Plant regulatory elements and uses thereof

Abstract

The present invention provides DNA molecules and constructs comprising their nucleotide sequences useful for modulating gene expression in plants and plant cells. Transgenic plants, plant cells, plant parts, seeds, and commodity products comprising the DNA molecules operably linked to heterologous transcribable polynucleotides are also provided, as are methods of use thereof.

Description

[0001] This application is a divisional application of the invention patent application entitled "Plant Regulatory Elements and Their Applications" filed on May 11, 2012, with application number 201280031326.X (application number 201710187051.3, filed on March 24, 2017, with invention title "Plant Regulatory Elements and Their Applications"). Citation of relevant applications

[0002] This application claims the benefit of U.S. Provisional Application No. 61 / 485,876, filed May 13, 2011, which is incorporated herein by reference in its entirety.

[0003] Introduction of sequence lists The sequence list contained in the file named "MONS304WO.txt" is hereby submitted electronically and incorporated by reference. The sequence list is 463 kilobytes (measured according to Microsoft Windows®) and was created on May 9, 2012. Invention Field This invention relates to the fields of plant molecular biology and plant genetic engineering, as well as DNA molecules that can be used to regulate gene expression in plants. background Regulatory elements are genetic elements that regulate gene activity by modulating the transcription of operatively linked, transcribed polynucleotide molecules. These elements include promoters, leader sequences, introns, and 3' untranslated regions, and are used in plant molecular biology and plant genetic engineering. Invention Overview This invention provides a source derived from melon ( Cucumber melon This invention provides novel gene regulatory elements for plants (such as promoters, leader sequences, and introns) commonly referred to as muskmelon plant varieties. The invention also provides DNA constructs containing said regulatory elements, transgenic plant cells, plants, and seeds. The sequences can be operatively linked to transcribed polynucleotide molecules, which may be heterologous to the regulatory sequences provided herein. The invention also provides methods for preparing and using said regulatory elements, DNA constructs containing said regulatory elements, and transgenic plant cells, plants, and seeds containing regulatory elements operatively linked to transcribed polynucleotide molecules.

[0004] Therefore, in one aspect, the present invention provides a DNA molecule, such as a set of regulatory expression elements for transcription, or a promoter, or a leader sequence, or an intron, comprising a polynucleotide sequence selected from the group consisting of: a) a sequence having at least 85% sequence identity with any one of SEQ ID NO: 1-199, 211, and 212; b) a sequence comprising any one of SEQ ID NO: 1-199, 211, and 212; and c) a fragment of any one of SEQ ID NO: 1-199, 211, and 212 exhibiting gene regulatory activity, wherein the DNA molecule is operatively linked to a heterologous transcribed polynucleotide molecule. In a specific embodiment, the set of regulatory expression elements for transcription, or a promoter, or a leader sequence, or an intron is at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 1-199, 211, and 212. In a particular embodiment, the heterologous transcribed polynucleotide molecule contains genes for agronomic purposes, genes that can provide herbicide resistance in plants, or genes that can provide plant resistance to pests.

[0005] The present invention also provides transgenic plant cells containing a set of transcriptional regulatory expression elements, or a promoter, or a leader sequence, or an intron, wherein the DNA molecule comprises a polynucleotide sequence selected from the group consisting of: a) a sequence having at least 85% sequence identity with any one of SEQ ID NO: 1-199, 211, and 212; b) a sequence comprising any one of SEQ ID NO: 1-199, 211, and 212; and c) a fragment exhibiting gene regulatory activity of any one of SEQ ID NO: 1-199, 211, and 212, wherein the DNA molecule is operatively linked to a heterologous transcribed polynucleotide molecule. Furthermore, the set of transcriptional regulatory expression elements, or the promoter, or the leader sequence, or the intron regulates gene expression. The transgenic plant cell may be a monocotyledonous or dicotyledonous plant cell.

[0006] The present invention further provides a transgenic plant or a portion thereof containing a set of transcriptional regulatory expression elements, or a promoter, or a leader sequence, or an intron, wherein the DNA molecule comprises a polynucleotide sequence selected from the group consisting of: a) a sequence having at least 85% sequence identity with any one of SEQ ID NO: 1-199, 211, and 212; b) a sequence comprising any one of SEQ ID NO: 1-199, 211, and 212; and c) a fragment exhibiting gene regulatory activity of any one of SEQ ID NO: 1-199, 211, and 212, wherein the DNA molecule is operatively linked to a heterologous transcribed polynucleotide molecule. In a specific embodiment, the transgenic plant may be a progeny plant of any generation containing the set of transcriptional regulatory expression elements, or a promoter, or a leader sequence, or an intron.

[0007] Furthermore, transgenic seeds containing a set of regulatory expression elements, promoters, leader sequences, or introns of DNA molecules such as transcriptional molecules are provided, said DNA molecules comprising polynucleotide sequences selected from the group consisting of: a) sequences having at least 85% sequence identity with any one of SEQ ID NO: 1-199, 211, and 212; b) sequences comprising any one of SEQ ID NO: 1-199, 211, and 212; and c) fragments exhibiting gene regulatory activity of any one of SEQ ID NO: 1-199, 211, and 212, wherein said DNA molecules are operatively linked to heterologous transcribed polynucleotide molecules.

[0008] In another aspect, the present invention provides a method for producing a commercial product from a transgenic plant, a transgenic plant portion, or a transgenic seed containing a set of regulatory expression elements, or a promoter, or a leader sequence, or an intron, such as a DNA molecule, the DNA molecule comprising a polynucleotide sequence selected from the group consisting of: a) a sequence having at least 85% sequence identity with any one of SEQ ID NO: 1-199, 211, and 212; b) a sequence comprising any one of SEQ ID NO: 1-199, 211, and 212; and c) a fragment of any one of SEQ ID NO: 1-199, 211, and 212 exhibiting gene regulatory activity, wherein the DNA molecule is operatively linked to a heterologous, transcribed polynucleotide molecule. In one embodiment, the commercial product is a protein concentrate, a protein isolate, a cereal, starch, a seed, a whole grain, flour, biomass, or seed oil.

[0009] In another aspect, the present invention provides goods comprising a set of regulatory expression elements, or promoters, or leader sequences, or introns of DNA molecules such as transcription, said DNA molecules comprising polynucleotide sequences selected from the group consisting of: a) sequences having at least 85% sequence identity with any one of SEQ ID NO: 1-199, 211, and 212; b) sequences comprising any one of SEQ ID NO: 1-199, 211, and 212; and c) fragments of any one of SEQ ID NO: 1-199, 211, and 212 exhibiting gene regulatory activity, wherein said DNA molecules are operatively linked to heterologous transcribed polynucleotide molecules.

[0010] In another aspect, the present invention provides a method for expressing a transcribed polynucleotide molecule in a transgenic plant using a DNA molecule such as a set of regulatory expression elements for transcription, or a promoter, or a leader sequence, or an intron, said DNA molecule having a DNA sequence that is at least 85% identical to any one of SEQ ID NO: 1-199, 211, and 212, or contains any one of SEQ ID NO: 1-199, 211, and 212 or consists of fragments of any one of SEQ ID NO: 1-199, 211, and 212; and cultivating said transgenic plant.

[0011] Brief description of the sequence SEQ ID NO: 1, 5, 7, 9, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 ,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,6 9, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 159, 162, 167, 168, 172, 175, 176, 177, 178, 181, 182, 183, 184, 185, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 211, and 212 are regulatory expression elements or EXP sequences of the genus *Calamus*, consisting of any of the following elements: promoter elements that operatively link a leader sequence element; promoter elements that operatively link a leader sequence element and an intron element; or promoter elements that operatively link a leader sequence element, operatively link an intron element, or operatively link a leader sequence element.

[0012] SEQ ID NO: 2, 6, 8, 10, 12, 163 and 169 are promoter elements.

[0013] SEQ ID NO: 3, 164, 166 and 170 are leader sequences.

[0014] SEQ ID NO: 4, 165 and 171 are intron sequences.

[0015] SEQ ID NO: 157, 160, 173, 179 and 186 are sequences in which the promoter is operatively connected to the leader sequence element.

[0016] SEQ ID NO: 158, 161, 174, 180 and 187 are sequences in which introns are operatively connected to leader sequence elements. Brief description of the attached figures Figure 1a-1f A comparison of promoter variant fragments corresponding to promoter elements isolated from melon is depicted. In particular, Figure 1a-1f The 2068 bp promoter sequence P-CUCme.Ubq1-1:1:15 (SEQ ID NO: 2) found in the transcriptional regulatory expression element group EXP-CUCme.Ubq1:1:1 (SEQ ID NO: 1) is compared with the promoter sequence P-CUCme.Ubq1-1:1:15 derived via the deletion of the 5' promoter. Deletion, for example, of the 5' end of P-CUCme.Ubq1-1:1:15, produces the following promoters: the 1459 bp promoter P-CUCme.Ubq1-1:1:16 (SEQ ID NO: 6) found in EXP-CUCme.Ubq1:1:2 (SEQ ID NO: 5); the 964 bp sequence P-CUCme.Ubq1-1:1:17 (SEQ ID NO: 8) contained in EXP-CUCme.Ubq1:1:3 (SEQ ID NO: 7); the 479 bp sequence P-CUCme.Ubq1-1:1:18 (SEQ ID NO: 10) contained in EXP-CUCme.Ubq1:1:4 (SEQ ID NO: 9); and the sequence P-CUCme.Ubq1-1:1:18 (SEQ ID NO: 10) contained in EXP-CUCme.Ubq1:1:5 (SEQ ID NO: 6). The 173bp sequence P-CUCme.Ubq1-1:1:19 (SEQ ID NO: 12) is found in NO: 11. Invention Details The invention disclosed herein provides polynucleotide molecules with favorable gene regulatory activity obtained from melon. The design, construction, and use of these polynucleotide molecules are described. The nucleotide sequences of these polynucleotide molecules are provided in SEQ ID NOs: 1-199, 211, and 212. For example, these polynucleotide molecules can influence the expression of transcribed polynucleotide molecules operably linked in plant tissues, thus selectively regulating gene expression or the activity of gene-encoding products in transgenic plants. The invention also provides methods for modifying, preparing, and using said polynucleotide molecules. The invention further provides compositions containing said promoters and / or other disclosed nucleotide sequences, transformation of host cells, transgenic plants, and seeds, as well as methods for preparing and using them.

[0018] The following definitions and methods are provided to better define the invention and to guide those skilled in the art in its implementation. Unless otherwise stated, these terms are to be understood according to their conventional usage by those skilled in the art.

[0019] 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 or polynucleotide molecule of deoxyribonucleotide bases, read from the 5' (upstream) end to the 3' (downstream) end. As used herein, the term "DNA sequence" refers to the nucleotide sequence of a DNA molecule.

[0020] As used herein, the term "separated DNA molecule" refers to a DNA molecule that is at least partially separated from other molecules that are normally associated with it in their natural or native state. In one embodiment, the term "separated" refers to a DNA molecule that is at least partially separated from some nucleic acids that are normally attached to the sides of the DNA molecule in their natural or native state. Thus, DNA molecules fused to regulatory or coding sequences that are normally unrelated to them (e.g., as a result of recombination techniques) are considered separated herein. When integrated into the chromosome of a host cell or present in a nucleic acid solution with other DNA molecules, such molecules are considered separated because they are not in their native state.

[0021] Many methods known in the art are used to isolate and manipulate the DNA molecules or fragments thereof disclosed in this invention. For example, PCR (polymerase chain reaction) technology can be used to amplify specific starting DNA molecules and / or generate variants of the original molecule. DNA molecules or fragments thereof can also be obtained by other techniques, such as direct synthesis of fragments by chemical means, as is typically performed using an automated oligonucleotide synthesizer.

[0022] As used herein, the term "sequence identity" refers to the degree of similarity between two optimized sequence alignments of polynucleotide sequences or two optimized sequence alignments of polypeptide sequences. Optimized sequence alignments are generated by manually comparing two sequences, such as a reference sequence and another sequence, to maximize the number of nucleotide matches with appropriate internal nucleotide insertions, deletions, or gaps in the sequence alignment. As used herein, the term "reference sequence" refers to the sequence provided as the polynucleotide sequence of SEQ ID NO: 1-199, 211, and 212.

[0023] As used herein, the terms "percentage sequence similarity" or "% similarity" or "% similarity" are similarity scores multiplied by 100. The "similarity score" of a sequence optimized for comparison with a reference sequence is the number of nucleotide matches in the optimized comparison divided by the total number of nucleotides in the reference sequence (e.g., the total number of nucleotides in the entire reference sequence). Therefore, one embodiment of the invention comprises a DNA molecule, when optimized for comparison with reference sequences provided herein as SEQ ID NO: 1-199, 211, and 212, a sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or about 99% similarity to said reference sequences. In a particular embodiment, such a sequence may be defined as a peptide having gene regulatory activity or encoding a peptide that acts to localize an operablely linked polypeptide within a cell.

[0024] Control element Regulatory elements are DNA molecules with gene regulatory activity, i.e., DNA molecules with the ability to influence the transcription and / or translation of operably linked transcribed polynucleotide molecules. Therefore, the term "gene regulatory activity" refers to the ability to influence the expression pattern of operably linked transcribed polynucleotide molecules by affecting their transcription and / or translation. As used herein, a set of regulatory expression elements (EXP) for transcription may consist of operably linked expression elements such as enhancers, promoters, leader sequences, and introns. Thus, a set of regulatory expression elements for transcription may include, for example, a promoter operably linked to a leader sequence, which is then operably linked to an intron sequence. The intron sequence may consist of a sequence starting from a point of first intron / exon splicing of the natural sequence and may further consist of a small leader sequence fragment containing a second intron / exon splicing point to provide appropriate intron / exon processing to facilitate transcription and appropriate processing of the resulting transcript. Leader sequences and introns can positively influence the transcription of operatively linked transcribed polynucleotide molecules and the translation of the resulting transcribed RNA. The pre-processed RNA molecule includes leader sequences and introns that can influence the post-transcriptional processing of the transcribed RNA and / or the export of the transcribed RNA molecule from the nucleus to the cytoplasm. Following post-transcriptional processing of the transcribed RNA molecule, the leader sequence can be retained as the final messenger RNA portion and can positively influence the translation of the messenger RNA molecule.

[0025] Regulatory elements, such as promoters, leader sequences, introns, and transcription termination regions, are DNA molecules that possess gene regulatory activity and act as integral parts of the overall gene expression in living cells. The term "regulatory element" refers to a DNA molecule with gene regulatory activity, that is, a DNA molecule capable of influencing the transcription and / or translation of transcribed polynucleotide molecules that are operatively linked. Therefore, isolated regulatory elements, such as promoters and leader sequences, that function in plants can be used to modify plant phenotypes through genetic engineering methods.

