked 5' to the synthetic promoter PAt.GSP442.nno:2 (SEQ ID NO: 2) and operably linked 5' to a synthetic leader, LAt.GSP442.nno:1 (SEQ ID NO: 3), operably linked 5' to a leader, L-At.Cyco-1:1:2 (SEQ ID NO: 40), which is operably linked 5' to an intron, I-At.Cyco:2 (SEQ ID NO: 33). The GUS transgene cassette contained 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 recyclable intron derived from the light-sensitive tissue-specific gene

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Potato ST-LS1 (Genbank Accession: X04753) functionally linked 5' to the synthetic 3' untranslated region of T-Zm.GST59.nno:1 (SEQ ID NO: 29). The resulting transformed cotton events were grown and tissue samples were prepared from leaf 4 node, petiole 8 node, petiole, leaf (source) and leaf (storage tissue); square bracts and square buds before fertilization; anther and ovary at anthesis, and boll wall 8 days post pollination (DAP) for qualitative and quantitative GUS expression.

Table 8 shows the average quantitative expression of GUS for each of the selected tissues stimulated by synthetic EXP, EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1, where nno means below the limit of detection.

Table 8. Average quantitative expression of GUS in stably transformed cotton plants stimulated by EXP-At.GSP571 .nno+At.GSP442.nno+At.Сусо:1

Stage Organ Average value 4 knot - sheet 177.74 8 knot - leaf, petiole NGO Sheet, storage tissue 108.39 Sheet, source 294.99 Before fertilization - square bracts 78,84 square buds 118.21 Blooming, dust cover 69, 19 Flowering, ovary 69.78 8 DAP Seed wall 159.58 boxes

As shown in Table 8, EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1 (SEQ ID NO: 23) was able to stimulate constitutive GUS expression in the sampled tissues. Expression in the petiole was below the detection limit. The highest expression was in the leaf (source) at node 8. Expression was relatively equal in the anther and ovary during anthesis. In addition, the synthetic 3'-UTR T-Zm.GST59.nno:1 (SEQ ID NO: 29) functioned similarly to the natural 3'-UTR in ensuring proper transcript termination and polyadenylation.

Example 9. Analysis of a synthetic EXP, EXP-At.GSP576.nno+At.Cyco:1, stimulating GUS expression in stably transformed soybean plants

Soybean plants were transformed with a vector, specifically a plant expression vector, containing a group of synthetic regulatory elements stimulating the expression of the β-glucuronidase (GUS) transgene. The resulting plants were analyzed for GUS protein expression to assess the effect of the selected group of synthetic regulatory elements on expression.

Soybean plants were transformed with a plant binary vector containing a synthetic EXP, EXP-At.GSP576.nno+At.Cyco:1 (SEQ ID NO: 45). The GUS transgene cassette also contained the 3' untranslated region of 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 soybean events were grown, and tissue samples from selected organs at several developmental stages were collected and analyzed to assess the qualitative and quantitative expression of GUS. GUS expression in stably transformed soybean plants stimulated by EXP-At.GSP576.nno+At.Cyco:1 is presented in Table 9.

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Table 9. Average quantitative expression of GUS in stably transformed soybean plants stimulated by EXP-At.GSP576.nno+At.Cyco: 1

Stage of development Organ Average value V5 Root 60, 95 Sheet (storage tissue) 97.43 Sheet (source) 181.64 R1 Root 82.4 Leaf (petiole) 208.28 Sheet (source) 214 Flowers 123.37 R3 Immature seeds 95.29 Pod 158.24 R5 Seed (cotyledon) 85, 97 R8 Seed (embryo) 67.4 Seed (cotyledon) 52.92

As shown in Table 9, EXP-At.GSP576.nno+At.Cyco:1 (SEQ ID NO: 45) provides constitutive expression in stably transformed soybean plants. The synthetic promoter P-At.GSP576.nno:4 (SEQ ID NO: 27) and the leader L-At.GSP576.nno:2 (SEQ ID NO: 28) drive constitutive expression of the operably linked transgene. As could be deduced from the previous examples in which the intron I-At.Cyco:2 (SEQ ID NO: 33) was operably linked to other constitutive synthetic promoters, I-At.Cyco:2 enhances or modulates the constitutive expression mediated by P-At.GSP576.nno:4 in at least some of the selected organs.

