Plant regulatory elements and uses thereof

Abstract

Recombinant DNA polynucleotides and constructs useful for modulating gene expression in plants and their nucleotide sequences are provided. Also provided are transgenic plants, plant cells, plant parts, and seeds comprising the recombinant DNA polynucleotides operably linked to a heterologous transcribable DNA polynucleotide. In addition, methods of use of the recombinant DNA polynucleotides and constructs are provided.

Description

Citation of relevant applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 601,354, filed November 21, 2023, which is incorporated herein by reference in its entirety.

[0002] Merging of sequence lists The 9,601-byte (as measured in Microsoft Windows®) sequence list contained in a file named “MONS584WO_ST26.xml” was created on November 7, 2024, and was simultaneously filed electronically (using the Patent Centre of the United States Patent and Trademark Office) and incorporated in its entirety by reference. Technical Field

[0003] This disclosure relates to the fields of plant molecular biology and plant genetic engineering. More specifically, this disclosure relates to DNA polynucleotides that can be used to regulate gene expression in plants. Background Technology

[0004] Regulatory elements are genetic elements that regulate gene activity by modulating the transcription of operatively linked transcribed DNA polynucleotides. These elements can include promoters, leader sequences, introns, and 3' untranslated regions (3' UTRs), and are used in plant molecular biology and plant genetic engineering. Summary of the Invention

[0005] This section provides a general overview of this disclosure and is not a full disclosure of its entire scope or all its features.

[0006] This document provides gene regulatory elements for use in plants. Several embodiments relate to recombinant DNA polynucleotides containing regulatory elements. Transgenic plant cells, plants, parts thereof, and seeds containing regulatory elements are also provided. In one embodiment, the regulatory element is operatively linked to a transcribed DNA polynucleotide. In some embodiments, the transcribed DNA polynucleotide may be heterologous to the regulatory DNA sequence. Thus, in a particular embodiment, the regulatory element DNA sequence provided herein can be defined as operatively linked to a heterologous transcribed DNA polynucleotide. Several embodiments relate to the use of regulatory elements and methods for preparing and using recombinant DNA polynucleotides containing regulatory elements, as well as transgenic plant cells, plants, and seeds containing regulatory elements operatively linked to transcribed DNA polynucleotides.

[0007] In some embodiments, a recombinant DNA polynucleotide is provided comprising a DNA sequence selected from the group consisting of: (a) a sequence having at least about 85% sequence identity with SEQ ID NO:1; (b) a sequence comprising SEQ ID NO:1; and (c) a fragment of SEQ ID NO:1, wherein the fragment contains gene regulatory activity; wherein the DNA sequence is operatively linked to a heterologous transcribed DNA polynucleotide. In a specific embodiment, the recombinant DNA polynucleotide comprises a DNA sequence having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the DNA sequence of SEQ ID NO:1.

[0008] In another aspect, this document provides transgenic plant cells comprising recombinant DNA polynucleotides comprising a DNA sequence selected from the group consisting of: (a) a sequence having at least about 85% sequence identity with SEQ ID NO:1; (b) a sequence comprising SEQ ID NO:1; and (c) a fragment of SEQ ID NO:1, wherein the fragment contains gene regulatory activity; wherein the DNA sequence is operatively linked to a heterologous transcribed DNA polynucleotide. In some embodiments, the transgenic plant cell may be a monocotyledonous plant cell. In other embodiments, the transgenic plant cell may be a dicotyledonous plant cell.

[0009] In another aspect, this document further provides a transgenic plant or a portion thereof comprising a recombinant DNA polynucleotide, said recombinant DNA polynucleotide comprising a DNA sequence selected from the group consisting of: a) a sequence having at least 85% sequence identity with SEQ ID NO:1; b) a sequence comprising SEQ ID NO:1; and c) a fragment of SEQ ID NO:1, said fragment comprising gene regulatory activity; wherein said DNA sequence is operatively linked to a heterologous transcribed DNA polynucleotide. In a specific embodiment, the transgenic plant may be a progeny plant of any generation comprising the recombinant DNA polynucleotide. A transgenic seed comprising the recombinant DNA polynucleotide is also provided, which, upon growth, produces such a transgenic plant.

[0010] Several embodiments relate to a method for producing a commercial product, comprising obtaining a transgenic plant or a portion thereof containing recombinant DNA polynucleotides as described herein (such as those comprising DNA sequences selected from SEQ ID NO:1 or fragments or variants thereof), and producing a commercial product therefrom. In one embodiment, the commercial product may be seeds, processed seeds, protein concentrates, protein isolates, starch, cereals, plant parts, seed oils, biomass, flour, and / or coarse flour.

[0011] Several embodiments relate to a method for producing transgenic plants comprising recombinant DNA polynucleotides as described herein, comprising transforming plant cells with the recombinant DNA polynucleotides to produce transformed plant cells and regenerating transgenic plants from the transformed plant cells. In a further embodiment, a method for expressing transcribed DNA polynucleotides is provided, the method comprising obtaining a transgenic plant as described herein and culturing the plant, wherein the transcribed DNA polynucleotides are expressed.

[0012] Sequence Summary SEQ ID NO:1 is the 400-nucleotide DNA sequence of the synthesized 3' UTR T-Zm.GST314.

[0013] SEQ ID NO:2 is a 1,213-nucleotide DNA sequence of the regulatory expression element set or EXP-At.Cyco, which contains a natural promoter operably linked to the 5' of a natural leader sequence and operably linked to a derivative of... Arabidopsis The 5' of the natural first intron of the cytochrome c oxidase subunit VIa gene (locus AT4G37830.3).

[0014] SEQ ID NO:3 is a 2,001-nucleotide synthetic coding sequence for plant expression of β-glucuronidase (GUS) containing a processable intron derived from the potato light-induced tissue-specific St-LS1 gene (GenBank accession number: X04753).

[0015] SEQ ID NO:4 is derived from Sea Island cotton ( Gossypium barbadense The 315-nucleotide DNA sequence of the 3' UTR T-Gb.FbL2 of the FbLate-2 gene (GenBank accession number U34401.1) was obtained. Detailed Implementation

[0016] Exemplary embodiments will now be described more fully. The descriptions and specific examples included herein are intended for illustrative purposes only and are not intended to limit the scope of this disclosure. The following definitions, descriptions, and methods will better define the invention and guide those skilled in the art in the practice of the invention. Unless otherwise stated, the terminology should be understood according to its conventional usage by those skilled in the art.

[0017] This document provides regulatory elements with gene-regulating activity in plants. The nucleotide sequences of such regulatory elements are provided as SEQ ID NO:1. These regulatory elements are capable of influencing the expression of operably linked, transcribed DNA polynucleotides in plant tissues, thus regulating the expression of operably linked transgenic genes in transgenic plants. Methods for modifying, producing, and using recombinant DNA polynucleotides containing the provided regulatory elements are also provided. Compositions comprising transgenic plant cells, plants, plant parts, and seeds are also provided, along with methods for preparing and using said compositions, wherein the transgenic plant cells, plants, plant parts, and seeds contain recombinant DNA polynucleotides comprising one or more regulatory elements as described herein.