[0026] Regulatory elements can influence (qualitatively and / or quantitatively) their expression patterns, for example, through their temporal, spatial, developmental, tissue, environmental, physiological, pathological, cell cycle, and / or chemical response patterns and any combination thereof, as well as through positive or negative and / or fundamental or other effects by quantitative or qualitative indicators. Promoters serve as regulatory elements that regulate the expression of operatively linked, transcribed polynucleotide molecules.

[0027] As used herein, "gene expression pattern" refers to any pattern in which operatively linked DNA molecules are transcribed into transcribed RNA molecules. Transcribed RNA molecules can be translated to produce protein molecules or provide antisense or other regulatory RNA molecules, such as dsRNA, tRNA, rRNA, miRNA, etc.

[0028] As used herein, the term "protein expression" refers to any pattern of translating transcribed RNA molecules into protein molecules. Protein expression can be characterized by its temporal, spatial, developmental, or morphological properties, as well as quantitative or qualitative indicators.

[0029] As used herein, the term "promoter" generally refers to a DNA molecule involved in recognizing and binding RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription. A promoter may first be isolated from the 5' untranslated region (5'UTR) of a copy of the gene's genome. Optionally, a promoter may be a synthetically generated or manipulated DNA molecule. A promoter may also be chimeric, i.e., a promoter generated by the fusion of two or more heterologous DNA molecules. Promoters that can be used to implement the present invention include any one of SEQ ID NO: 2, 6, 8, 10, 12, 163, and 169, or promoter elements, fragments, or variants thereof, contained in any one of SEQ ID No: 13-199, 211, and 212. In specific embodiments of the invention, such molecules and any variants or derivatives thereof described herein are further defined as having promoter activity, i.e., the ability to function as a promoter in host cells, such as in transgenic plants. In a further specific embodiment, a fragment may be defined as exhibiting promoter activity of the initiator promoter molecule from which it is derived, or a fragment may contain a "minimal promoter" that provides a basic level of transcription and consist of a TATA box or equivalent sequence for recognizing and binding to the RNA polymerase II complex for transcription initiation.

[0030] In one embodiment, a fragment of a promoter molecule is provided. As described above, the promoter fragment provides promoter activity and can be used alone or in combination with other promoters and promoter fragments for, for example, to construct a chimeric promoter. In a specific embodiment, a promoter fragment comprising at least about 50, 95, 150, 250, 500, 750, or at least about 1000 consecutive nucleotides of a polynucleotide molecule having the promoter activities disclosed herein is provided.

[0031] Compositions derived from any promoter present in SEQ ID Nos: 2, 6, 8, 10, 12, 163, and 169, or promoter elements contained in SEQ ID Nos: 13-199, 211, and 212, such as internal or 5' deletions, can be generated to enhance or alter expression, including by removing elements that have a positive or negative effect on expression; replicating elements that have a positive or negative effect on expression; and / or replicating or removing elements that have a tissue or cell-specific effect on expression. Compositions derived from any promoter present in SEQ ID Nos: 2, 6, 8, 10, 12, 163, and 169, or promoter elements contained in SEQ ID Nos: 13-199, 211, and 212 consisting of '3' deletions, wherein the TATA box element or its equivalent and downstream sequences are removed, can be used, for example, to prepare enhancer elements. Further deletions can be generated to remove all elements that have a positive or negative effect on expression, a tissue-specific effect, a cell-specific effect, or a timing-specific effect (e.g., but not limited to, circadian rhythm). Any promoter present in SEQ ID No: 2, 6, 8, 10, 12, 163, and 169, or promoter elements included in SEQ ID No: 13-199, 211, and 212, and fragments or enhancers derived therefrom, can be used to prepare chimeric transcriptional regulatory element compositions comprising any promoter present in SEQ ID No: 2, 6, 8, 10, 12, 163, and 169, or promoter elements included in SEQ ID No: 13-199, 211, and 212, and fragments or enhancers derived therefrom operatively linking other enhancers and promoters. The efficacy of modifications, duplications, or deletions in the desired expression of specific transgenes described herein can be confirmed by empirical tests, such as those described in the working examples herein, in stable and transient plant experiments, which may vary depending on the changes made and the target of the alterations in the starting molecule.

[0032] As used herein, the term "leader sequence" refers to a DNA molecule isolated from the 5' untranslated region (5'UTR) of a genomic copy of a gene and is generally defined as a nucleotide fragment between the transcription start site (TSS) and the protein-coding sequence start site. Alternatively, the leader sequence may be a synthetically generated or manipulated DNA element. Leader sequences can serve as 5' regulatory elements for regulating the expression of operatively linked, transcribed polynucleotide molecules. Leader sequence molecules can be used with heterologous promoters or with their native promoters. Thus, the promoter molecules of the present invention can be operatively linked with their native leader sequences or can be operatively linked with heterologous leader sequences. Leader sequences that can be used to implement the present invention include SEQ ID NO:3, 164, 166, and 170, or leader sequence elements, fragments, or variants thereof contained in SEQ ID No:13-199, 211, and 212. In specific embodiments, such sequences can be provided as capable of serving as leader sequences in host cells, such as transgenic plant cells. In one embodiment, such sequences are decoded to contain leader sequence activity.

[0033] The leader sequences (5'UTRs) present in SEQ ID Nos: 3, 164, 166, and 170, or the leader sequence elements included in any one of SEQ ID Nos: 13-199, 211, and 212, may consist of regulatory elements or may employ secondary structures capable of influencing the transcription or translation of transgenes. The leader sequences present in SEQ ID Nos: 3, 164, 166, and 170, or the leader sequence elements included in any one of SEQ ID Nos: 13-199, 211, and 212, can be used according to the present invention to prepare chimeric regulatory elements influencing the transcription or translation of transgenes. Furthermore, the leader sequences present in SEQ ID Nos: 3, 164, 166, and 170, or the leader sequence elements included in any one of SEQ ID Nos: 13-199, 211, and 212, can be used to prepare chimeric leader sequences influencing the transcription or translation of transgenes.

[0034] The introduction of exogenous genes into new plant hosts does not always lead to high expression of the introduced gene. Furthermore, when dealing with complex traits, it is sometimes necessary to regulate multiple genes with spatially or temporally different expression patterns. Introns can primarily provide this regulation. However, it has been shown that the multiple uses of the same intron in a transgenic plant are insufficient. In these cases, a set of basic control elements for constructing appropriate recombinant DNA elements is required. Since the existing set of introns known in the art for expression enhancement is limited, alternatives are needed.

[0035] Compositions derived from any intron present in SEQ ID Nos: 4, 165, and 171, or intronic elements contained in SEQ ID Nos: 13-199, 211, and 212, may consist of internal deletions or duplications of cis-regulatory elements; and / or when operable to link promoter + leader sequences or chimeric promoter + leader and coding sequences, alterations to the 5' and 3' sequences containing the intron / exon splicing site may be used to improve expression or expression specificity. Alterations to the 5' and 3' regions containing the intron / exon splicing site may also be made to reduce the likelihood of the introduction of false start and stop codons in the resulting transcript after messenger RNA processing and splicing. The introns may be empirically tested as described in the working examples to determine their effect on transgene expression.

[0036] According to the present invention, the presence of known promoter elements, i.e., DNA sequence characteristics such as TATA boxes and other known transcription factor binding site motifs, can be analyzed in the promoter or promoter fragment. Those skilled in the art can use this identification of known promoter elements to design variants with expression patterns similar to the original promoter.

[0037] As used herein, the term "enhancer" or "enhancer element" refers to a cis-acting transcriptional regulatory element, also known as a cis-element, which provides one aspect of the overall expression pattern but is generally insufficient to drive the transcription of operably linked polynucleotide sequences on its own. Unlike promoters, enhancer elements typically do not include a transcription start site (TSS) or TATA box or equivalent sequence. Promoters may naturally contain one or more enhancer elements that influence the transcription of operably linked polynucleotide sequences. Isolated enhancer elements may also be fused to promoters to produce chimeric promoter cis-elements, which provide one aspect of the overall regulation of gene expression. Promoters or promoter fragments may contain one or more enhancer elements that influence the transcription of operably linked genes. Many promoter enhancer elements are believed to bind DNA-binding proteins and / or influence DNA topology, creating local constructs that selectively allow or restrict RNA polymerase access to the DNA template or promote selective opening of the double helix at the transcription start site. Enhancer elements may function to bind transcription factors that regulate transcription. Some enhancer elements bind to more than one transcription factor, and transcription factors can interact with more than one enhancer domain through varying affinities. Enhancer elements can be identified using a variety of techniques, including deletion analysis (i.e., deletion of one or more nucleotides from the 5' end or within the promoter); DNA-binding protein analysis using DNase I footprinting, methylation interference, electrophoretic mobility shift assays, in vivo genomic footprinting via ligation-mediated PCR, and other conventional assays; or DNA sequence similarity analysis using known cis-element motifs or enhancer elements as target sequences or target motifs in conjunction with conventional DNA sequence comparison methods such as BLAST. The fine structure of enhancer domains can be further investigated through mutation (or substitution) of one or more nucleotides or by other conventional methods. Enhancer elements can be obtained through chemical synthesis or isolated from regulatory elements that include such elements, and they can be synthesized together with additional flanking nucleotides containing useful restriction enzyme sites to facilitate subsequent manipulation. Therefore, this invention covers the design, construction, and use of enhancer elements according to the methods disclosed herein for regulating the expression of operably linked, transcribed polynucleotide molecules.

[0038] In plants, the presence of some introns in a gene construct, relative to the construct lacking the introns, leads to increased mRNA and protein accumulation. This effect has been termed "intron-mediated enhancement" (IME) of gene expression (Mascarenhas et al., (1990) Plant Mol. Biol. 15:913-920). In maize genes (e.g., tubA1, Adh1, Sh1, Ubi1 (Jeon et al., (2000) Plant Physiol. 123:1005-1014; Callis et al., (1987) Genes Dev. 1:1183-1200; Vasil et al., (1989) Plant Physiol. 91:1575-1579; Christiansen et al., (1992) Plant Mol. Biol. 18:675-689) and in rice genes (e.g., salt, tpi: McElroy et al., Plant Cell 2:163-171 (1990); Xu et al., Plant Physiol. 123:1005-1014; Callis et al., (1987) Genes Dev. 1:1183-1200; Vasil et al., (1989) Plant Physiol. 123:1005-1014 ... Callis et al., (1987) Genes Dev. 1:1183-1200; Vasil et al., (1989) Plant Physiol. 123:1005-1014; Callis et al., (198 Introns known to stimulate expression in plants have been identified (106:459-467 (1994)). Similarly, introns from dicotyledonous genes, such as those from petunia (e.g., rbcS), potatoes (e.g., st-ls1), and Arabidopsis (e.g., ubq3 and pat1), have been found to increase gene expression rates (Dean et al., (1989) Plant Cell 1:201-208; Leon et al., (1991) Plant Physiol. 95:968-972; Norris et al., (1993) Plant Mol Biol 21:895-906; Rose and Last (1997) Plant J.11:455-464). Deletion or mutation within the splice site of an intron has been shown to reduce gene expression, suggesting that IME may require splicing (Mascarenhas et al., (1990) Plant Mol Biol.). 15:913-920; Clancy and Hannah, (2002) Plant Physiol. 130:918-929). However, point mutations within the splice site of the pat1 gene from Arabidopsis have shown that a certain IME in dicotyledonous plants does not require splicing itself (Rose and Beliakoff (2000) Plant Physiol. 122:535-542).

[0039] Enhanced gene expression via introns is not a universal phenomenon because some introns inserted into recombinant expression cassettes do not enhance expression (e.g., introns from dicotyledonous genes (rbcS from peas, lentil protein from beans, and stls-1 from potatoes) and introns from maize genes (the ninth intron of the adh1 gene and the first intron of the hsp81 gene)) (Chee et al., (1986) Gene 41:47-57; Kuhlemeier et al., (1988) MolGen Genet 212:405-411; Mascarenhas et al., (1990) Plant Mol. Biol. 15:913-920; Sinibaldi and Mettler (1992) In WE Cohn, K Moldave, eds, Progress in Nucleic Acid Research and Molecular Biology, Vol 42. Academic Press, New York, (pp. 229-257; Vancanneyt et al., 1990 Mol. Gen. Genet. 220:245-250). Therefore, not every intron can be used to manipulate the gene expression levels of non-endogenous or endogenous genes in transgenic plants. What characteristics or specific sequence features must be present in the intron sequence to enhance the expression rate of a given gene is unknown in the prior art; therefore, when used heterologously, it is impossible to predict whether a given plant intron will induce IME based on the prior art.

[0040] As used herein, the term "chimeric" refers to a single DNA molecule created by fusing a first DNA molecule with a second DNA molecule, wherein neither the first nor the second DNA molecule is typically found in this configuration, i.e., in fused configurations. Therefore, a chimeric DNA molecule is a novel DNA molecule rather than one typically found in nature. As used herein, the term "chimeric promoter" refers to a promoter created by performing this operation on a DNA molecule. Chimeric promoters can bind two or more DNA fragments; an example is the fusion of a promoter with an enhancer element. Therefore, this invention covers the design, construction, and use of chimeric promoters according to the methods disclosed herein for regulating the expression of operatively linked, transcribed polynucleotide molecules.

[0041] As used herein, the term "variant" refers to a second DNA molecule that is compositionally similar to but not identical to a first DNA molecule, while retaining conventional functionality, i.e., having the same or similar expression pattern as the first DNA molecule. A variant can be a shorter or truncated form of the first DNA molecule and / or a variation of the first DNA molecule's sequence, such as a DNA molecule with different restriction enzyme sites and / or internal deletions, substitutions, and / or insertions. "Variants" can also include regulatory elements having substituted, deleted, and / or inserted nucleotide sequences comprising one or more nucleotides of a reference sequence, wherein the derived regulatory element has stronger or weaker or equal transcriptional or translational activity than the corresponding parent regulatory molecule. The regulatory element "variants" can also encompass variants resulting from mutations naturally occurring in bacterial and plant cell transformations. In this invention, the polynucleotide sequences provided with SEQ ID Nos: 1-199, 211, and 212 can be used to generate variants that are compositionally similar to but not identical to the original regulatory element, while retaining conventional functionality, i.e., having the same or similar expression pattern as the original regulatory element. Such variant generation is well known to those skilled in the art based on this disclosure and is included within the scope of this invention. The "variant" of the chimeric regulatory element comprises constituent elements identical to the sequence of the reference chimeric regulatory element, but can be operatively linked by various methods known in the art, such as restriction enzyme digestion and ligation, cloning-independent ligation, modular assembly of PCR products during amplification, or direct chemical synthesis of the chimeric regulatory element, and other methods known in the art. The resulting "variant" chimeric regulatory element consists of constituent elements identical to the reference sequence but different in terms of one or more sequences used for operatively linking the constituent elements, or variants thereof. In this invention, reference sequences are provided for each of the polynucleotide sequences provided in SEQ ID Nos: 1-199, 211, and 212, wherein the constituent elements of the reference sequences can be linked by methods known in the art and can consist of one or more nucleotides naturally present in bacterial and plant cell transformations, or substitutions, deletions, and / or insertions of mutations.