Example 10. Analysis of a synthetic EXP, EXP-At.GSP576.nno+At.GSI17.nno:3, stimulating GUS expression in stably transformed soybean plants

Soybean plants are transformed with vectors, in particular plant expression vectors, containing groups of regulatory elements that stimulate expression of the β-glucuronidase (GUS) transgene. The resulting plants are analyzed for GUS protein expression to assess the effect of selected groups of regulatory elements on expression.

Soybean plants were 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:!:! (SEQ ID NO: 38). The GUS transgene cassettes also contained the 3' untranslated region of the Gossypium barbadense FbLate-2 gene (T-Gb.FbL2:1, SEQ ID NO: 36) operably linked 3' to the GUS coding sequence. The resulting soybean transformed events were grown, and tissue samples from selected organs at several developmental stages were collected and analyzed to assess qualitative and quantitative GUS expression. GUS expression in stably transformed soybean plants driven by EXP-At.GSP576.nno+At.GSI17.nno:3 is compared with that driven by EXP-A1Cyco:1:1. GUS expression in stably transformed soybean plants driven by EXP-At.GSP576.nno+At.GSI17.nno:3 demonstrates the ability of the synthetic PAt.GSP576.nno:4 promoter (SEQ ID NO: 27) and L-At.GSP576.nno:2 leader (SEQ ID NO: 28) to drive constitutive expression of a functionally linked transgene.

As shown in Examples 9 and 11, the synthetic promoter P-At.GSP576.nno:4 (SEQ ID NO: 27) and the leader L-At.GSP576.nno:2 (SEQ ID NO: 28) drive constitutive expression of the operably linked transgene. As shown in Example 4, the synthetic intron I-At.GSI17.nno:1 (SEQ ID NO: 17) enhanced or modulated transgene expression in multiple plant organs when operably linked to EXP-At.GSP564 (SEQ ID NO: 12). Likewise, it would be reasonable to expect that expression of the synthetic promoter P-At.GSP576.nno:4 and the leader L-At.GSP576.nno:2 would be enhanced or modulated in a similar manner.

Example 11. Analysis of a synthetic EXP, EXP-At.GSP576.nno+At.GSI17.nno:3, stimulating GUS expression in stably transformed cotton plants

Cotton plants were transformed with a vector, specifically a plant expression vector, containing a group of synthetic regulatory elements stimulating the expression of the β-glucuronidase (GUS) transgene. The resulting plants were analyzed for GUS protein expression to assess the effect of the selected group of synthetic regulatory elements on expression.

Cotton plants were transformed with a binary vector containing synthetic EXP, EXP-At.GSP576.nno+At.GSI17.nno:3 (SEQ ID NO: 26), as described previously in Example 10. The transgenic cas

- 28 048365 GUS sets also contained the 3'-untranslated region of the Gossypium barbadense FbLate-2 gene (TGb.FbL2:1, SEQ ID NO: 36) operably linked 3' to the GUS coding sequence. The resulting transformed cotton events were grown and tissue samples were obtained from the 4th node leaf, 8th node petiole, petiole, leaf (source), and leaf (storage tissue); square bracts and square buds before fertilization; anther and ovary at anthesis, and boll wall at 8 days post-pollination (DAP) for qualitative and quantitative GUS expression.

Table 10 shows the average quantitative GUS expression for each of the selected tissues stimulated by synthetic EXP-At.GSP576.nno+At.GS117.nno:3.