[0018] DNA polynucleotides This document provides DNA polynucleotides, their fragments and variants, and their corresponding DNA sequences. As used herein, the terms “DNA,” “DNA polynucleotide,” “DNA molecule,” and “nucleic acid molecule” refer to deoxyribonucleic acid (DNA) molecules. DNA polynucleotides are conventionally described from the 5' (upstream) end to the 3' (downstream) end. DNA polynucleotides may be of genomic or synthetic origin and / or contain recombinant or heterologous DNA polynucleotides or sequences. As used herein, the term “DNA sequence” refers to the nucleotide sequence of a DNA polynucleotide. Right now A sequence of consecutive nucleotides in a DNA molecule. Examples of DNA sequences disclosed herein include the DNA sequences of SEQ ID NO:1-4, or variants or fragments thereof, or the reverse complementary sequence of any of SEQ ID NO:1-4. As used herein with respect to DNA polynucleotides or nucleotides of DNA sequences or molecules, the terms “consecutive” and “contiguous” are interchangeable and synonymous, and refer to linked nucleotides in a DNA polynucleotide or DNA sequence, strand, or molecule without any gaps or breaks between them. The nomenclature used herein corresponds to Title 37 of the United States Code of Federal Regulations §1.822 and is set forth in WIPO Standard ST.26 (2021), Annex I, Tables 1 and 3.

[0019] As used herein, “recombinant DNA polynucleotide” is a DNA polynucleotide comprising a combination of DNA polynucleotides that would not naturally coexist without human intervention. For example, a recombinant DNA polynucleotide may be a DNA polynucleotide consisting of at least two DNA polynucleotides that are heterologous to each other, or a DNA polynucleotide containing a DNA sequence that deviates from the DNA sequence found in nature, or a DNA polynucleotide containing a synthetic DNA sequence, or a DNA polynucleotide that has been incorporated into the DNA of a host cell through genetic transformation or gene editing.

[0020] As used herein, a “synthetic nucleotide sequence” or “artificial nucleotide sequence” is a nucleotide sequence that is known to not exist in nature or not naturally occurring. Preferably, the synthetic nucleotide sequence shares little or no extended homology with the natural nucleotide sequence. In this context, extended homology generally refers to 100% sequence identity extending more than about 25 nucleotides of a continuous sequence. An example of a synthetic nucleotide sequence is 3' UTR T-Zm.GST314 (SEQ ID NO:1).

[0021] In this application, reference to "isolated DNA polynucleotide" or equivalent terms or phrases is intended to mean that the DNA polynucleotide is present alone or in combination with other compositions, but not in its natural environment. For example, any nucleic acid element (such as coding sequences, intron sequences, non-translational leader sequences, promoter sequences, transcription termination sequences) naturally present in the DNA of an organism's genome. Right now Elements (such as 3' UTRs) located within the genome of an organism and at their naturally occurring location within the genome are not considered "isolated". However, any element is considered "isolated" within the scope of this disclosure if each of these elements and any sub-parts thereof are not located within the genome of the organism and at their naturally occurring location within the genome. Similarly, any nucleotide sequence encoding an insecticidal protein or any naturally occurring insecticidal variant of that protein is not present in the DNA of a bacterium encoding the protein. For the purposes of this disclosure, synthetic nucleotide sequences encoding the amino acid sequence of a naturally occurring insecticidal protein will be considered isolated. For the purposes of this disclosure, any transgenic nucleotide sequence, such as a nucleotide sequence inserted into the genome of a plant or bacterial cell or present in DNA in an extrachromosomal vector, will be considered an isolated nucleotide sequence, whether present in a plasmid or similar structure used for transforming cells, present in the genome of a plant or bacterium, or present in a detectable amount in tissues, progeny, biological samples, or commercial products derived from plants or bacteria.

[0022] As used herein, the term "sequence identity" refers to the degree to which two optimally aligned polynucleotide sequences or two optimally aligned polypeptide sequences are identical. Optimal sequence alignment is achieved by aligning two sequences (e.g., 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. In some embodiments, the DNA sequence provided as SEQ ID NO:1 is used as a reference sequence.

[0023] As used herein, the terms “sequence identity percentage,” “sequence identity %,” “identity percentage,” and “identity %” are identity scores multiplied by 100. The “identity score” of a sequence that is best aligned to a reference sequence is the number of nucleotide matches in the best alignment, divided by the total number of nucleotides in the reference sequence, for example, the total number of nucleotides in the entire length of the reference sequence. Therefore, several embodiments involve DNA polynucleotides comprising a DNA sequence that, when best aligned with a reference sequence provided herein as SEQ ID NO:1, has at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity with the reference sequence from which it is derived. The DNA sequence disclosed herein may have the activity of the reference sequence from which it is derived, for example, it may have the activity of SEQ ID NO:1. Furthermore, the DNA sequence provided herein may contain the same or similar activity as the reference sequence from which it is derived, for example, it may contain the same or similar activity as SEQ ID NO:1.

[0024] Control element Regulatory elements, such as promoters, leader sequences (also known as 5' UTRs), enhancers, introns, and transcription termination regions (also known as 3' UTRs), play an indispensable role in the overall expression of genes in living cells. As used herein, the terms "regulatory element," "gene regulatory element," "expression element," and "regulatory expression element" refer to DNA polynucleotides with gene regulatory activity. As used herein, the term "transcriptional regulatory element" refers to a DNA polynucleotide with gene regulatory activity by influencing the transcription of the gene to which it is operably linked. As used herein, the term "gene regulatory activity" refers to the ability to influence the expression of operably linked transcribed DNA polynucleotides, for example, by influencing the transcription and / or translation of operably linked transcribed DNA polynucleotides. Regulatory elements that function in plants, such as promoters, leader sequences, enhancers, introns, and 3' UTRs, can be used to modify plant phenotypes through genetic engineering.

[0025] As used herein, a “regulatory expression element set” or “EXP” sequence may refer to a set of operatively linked regulatory elements, such as enhancers, promoters, leader sequences, and introns. For example, a regulatory expression element set may include, for instance, a promoter operatively linked to a 5’ of a leader sequence and a 5’ of an intron sequence.

[0026] As used in this article for regulatory elements, the terms "fragment" and "functional fragment" are interchangeable and refer to a segment or portion of a regulatory element that influences, regulates, or drives the expression of operatively linked, transcribed DNA polynucleotides. Right now It possesses gene regulatory activity. Fragments of regulatory elements can influence, regulate, or drive the expression of operatively linked transcribed DNA polynucleotides in a manner similar to that from which their reference sequence originates. According to one embodiment of this disclosure, a fragment of SEQ ID NO:1 that may possess gene regulatory activity is provided. In another embodiment, a fragment of SEQ ID NO:1 is provided, which may contain a reference sequence from which its origin originates. (Right now The gene regulatory activity is the same as or similar to that of SEQ ID NO:1. In an alternative embodiment, a gene regulatory fragment of SEQ ID NO:1 is provided, comprising at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 325, at least about 350, or at least about 375 consecutive nucleotides of SEQ ID NO:1.

[0027] Regulatory elements can be characterized by their gene expression patterns, such as positive and / or negative effects like constitutive expression or temporal, spatial, developmental, tissue, environmental, physiological, pathological, cell cycle, and / or chemical responses, and any combination thereof, as well as by quantitative or qualitative indicators. As used herein, a “gene expression pattern” is any pattern by which operatively linked DNA polynucleotides are transcribed into transcribed RNA molecules, resulting in the relative levels and abundance of transcribed RNA molecules in various plant tissues and cells during development. Transcribed RNA can be translated to produce proteins or to provide antisense or other regulatory RNAs, such as double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), small interfering RNA (siRNA), etc.