[0042] Construct As used herein, the term "construct" refers to any recombinant polynucleotide molecule derived from any source, capable of genome integration or autonomous replication, and containing a polynucleotide molecule (one or more of which are functionally or operationally linked), such as plasmids, granules, viruses, autonomously replicating polynucleotide molecules, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecules. As used herein, the term "vector" refers to any recombinant polynucleotide construct that can be used for transformation purposes (i.e., introducing heterologous DNA into host cells). The terms include expression cassettes isolated from any of the above-described molecules.

[0043] As used herein, the term "operably linked" means that a first molecule is linked to a second molecule, wherein the molecules are arranged such that the first molecule influences the function of the second molecule. The two molecules may or may not be part of a single, continuous molecule, and may be adjacent or not adjacent. For example, if a promoter regulates the transcription of a target transcribed polynucleotide molecule in a cell, then the promoter is operably linked to the transcribed polynucleotide molecule. Similarly, a leader sequence is operably linked to a coding sequence when it can be used as a leader sequence for a polypeptide encoded by that coding sequence.

[0044] In one embodiment, the construct of the present invention can be provided as a double Ti plasmid border DNA construct, which has T-DNA from Agrobacterium tumefaciens (…). Agrobacterium tumefaciens The right boundary (RB or AGRtu.RB) and left boundary (LB or AGRtu.LB) regions of the isolated Ti plasmid, along with the transformation molecules provided by *Agrobacterium tumefaciens* cells, allow T-DNA integration into the genome of plant cells (see, for example, U.S. Patent 6,603,061). The construct may also include plasmid backbone DNA fragments that provide replication function and antibiotic selectivity in bacterial cells, such as *E. coli* origins of replication like ori322, broad host-range origins of replication like oriV or oriRi, and coding regions encoding Tn7 aminoglycoside adenyl transferase (aadA) to provide selectable markers for resistance to spectinomycin or streptomycin, such as Spec / Strp, or gentamicin (Gm, Gent) selectable marker genes. For plant transformation, the host bacterial strain is typically *Agrobacterium tumefaciens* ABI, C58, or LBA4404; however, other strains known in the field of plant transformation may function in this invention.

[0045] Methods for assembling and introducing constructs into cells in a manner that transcribes transcribed polynucleotide molecules into functional mRNA molecules that are translated and expressed as protein products are known. For the practice of this invention, conventional compositions and methods for preparing and using constructs and host cells can be found, for example, *Molecular Cloning: A Laboratory Manual, 3rd edition Volumes 1, 2, and 3* (2000), J.F. Sambrook, D.W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press. Methods for preparing recombinant vectors particularly suitable for plant transformation include, but are not limited to, those described in their entirety in U.S. Patents 4,971,908, 4,940,835, 4,769,061, and 4,757,011. These types of vectors have also been reviewed in the scientific literature (see, for example, Rodriguez et al., Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston, (1988) and Glick et al., Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton, FL. (1993)). Typical vectors for expressing nucleic acids in higher plants are well known in the art and include vectors derived from tumor-inducing (Ti) plasmids of *Agrobacterium tumefaciens* (Rogers et al., Methods in Enzymology, 153:253-277 (1987)). Other recombinant vectors for plant transformation, including the pCaMVCN transformation control vector, have also been described in the scientific literature (see, for example, Fromm et al., Proc. Natl. Acad. Sci. USA, 82:5824-5828 (1985)).

[0046] Any of the constructs provided herein may include different regulatory elements. Any such regulatory element may be provided in combination with other regulatory elements. Such combinations may be designed or modified to produce desired regulatory properties. In one embodiment, the constructs of the present invention comprise at least one regulatory element operatively linked to a transcribed polynucleotide molecule operatively linked to a 3' transcription termination molecule.

[0047] The constructs of this invention may include any promoter or leader sequence provided herein or known in the art. For example, the promoters of this invention may be operatively linked to a heterologous untranslated 5' leader sequence, such as a heterologous untranslated 5' leader sequence derived from a heat shock protein gene (see, for example, U.S. Patent Nos. 5,659,122 and 5,362,865). Alternatively, the leader sequence of this invention may be operatively linked to a heterologous promoter such as the cauliflower mosaic virus 35S transcription promoter (see, U.S. Patent No. 5,352,605). The expression properties conferred by such operative linking of heterologous elements are not necessarily a superposition of the properties elucidated by each promoter and leader sequence, but are determined through empirical analysis of expression driven by operatively linked heterologous promoters and leader sequences.

[0048] As used herein, the term "intron" refers to a DNA molecule that can be isolated or identified from a genomic copy of a gene and is generally defined as a region of DNA spliced ​​during pre-translational mRNA processing. Alternatively, an intron can be a synthetically generated or manipulated DNA element. Introns may contain enhancer elements that influence the transcription of operably linked genes. Introns can be used as regulatory elements to regulate the expression of operably linked transcribed polynucleotide molecules. DNA constructs may contain introns, which may or may not be heterologous to the sequence of the transcribed polynucleotide molecule. Examples of introns in the art include the rice actin intron (US Patent No. 5,641,876) and the maize HSP70 intron (US Patent No. 5,859,347). Introns useful for carrying out the present invention include intronic elements contained in any one of SEQ ID Nos: 4, 165, and 171.

[0049] As used herein, the term "3' transcription termination molecule" or "3'UTR" refers to the DNA molecule used during transcription to generate the 3' untranslated region (3'UTR) of an mRNA molecule. The 3' UTR of an mRNA molecule can be generated through specific cleavage and 3' polyadenylation (i.e., poly-A tail). The 3'UTR can be operatively linked to and located downstream of a transcribed polynucleotide molecule and may include polynucleotides that provide polyadenylation signals and other regulatory signals that can influence transcription, mRNA processing, or gene expression. The poly-A tail is thought to play a role in mRNA stability and the initiation of translation. Examples of 3' transcription termination molecules include the 3' region of carmine synthase (see Fraley et al., Proc. Natl. Acad. Sci. USA, 80:4803-4807 (1983)); the 3' region of wheat hspl7; the 3' region of the small subunit of pea ribulose diphosphate carboxylase-oxygenase; the 3' region of cotton E6 (US Patent 6,096,950); the 3' region disclosed in WO0011200A2; and the 3' UTR of coixol (US Patent No. 6,635,806).

[0050] 3'UTRs are often found to have advantageous uses for specific gene recombination expression. In animal systems, the mechanisms of 3'UTRs are well understood (e.g., Zhao et al., Microbiol Mol Biol Rev 63:405-445 (1999); Proudfoot, Nature 322:562-565 (1986); Kim et al., Biotechnology Progress 19:1620-1622 (2003); Yonaha and Proudfoot, EMBO J. 19:3770-3777 (2000); Cramer et al., FEBS Letters 498:179-182 (2001); Kuerstem and Goodwin, Nature Reviews Genetics 4:626-637 (2003)). Effective termination of RNA transcription is required to prevent the transcription of unwanted, trait-irrelevant (downstream) sequences (which interfere with trait expression). Compared to irrelevant insertions, the arrangement of multiple gene expression cassettes in local proximity to each other (e.g., within a single T-DNA) can lead to suppression of gene expression of one or more genes within the construct (Padidam and Cao, BioTechniques 31:328-334 (2001)). This can, for example, prevent the achievement of adequate expression levels in cases where strong gene expression from all cassettes is desired.

[0051] In plants, well-defined polyadenylation signal sequences are unknown. Hasegawa et al., Plant J.33:1063-1072, (2003) could not identify them in forest tobacco (Tobacco mono). Nicotiana sylvestris The study investigated conserved polyadenylation signal sequences in in vitro and in vivo systems and determined the actual length of primary (non-polyadenylated) transcripts. Weak 3' UTRs have the potential to generate readthroughs, which can affect gene expression in adjacent expression cassettes (Padidam and Cao, BioTechniques 31:328-334 (2001)). Proper control of transcription termination can prevent readthroughs in downstream sequences (e.g., other expression cassettes) and can further allow efficient recycling of RNA polymerase, thereby enhancing gene expression. Effective termination of transcription (release of RNA polymerase from DNA) is essential for the restart of transcription and thus directly affects the overall transcript level. Following transcription termination, mature mRNA is released from the cytoplasmic synthesis and template sites. Eukaryotic mRNA accumulates in vivo in a multi-(A) form, making it difficult to detect transcription termination sites using conventional methods. However, predicting functional and effective 3' UTRs using bioinformatics methods is challenging because there are no conserved sequences that make effective 3' UTRs easily predictable.

[0052] From a practical point of view, the 3'UTR used in transgenic cassettes generally has the following characteristics that are advantageous. The 3'UTR will efficiently and effectively terminate the transcription of the transgene and prevent the transcript from reading through any adjacent DNA sequence or adjacent chromosome DNA where the T-DNA has been inserted, such that in the case of multiple cassettes remaining in a single T-DNA, the adjacent DNA sequence may be composed of another transgenic cassette. The 3'UTR should not cause a reduction in the transcriptional activity conferred by the promoter, leader sequence, and introns used to drive transgene expression. In plant biotechnology, the 3'UTR is commonly used to initiate the amplification reaction of reverse-transcribed RNA extracted from the transformed plant and to (1) assess the transcriptional activity or expression of the transgenic cassette that has been integrated into the plant chromosome; (2) assess the copy number inserted into the plant DNA; and (3) assess the conjugation of seeds formed after breeding. The 3'UTR is also used for the amplification reaction of DNA extracted from the transformed plant to characterize the integrity of the inserted cassette.

[0053] It can be based on the derivative of Xiaomi ( Italian setariaThe expression of sequence tags (ESTs) expressed in cDNA libraries composed of messenger RNA isolated from seeds, flowers, and other tissues of *L. Beauv.* was used to identify 3'UTRs useful for providing transgenic expression in plants. The cDNA libraries were constructed from tissues isolated from selected plant varieties using flower tissues, seeds, leaves, and roots. The obtained cDNAs were sequenced using various sequencing methods. The obtained ESTs were assembled into clusters using bioinformatics software such as clc_ref_assemble_complete version 2.01.37139 (CLC bio USA, Cambridge, Massachusetts 02142). Transcript abundance for each cluster was determined by counting the number of cDNA reads in each cluster. The identified 3'UTRs could consist of sequences derived from both cDNA and genomic DNA. The cDNA sequences were used to design primers, which were then coupled to GenomeWalker primers constructed according to the manufacturer's specifications. TM (Clontech Laboratories, Inc., Mountain View, CA) Libraries are used together to clone the 3' region of the corresponding genomic DNA sequence to provide a longer termination sequence. Relative transcript abundance analysis of direct or normalized counts of sequence reads observed in each tissue library can be used to infer characteristics regarding expression patterns. For example, some 3'UTRs can be found in transcripts with higher abundance observed in root tissues, contrary to leaves. This indicates that the transcript is highly expressed in roots, and that the characteristic of root expression can be attributed to transcriptional regulation by promoters, leader sequences, introns, or 3'UTRs. Empirical tests to identify 3'UTRs based on their expression characteristics in specific organs, tissues, or cell types can lead to the identification of 3'UTRs with enhanced expression in those specific organs, tissues, or cell types.

[0054] The construct and vector may also include a transport peptide encoding sequence expressing a linked peptide class for targeting protein products, particularly chloroplasts, leucoplasts, or other plastid organelles; mitochondria; peroxisomes; vacuoles, or extracellular sites. For a description of the use of chloroplast transport peptides, see U.S. Patent Nos. 5,188,642 and 5,728,925. Many chloroplast-localized proteins are expressed as precursors from nuclear genes and target chloroplasts via chloroplast transport peptides (CTPs). Examples of such isolated chloroplast proteins include, but are not limited to, those described in U.S. Patent No. 7,193,133 related to the small subunit (SSU) of ribulose-1,5,2-bisphosphate carboxylase, ferricredoxin, ferricredoxin oxidoreductase, light-harvesting complex proteins I and II, thioredoxin F, enolpyruvate shikimate phosphate synthase (EPSPS), and transport peptides. It has been demonstrated in vivo and in vitro that non-chloroplast proteins can be targeted to chloroplasts by using protein fusion with a heterologous CTP, and that the CTP is sufficient to target the protein to the chloroplast. The introduction of suitable chloroplast transport peptides such as Arabidopsis EPSPS CTP (CTP2) (see, Klee et al., Mol. Gen. Genet., 210:437-442 (1987)) or Petunia EPSPS CTP (CTP4) (see, della-Cioppa et al., Proc. Natl. Acad. Sci. USA, 83:6873-6877 (1986)) has been shown to target heterologous EPSPS protein sequences to chloroplasts in transgenic plants (see, U.S. Patent Nos. 5,627,061, 5,633,435 and 5,312,910 and EP 0218571, EP 189707, EP508909 and EP 924299).

[0055] Transcribed polynucleotide molecules As used herein, the term "transcribed polynucleotide molecule" means any DNA molecule capable of being transcribed into an RNA molecule, including but not limited to those having protein-coding sequences and those that generate RNA molecules having sequences for gene repression. "Transgenic" means a transtranscribed polynucleotide molecule that is at least heterologous to the host cell in terms of its position in the genome and / or a transtranscribed polynucleotide molecule artificially introduced into the host cell genome in present or any previous generation of cells.

[0056] The promoter of the present invention can be operatively linked to a transcribed polynucleotide molecule that is heterologous to the promoter molecule. As used herein, the term "heterologous" means a combination of two or more polynucleotide molecules that is not normally present in nature. For example, the two molecules may be derived from different species and / or the two molecules may be derived from different genes, for example, different genes from the same species or the same gene from different species. Thus, if such a combination is not normally present in nature, the promoter is heterologous to the operatively linked transcribed polynucleotide molecule, i.e., the combination of the transcribed polynucleotide molecule and the promoter molecule is not naturally occurring and operatively linked.

[0057] Transcribed polynucleotide molecules can generally be any DNA molecule required to express an RNA transcript. This expression of the RNA transcript leads to the translation of the resulting mRNA molecule and the expression of the protein. Optionally, for example, the transcribed polynucleotide molecule can be designed to ultimately cause a reduction in the expression of a specific gene or protein. In one embodiment, this can be achieved by using a transcribed polynucleotide molecule oriented in an antisense direction. In short, when an antisense transcribed polynucleotide molecule is transcribed, the RNA product hybridizes with and sequesters the complementary RNA molecule in the cell. This double-helix RNA molecule cannot be translated into protein by the cell's translational mechanisms and is degraded in the cell. Any gene can be negatively regulated in this way.