Table 10. Average quantitative expression of GUS in stably transformed cotton plants stimulated by EXP-At.GSP576.nno+At.GSI17.nno:3

Stage Organ Average value 4 knot - sheet 579,03 8 knot - leaf, petiole 301,57 Sheet, storage fabric 159, 4 Sheet, source 577,11 Before fertilization - square bracts 262.66 square buds 223.59 Blooming, dust cover 171.2 Flowering, ovary 109 8 DAP Wall of the seed box 433,64

As shown in Table 10, EXP-At.GSP576.nno+At.GSI17.nno:3 (SEQ ID NO: 26) drove constitutive expression of the GUS transgene in stably transformed cotton plants. The highest expression was observed in the 4th node leaf, 8th node leaf (source) and boll wall at 8 DAP. The synthetic promoter B-AtGSP57 6.nno:4 (SEQ ID NO: 27) and leader L-At.GSP576.nno:2 (SEQ ID NO: 28) were able to stimulate constitutive expression of the functionally linked transgene in stably transformed cotton plants. As shown in Example 4, the synthetic intron I-At.GSI17.nno:1 (SEQ ID NO: 17) enhanced or modulated transgene expression in multiple plant organs when operably linked to EXP-At.GSP564 (SEQ ID NO: 12). Similarly, it would be reasonable to expect that expression of the synthetic promoter P-At.GSP576.nno:4 and leader L-At.GSP576.nno:2 would be enhanced or modulated in a similar manner in stably transformed cotton plants.

Example 12. Enhancer elements derived from a regulatory element

Enhancers are derived from promoter elements shown as SEQ ID NOs: 2, 5, 13, 20, 25, 27, 31 and 39. An enhancer element may consist of one or more cis-regulatory elements that, when operably linked 5' or 3' to a promoter element or operably linked 5' or 3' to additional enhancer elements that are operably linked to a promoter, can enhance or modulate the expression levels of a transcribed DNA molecule or ensure the expression of a transcribed DNA molecule in a particular cell type or plant organ or at a particular time in development or circadian rhythm. Enhancers are obtained by deleting the TATA box or functionally similar elements and any downstream sequence from the promoters that allows initiation of transcription from the promoters shown as SEQ ID NOs: 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 obtained from the synthetic promoter PAt.GSP571.nno:5 (SEQ ID NO: 5) and consists of nucleotides 1 through 422 of P-At.GSP571.nno:5, excluding the 3' downstream sequence that also contains the TATA box of the synthetic promoter.

Further refinement of the enhancer element may be required and has been supported empirically. 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 positions of each element. Some promoter elements will have multiple TA elements

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TA-box or TATA-box-like elements and potentially multiple transcription start sites. In such circumstances, it may be necessary to first determine where the first TSS is and then begin designing enhancers using the first TSS to prevent transcription from initiating at the putative enhancer element.

Enhancer elements derived from the synthetic promoter elements shown as SEQ ID NOs: 2, 5, 13, 20, 25, 27, 31 and 39 are cloned using methods known in the art to operably link 5' or 3' to a promoter element or operably link 5' or 3' to additional enhancer elements operably linked to a promoter. Alternatively, the enhancer elements can be cloned using methods known in the art to provide a larger enhancer element that consists of two or more copies of an enhancer and cloned using methods known in the art to operably link 5' or 3' to a promoter element or operably link 5' or 3' to additional enhancer elements that are operably linked to a promoter producing a chimeric transcriptional regulatory element. Enhancer elements may also be cloned using methods known in the art to operably link 5' to a promoter element derived from an organism of another genus, or to operably link 5' or 3' to additional enhancer elements derived from organisms of another genus that are operably linked to a promoter derived from an organism of the same or another genus, resulting in the creation of a chimeric regulatory element. A plant transformation vector for expression of GUS may be constructed using methods known in the art similar to the constructs described in Example 2, wherein the resulting plant expression vectors comprise 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; a second transgene cassette for testing an enhancer element, consisting of an enhancer element operably linked 5' or 3' to a promoter element or operably linked 5' or 3' to additional enhancer elements, which in turn are operably linked to a promoter, which is operably linked 5' to a leader element operably linked to the coding sequence for β-glucuronidase (GUS, GOI-Ec.uidA+St.LS1:1:1, SEQ ID NO: 42), containing a processable intron obtained from the light-inducible tissue-specific ST-LS1 gene of potato (Genbank Accession: X04753), operably linked to the 3' termination region and the left border region from A. tumefaciens (B-AGRtu.left border). The resulting plasmids are used to transform soybean plants or other plants by the methods described in the examples. Alternatively, protoplast cells derived from soybean plants or other genera are transformed using methods known in the art to perform transient expression assays.