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

[0029] Promoters serve as regulatory elements for controlling the expression of operably linked, transcribed DNA polynucleotides. As used herein, the term "promoter" generally refers to a DNA polynucleotide involved in recognizing and binding RNA polymerases (e.g., RNA polymerase II) and other proteins (such as trans-acting transcription factors) to initiate transcription. Promoters may initially separate from the 5' untranslated region (5' UTR) of a genomic copy of a gene. In some embodiments, the promoter is operably linked to the 5' of a leader sequence. Promoters may be synthetically generated or manipulated DNA polynucleotides. Promoters may also be chimeric. Chimeric promoters are generated by the fusion of two or more heterologous DNA polynucleotides.

[0030] It is possible to analyze whether the promoter or promoter fragment described herein contains known promoter elements, such as DNA sequence characteristics, TATA boxes, and other known transcription factor binding site motifs. Those skilled in the art can use the identification of such known promoter elements to design promoter variants that have similar expression patterns to the original promoter.

[0031] As used herein, the term "leader sequence" refers to a DNA polynucleotide isolated from the untranslated 5' region (5' UTR) of a gene and is generally defined as a nucleotide segment between the transcription start site (TSS) and the protein-coding sequence start site. Alternatively, a leader sequence may be a synthetically generated or manipulated DNA element. A leader sequence can serve as a 5' regulatory element for regulating the expression of operatively linked, transcribed DNA polynucleotides. A leader polynucleotide may be used with a heterologous promoter or with its natural promoter. In specific embodiments, such DNA sequences may be defined as capable of serving as leader sequences in host cells, including, for example, transgenic plant cells. In one embodiment, such sequences are decoded to contain leader sequence activity.

[0032] As used herein, the term "intron" refers to a DNA polynucleotide that can be isolated from or identified from a gene and can be generally defined as a region spliced ​​during pre-translational messenger RNA (mRNA) processing. Alternatively, an intron can be a synthetically produced or modified DNA element. Introns can contain enhancer elements that enable transcription of operably linked genes. Introns can serve as regulatory elements for regulating the expression of operably linked transcribed DNA polynucleotides. Constructs may contain introns, and introns may or may not be heterologous relative to transcribed DNA polynucleotides. Examples of introns include the rice actin intron and the maize HSP70 intron.

[0033] In plants, the presence of introns in gene constructs, relative to those lacking introns, leads to increased mRNA and protein accumulation. This effect is known as "intron-mediated enhancement" (IME) of gene expression. Known introns stimulating expression in plants have been identified in maize genes (e.g., tubA1, Adh1, Sh1, and Ubi1), rice genes (e.g., tpi), and dicotyledonous genes such as those from petunia (e.g., rbcS), potato (e.g., st-ls1), and Arabidopsis (e.g., rbcS). Arabidopsis thaliana Those (e.g., ubq3 and pat1). Studies have shown that deletions or mutations within intron splicing sites reduce gene expression, indicating that IMEs may require splicing. However, IMEs in dicotyledonous plants have been confirmed by point mutations within the splicing site of the Arabidopsis pat1 gene. Repeated use of the same intron in a single plant has shown drawbacks. In those cases, it is necessary to collect basic control elements to construct appropriate recombinant DNA elements.

[0034] As used herein, the terms “3’ transcription termination polynucleotide,” “3’ untranslated region,” and “3’ UTR” refer to DNA polynucleotides used during transcription of the untranslated region of the 3’ portion of mRNA. The 3’ untranslated region of mRNA (also known as the poly(A) tail) can be generated through specific cleavage and 3’ polyadenylation. The 3’ UTR can be operatively linked to and downstream of a transcribed DNA polynucleotide and can include polyadenylation signals and other regulatory signals capable of influencing transcription, mRNA processing, or gene expression. The poly(A) tail is thought to play a role in mRNA stability and translation initiation. Examples of 3’ transcription termination polynucleotides include the 3’ region of carmine synthase, the 3’ region of wheat hsp17, the 3’ region of the pea rubisco small subunit, the 3’ region of cotton E6, and the 3’ UTR of coixin.

[0035] 3' UTRs are generally beneficial for the recombination expression of specific DNA polynucleotides. Weak 3' UTRs can lead to readthrough during transcription, which may affect the expression of DNA polynucleotides located in adjacent expression cassettes. Proper control of transcription termination prevents readthrough of downstream DNA sequences (e.g., other expression cassettes) and can further allow for efficient recycling of RNA polymerase to improve gene expression. Efficient termination of transcription (release of RNA polymerase II from DNA) is a prerequisite for restarting transcription, thus directly affecting the overall transcriptional level. After transcription termination, mature mRNA is released from the site of synthesis and transported to the cytoplasm. Eukaryotic mRNA exist It accumulates in vivo in the form of poly(A), making it difficult to detect transcription termination sites using conventional methods. However, it is difficult to predict functional and efficient 3' UTRs using bioinformatics methods because there is no conserved DNA sequence that allows for easy prediction of efficient 3' UTRs.

[0036] From a practical perspective, the following characteristics are generally beneficial for the 3' UTR used in expression cassettes. First, the 3' UTR should be able to efficiently and effectively terminate the transcription of transcribed DNA polynucleotides (e.g., transgenes) and prevent the transcript from reading through any adjacent DNA sequence that may contain another expression cassette, such as in cases where multiple expression cassettes are present on a single transfer DNA (T-DNA) or on adjacent chromosomal DNA that has already been inserted into T-DNA during plant transformation. Second, the 3' UTR should not result in a reduction of the transcriptional activity conferred by the promoters, leader sequences, enhancers, and introns used to drive the expression of transcribed DNA polynucleotides. Finally, in plant biotechnology, the 3' UTR is commonly used to initiate the amplification reaction of reverse-transcribed RNA extracted from transformed plants and is used for: (1) assessing transcriptional activity or expression after the expression cassette is integrated into the plant chromosome; (2) assessing the copy number of the insertion within the plant DNA; and (3) assessing the conjugation of the resulting seeds after breeding. The 3' UTR is also used for the amplification reaction of DNA extracted from transformed plants to characterize the integrity of the insertion cassette. A 3' UTR that can be used in combination with a further regulating element (e.g., the regulating element presented as SEQ ID NO:2) is provided as SEQ ID NO:1. A 3' UTR that can also be used in combination with a further regulating element is a fragment and variant of SEQ ID NO:1, wherein the fragment and variant may contain the same or similar activity as SEQ ID NO:1.

[0037] As used herein, the term "reinforcer" or "reinforcer element" refers to Cis The control element, also known as Shun Mode Enhancer elements are elements that impart aspects of the overall expression pattern, but are generally insufficient to drive the transcription of operably linked transcribed DNA polynucleotides on their own. Unlike promoters, enhancer elements typically do not include a transcription start site (TSS) or TATA box or equivalent DNA sequence. Promoters or promoter fragments may naturally contain one or more enhancer elements that influence the transcription of operably linked DNA sequences. Enhancer elements may also be fused to promoters to produce chimeric promoters. Cis Elements that impart aspects of overall regulation of gene expression.