[0058] Therefore, one embodiment of the invention is a regulatory element of the invention, such as those provided with SEQ ID NO: 1-199, 211, and 212, which is operatively linked to a transcribed polynucleotide molecule such that when said construct is introduced into the genome of a plant cell, the transcription of the transcribed polynucleotide molecule is regulated at a desired level or in a desired pattern. In one embodiment, the transcribed polynucleotide molecule comprises a protein-coding region of a gene, and the promoter influences the transcription of an RNA molecule that is translated and expressed as a protein product. In another embodiment, the transcribed polynucleotide molecule comprises an antisense region of a gene, and the promoter influences the transcription of antisense RNA molecules, double-stranded RNA, or other similar repressive RNA molecules to inhibit the expression of a target-specific RNA molecule in the target host cell.

[0059] Agronomic Genes Transcribed polynucleotide molecules can be agronomically targeted genes. As used herein, the term "agronomically targeted gene" refers to a transcribed polynucleotide molecule that, when expressed in a specific plant tissue, cell, or cell type, confers desired characteristics related to plant morphology, physiology, growth, development, yield, product, nutrient distribution, disease or pest resistance, and / or environmental or chemical tolerance. Agronomically targeted genes include, but are not limited to, those encoding yield proteins, stress resistance proteins, developmental control proteins, tissue differentiation proteins, meristematic proteins, environmental response proteins, senescence proteins, hormone response proteins, abscission proteins, source proteins, sink proteins, flower control proteins, seed proteins, herbicide resistance proteins, disease resistance proteins, fatty acid biosynthesizers, tocopherol biosynthesizers, amino acid biosynthesizers, insecticidal proteins, or any other agent, such as targeting antisense or RNAi molecules that inhibit a specific gene. The products of agronomically targeted genes can function within plants to influence plant physiology or metabolism, or can function as insecticides as food for plant-feeding pests.

[0060] In one embodiment of the invention, the promoter of the invention is introduced into the construct such that the promoter is operatively linked to a transcribed polynucleotide molecule serving as an agronomic target gene. Expression of the agronomic target gene is desired to confer beneficial agronomic traits. Beneficial agronomic traits may include, for example, but not limited to, herbicide tolerance, insect control, improved yield, fungal disease resistance, viral resistance, nematode resistance, bacterial disease resistance, plant growth and development, starch production, improved oil production, high oil production, improved fatty acid content, high protein production, fruit ripening, improved animal and human nutrition, biopolymers, environmental stress resistance, medicinal peptides and secretible peptides, improved processing traits, improved digestibility, enzyme production, flavor, nitrogen fixation, hybrid seed production, fiber production, and biofuel production. Examples of agronomically targeted genes include those exhibiting herbicide resistance (US 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) and increased yield (US Patent Nos. USRE38,446, 6,716,474, 6,663,906, 6,476,295, 6,441,277, and 6,000). ,423,828, 6,399,330, 6,372,211, 6,235,971, 6,222,098 and 5,716,837), insect control (US Patent Nos. 6,809,078, 6,713,063, 6,686,452, 6,657,046, 6,645,497, 6,642,030, 6,639,054, 6,620,988, 6,593,293, 6,555,655, 6,538,109, 6,537,756, 6,521,442, 6,501,009, 6,468,523, 6,326,351, 6,313,378, 6,284,949, 6,281,016, 6,248,536, 6,242,241, 6,221,649, 6,177,615, 6,156,573, 6,153,814, 6,110,464, 6,093,695, 6,063,756, 6,063,597 6,023,013, 5,959,091, 5,942,664, 5,942,658, 5,880,275, 5,763,245 and 5,763,241), fungal disease resistance (US Patent Nos. 6,653,280, 6,573,361, 6,506,962, 6,316,407, 6,215,048, 5,516,671, 5,773,696, 6,121,436, 6,316,407 and 6,506,962), virus resistance (US Patent Nos. 6,617,496, 6,608,241, 6,015,940, 6,013,864, 5,850,023 and 5,304,730), nematode resistance (US Patent No. 6,228,992), bacterial disease resistance (US Patent No. 5,516,671), plant growth and development (US Patent Nos. 6,723,897 and 6,518,488), starch production (US Patent Nos. 6,538,181, 6,538,179, 6,538,178, 5,750,876, 6,476,295), improved oil production (US Patent Nos. 6,617,496, 6,608,241, 6,015,940, 6,013,864, 5,850,023 and 5,304,730), US Patent Nos. 6,444,876; 6,426,447 and 6,380,462), high oil production (US Patent Nos. 6,495,739, 5,608,149, 6,483,008 and 6,476,295), improved fatty acid content (US Patent Nos. 6,828,475, 6,822,141, 6,770,465, 6,706,950, 6,660,849, 6,596,538, 6,589,767, 6,537,750, 6,489,461 and 6,459,018), and high protein production (US Patent No. 6,380,466). Fruit ripening (US Patent No. 5,512,466), improved animal and human nutrition (US Patent Nos. 6,723,837, 6,653,530, 6,5412,59, 5,985,605, and 6,171,640), biopolymers (US Patent Nos. USRE 37,543, 6,228,623, 5,958,745, and 6,946,588), environmental stress resistance (US Patent No. 6,072,103), medicinal peptides and secretible peptides (US Patent Nos. 6,812,379, 6,774,283, 6,140,075, and 6,080,560), and improved Processing properties (US Patent No. 6,476,295), improved digestibility (US Patent No. 6,531,648), low raffinose (US Patent No. 6,166,292), industrial enzyme production (US Patent No. 5,543,576), improved flavor (US Patent No. 6,011,199), nitrogen fixation (US Patent No. 5,229,114), hybrid seed production (US Patent No. 5,689,041), fiber production (US Patent Nos. 6,576,818, 6,271,443, 5,981,834, and 5,869,720), and biofuel production (US Patent No. 5,998,700).

[0061] Optionally, the agronomic target gene can influence the aforementioned plant characteristics or phenotypes by encoding a targeted regulatory RNA molecule that induces gene expression of an endogenous gene, for example, through antisense (see, for example, U.S. Patent 5,107,065), repressive RNA ("RNAi", including gene expression mediated by miRNA-, siRNA-, trans-acting siRNA-, and phased-controlled sRNA-, for example, as described in published applications US2006 / 0200878 and US2008 / 0066206 and U.S. Patent Application 11 / 974,469), or co-repressive-mediated mechanisms. The RNA can also be a catalytic RNA molecule engineered to cleave the desired endogenous mRNA product (e.g., a ribozyme or riboswitch; see, for example, US2006 / 0200878). Therefore, any transcribed polynucleotide molecule encoding a transcribed RNA molecule that influences agronomically important phenotypes or target morphological changes can be used to implement the present invention. Methods for constructing constructs and introducing them into cells by transcribing transcribed polynucleotide molecules into molecules capable of inducing gene repression are known in the art. For example, post-transcriptional gene repression using constructs and antisense-directed transcribed polynucleotide molecules to regulate gene expression in plant cells is disclosed in U.S. Patent Nos. 5,107,065 and 5,759,829, and post-transcriptional gene repression using constructs and sense-directed transcribed polynucleotide molecules to regulate gene expression in plants is disclosed in U.S. Patent Nos. 5,283,184 and 5,231,020. Expression of transcribed polynucleotides in plant cells can also be used to repress plant pests that consume plant cells, such as compositions isolated from coleopteran pests (U.S. Patent Publication No. US20070124836) and compositions isolated from nematode pests (U.S. Patent Publication No. US20070250947). Plant pests include, but are not limited to, arthropod pests, nematode pests, and fungal or microbial pests. Exemplary transcribed polynucleotide molecules incorporating the constructs of this invention include, for example, DNA molecules or genes from species different from the target species, or genes originating in or present in the same species but introduced into recipient cells through genetic engineering methods rather than conventional propagation or breeding techniques. Types of polynucleotide molecules include, but are not limited to, polynucleotide molecules already present in plant cells, polynucleotide molecules from another plant, polynucleotide molecules from different organisms, or externally generated polynucleotide molecules, such as polynucleotide molecules containing antisense genetic messages, or polynucleotide molecules encoding artificial, synthetic, or other modified forms of transgenic genes.

[0062] Selective tagging As used herein, the term "marker" refers to any transcribed polynucleotide molecule whose expression or non-expression can be screened or scored in some way. Marker genes used to carry out the present invention include, but are not limited to, transcribed polynucleotide molecules encoding β-glucuronidase (GUS as described in U.S. Patent No. 5,599,670), green fluorescent protein and its variants (GFP as described in U.S. Patent Nos. 5,491,084 and 6,146,826), proteins conferring antibiotic resistance, or proteins conferring herbicide resistance. Available antibiotic resistance markers include those encoding proteins conferring resistance to kanamycin (nptII), hygromycin B (aph IV), streptomycin or spectinomycin (aad, spec / strep), and gentamicin (aac3 and aacC4). Herbicides that have been proven to be tolerant to transgenic plants and can be applied to the methods of this invention include, but are not limited to: amino-methylphosphonic acid, glyphosate, glufosinate, sulfonylurea, imidazolinone, bromobenzonitrile, delapon, dicamba, cyclohexanedione, protoporphyrinogen oxidase inhibitors, and isoxaflutole herbicides.Transcribed polynucleotide molecules encoding proteins involved in herbicide tolerance include, but are not limited to, transcribed polynucleotide molecules encoding 5-enolpyruvate shikimate-3-phosphate synthase (EPSPS for glyphosate tolerance described in U.S. Patent Nos. 5,627,061, 5,633,435, 6,040,497, and 5,094,945); transcribed polynucleotide molecules encoding glyphosate oxidoreductase and glyphosate-N-acetyltransferase (GOX described in U.S. Patent No. 5,463,175, GAT described in U.S. Patent Publication No. 20030083480, and dicamba monooxygenase in U.S. Patent Publication No. 20030135879); transcribed polynucleotide molecules encoding bromobenzonitrile hydrolase (Bxn for bromobenzonitrile tolerance described in U.S. Patent No. 4,810,648); as described in Misawa et al., Plant Journal. The transcribed polynucleotide molecules encoding phytohexene desaturase (crtI) for tolerance to dapoxetine are described in 4:833-840 (1993) and Misawa et al., Plant Journal 6:481-489 (1994); the transcribed polynucleotide molecules encoding acetylhydroxy acid synthase (AHAS, also known as ALS) for tolerance to sulfonylurea herbicides are described in Sathasiivan et al., Nucl. AcidsRes. 18:2188-2193 (1990); and the bar gene for tolerance to glufosinate and phosmet is described in DeBlock et al., embo EMBO Journal 6:2513-2519 (1987). The promoter molecule of this invention can express a transcribed polynucleotide molecule that encodes phosphinothricin acetyltransferase, glyphosate-resistant EPSPS, aminoglycoside phosphotransferase, hydroxyphenylpyruvate dehydrogenase, hygromycin phosphotransferase, neomycin phosphotransferase, sphagnum dehalogenase, bromobenzonitrile-resistant nitrile hydrolase, anthranilate synthase, aryloxyalkylene ester dioxygenase, acetyl-CoA carboxylase, glyphosate oxidoreductase, and glyphosate-N-acetyltransferase.

[0063] Included in the term "selective marker" are genes encoding secretible markers, the secretion of which can serve as a means of identifying or selecting transformed cells. Examples include markers encoding secretible antigens that can be identified through antibody interactions or even markers encoding secretible enzymes that can catalyze detection. Selectively secreted marker proteins fall into several categories, including small, diffusible proteins that are detectable (e.g., by ELISA), small active enzymes that can be detected in extracellular solution (e.g., α-amylase, β-lactamase, glufosinate-transferase), or proteins that are inserted into or embedded in the cell wall (e.g., proteins including leader sequences found, for example, in extended expression units or tobacco-pathogenic proteins also known as tobacco PR-S). Other possible selective marker genes will be apparent to those skilled in the art and are covered herein.

[0064] Cell transformation The present invention also relates to a method for producing transformed cells and plants comprising promoters operatively linked to transcribed polynucleotide molecules.

[0065] The term "transformation" refers to the introduction of nucleotides into a recipient host. As used herein, the term "host" refers to bacteria, fungi, or plants, including any cell, tissue, organ, or progeny of bacteria, fungi, or plants. Specific target plant tissues and cells include protoplasts, callus, roots, tubers, seeds, stems, leaves, seedlings, embryos, and pollen.

[0066] As used herein, the term "transformed" refers to a cell, tissue, organ, or organism in which a foreign polynucleotide molecule, such as a construct, has been introduced. The introduced polynucleotide molecule can be integrated into the genomic DNA of the recipient cell, tissue, organ, or organism, and thus the introduced polynucleotide molecule is inherited by subsequent offspring. "Transgenic" or "transformed" cells or organisms also include the offspring of the cells or organisms, as well as offspring generated by breeding procedures that use such transgenic organisms as maternal hybrids and exhibit altered phenotypes resulting from the presence of the foreign polynucleotide molecule. The term "transgenic" refers to bacteria, fungi, or plants containing one or more heterologous polynucleotide molecules.

[0067] There are many methods for introducing polynucleotide molecules into plant cells. These methods generally involve selecting suitable host cells, transforming the host cells using a recombinant vector, and obtaining the transformed host cells. Suitable methods include bacterial infection (e.g., Agrobacterium), binary bacterial artificial chromosome vectors, direct delivery of DNA (e.g., via PEG-mediated transformation), drying / inhibition-mediated DNA uptake, electroporation, stirring with silicon carbide fibers, and acceleration with DNA-coated particles (reviewed in Potrykus et al., Ann. Rev. Plant Physiol. Plant Mol. Biol., 42:205 (1991)).

[0068] Any transformation method can be used to transform host cells using one or more promoters and / or constructs of the present invention. The host cell can be any cell or organism such as plant cells, algal cells, fungal cells, fungi, bacterial cells, or insect cells. Preferred hosts and transformed cells include those derived from plants, Aspergillus species (…). Aspergillus ), yeast, insects, bacteria and algae cells.

[0069] Regenerated transgenic plants can self-pollinate to provide homozygous transgenic plants. Optionally, pollen obtained from regenerated transgenic plants can be hybridized with inbred lines of non-transgenic plants, preferably agronomically important species. Descriptions of breeding methods commonly used for different traits and crops can be found in one of several reference books, 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, 369-399 (1979); Sneep and Hendriksen, Plant Breeding Perspectives, Wageningen (ed), Center for Agricultural Publishing and Documentation (1979); Fehr, Soybeans: Improvement, Production and Uses, 2nd Edition, Monograph, 16:249 (1987); Fehr, Principles of Variety Development, Theory and Technique, (Vol. 1) and Crop Species Soybean (Vol. 2), Iowa State Univ., Macmillan Pub. Co., NY, 360-376 (1987). Conversely, pollen from non-GMO plants can be used to pollinate regenerated GMO plants.