GUS expression stimulated by a regulatory element containing one or more enhancers is assessed by performing stable or transient expression assays in plants to determine the effect of the enhancer element on the expression of the transcribed DNA molecule. Modifications of one or more enhancer elements or duplication of one or more enhancer elements can be made based on empirical experiments and the resulting regulation of gene expression that is observed using each composition of regulatory elements. Changing the relative positions of one or more enhancers in the resulting regulatory or chimeric regulatory elements can affect the transcriptional activity or specificity of the regulatory or chimeric regulatory element and is determined empirically to identify the best enhancers for the desired expression profile of the transgene in soybean or other plant genus.

Example 13. Analysis of the effect on GUS expression caused by the synthetic 3'-untranslated region, T-Zm.GST7.nno:2, in stably transformed soybean plants

Soybean plants were transformed with a vector, specifically plant expression vectors, containing groups of regulatory elements that stimulate the expression of the β-glucuronidase (GUS) transgene. The resulting plants were analyzed for GUS protein expression to assess the effect of the selected regulatory elements on expression.

Soybean plants were transformed with two binary vectors containing EXP-At.GSP571 (SEQ ID NO: 4), which drives GUS expression. The GUS transgene cassettes also contained either the endogenous 3'-untranslated region T-Mt.Sali3-2-1:2:1 (SEQ ID NO: 34) or the synthetic 3'-untranslated region T-Zm.GST7.nno:2 (SEQ ID NO: 44). GUS protein expression was quantified in organs of stably transformed soybean plants transformed with the two constructs. GUS expression was compared between the constructs. The following Table 11 shows the average GUS expression modulated by the synthetic 3'-UTR TZm.GST7.nno:2 relative to that modulated by the endogenous 3'-UTR T- 30 048365

Mt.Sali3-2-1:2:1, where N/D means not determined and BLDL means below detection limit.

Table 11. Average quantitative expression of GUS in stably transformed soybean plants

Stage of development Organ T-Mt.Sali3- 2-1:2:1 (SEQ ID NO:34) T- Ζιη. GST7. nno :2 (SEQ ID NO:44) Weakening (times) V5 Root 40 NGO Sheet (storage fabric) 650 88 7.4 Sheet (source) 1379 278 5, 0 R1 Root 110 72 1,5 Leaf (petiole) 951 199 4.8 Sheet (source) 1995 642 3, 1 Flowers 703 139 5, 1 R3 Immature seeds 75 NGO Pod 852 386 2,2 R5 Seed (cotyledon) 650 174 3.7 R8 Seed (embryo) 1153 H/O Seed (cotyledon) 2449 H/O

As shown in Table 11, the synthetic 3'-UTR, T-Zm.GST7.nno:2, attenuated expression relative to the 3'-UTR, T-Mt.Sali3-2-1:2:1, in all tissues analyzed. The degree of attenuation varied for each tissue from 1.5-fold in R1 roots to 7.4-fold in V5 leaves (source). The use of the 3'-UTR to attenuate expression in stably transformed plants has broad applications. For example, the 3'-UTR can be used in combination with other regulatory elements such as promoters, leaders, and introns to fine-tune transgene expression, particularly in cases where high expression may result in non-phenotypic effects that are detrimental to the transformed plant. Analysis of the resulting GUS transcript confirmed proper transcript termination mediated by the synthetic 3'-untranslated region of T-Zm.GST7.nno:2. The synthetic 3'-untranslated region of T-Zm.GST7.nno:2 is able to modulate expression and ensure proper transcription termination in stably transformed soybean plants.