[0038] As used herein, the term "variant" refers to a second DNA polynucleotide (such as a regulatory element) that is similar to but not identical to a first DNA polynucleotide, and wherein the second DNA polynucleotide retains the general functionality of the first DNA polynucleotide, for example, the same or similar expression pattern, such as through more or less equivalent transcriptional activity. A variant may be a shortened or truncated form of the first DNA polynucleotide or a modified form of the sequence of the first DNA polynucleotide, such as a DNA polynucleotide with different restriction enzyme sites and / or internal deletions, substitutions, or insertions. "Variant" may also cover a regulatory element having a nucleotide sequence comprising one or more modifications, such as substitution, duplication, deletion, and / or insertion, of one or more nucleotides of a reference sequence, wherein the derived regulatory element has more or less or equivalent transcriptional or translational activity compared to the corresponding parental regulatory polynucleotide or element. Regulatory element "variants" will also cover variants resulting from mutations naturally occurring in bacterial and plant cell transformations. In some embodiments, the DNA polynucleotide sequence provided as SEQ ID NO:1 can be used to generate variants whose composition is similar to the original regulatory element ( Right now The DNA sequences of SEQ ID NO:1 are similar but not identical, while still maintaining the general functionality of the original regulatory elements, such as the same or similar expression patterns. In view of this disclosure, the generation of such variants is entirely within the scope of ordinary skill in the art and is as considered herein.

[0039] The efficacy of the modifications described herein (such as substitution, duplication, deletion, and / or insertion) in the desired expression of a specific transgene can be empirically tested in stable and transient plant assays (such as those described in the working examples herein) to validate the results, which may vary depending on the alterations made in the starting DNA polynucleotides and the target of the alterations.

[0040] Construct As used herein, the term "construct" means any recombinant DNA polynucleotide or molecule, such as plasmids, granules, viruses, bacteriophages, or linear or circular DNA or RNA polynucleotides, derived from any source, capable of genome integration or autonomous replication, containing DNA polynucleotides, wherein at least one DNA polynucleotide has been functionally linked. Right now (It can be operatively linked) to another DNA polynucleotide. As used herein, the term "vector" means a device that can be used for transformation purposes (…). Right now Any construct that introduces heterologous DNA or RNA into a host cell. Constructs typically contain one or more expression cassettes. As used herein, an "expression cassette" refers to a recombinant DNA polynucleotide that contains at least one transcribed DNA polynucleotide operably linked to one or more regulatory elements (typically at least a promoter and a 3' UTR).

[0041] As used herein, the term "operably linked" refers to the linking of a first DNA polynucleotide to a second DNA polynucleotide, wherein the first and second DNA polynucleotides are arranged such that the first DNA polynucleotide influences the function of the second DNA polynucleotide. The two DNA polynucleotides may or may not be part of a single, consecutive DNA polynucleotide, and may or may not be adjacent. For example, if a promoter regulates the transcription of a transcribed DNA polynucleotide of interest in a cell, the promoter is operably linked to said transcribed DNA polynucleotide. For example, if a leader sequence is capable of influencing the transcription or translation of a DNA sequence, it is operably linked to the DNA sequence. For example, if a 3' UTR regulates and / or terminates the transcription of a transcribed DNA polynucleotide of interest in a cell, the 3' UTR is operably linked to said transcribed DNA polynucleotide.

[0042] In some embodiments, one or more regulatory elements operatively linked to transcribed DNA polynucleotides, as described herein, are provided in a dual tumor-inducing (Ti) plasmid boundary construct having T-DNA contained in the plasmid. root Agrobacterium tumefaciens The right boundary (RB or AGRtu.RB) and left boundary (LB or AGRtu.LB) regions of the separated Ti plasmid, wherein the one or more regulatory elements are associated with the region formed by the separation of Ti plasmids. Agrobacterium rhizogenes Together with transport molecules provided by the cell, T-DNA is allowed to integrate into the plant cell genome. See For example, U.S. Patent 6,603,061). The construct may also contain plasmid backbone DNA segments that provide replication function and antibiotic selection in bacterial cells, such as those found in *E. coli* (…). Escherichia coli Origin of replication, such as ori322; broad host-range origin of replication, such as oriV or oriRi; and coding regions for selective markers, such as Tn7 aminoglycoside adenosine transferases that confer resistance to spectinomycin or streptomycin. aadA The Spec / Strp gene, or the gentamicin (Gm, Gent) selective marker gene. For plant transformation, the host bacterial strain is usually... Agrobacterium rhizogenes ABI, C58, or LBA4404, however, other strains known to technicians in the field of plant transformation can also be effective.

[0043] Methods for assembling and introducing constructs into cells in a manner that causes transcribed DNA polynucleotides to be transcribed into functional mRNAs translated and expressed as proteins are known in the art. Compositions and methods for preparing and using constructs and host cells are well known to those skilled in the art. Typical vectors that can be used to express nucleic acids in plants are well known in the art and include those derived from… Agrobacterium rhizogenes The vector for the Ti plasmid and the pCaMVCN delivery control vector.

[0044] The construct may contain a variety of regulatory elements, including any of those provided herein. Any such regulatory element may be provided in combination with other regulatory elements. Such combinations may be designed or modified to produce desired regulatory characteristics. In one embodiment, the construct may contain at least one regulatory element operatively linked to a transcribed DNA polynucleotide operatively linked to a 3' UTR. The construct disclosed herein may contain at least one regulatory element operatively linked to a transcribed DNA polynucleotide operatively linked to SEQ ID NO:1. In another embodiment, the construct disclosed herein may contain at least one regulatory element operatively linked to a transcribed DNA polynucleotide operatively linked to a fragment or variant of SEQ ID NO:1.

[0045] The expression cassette may also include a transport peptide encoding a peptide that can be used to operatively link a protein to subcellular targeting, particularly to chloroplasts, leucoplasts, or other plastid organelles; mitochondria; peroxisomes; vacuoles; or extracellular locations. Many chloroplast-localizing proteins are expressed as precursors by nuclear genes and target chloroplasts via chloroplast transport peptides (CTPs). Examples of such isolated chloroplast proteins include, but are not limited to, those associated with: the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase, ferricredoxin, ferricredoxin oxidoreductase, light-harvesting complex proteins I and II, thioredoxin F, and enolpyruvate shikimate phosphate synthase (EPSPS). Chloroplast transport peptides are described, for example, in U.S. Patent No. 7,193,133. It has been demonstrated that non-chloroplast proteins can target chloroplasts via the expression of a heterologous CTP operatively linked to a transgene encoding a non-chloroplast protein.

[0046] Transcribed DNA polynucleotides As used herein, the term "transcribed DNA polynucleotide" refers to any DNA polynucleotide capable of being transcribed into RNA, including but not limited to those having a protein-coding sequence (e.g., ，Polynucleotides that encode mRNA, polynucleotides that encode guide RNA (gRNA), and polynucleotides that produce RNA with sequences that can be used for gene repression (e.g., siRNA, miRNA, and dsRNA). Types of DNA polynucleotides may include, but are not limited to, DNA polynucleotides from the same plant, DNA polynucleotides from another plant, DNA polynucleotides from different organisms, or synthetic DNA polynucleotides (such as DNA polynucleotides containing antisense information of a gene) or DNA polynucleotides encoding artificial, synthetic, or otherwise modified forms of transgenic DNA polynucleotides. Examples of transcribed DNA polynucleotides used for incorporation into constructs as described herein include, for example, DNA polynucleotides or genes from a species different from the species into which the DNA polynucleotide is incorporated, or genes derived from or present in the same species but incorporated into recipient cells through genetic engineering methods rather than classical breeding techniques.

[0047] As used herein, the term "heterologous transcribed DNA polynucleotide" refers to a transcribed DNA polynucleotide that is heterologous to one or more regulatory elements to which it is operatively linked.

[0048] "Transgenic" refers to a heterologous transcribed DNA polynucleotide in the host cell genome that is at least relative to its position in the host cell genome and / or artificially incorporated into the host cell genome in the current or any previous generation of cells.