[0070] The presence of target genes in transformed plants and the expression levels and / or distribution conferred by the regulatory elements of this invention can be analyzed. Those skilled in the art are aware of many methods that can be used to analyze transformed plants. For example, plant analysis methods include, but are not limited to, Southern blotting or northern blotting, PCR-based methods, biochemical analysis, phenotypic screening, field assessment, and immunodiagnostic assays. The expression of transcribed polynucleotide molecules can be measured using TaqMan® (Applied Biosystems, Foster City, CA) reagents and methods described by the manufacturer, and PCR cycle times determined using the TaqMan® Testing Matrix. Alternatively, Invader® (Third Wave Technologies, Madison, WI) reagents and methods described by the manufacturer can be used for transgenic expression.

[0071] The seeds of the plants of the present invention can be harvested from fertile transgenic plants and used to grow the plant offspring transformed by the present invention, including plant hybrids that contain the constructs of the present invention and express agronomic target genes.

[0072] This invention also provides parts of the plant described in this invention. Plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovules, and pollen. This invention also includes and provides plant cells transformed with nucleic acid molecules comprising the nucleic acid molecules of this invention.

[0073] Transgenic plants can transfer transgenic polynucleotide molecules to their offspring. Offspring include any regenerable plant parts or seeds containing transgenes derived from the ancestral plant. For the transformed polynucleotide molecule, the transgenic plant is preferably homozygous and, as a result of sexual reproduction, transfers the sequence to all offspring. Offspring can grow from seeds generated from the transgenic plant. These additional plants can then self-pollinate to generate true breeding lines of the plant. Among other things, gene expression in the offspring from these plants is evaluated. Gene expression can be detected using several conventional methods such as Western blotting, Northern blotting, immunoprecipitation, and ELISA.

[0074] The invention has now been generally described, and will be more readily understood by referring to the embodiments provided below by way of example, which, unless otherwise specified, are not intended to limit the invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques discovered by the inventors that are well-suited for implementing the invention. However, those skilled in the art will understand that many changes can be made to the specific embodiments disclosed in accordance with the invention, and similar or related results can still be obtained without departing from the spirit and scope of the invention; therefore, all content set forth or shown in the accompanying drawings should be understood as exemplary, not restrictive. Detailed Implementation

[0075] Example 1: Identification and Cloning of Control Elements Novel transcriptional regulatory elements or transcriptional expression element set (EXP) sequences were identified and isolated from the genomic DNA of the dicotyledonous melon species WSH-39-1070AN.

[0076] Selecting transcriptional regulatory elements based on proprietary and public microarray data derived from soybean ( Glycine max Transcriptional profiling experiments were conducted in Arabidopsis thaliana and homology-based searches were performed using known dicotyledonous plant sequences to query proprietary melon sequences.

[0077] Using the identified sequences, bioinformatics analysis was performed to identify regulatory elements in the amplified DNA, followed by identification of transcription start sites (TSS) and any bidirectional, intronic, or upstream coding sequences present in the sequence. Using the analysis results, regulatory elements were identified within the DNA sequence, and primers were designed to amplify these elements. The corresponding DNA molecules for each regulatory element were amplified using standard polymerase chain reaction conditions, primers containing unique restriction enzyme sites, and genomic DNA isolated from melon. The resulting DNA fragments were ligated into a basic plant expression vector using standard restriction enzyme digestion and DNA ligation methods compatible with the restriction sites.

[0078] The regulatory elements TSS and intron / exon splicing sites can be analyzed using transformed plant protoplasts. In short, the protoplasts are transformed with the plant expression vector containing a cloning DNA fragment operatively linked to a heterologous transcribed polynucleotide molecule, and the regulatory elements TSS and intron / exon splicing sites are confirmed using a 5′ RACE system, version 2.0 (Invtrogen, Carlsbad, California 92008), for rapid cDNA end amplification, by analyzing the sequence of the resulting mRNA transcript.

[0079] The sequences of the regulatory expression elements (EXPs) encoding ubiquitin 1 transcription were analyzed as described above, and each EXP was decomposed into its corresponding promoter, leader sequence, and intron. The sequences of the identified EXPs are provided herein as SEQ ID Nos: 1, 5, 7, 9, and 11, and are listed in Table 1 below. The corresponding ubiquitin 1 promoters are provided herein as SEQ ID Nos: 2, 6, 8, 10, and 12. The ubiquitin 1 leader sequences and introns are provided herein as SEQ ID Nos: 3 and 4, respectively.

[0080] SEQ ID Nos: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 7 4, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 12 7, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 159, 162, 167, 168, 172, 175, 176, 177, 178, 181, 182, 183, 184, 185, 188, 189, 190, 1 Sequences 91, 192, 193, 194, 195, 196, 197, 198, 199, 211, and 212 provide sequences encoding regulatory expression element sets or EXP sequences for transcription of other *Cucumis* species, and these sequences are also listed in Table 1 below. The EXP sequences consist of promoter elements operatively linked to a leader sequence element; or promoter elements operatively linked to a leader sequence element and an intron element; or promoter elements operatively linked to a leader sequence element, operatively linked to an intron element, and operatively linked to a leader sequence element. Additional promoter elements are provided with SEQ ID Nos. 163 and 169. Additional leader sequence elements are provided with SEQ ID Nos. 164, 166, and 170. Additional intron elements are provided with SEQ ID Nos. 165 and 171. Elements in which the promoter is operatively linked to a leader sequence element are provided with SEQ ID Nos. 157, 160, 173, 179, and 186. Elements wherein introns operatively connect leader sequence elements are provided in SEQ ID Nos: 158, 161, 174, 180 and 187.These sequences were selected and cloned based on experimental results, such as promoter-driven transcript distribution or expression from homologous genes from different species, based on the subset of sequences provided with SEQ ID Nos: 13-199, 211, and 212, according to desired expression patterns such as constitutive expression, root expression, above-ground expression, or seed expression. The actual activity conferred by the *Calamus* genus sequences is determined empirically and is not necessarily the same as the activity of regulatory elements derived from homologous genes from species other than *Calamus* when used for transformation of plant host cells and whole transgenic plants.

[0081] Table 1. Transcriptional regulatory elements, promoters, leader sequences, and introns isolated from melon.

[0082] As shown in Table 1, for example, the transcriptional regulatory expression element set (EXP) represented by EXP-CUCme.Ubq1:1:1 (SEQ ID NO: 1), which has components isolated from melon, includes a promoter element P-CUCme.Ubq1-1:1:15 (SEQ ID NO: 2) with a size (bp) of 2068 base pairs, operably linked to the leader sequence element L-CUCme.Ubq1-1:1:1 (SEQ ID NO: 3) and operably linked to the intron element I-CUCme.Ubq1-1:1:1 (SEQ ID NO: 4). The transcriptional regulatory expression element set (EXP) represented by EXP-CUCme.Ubq1:1:2 (SEQ ID NO: 5), which contains components isolated from melon, comprises a 1459 bp promoter element P-CUCme.Ubq1-1:1:16 (SEQ ID NO: 6) operably linked 5' to the leader sequence element L-CUCme.Ubq1-1:1:1 (SEQ ID NO: 3) and operably linked 5' to the intron element I-CUCme.Ubq1-1:1:1 (SEQ ID NO: 4). The transcriptional regulatory expression element set (EXP) represented by EXP-CUCme.Ubq1:1:3 (SEQ ID NO: 7), which contains components isolated from melon, comprises a 964 bp promoter element P-CUCme.Ubq1-1:1:17 (SEQ ID NO: 8) operably linked 5' to the leader sequence element L-CUCme.Ubq1-1:1:1 (SEQ ID NO: 3) and operably linked 5' to the intron element I-CUCme.Ubq1-1:1:1 (SEQ ID NO: 4). The transcriptional regulatory expression element set (EXP) represented by EXP-CUCme.Ubq1:1:4 (SEQ ID NO: 9), which contains components isolated from melon, comprises a 479 bp promoter element P-CUCme.Ubq1-1:1:18 (SEQ ID NO: 10) operably linked 5' to the leader sequence element L-CUCme.Ubq1-1:1:1 (SEQ ID NO: 3) and operably linked 5' to the intron element I-CUCme.Ubq1-1:1:1 (SEQ ID NO: 4).The transcriptional regulatory expression element set (EXP) represented by EXP-CUCme.Ubq1:1:5 (SEQ ID NO: 11), which contains components isolated from melon, comprises a 173 bp promoter element P-CUCme.Ubq1-1:1:19 (SEQ ID NO: 12) operably linked 5' to the leader sequence element L-CUCme.Ubq1-1:1:1 (SEQ ID NO: 3) and operably linked 5' to the intron element I-CUCme.Ubq1-1:1:1 (SEQ ID NO: 4).

[0083] The comparison of the ubiquitin-1 promoter sequence provides for Figure 1a-1f In addition, promoter elements P-CUCme.Ubq1-1:1:16 (SEQ ID NO: 6), P-CUCme.Ubq1-1:1:17 (SEQ ID NO: 8), P-CUCme.Ubq1-1:1:18 (SEQ ID NO: 10), and P-CUCme.Ubq1-1:1:19 (SEQ ID NO: 12) were constructed by introducing different lengths of missing 5' ends from the promoter P-CUCme.Ubq1-1:1:15 (SEQ ID NO: 2).

[0084] Example 2: Analysis of the regulatory elements driving GUS in soybean cotyledon protoplasts Soybean cotyledonary protoplasts were transformed with a plant expression vector containing a set of test transcriptional regulatory expression elements that drive the expression of the β-glucuronidase (GUS) transgene, and compared with GUS expression in leaf protoplasts, wherein the expression of GUS is driven by a known constitutive promoter.

[0085] Transgenic expression driven by EXP-CUCme.Ubq1:1:1 (SEQ ID NO: 1), EXP-CUCme.Ubq1:1:2 (SEQ ID NO: 5), EXP-CUCme.Ubq1:1:3 (SEQ ID NO: 7), EXP-CUCme.Ubq1:1:4 (SEQ ID NO: 9), and EXP-CUCme.Ubq1:1:5 (SEQ ID NO: 11) was compared with expression from known constitutive promoters. Each plant expression vector consists of a right boundary region from *Agrobacterium tumefaciens*, a first transgenic cassette, a second transgenic selection cassette, and a left boundary region from *Agrobacterium tumefaciens*, wherein the first transgenic cassette comprises a β-glucuronidase (GUS, SEQ ID NO: 206) operably linked 5' to a processing intron derived from the potato light-induced tissue-specific ST-LS1 gene (gene bank accession number: X04753) using a coding sequence operably linked 5' to a gene from *Cotton Island* (*Cotton Island cotton*). Gossypium barbadense E6 gene (T-Gb.E6-3b:1:1, SEQ ID NO: 204), pea ( Pea agricultural product The EXP sequence of the 3' termination region of the RbcS2-E9 gene (T-Ps.RbcS2-E9-1:1:6, SEQ ID NO: 203) or the FbLate-2 gene of sea island cotton (T-Gb.FbL2-1:1:1, SEQ ID NO: 205) or a known constitutive promoter composition was used; the second transgenic selection cassette was used to select plant cells conferred resistance to the herbicide glyphosate (driven by the Arabidopsis actin 7 promoter) or the antibiotic kanamycin. A promoterless control plant expression vector (pMON124912) was used as a negative control for expression. The above-tested and constitutive expression element sets were cloned into plant expression vectors as shown in Table 2 below.

[0086] Table 2. Plant expression vectors and corresponding expression element sets and 3'UTR

[0087] In addition, two plasmids were constructed for co-transformation and data normalization. One transformation control plasmid was generated from the driver firefly (…). Photinus pyralis The transformation control plasmid consists of a constitutive promoter expressing a luciferase-coding sequence (FLuc, SEQ ID NO: 207), said constitutive promoter being operatively linked at 5' to the 3' termination region of the *Agrobacterium tumefaciens* carmine synthase gene (T-AGRtu.nos-1:1:13, SEQ ID NO: 209). Another transformation control plasmid is derived from *Dendrobium nobile* (*Dendrobium tumefaciens*). Renilla reniformisThe constitutive promoter for the expression of the luciferase coding sequence (RLuc, SEQ ID NO: 208) is operatively linked at 5' to the 3' termination region of the rutin base synthase gene from Agrobacterium tumefaciens.

[0088] Soybean cotyledon protoplast cells were transformed using the PEG transformation method with plant expression vectors pMON80585, pMON109584, pMON118756, pMON124912, pMON138776, pMON138777, pMON138778, pMON138779, and pMON138780. Equimolar amounts of each of the two transformation control plasmids and the test plant expression vector were used to transform protoplast cells. GUS and luciferase activities were measured. GUS and luciferase were measured by placing aliquots of the lysate of the transformed cells into two separate well trays. One tray was used for GUS measurement, while the other was used for dual-luciferase detection using a dual-luciferase reporter gene detection system (Promega Corp., Madison, WI; see, for example, Promega Notes Magazine, No: 57, 1996, p. 02). Three or four replicates were used per transformation for sample measurement. The average values ​​of GUS and luciferase are shown in Table 3 below.

[0089] Table 3. Mean expression of GUS and luciferase and GUS / luciferase ratio

[0090] To compare the relative activity of each promoter in soybean cotyledon protoplasts, GUS values ​​were expressed as the ratio of GUS to luciferase activity and normalized according to the observed expression levels of the constitutive expression element sets EXP-At.Act7:1:11 and EXP-CaMV.35S-enh+Ph.DnaK:1:3. Table 4 below shows the ratio of GUS to firefly luciferase (FLuc) normalized according to EXP-At.Act7:1:11 and EXP-CaMV.35S-enh+Ph.DnaK:1:3. Table 5 below shows the ratio of GUS to Renilla luciferase (RLuc) normalized according to EXP-At.Act7:1:11 and EXP-CaMV.35S-enh+Ph.DnaK:1:3.