Example 14. Analysis of the synthetic 3'-untranslated regions of T-Zm.GST7.nno:2 and TZm.GST59.nno:1 for GUS expression in maize protoplasts

Maize leaf protoplasts were transformed with vectors, specifically expression vectors containing the tested regulatory elements that stimulate expression of the β-glucuronidase (GUS) transgene. The resulting transformed maize leaf protoplasts were analyzed for GUS protein expression to assess the effect of the selected regulatory elements on expression.

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

The plasmid used for co-transformation of protoplasts and data normalization was also constructed using methods known in the art. It contained trans

- 31 048365 a gene cassette consisting of EXP-CaMV.35S (SEQ ID NO: 46) operably linked 5' to the coding sequence encoding the NanoLuc® luciferase fluorescent protein (Promega, Madison, WI 53711), Nluc (SEQ ID NO: 49), which was operably linked 5' to the 3' untranslated region of T-Os.LTP:1 (SEQ ID NO: 48).

Maize leaf protoplasts were transformed using a PEG-based transformation method similar to those known in the art. Protoplast cells were transformed on a ninety-six-well plate. Twelve micrograms of test vector DNA or control vector DNA and six micrograms of NanoLuc® vector DNA were used to transform 3.2 x 10 ⁵ protoplasts per well. Following transformation, protoplasts were incubated at 25°C in the dark for 16 to 20 h. Following incubation, protoplasts were lysed and the lysate was used to measure GUS and luciferase expression. To lyse the cells, the plate was pelleted, washed, resuspended in a smaller volume and transferred to dedicated tubes in strips. The tubes were centrifuged again and the supernatant was aspirated, leaving a pellet of protoplast cells. 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). Cells were lysed by vigorously pipetting the cells several times, vortexing, and allowing the tubes to incubate on ice for five minutes. The lysate was then centrifuged to pellet cell debris. The resulting lysate was then transferred to a clean plate.

Luciferase activity was assayed using NanoGlo® Luciferase Assay Substrate (Promega, Madison, WI 53711) in QB buffer. Briefly, a small volume of lysate, QB buffer, and Nano-Glo® Luciferase Assay Substrate solution in QB were mixed together in white ninety-six-well plates. Fluorescence was then measured using a PHERAstar® Plate Reader (BMG LABTECH Inc., Cary, NC 27513).

GUS activity was assayed using the fluorogenic substrate 4-methylumbelliferyl-b-O-glucuronide (MUG) in a total reaction volume of 50 μl. The reaction product, 4-methylumbelliferone (4-MU), is maximally fluorescent at high pH, where the hydroxyl group is ionized. Addition of a stock solution of sodium carbonate both stops the assay and adjusts the pH for quantification of the fluorescent product. An aliquot of lysate was mixed with an aliquot of MUG dissolved in QB buffer and incubated at 37°C. A small aliquot of the lysate/MUG reaction mixture was removed and added to stop buffer at three different time points: (1) immediately after mixing the lysate/MUG reaction mixture (time defined as time zero minutes); (2) after 20 min; and (3) after 60 min. Fluorescence was measured with excitation at 355 nm, emission at 460 nm using a PHERAstar® plate reader (BMG LABTECH Inc., Cary, NC 27513).

At least two plates were used for the transformation process, with four to eight transformations per plate for each expression vector. On each plate, four or eight wells of each construct were transformed. An aliquot was taken from each transformation for MUG analysis, and the hydrolyzed MUG value, nM, was taken from the standard curve for the plate. An aliquot of each transformation was also taken for NanoLuc® readout (NanoLuc® RLU). The average hydrolyzed MUG (nM)/NanoLuc® RLU for each expression vector was normalized to the expression vector EXP-CaMV.35S/I-Zm.DnaK:1/TOs.LTP:1, which was set to 100%. Table 1 12 shows the average of the means for all wells used in transformation for each expression vector containing the synthetic 3'-untranslated regions T-Zm.GST7.nno:2 and T-Zm.GST59.nno:1 and for the controls.