[0049] Regulatory elements, such as 3' UTRs (e.g., SEQ ID NO:1) or fragments or variants thereof, can be operatively linked to a transcribed DNA polynucleotide that is heterologous to the regulatory element. As used herein, the term "heterologous" refers to a combination of two or more DNA polynucleotides (or nucleotide sequences or DNA sequences) that is not normally found in nature. For example, two DNA polynucleotides (or nucleotide sequences or DNA sequences) may be derived from different species and / or two DNA polynucleotides (or nucleotide sequences or DNA sequences) may be derived from different genes, for example, different genes from the same species or the same gene from different species. Therefore, if such combinations are not normally found in nature, Right now If the transcribed DNA polynucleotide is not naturally operatively linked to the regulatory sequence, then the regulatory element is heterologous relative to the operatively linked transcribed DNA polynucleotide.

[0050] Transcriptable DNA polynucleotides can generally be any DNA polynucleotide for the desired expression of a transcript. Such expression of the transcript can lead to the translation of the resulting mRNA, thereby resulting in protein expression. Alternatively, for example, a transcriptable DNA polynucleotide can be programmed to ultimately result in reduced expression of a specific gene or protein. In one embodiment, this can be achieved by using a transcriptable DNA polynucleotide oriented in an antisense direction. Those skilled in the art are familiar with the use of such antisense techniques. Any gene can be negatively regulated in this manner, and in one embodiment, the transcriptable DNA polynucleotide can be programmed to repress a specific gene through the expression of dsRNA, siRNA, or miRNA.

[0051] Therefore, one embodiment provides a recombinant DNA polynucleotide comprising a regulatory element (such as a regulatory element provided as SEQ ID NO:1 or a fragment or variant thereof), said regulatory element being operatively linked to a heterotranscribed DNA polynucleotide such that when the construct is integrated into the genome of a plant cell or a transgenic plant cell, the transcription of the transtranscribed DNA polynucleotide is regulated at a desired level or in a desired pattern. In one embodiment, the transtranscribed DNA polynucleotide comprises a protein-coding region of a gene, and in another embodiment, the transtranscribed DNA polynucleotide comprises an antisense region of a gene.

[0052] Genes with agronomical value Transcribed DNA polynucleotides may contain genes of agronomic value. As used herein, the term "gene of agronomic value" refers to a transcribed DNA polynucleotide that confers desired properties when expressed in a particular plant tissue, cell, or cell type. The product of a gene of agronomic value can function within a plant to influence plant morphology, physiology, growth, development, yield, cereal composition, nutritional status, disease or pest resistance, and / or environmental or chemical tolerance, or can act as an insecticide in the diet of plant-feeding pests. In one embodiment, a regulatory element (such as a regulatory element provided as SEQ ID NO:1 or a fragment or variant thereof) is incorporated into the construct such that the regulatory element is operatively linked to the transcribed DNA polynucleotide that is the gene of agronomic value. In transgenic plants containing such constructs, expression of the gene of agronomic value can confer beneficial agronomic traits. Beneficial agronomic traits may include, but are not limited to, herbicide tolerance, insect control, improved yield, disease resistance, pathogen resistance, improved plant growth and development, improved starch content, improved oil content, improved fatty acid content, improved protein content, improved fruit ripeness, enhanced animal and human nutrition, biopolymer production, environmental stress resistance, medicinal peptides, improved processing quality, improved flavor, hybrid seed production utility, improved fiber production, enhanced carbon sequestration and / or desired biofuel production.

[0053] Examples of genes known in the art to have agronomical value include the following genes: 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. USRE 38,446; 6,716,474; 6,663,906; 6,476,295; 6,441,277; 6,423,828; 6,39...). US Patent Nos. 9,330, 6,372,211, 6,235,971, 6,222,098, and 5,716,837; and 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, and 6,501,009; No. 6,468,523; No. 6,326,351; No. 6,313,378; No. 6,284,949; No. 6,281,016; No. 6,248,536; No. 6,242,241; No. 6,221,649; No. 6,177,615; No. 6,156,573; No. 6,153,814; No. 6,110,464; No. 6,093,695; No. 6,063,756; No. 6,063,597; No. 6,023,013; No. 5,959,091; No. 5,942,664; No. 5,942,658; No. 5,880 U.S. Patent Nos. 275, 5,763,245, and 5,763,241; fungal disease resistance (U.S. Patent Nos. 6,653,280, 6,573,361, 6,506,962, 6,316,407, 6,215,048, 5,516,671, 5,773,696, 6,121,436, 6,316,407, and 6,506,962); and viral resistance (U.S. Patent Nos. 6,617,496, 6,608,241, 6,015,940, 6,013,864, and 5,850,023);U.S. Patent No. 5,304,730), nematode resistance (U.S. Patent No. 6,228,992), bacterial disease resistance (U.S. Patent No. 5,516,671), plant growth and development (U.S. Patent Nos. 6,723,897 and 6,518,488), starch production (U.S. Patent Nos. 6,538,181; 6,538,179; 6,538,178; 5,750,876; 6,476,295), improved oil production (U.S. Patent Nos. 6,444,876; 6,426,447; and 6,380,462), high-oil production (U.S. Patent No. 6,495). (US Patent Nos. 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), high protein production (US Patent No. 6,380,466), fruit ripening (US Patent No. 5,512,466), enhanced 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; and 5,958,745 and 6,946,588), environmental stress resistance (US Patent No. 6,072,103), pharmaceutical peptides and secretible peptides (US Patent Nos. 6,812,379; 6,774,283; 6,140,075; and 6,080,560), improved processing properties (US Patent No. 6,723,837; 6,653,530; 6,5412,59; 5,985,605; and 6,171,640). This includes technologies such as: 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).

[0054] Alternatively, genes with agronomical value can influence the aforementioned plant characteristics or phenotypes by encoding RNA that targets and regulates the expression of endogenous genes, for example, through antisense RNA (…). See For example, U.S. Patent 5,107,065; repressive RNA (“RNAi”), including mechanisms that regulate gene expression through miRNA, siRNA, trans-acting siRNA, and phased sRNA, such as those described in published applications US 2006 / 0200878 and US 2008 / 0066206 and U.S. Patent Application 11 / 974,469; or co-repressive mechanisms. RNA may also be catalytic RNA (e.g., ribozymes or riboswitch) engineered to cleave desired endogenous mRNA products; See For example, US 2006 / 0200878). Methods for constructing constructs and introducing them into cells in a manner that causes transcribed DNA polynucleotides to be transcribed into molecules capable of causing gene repression are known in the art.

[0055] Selective markers Transcribed DNA polynucleotides encoding selectivity markers may also be used in conjunction with regulatory elements, such as those provided as SEQ ID NO:1 or fragments or variants thereof. As used herein, the term "selectivity marker" refers to any transcribed DNA polynucleotide whose expression or lack thereof in a transgenic plant, tissue, or cell can be screened and / or scored in some manner. Selectivity markers (also known as reporter genes) and their associated selection and screening techniques are known in the art and include, but are not limited to, transcribed DNA polynucleotides encoding β-glucuronidase (GUS), green fluorescent protein (GFP), proteins conferring antibiotic resistance, and proteins conferring herbicide resistance. An example of a reporter transgenic is provided as SEQ ID NO:3.