[0091] Table 4. Ratio of GUS to firefly luciferase (FLuc) normalized according to EXP-At.Act7:1:11 and EXP-CaMV.35S-enh+Ph.DnaK:1:3

[0092] Table 5. Ratios of GUS to Renal luciferase (RLuc) normalized according to EXP-At.Act7:1:11 and EXP-CaMV.35S-enh+Ph.DnaK:1:3

[0093] As shown in Tables 4 and 5 above, each of the expression element groups EXP-CUCme.Ubq1:1:1 (SEQ ID NO: 1), EXP-CUCme.Ubq1:1:2 (SEQ ID NO: 5), EXP-CUCme.Ubq1:1:3 (SEQ ID NO: 7), EXP-CUCme.Ubq1:1:4 (SEQ ID NO: 9), and EXP-CUCme.Ubq1:1:5 (SEQ ID NO: 11) demonstrated the ability to drive transgene expression in soybean cotyledon protoplasts. Their expression levels in this assay were higher than those of EXP-At.Act7:1:11, and were 2.9 to 5.8 times (FLuc) or 3 to 7 times (RLuc) higher than those of EXP-At.Act7:1:11. The expression was comparable to or higher than that observed in EXP-CaMV.35S-enh+Ph.DnaK:1:3. The expression level was 0.8 to 1.7 times (FLuc) or 1 to 2.4 times (RLuc) higher than that observed in EXP-CaMV.35S-enh+Ph.DnaK:1:3.

[0094] Example 3: Analysis of the regulatory elements driving GUS in bombarded soybean leaves and roots Soybean leaves and roots were transformed with a plant expression vector containing a set of test transcriptional regulatory expression elements that drive the expression of the β-glucuronidase (GUS) transgene, and compared with GUS expression in roots and leaves, wherein the expression of GUS is driven by a known constitutive promoter.

[0095] Transgenic expression driven by EXP-CUCme.Ubq1:1:1 (SEQ ID NO: 1), EXP-CUCme.Ubq1:1:2 (SEQ ID NO: 5), EXP-CUCme.Ubq1:1:3 (SEQ ID NO: 7), EXP-CUCme.Ubq1:1:4 (SEQ ID NO: 9), and EXP-CUCme.Ubq1:1:5 (SEQ ID NO: 11) in soybean leaves and roots bombarded with particles was compared with expression from known constitutive promoters. The plant expression vectors used for transforming leaves and roots were the same as those shown in Table 2 of Example 2 above.

[0096] The plant expression vectors pMON80585, pMON109584, pMON118756, pMON124912, pMON138776, pMON138777, pMON138778, pMON138779, and pMON138780 were used to transform soybean leaves and roots using the particle bombardment transformation method.

[0097] In summary, A3244 soybean seeds were surface-sterilized and germinated in trays using a photocycle of 16 hours light and 8 hours dark. After approximately 13 days, leaf and root tissues were harvested from the seedlings under aseptic conditions for bombardment. The tissue samples were randomly placed on Petri dishes containing plant culture medium. 0.6-micron gold particles (catalog #165-2262 Bio-Rad, Hercules, CA) coated with 10 μg of plasmid DNA were used for bombardment. Large vectors were loaded onto the DNA-coated gold particles (catalog #165-2335 Bio-Rad, Hercules CA). Transformation was performed using a PDS 1000 / He gene gun (catalog #165-2257 Bio-Rad, Hercules CA). The bombarded root and leaf tissues were cultured in the dark at 26°C for 24 hours. Following this overnight culture, the tissues were stained overnight at 37°C in a solution for GUS expression. After overnight staining, the tissues were soaked overnight in 70% ethanol to remove chlorophyll and reveal GUS staining. The tissues were then photographed and each construct was assigned a gradation scale of "0", "+" to "++++++" reflecting GUS expression levels (0 for no expression, and + to ++++++ for low to high expression).

[0098] The expression of GUS transgenes shown in each tissue was used to infer the relative potential level and specificity of each element's ability to drive transgene expression in stably transformed maize plants. The average gradation of GUS expression is provided in Table 6 below.

[0099] Table 6. GUS expression levels in leaves and roots bombarded by particles

[0100] As can be seen from Table 6 above, each of the expression element groups EXP-CUCme.Ubq1:1:1 (SEQ ID NO: 1), EXP-CUCme.Ubq1:1:2 (SEQ ID NO: 5), EXP-CUCme.Ubq1:1:3 (SEQ ID NO: 7), EXP-CUCme.Ubq1:1:4 (SEQ ID NO: 9), and EXP-CUCme.Ubq1:1:5 (SEQ ID NO: 11) demonstrates the ability to drive transgene expression in particle-bombarded transformed leaf and root tissues.

[0101] Example 4: Analysis of regulatory elements driving GUS in soybean cotyledon protoplasts Soybean cotyledonary protoplasts were transformed with a plant expression vector containing a set of test transcriptional regulatory expression elements that drive the expression of the β-glucuronidase (GUS) transgene, and compared with GUS expression in leaf protoplasts, wherein the expression of GUS is driven by a known constitutive promoter.

[0102] It will be composed of P-CUCme.1-1:1:1rc (SEQ ID NO: 155), P-CUCme.2-1:1:1 (SEQ ID NO: 14), P-CUCme.3-1:1:3 (SEQ ID NO: 15), EXP-CUCme.4:1:1 (SEQ ID NO: 156), EXP-CUCme.5:1:1 (SEQ ID NO: 159), P-CUCme.6-1:1:1 (SEQ ID NO: 18), P-CUCme.8-1:1:2 (SEQ ID NO:19), P-CUCme.9-1:1:2 (SEQ ID NO: 20), P-CUCme.10-1:1:1 (SEQ ID NO: 21), EXP-CUCme.eEF1a:1:1 (SEQ ID NO: 162), P-CUCme.15-1:1:2 (SEQ ID NO: 23), P-CUCme.16a-1:1:2 (SEQ ID NO: 24), P-CUCme.17-1:1:2 (SEQ ID NO: 26), P-CUCme.18-1:1:2 (SEQID NO: 27), P-CUCme.19-1:1:3 (SEQ ID NO: 167), P-CUCme.20-1:3 (SEQ ID NO: 211), P-CUCme.21-1:1:1 (SEQ ID NO: 30), P-CUCme.22-1:1:3 (SEQ ID NO: 31), EXP-CUCme.SAMS2:1:1 (SEQ ID NO: 168), P-CUCme.26-1:1:2 (SEQ ID NO: 33), P-CUCme.28-1:1:2 (SEQ ID NO: The expression of transgenes driven by 34) and EXP-CUCme.29:1:2 (SEQ ID NO: 212) was compared with the expression from known constitutive expression element groups.Each plant expression vector consists of a right boundary region from *Agrobacterium tumefaciens*, a first transgenic cassette, a second transgenic selection cassette, and a left boundary region from *Agrobacterium tumefaciens*. The first transgenic cassette comprises a 5' region operably linked to a coding sequence of a β-glucuronidase (GUS, SEQ ID NO: 206) containing a processable intron derived from the potato light-induced tissue-specific ST-LS1 gene (gene bank accession number: X04753), and a 5' region operably linked to the coding sequence of an E6 gene from *Cotton Island cotton* (T-Gb.E6-3b:1:1, SEQ ID NO: 204), a *pea RbcS2-E9* gene (T-Ps.RbcS2-E9-1:1:6, SEQ ID NO: 203), or a *Cotton Island cotton* FbLate-2 gene (T-Gb.FbL2-1:1:1, SEQ ID NO: 204). The test promoter or a known constitutive promoter is composed of the 3' termination region of NO: 205; the second transgenic selection cassette is used to select plant cells that are conditioned for the herbicide glyphosate (driven by the Arabidopsis actin 7 promoter) or the antibiotic kanamycin. A promoterless control plant expression vector (pMON124912) is used as a negative control for expression. The above test and constitutive expression element sets are cloned into plant expression vectors as shown in Table 7 below.

[0103] Table 7. Plant expression vectors and corresponding expression element sets and 3'UTR

[0104] In addition, two plasmids were constructed for co-transformation and data normalization. One transformation control plasmid was generated from the driver firefly (…). Photinus pyralis The transformation control plasmid consists of a constitutive promoter expressing a luciferase-coding sequence (FLuc, SEQ ID NO: 207), said constitutive promoter being operatively linked at 5' to the 3' termination region of the *Agrobacterium tumefaciens* carmine synthase gene (T-AGRtu.nos-1:1:13, SEQ ID NO: 209). Another transformation control plasmid is derived from *Dendrobium nobile* (*Dendrobium tumefaciens*). Renilla reniformis The constitutive promoter for the expression of the luciferase coding sequence (RLuc, SEQ ID NO: 208) is operatively linked at 5' to the 3' termination region of the rutin base synthase gene from Agrobacterium tumefaciens.

[0105] The PEG transformation method was used with plant expression vectors pMON80585, pMON109584, pMON118756, pMON124912, pMON140818, pMON140819, pMON140820, and pMON140821. pMON140822, pMON140823, pMON140824, pMON140825, pMON140826, pMON140827, pMON140828, pMON140829, pMON140830, pMON140831, pMON140832, pMON140833, pMON140834, pMON140835, pMON140836, pMON140837, pMON140838, and pMON140839 were used to transform soybean cotyledon protoplast cells. Equimolar amounts of each of the two transformation control plasmids and the test plant expression vector were also used to transform protoplast cells. GUS and luciferase activities were measured. GUS and luciferase activities were measured by placing aliquots of the lysis formulation of the transformed cells into two separate well trays. One tray was used for GUS measurement, while the other tray was used for dual-luciferase detection using a dual-luciferase reporter gene detection system (Promega Corp., Madison, WI; see, for example, Promega Notes Magazine, No: 57, 1996, p. 02). Three or four replicas were used for sample measurement per transformation. The average values ​​for GUS and luciferase are shown in Table 8 below.

[0106] Table 8. Mean GUS and luciferase expression and GUS / luciferase ratio

[0107] To compare the relative activity of each promoter in soybean cotyledon protoplasts, GUS values ​​were expressed as the ratio of GUS to luciferase activity and normalized according to the observed expression levels of the constitutive expression element sets EXP-At.Act7:1:11 and EXP-CaMV.35S-enh+Ph.DnaK:1:3. Table 9 below shows the ratio of GUS to firefly luciferase (FLuc) normalized according to EXP-At.Act7:1:11 and EXP-CaMV.35S-enh+Ph.DnaK:1:3. Table 10 below shows the ratio of GUS to Renilla luciferase (RLuc) normalized according to EXP-At.Act7:1:11 and EXP-CaMV.310S-enh+Ph.DnaK:1:3.

[0108] Table 9. Ratio of GUS to firefly luciferase (FLuc) normalized according to EXP-At.Act7:1:11 and EXP-CaMV.35S-enh+ Ph.DnaK:1:3

[0109] Table 10. Ratio of GUS to Renal luciferase (RLuc) normalized according to EXP-At.Act7:1:11 and EXP-CaMV.35S-enh+ Ph.DnaK:1:3

[0110] As can be seen from Tables 9 and 10, most of the tested expression element sets demonstrated the ability to drive transgene expression in soybean cotyledon protoplast cells. In this assay, one expression element set, EXP-CUCme.4:1:1 (SEQ ID NO:156), showed a higher transgene expression level than EXP-At.Act7:1:11.

[0111] Example 5: Analysis of the regulatory elements driving GUS in bombarded soybean leaves and roots Soybean leaves and roots were transformed with a plant expression vector containing a set of test transcriptional regulatory expression elements that drive the expression of the β-glucuronidase (GUS) transgene, and compared with GUS expression in roots and leaves, wherein the expression of GUS is driven by a known constitutive promoter.

[0112] The following compounds will be present in soybean leaves and roots bombarded with particles: P-CUCme.1-1:1:1rc (SEQ ID NO: 155), P-CUCme.2-1:1:1 (SEQ ID NO: 14), P-CUCme.3-1:1:3 (SEQ ID NO: 15), EXP-CUCme.4:1:1 (SEQ ID NO: 156), EXP-CUCme.5:1:1 (SEQ ID NO: 159), P-CUCme.6-1:1:1 (SEQ ID NO: 18), P-CUCme.8-1:1:2 (SEQ ID NO: 19), P-CUCme.9-1:1:2 (SEQ ID NO: 20), P-CUCme.10-1:1:1 (SEQ ID NO: 21), and EXP-CUCme.eEF1a:1:1 (SEQ ID NO: 155). 162), P-CUCme.15-1:1:2 (SEQ ID NO: 23), P-CUCme.16a-1:1:2 (SEQ ID NO: 24), P-CUCme.17-1:1:2 (SEQ ID NO: 26), P-CUCme.18-1:1:2 (SEQ ID NO: 27), P-CUCme.19-1:1:3 (SEQ ID NO: 167), P-CUCme.20-1:3 (SEQ ID NO: 211), P-CUCme.21-1:1:1 (SEQ ID NO: 30), P-CUCme.22-1:1:3 (SEQ ID NO: 31), EXP-CUCme.SAMS2:1:1 (SEQ ID NO: 168), P-CUCme.26-1:1:2 (SEQ ID NO: The expression of transgenes driven by 33), P-CUCme.28-1:1:2 (SEQ ID NO: 34), and EXP-CUCme.29:1:2 (SEQ ID NO: 212) was compared with that from known constitutive expression element groups. The plant expression vectors used for transforming leaves and roots were the same as those shown in Table 7 of Example 4 above.

[0113] Particle bombardment transformation was used, employing plant expression vectors pMON80585, pMON109584, pMON118756, pMON124912, pMON140818, pMON140819, pMON140820, pMON140821, pMON140822, pMON140823, pMON140824, pMON140825, and pMON14. 0826, pMON140827, pMON140828, pMON140829, pMON140830, pMON140831, pMON140832, pMON140833, pMON140834, pMON140835, pMON140836, pMON140837, pMON140838 and pMON140839 were used to convert soybean leaves and roots.

[0114] In short, A3244 soybean seeds were surface-sterilized and germinated in trays using a photocycle of 16 hours light and 8 hours dark. After approximately 13 days, leaf and root tissues were harvested from the seedlings under aseptic conditions and used for bombardment. The tissue samples were randomly placed on Petri dishes containing plant culture medium. 0.6-micron gold particles (catalog #165-2262 Bio-Rad, Hercules, CA) coated with 10 μg of plasmid DNA were used for bombardment. Large vectors were loaded onto the DNA-coated gold particles (catalog #165-2335 Bio-Rad, Hercules CA). Transformation was performed using a PDS 1000 / He gene gun (catalog #165-2257 Bio-Rad, Hercules CA). The bombarded root and leaf tissues were cultured in the dark at 26°C for 24 hours. Following this overnight culture, the tissues were stained overnight at 37°C in a solution for GUS expression. After overnight staining, the tissues were soaked overnight in 70% ethanol to remove chlorophyll and reveal GUS staining. The tissues were then photographed and each construct was assigned a gradation scale of "0", "+" to "++++++" reflecting GUS expression levels (0 for no expression, and + to ++++++ for low to high expression).

[0115] The expression of GUS transgenes shown in each tissue was used to infer the relative potential level and specificity of each element's ability to drive transgene expression in stably transformed maize plants. The mean gradation of GUS expression is provided in Table 11 below.