Table 12. Average hydrolyzed MUG (nM)/NanoLuc® RLU values ____________________for each expression vector____________________

3'-untranslated region Average of average values Stderr T-Os.LTP:1 100,00 8.09 No 3' untranslated region 51.95 4.71 T-Zm.GST59.nno:1 505,45 37.75 T-Zm.GST7.nno:2 345.31 40.73

As shown in Table 12, the expression vector lacking the 3'-untranslated region provided lower expression than the T-Os.LTP:1 control. Expression was enhanced by the synthetic 3'-untranslated regions T-Zm.GST7.nno:2 and T-Zm.GST59.nno:1 compared to the T-Os.LTP:1 control. Transcript analysis showed proper termination mediated by the synthetic 3'-untranslated regions T-Zm.GST7.nno:2 and T-Zm.GST59.nno:1. The synthetic 3'-untranslated regions of T

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Zm.GST7.nno:2 and T-Zm.GST59.nno:1 are able to modulate expression and ensure proper transcription termination in transformed maize leaf protoplasts.

Example 15. Analysis of regulatory elements stimulating GUS in cotton leaf protoplasts

Cotton leaf protoplasts were transformed with vectors, in particular expression vectors containing groups of regulatory elements stimulating the expression of the β-glucuronidase (GUS) transgene. The resulting transformed cotton leaf protoplasts were analyzed for GUS protein expression to assess the effect of the selected groups of regulatory elements on expression.

Cotton protoplasts derived from leaf tissue were transformed with expression vectors containing synthetic expression elements and compared with expression elements known in the art. Separate experiments were performed to assess the activity of EXP, EXP-At.GSP571 (SEQ ID NO: 4), EXP-AtGSP571.nno+At.GSI21.nno:10 (SEQ ID NO: 8), EXPAtGSP571.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 expression vectors and operably linked to the GUS coding sequence, GOIEc.uidA+St.LSI:1:1 (SEQ ID NO: 42), which contained the intron being processed. Control expression vectors contained various configurations of known expression elements.

Two plasmids for co-transformation and data normalization were also constructed using methods known in the art. Each plasmid contained a specific luciferase coding sequence that was 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-Enhm+Zm.DnaK: 1:1, SEQ ID NO: 53), operably linked 5' to the firefly (Photinus pyralis) luciferase coding sequence (LUCIFERASE:1:3, SEQ ID NO: 54), operably linked 5' to the 3' untranslated region 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 sequence encoding Renilla reniformis luciferase (CR-Ren.hRenilla Lucife-0:0:1, SEQ ID NO: 57), operably linked 5' to the 3'-untranslated region from the Agrobacterium tumefaciens nopaline synthase gene (T-AGRtu.nos-1:1:13, SEQ ID NO: 55).

Cotton leaf protoplasts were transformed using a PEG-based transformation method known in the art. The protoplasts were transformed with plasmids, pFLUC and pRLUC, and equimolar amounts of EXP expression vectors. GUS and luciferase measurements were performed by placing aliquots of the lysed cell preparation transformed as described above into two different shallow-well plates. One plate was used for GUS measurements and the second for the Dual-Luciferase assay using the Dual-Luciferase Reporter Gene 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 presented in Example 14. Average GUS/FLUC values were calculated similar to Example 14 but were not normalized to the EXP control vectors.

EXP sequences, 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 5' to the GUS coding sequence (SEQ ID NO: 42), operably linked 5' to the 3' untranslated region of T-Mt.Sali3-2-1:2:1 (SEQ ID NO: 34). Two control plant expression vectors were constructed using an EXP sequence, EXP-At.Bglu21+At.Cyco:2 (SEQ ID NO:50), known to be poorly expressed in cotton leaf protoplasts, and an EXP sequence, EXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO:51), which is well expressed in cotton leaf protoplasts. The control EXP sequences were operably linked to the same 3' UTR and GUS sequence. In addition, the plant expression vector containing the GUS transgene cassette containing EXP, EXP-At.GSP571 (SEQ ID NO: 4), operably linked to GUS, contains a synthetic 3'-untranslated region, T-Zm.GST7.nno:2 (SEQ ID NO: 44) to assess the activity of the synthetic 3'-untranslated region. The average GUS/FLUC values for several transformations are presented in Table 13.