[0056] The use of reporter gene assays (also known as reporter transgene assays) to determine the gene regulatory activity (also known as expression profiling) of regulatory elements is well known in the art (e.g. ， Clark et al. Molecular Biology, Unit 4, Chapter 21, Third Edition, Academic Press, Elsevier Inc., 2019. As used herein, the term "reporter assay" refers to the determination of a specific regulatory element by first using a reporter gene (such as a transgene encoding a β-glucuronidase (GUS) protein) as a heterologous transcribed DNA polynucleotide operablely linked to a specific regulatory element. (For example, methods for gene regulatory activity (or expression profile) of promoters or 3' UTRs. In one embodiment, a reporter gene (such as a GUS gene) may be used as a heterologous transcribed DNA polynucleotide in a reporter gene assay, said heterologous transcribed DNA polynucleotide being operatively linked to SEQ ID NO:1 or a fragment or variant thereof, to determine the gene regulatory activity of SEQ ID NO:1. In a subsequent reporter gene assay, qualitative and quantitative GUS analyses may be used to evaluate the gene regulatory activity (or expression profile) of regulatory elements in selected plant organs and / or tissues of transformed plants. It should be understood that the gene regulatory activity (or expression profile) of a regulatory element (e.g., a promoter or 3' UTR) determined by using a reporter gene assay (e.g., a GUS assay) is the same as, substantially the same as, or substantially similar to other operatively linked transcribed DNA polynucleotides other than GUS. In one embodiment, the other operatively linked transcribed DNA polynucleotides may be genes of agronomical value, including but not limited to those described herein.

[0057] Genome editing Several embodiments relate to a recombinant DNA construct comprising one or more expression cassettes, said expression cassettes containing a sequence having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or higher with SEQ ID NO:1 or a fragment or variant thereof, said SEQ ID NO:1 or a fragment or variant thereof being operatively linked to a heterologous DNA sequence encoding a site-specific genome-modifying enzyme and / or one or more associated proteins for performing genome modification. These one or more site-specific genome-modifying enzyme expression cassettes may be present in the same molecule or vector (cis) with a donor template for template editing, or in separate molecules or vectors (trans). Several editing methods are known in the art that involve modifying different sequence-specific genome-modifying enzymes (or complexes of proteins and / or guide RNA) of genomic DNA. In some embodiments, site-specific genome-modifying enzymes modify the genome by inducing double-strand breaks (DSBs) or nicks at desired genomic sites or loci. In some embodiments, during the process of repairing DSBs or nicks introduced by the genome-modifying enzyme, donor template DNA may be integrated into the genome at the site of the DSB or nick. In some embodiments, during the process of repairing DSBs or nicks introduced by the genome-modifying enzyme, insertion or deletion mutations (insertions or deletions) may be introduced into the genome. In some embodiments, the site-specific genome-modifying enzyme comprises cytidine deaminase. In some embodiments, the site-specific genome-modifying enzyme comprises adenine deaminase. In this disclosure, site-specific genome-modifying enzymes include endonucleases, recombinases, transposases, deaminases, helicases, reverse transcriptases, and any combination thereof.

[0058] Several embodiments involve gene regulatory elements as described herein, operatively linked to heterologous transcribed DNA molecules encoding one or more components of a genome editing system. Genome editing systems can be used to introduce one or more insertions, deletions, substitutions, base modifications, translocations, or inversions into the genome of a host cell. In some embodiments, gene regulatory elements as described herein are operatively linked to heterologous transcribed DNA polynucleotides encoding sequence-specific DNA-binding domains (such as CRISPR-Cas effector proteins, zinc finger proteins, or transcription activator (TAL) proteins). In some embodiments, the sequence-specific DNA-binding domain may be a fusion protein. In some embodiments, gene regulatory elements as described herein are operatively linked to heterologous transcribed DNA polynucleotides encoding CRISPR-Cas effector proteins. In some embodiments, the CRISPR-Cas effector protein is selected from type I, type II, type III, type IV, type V, or type VI CRISPR-Cas systems. In some embodiments, the gene regulatory element, as described herein, is operatively linked to a heterologous transcribed DNA polynucleotide encoding a guide RNA. As used herein, “guide RNA” or “gRNA” refers to RNA that recognizes a target DNA sequence and guides or “directs” a CRISPR effector protein to the target DNA sequence. A guide RNA comprises a region complementary to the target DNA (called CRISPR RNA or crRNA or spacer) and a region that binds a CRISPR effector protein (called tracrRNA). The guide RNA may be a single RNA molecule (single guide RNA, “sgRNA”) or two separate RNA molecules (dual guide RNA, “dgRNA”). In some embodiments, the gRNA may further comprise a reverse transcriptase lead editing (PE) guide RNA (“PEgRNA”).

[0059] Several embodiments involve gene regulatory elements as described herein, operatively linked to heterologous transcribed DNA polynucleotides encoding one or more components of a CRISPR-Cas genome editing system comprising CRISPR-Cas effector proteins. Examples of CRISPR-Cas effector proteins include, but are not limited to, Cas9, Cas12b, C2c3, C2c4, C2c5, C2c8, C2c9, C2c10, Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', Cas3'', Cas4, Cas5, Cas6, Cas7, Cas8, and Cas9. (Also known as Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), Csf5, Cas14a, Cas14b, and Cas14c effector proteins. In one embodiment, the CRISPR-Cas nuclease may be a Cas12a effector protein. In some embodiments, the gene regulatory element as described herein is operatively linked to a CRISPR-Cas effector protein containing a mutation in its nuclease active site (e.g., the RuvC, HNH, and / or NUC domain). CRISPR-Cas effector proteins that have a mutation in their nuclease active site and therefore no longer contain nuclease activity are generally referred to as “dead,” such as dCas. In some embodiments, the domains or peptides of a CRISPR-Cas effector protein with a mutation in its nuclease active site may have impaired or reduced activity compared to the same CRISPR-Cas effector protein without the mutation. In some embodiments, the gene regulatory element as described herein is operatively linked to a CRISPR-Cas effector protein with a mutation in its nuclease active site to produce a nicking enzyme activity operatively linked to a reverse transcriptase.

[0060] Cell transformation Methods for generating transformed cells, plant cells, and plants are also provided, the transformed cells, plant cells, and plants comprising one or more regulatory elements operatively linked to transcribed DNA polynucleotides, such as regulatory elements provided as SEQ ID NO:1 or fragments or variants thereof.

[0061] The term "transformation" refers to the introduction of DNA polynucleotides 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. Plant tissues and cells of particular interest include protoplasts, callus, roots, tubers, seeds, stems, leaves, seedlings, embryos, and pollen.

[0062] As used herein, the term "transformation" refers to a cell, tissue, organ, or organism into which a foreign DNA polynucleotide (such as, for example, a construct as described herein) has been introduced. The introduced DNA polynucleotide may be integrated into the genomic DNA of the recipient cell, tissue, organ, or organism, such that the introduced DNA polynucleotide is inherited by subsequent offspring. "Transgenic" or "transformed" cells or organisms also include the offspring of the cells or organisms and offspring produced in breeding programs that use such transgenic plants as parents in hybridization and exhibit altered phenotypes caused by the presence of external DNA molecules. The introduced DNA polynucleotide may also be transiently introduced into the recipient cell, such that the introduced DNA polynucleotide is not inherited by subsequent offspring. The term "transgenic" refers to a bacterium, fungus, or plant containing one or more heterologous DNA polynucleotides.

[0063] Numerous methods well-known to those skilled in the art exist for introducing DNA polynucleotides into plant cells. These methods typically include the steps of selecting a suitable host cell, transforming the host cell with a vector, and obtaining the transformed host cell. Methods and materials for transforming plant cells by introducing plant constructs into the plant genome can include any well-known and proven methods. Suitable methods include, but are not limited to, bacterial infection (e.g., Agrobacterium (e.g., binary BAC vectors, direct DNA delivery (e.g., PEG-mediated transformation, drying / inhibition-mediated DNA uptake, electroporation, acceleration using silicon carbide fibers for stirring and DNA-coated particles), gene editing (e.g., CRISPR-Cas system), etc.)