[0116] Table 11. GUS expression levels in leaves and roots after particle bombardment

[0117] As can be seen from Table 11 above, all but one of the expression element groups showed the ability to drive transgene expression in soybean leaf and root tissues bombarded by particles. In this assay, the two expression element groups P-CUCme.28-1:1:2 (SEQ ID NO: 34) and EXP-CUCme.4:1:1 (SEQ ID NO: 156) showed similar or higher expression levels compared to expression driven by EXP-CaMV.35S-enh+Ph.DnaK:1:3.

[0118] Example 6: Analysis of the regulatory elements driving GUS in soybean cotyledon protoplasts using transgenic cassette amplicon Soybean cotyledonary protoplasts were transformed with a transgenic cassette amplicon containing a set of transcriptional regulatory expression elements that drive the expression of the β-glucuronidase (GUS) transgene, and the expression was compared with GUS expression in leaf protoplasts, wherein GUS expression is driven by a known constitutive promoter. The transgenic cassette amplicon consists of an EXP sequence operably linked to a GUS coding sequence (GUS, SEQ ID NO: 206) and an EXP sequence operably linked to a 3'UTR (T-Gb.FbL2-1:1:1, SEQ ID NO: 205). Average GUS expression was compared with the control EXP elements P-CaMV.35S-enh-1:1:102 / L-CaMV.35S-1:1:2 (SEQ ID NO: 210) and EXP-At.Atntt1:1:2 (SEQ ID NO: 200).

[0119] Furthermore, plasmids for co-transformation and data normalization were used in a similar manner to that described above in Example 2. The transformation control plasmid was derived from *Driver firefly* (…). Photinus pyralis The constitutive promoter for the expression of the luciferase coding sequence (FLuc, SEQ ID NO: 205) is operatively linked at 5' to the 3' termination region of the Agrobacterium tumefaciens carmine synthase gene (T-AGRtu.nos-1:1:13, SEQ ID NO: 209).

[0120] Table 12 below shows the average GUS expression conferred by each transgenic amplicon. Table 13 below shows the ratio of GUS to firefly luciferase (FLuc) normalized according to EXP-At.Atntt1:1:2 and P-CaMV.35S-enh-1:1:102 / L-CaMV.35S-1:1:2.

[0121] Table 12. Mean GUS and luciferase expression and GUS / luciferase ratio

[0122] Table 13. Ratio of GUS to firefly luciferase (FLuc) normalized according to EXP-At.Atntt1:1:2 and P-CaMV.35S-enh-1:1:102 / L-CaMV.35S-1:1:2

[0123] As can be seen from Table 12 above, not all EXP sequences showed the ability to drive transgene expression when compared with the promoter-free control. However, the EXP sequences CumMe_WSM_SF16429.G5670 (SEQ ID NO: 40), P-CUCme.CumMe_WSM_SF16444.G5140-1:1:1 (SEQ ID NO: 175), P-CUCme.CumMe_WSM_SF16563.G5560-1:1:1 (SEQ ID NO: 176), CumMe_WSM_SF17051.G5470 (SEQ ID NO: 48), P-CUCme.CumMe_WSM_SF17111.G5790-1:1:1 (SEQ ID NO: 177), P-CUCme.WSM_SF17252.G7330-1:1:1 (SEQ ID NO: 179), CumMe_WSM_SF17866.G6050 (SEQ ID NO: 62). 71), P-CUCme.CumMe_WSM_SF18634.G5190-1:1:1(SEQ ID NO: 183), CumMe_WSM_SF18986.G6110(SEQ ID NO: 79), EXP-CUCme.WSM_SF19064.G5690:1:1(SEQ ID NO: 185), P-CUCme.CumMe_WSM_SF19647.G5760-1:1:1 (SEQ ID NO: 188), P-CUCme.CumMe_WSM_SF19839.G5090-1:1:1 (SEQ ID NO: 189), CumMe_WSM_SF19902.G5260 (SEQ ID NO: 87), P-CUCme.CumMe_WSM_SF20132.G5560-1:1:1(SEQ ID NO: 190), CumMe_WSM_SF20359.G5870(SEQ ID NO: 92), CumMe_WSM_SF206458.G5970(SEQ ID NO: 98), CumMe_WSM_SF206534.G5200 (SEQ ID NO: 99), CumMe_WSM_SF22008.G5670 (SEQ ID NO: 108), CumMe_WSM_SF22355.G5310 (SEQ ID NO: 113), P-CUCme.CumMe_WSM_SF22531.G5120-1:1:1 (SEQ ID NO: 192), EXP-CUCme.WSM_SF19064.G5690:1:1(SEQ ID NO: 193), P-CUCme.CumMe_WSM_SF23906.G6180-1:1:1(SEQ ID NO: 194), CumMe_WSM_SF24045.G5400(SEQ ID P-CUCme.CumMe_WSM_SF25141.G5160-1:1:2 (SEQ ID NO: 195), P-CUCme.CumMe_WSM_SF25936.G5450-1:1:1 (SEQ ID NO: 197), CumMe_WSM_SF28729.G5340 (SEQ ID NO: 134), CumMe_WSM_SF31264.G5380 (SEQ ID NO: 136), and P-CUCme.CumMe_WSM_SF35856.G5150-1:1:1 (SEQ ID NO: 198) showed the ability to drive transgene expression in soybean cotyledon protoplasts at levels similar to or higher than EXP-At.Atntt1:1:2. As shown in Table 13 above, in this assay, the EXP sequence P-CUCme.CumMe_WSM_SF19647.G5760-1:1:1 (SEQ ID NO: 188) demonstrated an ability to drive transgene expression at a higher level than EXP-At.Atntt1:1:2.

[0124] Example 7: Analysis of the regulatory elements driving GUS in cotton leaf protoplasts Cotton leaf protoplasts were transformed with a plant expression vector containing a set of test transcriptional regulatory expression elements that drive the expression of the β-glucuronidase (GUS) transgene, and compared with GUS expression in leaf protoplasts, wherein GUS expression is driven by a known constitutive promoter.

[0125] It will be composed of P-CUCme.1-1:1:1rc (SEQ ID NO: 155), P-CUCme.2-1:1:1 (SEQ ID NO: 14), P-CUCme.3-1:1:3 (SEQ ID NO: 15), EXP-CUCme.4:1:1 (SEQ ID NO: 156), P-CUCme.6-1:1:1 (SEQ ID NO: 18), P-CUCme.8-1:1:2 (SEQ ID NO: 19), P-CUCme.9-1:1:2 (SEQ ID NO: 20), P-CUCme.10-1:1:1 (SEQ ID NO: 21), EXP-CUCme.eEF1a:1:1 (SEQ ID NO:162), P-CUCme.15-1:1:2 (SEQ ID NO: 23), P-CUCme.16a-1:1:2 (SEQ ID NO: 24), P-CUCme.17-1:1:2 (SEQ ID NO: 26), P-CUCme.18-1:1:2 (SEQ ID NO: 27), P-CUCme.19-1:1:3 (SEQ ID NO: 167), P-CUCme.20-1:3 (SEQ ID NO: 211), P-CUCme.21-1:1:1 (SEQ ID NO: 30), P-CUCme.22-1:1:3 (SEQ ID NO: 31), EXP-CUCme.SAMS2:1:1 (SEQ ID NO:168), P-CUCme.26-1:1:2 (SEQ ID NO: 33), P-CUCme.28-1:1:2 (SEQ ID NO: 34) and EXP-CUCme.29:1:2 (SEQ ID NO: 212) The expression of the transgene driven by the gene was compared with that from known constitutive expression element groups.Each plant expression vector consists of a right boundary region from *Agrobacterium tumefaciens*, a first transgenic cassette, a second transgenic selection cassette, and a left boundary region from *Agrobacterium tumefaciens*. The first transgenic cassette comprises a 5' region operably linked to a coding sequence of a β-glucuronidase (GUS, SEQ ID NO: 206) containing a processable intron derived from the potato light-induced tissue-specific ST-LS1 gene (gene bank accession number: X04753), and a 5' region operably linked to the coding sequence of an E6 gene from *Cotton Island cotton* (T-Gb.E6-3b:1:1, SEQ ID NO: 204), a *pea RbcS2-E9* gene (T-Ps.RbcS2-E9-1:1:6, SEQ ID NO: 203), or a *Cotton Island cotton* FbLate-2 gene (T-Gb.FbL2-1:1:1, SEQ ID NO: 204). The test promoter or a known constitutive promoter is composed of the 3' termination region of NO: 205; the second transgenic selection cassette is used to select plant cells that are resistant to the herbicide glyphosate (driven by the Arabidopsis actin 7 promoter) or the antibiotic kanamycin. A promoterless control plant expression vector (pMON124912) is used as a negative control for expression. The above test and constitutive expression element sets are cloned into plant expression vectors as shown in Table 14 below.

[0126] Table 14. Plant expression vectors and corresponding expression element sets and 3'UTR

[0127] In addition, two plasmids were constructed for co-transformation and data normalization. One transformation control plasmid was generated from the driver firefly (…). Photinus pyralis The constitutive promoter for the expression of the luciferase-coding sequence (FLuc, SEQ ID NO: 205) is operatively linked at 5' to the 3' termination region of the *Agrobacterium tumefaciens* carmine synthase gene (T-AGRtu.nos-1:1:13, SEQ ID NO: 209). Another transformation control plasmid contains a driver for *Gnaphalium affine* (FLuc, SEQ ID NO: 205). Renilla reniformis The constitutive promoter for the expression of the luciferase coding sequence (RLuc, SEQ ID NO: 206) is operatively linked at 5' to the 3' termination region of the rutin base synthase gene from Agrobacterium tumefaciens.

[0128] The PEG transformation method was used to transform the plant expression vectors pMON80585, pMON109584, pMON118756, pMON124912, pMON140818, pMON140819, pMON140820, pMON140821, pMON140822, pMON140823, pMON140824, pMON140825, and pMON1408. 26. pMON140827, pMON140828, pMON140829, pMON140830, pMON140831, pMON140832, pMON140833, pMON140834, pMON140835, pMON140836, pMON140837, pMON140838, and pMON140839 were used to transform cotton leaf protoplast cells. Equimolar amounts of each of the two transformation control plasmids and the test plant expression vector were used to transform protoplast cells. GUS and luciferase activities were measured. GUS and luciferase activities were measured by placing equal aliquots of the lysis formulation of the transformed cells as described above into two separate well trays. One tray was used for GUS measurement, while the other tray was used for dual-luciferase detection using a dual-luciferase reporter gene assay system (PromegaCorp., Madison, WI; see, for example, Promega Notes Magazine, No: 57, 1996, p. 02). Four replicates were used for each transformation for sample measurement. The average values ​​for GUS and luciferase are shown in Table 15 below.

[0129] Table 15. Mean GUS and luciferase expression and GUS / luciferase ratio

[0130] To compare the relative activity of each promoter in cotton leaf protoplasts, GUS values ​​were expressed as the ratio of GUS to luciferase activity and normalized according to the observed expression levels of the constitutive expression element sets EXP-At.Act7:1:11 and EXP-CaMV.35S-enh+ Ph.DnaK:1:3. Table 16 below shows the ratio of GUS to firefly luciferase (FLuc) normalized according to EXP-At.Act7:1:11 and EXP-CaMV.35S-enh+ Ph.DnaK:1:3. Table 17 below shows the ratio of GUS to Renilla luciferase (RLuc) normalized according to EXP-At.Act7:1:11 and EXP-CaMV.317S-enh+ Ph.DnaK:1:3.

[0131] Table 16. Ratio of GUS to firefly luciferase (FLuc) normalized according to EXP-At.Act7:1:11 and EXP-CaMV.35S-enh+ Ph.DnaK:1:3

[0132] Table 17. Ratio of GUS to Renal luciferase (RLuc) normalized according to EXP-At.Act7:1:11 and EXP-CaMV.35S-enh+ Ph.DnaK:1:3

[0133] As can be seen from Tables 16 and 17, most of the tested expression element sets demonstrated the ability to drive transgene expression in cotton leaf protoplast cells. In this assay, one expression element set, EXP-CUCme.4:1:1 (SEQ ID NO:156), showed a higher transgene expression level than EXP-At.Act7:1:11.

[0134] Example 8: Analysis of the regulatory elements driving GUS in cotton leaf protoplasts using transgenic cassette amplicon Cotton leaf protoplasts were transformed with a transgenic cassette amplicon containing a set of transcriptional regulatory expression elements that drive the expression of the β-glucuronidase (GUS) transgene, and the expression was compared with GUS expression in leaf protoplasts, wherein GUS expression is driven by a known constitutive promoter. The transgenic cassette amplicon consists of an EXP sequence operably linked to a GUS coding sequence (GUS, SEQ ID NO: 206) and an EXP sequence operably linked to a 3'UTR (T-Gb.FbL2-1:1:1, SEQ ID NO: 205). Average GUS expression was compared with the control EXP elements P-CaMV.35S-enh-1:1:102 / L-CaMV.35S-1:1:2 (SEQ ID NO: 210) and EXP-At.Atntt1:1:2 (SEQ ID NO: 200).

[0135] Furthermore, plasmids for co-transformation and data normalization were used in a similar manner to that described above in Example 2. The transformation control plasmid was derived from *Driver firefly* (…). Photinus pyralis The constitutive promoter for the expression of the luciferase coding sequence (FLuc, SEQ ID NO: 205) is operatively linked at 5' to the 3' termination region of the Agrobacterium tumefaciens carmine synthase gene (T-AGRtu.nos-1:1:13, SEQ ID NO: 209).

[0136] Table 18 below shows the average GUS expression conferred by each transgenic amplicon. Table 19 below shows the ratio of GUS to firefly luciferase (FLuc) normalized according to EXP-At.Atntt1:1:2 and P-CaMV.35S-enh-1:1:102 / L-CaMV.35S-1:1:2.

[0137] Table 18. Mean GUS and luciferase expression and GUS / luciferase ratio

[0138] Table 19. Ratio of GUS to firefly luciferase (FLuc) normalized according to EXP-At.Atntt1:1:2 and P-CaMV.35S-enh-1:1:102 / L-CaMV.35S-1:1:2

[0139] As can be seen from Table 18 above, not all EXP sequences showed the ability to drive transgene expression when compared with the promoter-free control. However, the EXP sequences P-CUCme.CumMe_WSM_SF16444.G5140-1:1:1 (SEQ ID NO: 175) and P-CUCme.CumMe_WSM_SF19839.G5090-1:1:1 (SEQ ID NO: 189) showed the ability to drive transgene expression in soybean cotyledon protoplasts at levels similar to or higher than EXP-At.Atntt1:1:2. As shown in Table 19 above, in this assay, the EXP sequence P-CUCme.CumMe_WSM_SF19839.G5090-1:1:1 (SEQ ID NO: 189) showed the ability to drive transgene expression at a higher level than EXP-At.Atntt1:1:2.