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Table 13. Average GUS/FLUC values for transformed cotton leaf protoplasts

EXP EXP SEQ ID NO: 3'- untranslated May area 3' untranslated region, SEQ ID NO: Average GUS/FLUC EXP- At.Bglu21+At.Suso:2 50 T-Mt.Sali3- 2-1:2:1 34 0.09 EXP-CaMV.35Senh+Ph.DnaK:1:3 51 T-Mt.Sali3- 2-1:2:1 34 1.70 EXP-At.GSP571 4 T-Mt.Sali3- 2-1:2:1 34 0.56 EXP- At.GSP571.nno+At.GS 121.nno:10 8 T-Mt.Sali3- 2-1:2:1 34 1,02 EXP- At.GSP571.nno+At.GS 10 T-Mt.Sali3- 2-1:2:1 34 0.95

1102.nno:1 EXP-At.GSP571 4 T- Zm.GST7.nno: 2 44 0.46

As shown in Table 13, EXP-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) showed expression in cotton leaf protoplasts. The synthetic 3'-untranslated region of T-Zm.GST7.nno:10 (SEQ ID NO: 44) functioned similarly to the endogenous 3'-untranslated region of T-Mt.Sali3-2l:2:1.

The EXP sequence, EXP-At.GSP564.nno+At.GSI17.nno:2 (SEQ ID NO: 16) was cloned into plant expression vectors operably linked 5' to the GUS coding sequence (SEQ ID NO: 42) operably linked 5' to the endogenous 3' untranslated region of T-Mt.Oxr-1:2:1 (SEQ ID NO: 35). Two control plant expression vectors were constructed using the EXP, EXP-Gm.Sphas1:1:1 (SEQ ID NO: 52), which is known to be poorly expressed in cotton leaf protoplasts, and the EXP sequence, EXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO:

51), which is well expressed in cotton leaf protoplasts. Control sequences

EXP values were functional with the region and GUS. Average G values Table 14. Average GUi values EXP EXP-Gm.Sphasl:1:1 EXP-CaMV.35S- enh+Ph.DnaK:1:3 knitted with US/FLUC S/FLUC for EXP SEQ ID NO: 52 51 one and the same for several I transform 3' - non-translatable area T-Mt.Ohg- 1:2:1 T-Mt.Ohg- 1:2:1 e sequences of transformed protope 3'-untranslated region, SEQ ID NO: 35 35 3'-ness is represented by the last leaf Average value GUS/FLUC 0.01 2.30 broadcast 1ы in table. 14. :in cotton EXP- At.GSP564.nno+At. GSI17.nno:2 16 T-Mt.Ohg- 1:2:1 35 0.34

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

The EXP sequence, EXP-At.GSP579.nno+At.GSI102.nno:3 (SEQ ID NO: 22), was cloned into plant expression vectors operably linked 5' to the GUS coding sequence (SEQ ID NO: 42) operably linked 5' to the endogenous 3' untranslated region of T-Mt.RD221:2:1 (SEQ ID NO: 37). Two control plant expression vectors were constructed using an EXP, EXP-Gm.Sphas1:1:1 (SEQ ID NO: 52), known to be poorly expressed in cotton leaf protoplasts, and an EXP sequence, EXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO: 51), known to be well expressed in cotton leaf protoplasts. The control post

- 34,048,365 EXP sequences were functionally linked to the same 3'UTR sequence and GUS. The average GUS/FLUC values for several transformations are presented in Table 15.