[0064] The host cell can be any cell or organism, such as plant cells, algal cells, fungal cells, bacterial cells, or insect cells. In a specific embodiment, the host cell and the transformed cell may include cells derived from a crop plant. In a further specific embodiment, the host cell and the transformed cell may include cells derived from a soybean plant.

[0065] The transgenic plant can then be regenerated from the transgenic plant cells as described herein. Seeds can be produced from this transgenic plant using conventional breeding techniques or self-pollination. Such seeds and the resulting offspring plants grown from such seeds will contain recombinant DNA polynucleotides as described herein (such as recombinant DNA polynucleotides containing sequences presented as SEQ ID NO:1 or fragments or variants thereof), and will therefore be transgenic.

[0066] Transgenic plants can self-pollinate to provide seeds of homozygous transgenic plants (homozygous for recombinant DNA polynucleotides as described herein), or hybridize with non-transgenic plants or different transgenic plants to provide seeds of heterozygous transgenic plants (heterozygous for recombinant DNA polynucleotides as described herein). Both homozygous and heterozygous transgenic plants are referred to herein as “progeny plants.” Progeny plants are transgenic plants that originated from the original transgenic plant and contain recombinant DNA polynucleotides as described herein. Seeds produced using transgenic plants can be harvested and used to cultivate generations of transgenic plants. Right now Progeny plants containing recombinant DNA polynucleotides as described herein and expressing genes of agronomical value. Descriptions of breeding methods commonly used for different 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 (Volume 1) and Crop Species Soybean (Volume 2), Iowa State Univ., Macmillan Pub. Co., NY, 360-376 (1987).

[0067] The analysis can determine the presence of one or more genes of interest in transformed plants, as well as the expression levels and / or profiles conferred by regulatory elements (such as those provided as SEQ ID NO:1 or fragments or variants thereof). Those skilled in the art are aware of a variety of methods that can be used to analyze transformed plants. For example, methods for plant analysis include, but are not limited to, Southern or Northern blotting, PCR-based methods, biochemical analysis, phenotypic screening methods, field evaluation, and immunodiagnostic assays. Expression of transcribed DNA polynucleotides can be measured using TaqMan® (Applied Biosystems, FosterCity, CA) reagents and as described by the manufacturer, and PCR cycle times can be determined using a TaqMan® test matrix. Alternatively, transgenic expression can be evaluated using Invader® (Third Wave Technologies, Madison, WI) reagents and as described by the manufacturer.

[0068] Plant parts as described herein are also provided. Plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovules, and pollen. Plant parts may be viable, non-viable, renewable, and / or non-renewable. Transformed plant cells containing DNA polynucleotides as described herein, such as DNA polynucleotides provided as SEQ ID NO:1 or fragments or variants thereof. Transformed or transgenic plant cells include renewable and / or non-renewable plant cells.

[0069] Commercial products produced from genetically modified plants or parts thereof may contain recombinant DNA polynucleotides as described herein (such as recombinant DNA polynucleotides provided as SEQ ID NO:1 or fragments or variants thereof). In some embodiments, the commercial product may contain a detectable amount of DNA comprising a DNA sequence present as SEQ ID NO:1 or fragments or variants thereof. As used herein, “commercial product” means any composition or product comprising material derived from genetically modified plants, seeds, plant cells, or plant parts containing recombinant DNA polynucleotides as described herein, such as recombinant DNA polynucleotides provided as SEQ ID NO:1 or fragments or variants thereof. Commercial products include, but are not limited to, processed seeds, cereals, plant parts, and flours. Commercial products containing detectable amounts of DNA corresponding to recombinant DNA polynucleotides as described herein, such as recombinant DNA polynucleotides provided as SEQ ID NO:1 or fragments or variants thereof are considered. Detection of one or more of such DNA in a sample can be used to determine the content or origin of the commercial product. Any standard method for detecting DNA polynucleotides, including the detection methods disclosed herein, may be used.

[0070] The definitions and methods provided define this disclosure and guide those skilled in the art in practicing it. Unless otherwise stated, terminology should be understood according to its conventional usage by those skilled in the art. Definitions of commonly used terms and methods in molecular biology can also be found, for example, in Clark... et al. Molecular Biology, 3rd Edition, AcademicPress, Elsevier Inc., 2019; Alberts et al. , Molecular Biology of The Cell, 5th Edition, Garland Science Publishing, Inc.: New York, 2007; Rieger et al. , Glossary ofGenetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; King et al. , A Dictionary of Genetics, 6th ed., Oxford University Press: New York, 15 2247; and Lewin, Genes IX, Oxford University Press: New York, 2007.

[0071] Implementation Plan To further illustrate, additional exemplary non-limiting embodiments of this disclosure are described below.

[0072] Implementation scheme 1 is a recombinant DNA polynucleotide comprising a DNA sequence selected from the group consisting of: a) A sequence that has at least 85% sequence identity with SEQ ID NO:1; b) A sequence containing SEQ ID NO:1; and c) The fragment of SEQ ID NO:1, wherein the fragment contains gene regulatory activity; The DNA sequence therein is operatively linked to a heterologous transcribed DNA polynucleotide.

[0073] Implementation scheme 2 is a recombinant DNA polynucleotide as described in implementation scheme 1, wherein the DNA sequence has at least 90% sequence identity with the DNA sequence of SEQ ID NO:1.

[0074] Embodiment 3 is a recombinant DNA polynucleotide as described in any one of Embodiments 1 or 2, wherein the DNA sequence has at least 95% sequence identity with the DNA sequence of SEQ ID NO:1.

[0075] Implementation scheme 4 is a recombinant DNA polynucleotide as described in any one of implementation schemes 1 to 3, wherein the DNA sequence contains gene regulatory activity.

[0076] Implementation scheme 5 is a recombinant DNA polynucleotide as described in any one of implementation schemes 1 to 4, wherein the heterotranscribed DNA polynucleotide contains a gene of agronomic value.

[0077] Implementation scheme 6 is a recombinant DNA polynucleotide as described in implementation scheme 5, wherein the agronomically valuable gene confers herbicide tolerance to the plant.

[0078] Implementation scheme 7 is a recombinant DNA polynucleotide as described in implementation scheme 5, wherein the agronomically valuable gene confers plant pest resistance.

[0079] Embodiment 8 is a recombinant DNA polynucleotide as described in any one of Embodiments 1 to 4, wherein the heterologous transcribed DNA polynucleotide encodes dsRNA, miRNA, or siRNA.

[0080] Implementation scheme 9 is a transgenic plant cell containing recombinant DNA polynucleotides, said recombinant DNA polynucleotides comprising DNA sequences selected from the group consisting of: a) A sequence that has at least 85% sequence identity with SEQ ID NO:1; b) A sequence containing SEQ ID NO:1; and c) The fragment of SEQ ID NO:1, wherein the fragment contains gene regulatory activity; The DNA sequence therein is operatively linked to a heterologous transcribed DNA polynucleotide.

[0081] Implementation scheme 10 is a transgenic plant cell as described in implementation scheme 9, wherein the transgenic plant cell is a monocotyledonous plant cell.

[0082] Implementation scheme 11 is a transgenic plant cell as described in implementation scheme 9, wherein the transgenic plant cell is a dicotyledonous plant cell.