[0140] Example 9: Analysis of the regulatory elements driving GUS in stable-converting soybean Soybean plants were transformed with a plant expression vector containing an EXP sequence that drives the expression of the β-glucuronidase (GUS) transgene.

[0141] The following compounds were qualitatively and quantitatively detected by examining stained tissue sections: EXP-CUCme.Ubq1:1:1 (SEQ ID NO: 1), EXP-CUCme.Ubq1:1:3 (SEQ ID NO: 7), P-CUCme.1-1:1:1rc (SEQ ID NO: 155), P-CUCme.2-1:1:1 (SEQ ID NO: 14), P-CUCme.3-1:1:3 (SEQ ID NO: 15), EXP-CUCme.4:1:1 (SEQ ID NO: 156), EXP-CUCme.5:1:1 (SEQ ID NO: 159), P-CUCme.6-1:1:1 (SEQ ID NO: 18), P-CUCme.8-1:1:2 (SEQ ID NO: 19), and P-CUCme.9-1:1:2 (SEQ ID NO: 18). NO:20), P-CUCme.10-1:1:1 (SEQ ID NO: 21), EXP-CUCme.eEF1a:1:1 (SEQ ID NO: 162), P-CUCme.15-1:1:2 (SEQ ID NO: 23), P-CUCme.17-1:1:2 (SEQ ID NO: 26), P-CUCme.18-1:1:2 (SEQ ID NO: 27), P-CUCme.19-1:1:3 (SEQ ID NO: 167), P-CUCme.20-1:3 (SEQ IDNO: 211), P-CUCme.21-1:1:1 (SEQ ID NO: 30), EXP-CUCme.SAMS2:1:1 (SEQ ID NO: 168), P-CUCme.26-1:1:2 (SEQ ID NO: 33), EXP-CUCme.29:1:2 (SEQ ID NO: 212), P-CUCme.CumMe_WSM_SF25355.G5000-1:1:1 (SEQ ID NO: 196), P-CUCme.CumMe_WSM_SF17111.G5790-1:1:1 (SEQ ID NO: 177), P-CUCme.CumMe_WSM_SF22531.G5120-1:1:1 (SEQ ID NO: 192), P-CUCme.CumMe_WSM_SF18488.G5340-1:1:1 (SEQ ID NO: 181), P-CUCme.CumMe_WSM_SF23760.G5200-1:1:1 (SEQ ID NO: 193), EXP-CUCme.WSM_SF19064.G5690:1:1 (SEQ ID NO: 185), P-CUCme.WSM_SF17252.G7330-1:1:1 (SEQ ID NO:179), P-CUCme.CumMe_WSM_SF18634.G5190-1:1:1 (SEQ ID NO: 183), P-CUCme.CumMe_WSM_SF19647.G5760-1:1:1 (SEQ ID NO: 188), P-CUCme.CumMe_WSM_SF25936.G5450-1:1:1 (SEQ ID NO: 197), P-CUCme.CumMe_WSM_SF19839.G5090-1:1:1 (SEQ ID NO: Expression of GUS transgenes driven by 189), CumMe_WSM_SF206458.G5970 (SEQ ID NO: 98) and P-CUCme.CumMe_WSM_SF18716.G5860-1:1:1 (SEQ ID NO: 184). Each plant expression vector consists of a right boundary region from *Agrobacterium tumefaciens*, a first transgenic cassette, a second transgenic selection cassette, and a left boundary region from *Agrobacterium tumefaciens*. The first transgenic cassette comprises an EXP sequence operably linked 5' to the coding sequence of a β-glucuronidase (GUS, SEQ ID NO: 206) containing a processable intron derived from the potato light-induced tissue-specific ST-LS1 gene (gene bank accession number: X04753), and operably linked 5' to the 3' termination region of the *Cotton Island cotton* FbLate-2 gene (T-Gb.FbL2-1:1:1, SEQ ID NO: 205). The second transgenic selection cassette is used to select plant cells transformed to confer resistance to the herbicide glyphosate (driven by the *Arabidopsis thaliana* actin 7 promoter).

[0142] The EXP sequences described above were cloned into plant expression constructs as shown in Tables 20-23 below, and then used to transform soybean plants using Agrobacterium-mediated transformation. GUS expression was qualitatively and quantitatively detected using tissue sections from selected tissues.

[0143] Histochemical GUS analysis was used for qualitative expression analysis in transformed plants. Whole tissue sections were cultured with GUS staining solution X-Gluc (5-bromo-4-chloro-3-indolyl-β-glucuronide) (1 mg / mL) for an appropriate duration, rinsed, and visually examined for blue staining. GUS activity was qualitatively determined by direct visual examination or microscopic examination of selected plant organs and tissues. Expression in R0 generation plants was examined in Vn5 roots, R1 roots, Vn5 sarcopter leaves, Vn5 progenitor leaves, R1 progenitor leaves, R1 petioles, yellow pod embryos, yellow pod cotyledons, R3 immature seeds, R3 pods, R5 cotyledons, and R1 flowers.

[0144] For quantitative analysis, total protein was extracted from selected transformed maize plant tissue. One microgram of total protein was used with a fluorescent substrate, 4-methyleumbelliferyl-β-D-glucuronide (MUG), in a total reaction volume of 50 microliters. The reaction product, 4-methyleumbelliferyl (4-MU), exhibits maximum fluorescence at high pH, ​​where the hydroxyl group is ionized. The detection was stopped by adding an alkaline solution of sodium carbonate, and the pH was adjusted to quantify the fluorescent product. Using a Fluoromax-3 (Horiba; Kyoto, Japan) with a Micromax reader, the slit width was set to excitation at 2 nm and emission at 3 nm, and fluorescence was measured at excitation at 365 nm and emission at 445 nm.

[0145] Tables 20 and 21 below show the average quantitative expression levels measured in R0 generation plant tissues. In both tables, tissues that were not detected are shown as blank cells.

[0146] Table 20. Mean GUS expression in Vn5 roots, R1 roots, Vn5 sapodilla leaves, Vn5 progenitor leaves, R1 progenitor leaves, and R1 petioles of soybean plants transformed in the R0 generation.

[0147] Table 21. Mean GUS expression in yellow pod embryos, yellow pod cotyledons, R3 immature seeds, R3 pods, R5 cotyledons, and R1 flowers of soybean plants transformed from R0 generation.

[0148] As can be seen from Tables 20 and 21, depending on the EXP sequence used to drive expression, the EXP sequences are: EXP-CUCme.Ubq1:1:1 (SEQ ID NO: 1), EXP-CUCme.Ubq1:1:3 (SEQ ID NO: 7), P-CUCme.1-1:1:1rc (SEQ ID NO: 155), P-CUCme.2-1:1:1 (SEQ ID NO: 14), EXP-CUCme.4:1:1 (SEQ ID NO: 156), EXP-CUCme.5:1:1 (SEQ ID NO: 159), P-CUCme.6-1:1:1 (SEQ ID NO: 18), P-CUCme.8-1:1:2 (SEQ ID NO: 19), P-CUCme.9-1:1:2 (SEQ ID NO: 20), and P-CUCme.10-1:1:1 (SEQ ID NO: 155). 21), EXP-CUCme.eEF1a:1:1 (SEQ ID NO: 162), P-CUCme.15-1:1:2 (SEQ ID NO:23), P-CUCme.17-1:1:2 (SEQ ID NO: 26), P-CUCme.18-1:1:2 (SEQ ID NO: 27), P-CUCme.19-1:1:3 (SEQ ID NO: 167), P-CUCme.20-1:3 (SEQ ID NO: 211), P-CUCme.21-1:1:1 (SEQ ID NO: 30), EXP-CUCme.SAMS2:1:1 (SEQ ID NO: 168), P-CUCme.26-1:1:2(SEQ ID NO: 33), EXP-CUCme.29:1:2(SEQ ID NO: 212), P-CUCme.CumMe_WSM_SF25355.G5000-1:1:1 (SEQ ID NO: 196), P-CUCme.CumMe_WSM_SF17111.G5790-1:1:1 (SEQ ID NO:177), P-CUCme.CumMe_WSM_SF22531.G5120-1:1:1 (SEQ ID NO: 192), P-CUCme.CumMe_WSM_SF18488.G5340-1:1:1 (SEQ ID NO: 181), P-CUCme.CumMe_WSM_SF23760.G5200-1:1:1 (SEQ ID NO: 193), EXP-CUCme.WSM_SF19064.G5690:1:1 (SEQ ID NO: 185), P-CUCme.WSM_SF17252.G7330-1:1:1 (SEQ ID NO: 179), P-CUCme.CumMe_WSM_SF18634.G5190-1:1:1 (SEQ ID NO: 183), P-CUCme.CumMe_WSM_SF19647.G5760-1:1:1 (SEQ ID NO: 188), P-CUCme.CumMe_WSM_SF25936.G5450-1:1:1 (SEQ ID NO: 197), P-CUCme.CumMe_WSM_SF19839.G5090-1:1:1 (SEQ ID NO: 189), CumMe_WSM_SF206458.G5970 (SEQ ID NO: 98) and P-CUCme.CumMe_WSM_SF18716.G5860-1:1:1 (SEQ ID NO: 184) quantitatively demonstrated the ability to drive transgene expression in some or all of the detected tissues.

[0149] Histological analysis of the selected tissue sections further provided evidence of numerous EXP sequence expressions. Although quantitative analysis showed fairly low expression levels, EXP-CUCme.Ubq1:1:1 (SEQ ID NO: 1) and EXP-CUCme.Ubq1:1:3 (SEQ ID NO: 7) showed constitutive expression patterns observed in all tissues. This type of driven expression pattern requiring constitutively low levels of transgenic expression is likely the most accidental. Expression driven by P-CUCme.1-1:1:1rc (SEQ ID NO: 155) was observed in the vascular bundles and xylem of the saphenous and progenitor leaves, as well as in the root bark, phloem, xylem, endodermis, stele, and apex. Expression driven by EXP-CUCme.4:1:1 (SEQ ID NO: 156) was observed in all tissues, with the highest expression observed during the reproductive period of the plant. Expression driven by P-CUCme.10-1:1:1 (SEQ ID NO: 21) was observed only in the V5 follicles and R1 anthers. Expression driven by EXP-CUCme.eEF1a:1:1 (SEQ ID NO: 162) showed the constitutive expression pattern observed in the yellow pod embryos and cotyledons. Yellow pod embryo activity in the R1 generation was 5-fold higher than in the R0 generation (see Table 23 below). Expression driven by P-CUCme.15-1:1:2 (SEQ ID NO: 23), P-CUCme.17-1:1:2 (SEQ ID NO: 26), and P-CUCme.18-1:1:2 (SEQ ID NO: 27) showed histological constitutive expression levels. Expression driven by P-CUCme.19-1:1:3 (SEQ ID NO: 167) showed a histological constitutive expression pattern, except in the V5 roots and R1 petioles. R3 pods showed the highest expression.

[0150] Except for expression in the V5 root, expression driven by P-CUCme.20-1:3 (SEQ ID NO: 211) showed a histological constitutive expression pattern. The highest expression was observed in the cotyledons of the R8 stage. Expression driven by EXP-CUCme.SAMS2:1:1 (SEQ ID NO: 168) showed a constitutive expression pattern with histologically observed expression in all tissues. Increased GUS expression was observed in the R1 generation (see Tables 22 and 23 below). The R1 stage flowers and petioles showed the highest expression levels in soybean. Expression driven by P-CUCme.CumMe_WSM_SF22531.G5120-1:1:1 (SEQ ID NO: 192) showed a histological constitutive expression pattern with the highest expression in the cotyledons and embryos of the R8 stage. Expression driven by P-CUCme.CumMe_WSM_SF18488.G5340-1:1:1 (SEQ ID NO: 181) showed constitutive expression levels, while quantitative high expression was observed in yellow pod embryos.

[0151] R0 generation plants transformed with plasmid constructs containing EXP-CUCme.eEF1a:1:1 (SEQ ID NO: 162) and EXP-CUCme.SAMS2:1:1 (SEQ ID NO: 168) produced seeds, and GUS expression in R1 generation plants was analyzed. Expression in Vn5 roots, Vn5 saphenous leaves, Vn5 progenitor leaves, R1 progenitor leaves, R1 petioles, yellow pod embryos, yellow pod cotyledons, R3 immature seeds, R3 pods, R5 cotyledons, and R1 flowers was analyzed. Tables 22 and 23 show the mean GUS expression measured in each tissue of the R1 generation transformed plants.

[0152] Table 22. Mean GUS expression in Vn5 roots, Vn5 saponins, Vn5 source leaves, R1 source leaves, and R1 petioles of R1-transformed soybean plants.

[0153] Table 23. Mean GUS expression in yellow pod embryos, yellow pod cotyledons, R3 immature seeds, R3 pods, R5 cotyledons, and R1 flowers of soybean plants transformed from R1 generation.

[0154] As can be seen from Tables 22 and 23 above, the expression driven by EXP-CUCme.eEF1a:1:1 (SEQ ID NO: 162) and EXP-CUCme.SAMS2:1:1 (SEQ ID NO: 168) in the R1 generation shows that increased constitutive expression levels relative to the R0 generation are observed in many tissues of the R1 generation.

[0155] The principles of the invention have been elucidated and described. Changes in the arrangement and details of the invention may be made without departing from these principles, as will be apparent to those skilled in the art. All variations within the spirit and scope of the claims are intended to be included within the scope of this invention. All publications and disclosed patent documents cited herein are incorporated by reference, as if specifically and individually indicated that each individual publication or patent application is incorporated by reference.

Claims

1. A DNA molecule exhibiting gene regulatory activity, comprising a polynucleotide sequence consisting of a fragment of at least 500 consecutive nucleotides comprising SEQ ID NO: 211; The polynucleotide sequence thereon is operatively linked to a heterologous transcribed polynucleotide molecule.

2. The DNA molecule of claim 1, wherein the heterologous transcribed polynucleotide molecule comprises a gene for agricultural purposes.

3. The DNA molecule of claim 2, wherein the agronomically intended gene confers herbicide tolerance in plants.

4. The DNA molecule of claim 2, wherein the agronomically intended gene confers pest resistance in the plant.

5. A method for producing a commodity, comprising: a) Obtaining a transgenic plant or a portion thereof comprising the DNA molecule of claim 1; and b) The goods are thus produced.

6. The method of claim 5, wherein the commodity is a protein concentrate, a protein isolate, a grain, starch, a seed, a coarse flour, a flour, a biomass, or a seed oil.

7. A method for expressing a transcribed polynucleotide molecule, comprising: a) Obtaining a transgenic plant comprising the DNA molecule of claim 1; and b) Cultivate the transgenic plant in which the transcribed polynucleotide is expressed.

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