Table 15. Average GUS/FLUC values for transformed cotton leaf protoplasts

EXP EXP SEQ ID NO: 3' - untranslated area 3'- untranslated region, SEQ ID NO: Average GUS/FLUC EXP-Gm.Sphasl:1:1 52 T-Mt.RD22- 1:2:1 37 0, 01 EXP-CaMV.35S- enh+Ph.DnaK:1:3 51 T-Mt.RD22- 1:2:1 37 2.88 EXP- At. GSP57 9.nno+At. GSI102.nno:3 22 T-Mt.RD22- 1:2:1 37 1, 19

As shown in Table 15, the synthetic EXP, EXP-At.GSP579.nno+At.GSI102.nno:3 (SEQ ID NO: 22), demonstrated expression in cotton leaf protoplasts.

It will be apparent to those skilled in the art from the illustrations and descriptions of the principles of the present invention that the invention may be modified in detail and arrangement without departing from such principles. The claims include all modifications that fall within the spirit and scope of the claims. All publications and published patent documents cited herein are hereby incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims (18)

1. A recombinant DNA molecule having promoter activity in a plant or part thereof, comprising a DNA sequence selected from the group consisting of: a) a sequence at least 85% identical to any of the full-length sequences of SEQ ID NO: 8, 4, 5 and 9; b) a sequence comprising any one of SEQ ID NOs: 8, 4, 5 and 9; and c) a fragment containing at least 100 consecutive nucleotides of any of the sequences of SEQ ID NO: 8, 4, 5 and 9, wherein this fragment has gene regulatory activity. 2. A recombinant DNA molecule according to claim 1, characterized in that the DNA sequence is functionally linked to a heterologous transcribed DNA molecule. 3. A recombinant DNA molecule according to claim 1, characterized in that the DNA sequence is at least 90% identical to the full-length DNA sequence of any of SEQ ID NOs: 8, 4, 5, and 9. 4. A recombinant DNA molecule according to claim 1, characterized in that the DNA sequence is at least 95% identical to the full-length DNA sequence of any of SEQ ID NOs: 8, 4, 5, and 9. 5. A recombinant DNA molecule according to claim 1, characterized in that the DNA sequence has gene-regulatory activity. 6. A recombinant DNA molecule according to claim 2, characterized in that the heterologous transcribed DNA molecule contains a gene of agronomic interest. 7. A recombinant DNA molecule according to claim 6, characterized in that the gene of agronomic interest imparts herbicide resistance to plants. 8. A recombinant DNA molecule according to claim 6, characterized in that the gene of agronomic interest imparts pest resistance to plants. 9. A transgenic plant cell containing a recombinant DNA molecule according to claim 1, wherein said recombinant DNA molecule has promoter activity in said plant cell. 10. A transgenic plant cell according to claim 9, characterized in that the DNA sequence is functionally linked to a heterologous transcribed DNA molecule. 11. A transgenic plant cell according to claim 9, characterized in that said transgenic plant cell is a monocotyledonous plant cell. 12. A transgenic plant cell according to claim 9, characterized in that said transgenic plant cell is a dicotyledonous plant cell. 13. A transgenic plant or part thereof containing a recombinant DNA molecule according to claim 1, wherein said recombinant DNA molecule has promoter activity in said plant or its - 35 048365 parts. 14. A plant that is a descendant of a transgenic plant according to claim 13, or a part thereof, characterized in that the descendant plant or part thereof contains a recombinant DNA molecule, wherein said recombinant DNA molecule has promoter activity in said descendant plant or part thereof. 15. Transgenic seed containing a recombinant DNA molecule according to claim 1. 16. A method for obtaining a food or feed product, comprising obtaining a transgenic plant or part thereof according to paragraph 13 and obtaining a food or feed product from it. 17. The method according to claim 16, characterized in that the food or feed product is selected from the group consisting of protein concentrate, protein isolate, grain, starch, seeds, cereals, flour, biomass and seed oil. 18. A method for expressing a transcribed DNA molecule, comprising obtaining a transgenic plant according to claim 13 and cultivating said plant in which the transcribed DNA is expressed.