[0083] Implementation Scheme 12 is a transgenic plant or a portion thereof, said transgenic plant or a portion thereof comprising any one of Implementation Schemes 1 to 8.

[0084] Embodiment 13 is a progeny plant or a portion thereof of the transgenic plant as described in Embodiment 12, wherein the progeny plant or a portion thereof contains any one of the recombinant DNA polynucleotides described in Embodiments 1 to 8.

[0085] Implementation scheme 14 is a transgenic seed, wherein the seed contains recombinant DNA polynucleotides as described in any one of implementation schemes 1 to 8.

[0086] Implementation scheme 15 is a method of producing a commercial product, which includes obtaining the genetically modified plant or a portion thereof as described in implementation scheme 12 or the progeny plant or a portion thereof as described in implementation scheme 13 and producing the commercial product therefrom.

[0087] Implementation scheme 16 is the method as described in implementation scheme 15, wherein the commercial product is selected from the group consisting of: seeds, processed seeds, protein concentrates, protein isolates, starch, grains, plant parts, seed oils, biomass, flour and coarse flour.

[0088] Implementation Scheme 17 is a method for producing the transgenic plant described in Implementation Scheme 12, comprising: a) Transforming plant cells with the recombinant DNA polynucleotides described in any one of embodiments 1 to 8 to produce transformed plant cells; and b) Regenerating transgenic plants from the transformed plant cells.

[0089] Implementation scheme 18 is a method for expressing transcribed DNA polynucleotides, which includes obtaining the transgenic plant of implementation scheme 12 or the progeny plant of implementation scheme 13 or a portion thereof and culturing the plant, wherein the transcribed DNA polynucleotides are expressed.

[0090] Implementation scheme 19 is an isolated recombinant DNA molecule characterized by comprising the DNA sequence of SEQ ID NO:1, wherein the DNA sequence is operatively linked to a heterologous transcribed polynucleotide molecule.

[0091] Implementation scheme 20 is an isolated recombinant DNA molecule as described in implementation scheme 19, characterized in that the heterologous transcribed DNA polynucleotide molecule contains a gene of agronomic value.

[0092] Implementation scheme 21 is the isolated recombinant DNA molecule as described in implementation scheme 20, characterized in that the agronomically valuable gene confers herbicide tolerance to the plant.

[0093] Implementation scheme 22 is the isolated recombinant DNA molecule as described in implementation scheme 20, characterized in that the agronomically valuable gene confers plant pest resistance.

[0094] Implementation scheme 23 is a method for producing transgenic plants, characterized in that, in addition to the plants obtained by said method, it includes: a) Transforming plant cells with the isolated recombinant DNA molecules described in Implementation Scheme 19 to produce transformed plant cells; and b) Regenerating transgenic plants from the transformed plant cells.

[0095] Implementation scheme 24 is a construct characterized by comprising the isolated recombinant DNA molecule described in implementation scheme 19.

[0096] The embodiments described herein can be more readily understood by referring to the following examples, which are provided by way of illustration and are not intended to be limiting unless otherwise specified. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in practicing the invention. However, given this disclosure, those skilled in the art will understand that many variations can be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention. Therefore, all matters set forth or illustrated in the drawings should be understood as illustrative and not limiting.

[0097] Example Example 1 Design, synthesis and cloning of 3' UTR T-Zm.GST314 (SEQ ID NO:1) Novel synthetic transcriptional regulatory elements, such as the synthetic 3' UTR T-Zm.GST314 (SEQ ID NO:1), are synthetic expression elements designed using algorithmic methods. These computationally derived synthetic transcriptional regulatory elements are chemically synthesized and cloned using techniques known in the art. Hundreds of synthetic 3' UTRs were designed and tested in protoplasts and stably transformed plants to identify those synthetic 3' UTRs that provide desired properties, such as protein expression levels, expression patterns, and proper termination and polyadenylation of the transcript. The synthetic 3' UTRs disclosed herein are characterized by their effect on gene expression and the proper termination of operatively linked heterologous transcribed DNA polynucleotide transcripts.

[0098] The designed synthetic 3' UTR T-Zm.GST314 (SEQ ID NO:1) does not exhibit extended homology with any known existing nucleic acid sequence, but influences the transcription of the operably linked coding sequence in the same manner as naturally occurring 3' UTRs. Using methods known in the art, the synthetic 3' UTR T-Zm.GST314 was cloned into a binary plant transformation vector, operably linked to the β-glucuronidase (GUS) coding sequence, and its expression levels and patterns in stably transformed maize plants were evaluated.

[0099] Example 2 Analysis of the synthesis of 3' UTR T-Zm.GST314 (SEQ ID NO:1): its effect on transgenic expression in stably transformed soybean plants. Soybean plants were transformed with a plant binary expression vector construct containing a transcriptional regulatory element driving β-glucuronidase (GUS) transgene expression and the synthetic 3' UTR T-Zm.GST314 (SEQ ID NO:1) presented in Example 1. GUS protein expression in the resulting plants was analyzed to assess the effect of the synthetic 3' UTR T-Zm.GST314 on transgene expression.

[0100] Soybean plants were transformed with a plant binary GUS expression vector construct. The synthetic 3'UTR T-Zm.GST314 was cloned into a basic plant expression vector using methods known in the art. The resulting plant expression vector contained GUS derived from... Agrobacterium rhizogenes The left boundary region; a first transgenic selection cassette for selecting transformed plant cells, the first selection cassette conferring resistance to the antibiotic spectinomycin; a second transgenic cassette for evaluating the activity of the synthetic 3' UTR T-Zm.GST314, the second transgenic cassette containing EXP EXP-At.Cyco (SEQ ID NO:2), the EXP being operatively linked to the 5' of a synthetic coding sequence designed for expression in plant cells encoding β-glucuronidase (GUS, SEQ ID NO:3) and containing a processable intron derived from the potato light-inducible tissue-specific ST-LS1 gene (GenBank accession number: X04753), the EXP being operatively linked to the 5' of the synthetic 3' UTR T-Zm.GST314 (SEQ ID NO:1), followed by a sequence from... Agrobacterium rhizogenesThe right boundary region. A GUS construct used as a control was also used to transform soybean plants, the construct comprising an expression cassette driven by the same EXP-At.Cyco (SEQ ID NO:2) for GUS expression, wherein the 3' UTR T-Gb.FbL2 (SEQ ID NO:4) is instead operatively linked to the 3' of the GUS synthesis coding sequence (SEQ ID NO:3).

[0101] As is well known in the art, the binary transformation vector construct described above can be used via... Agrobacterium Mediated transformation is used to transform soybean plant cells. The resulting transformed plant cells are induced to form complete soybean plants.

[0102] Qualitative and quantitative GUS analysis was used to evaluate the activity of expression elements in selected plant organs and tissues of transformed plants. For qualitative analysis of GUS expression by histochemical staining, whole or sectioned tissues were incubated with a GUS staining solution containing 1 mg / mL X-Gluc (5-bromo-4-chloro-3-indolyl-β-glucuronide) at 37°C for 5 hours and destained with 35% EtOH and 50% acetic acid. GUS expression was qualitatively determined by visual examination of blue staining in selected plant organs or tissues under a dissecting microscope or a combination microscope.

[0103] To quantify GUS expression via enzymatic assay, total protein was extracted from selected tissues of transformed soybean plants. One to two micrograms of total protein were incubated with 1 mM of the fluorescent substrate 4-methylumbelliferyl-β-D-glucuronide (MUG) in a total reaction volume of 50 μL. After incubation at 37 °C for 1 h, the reaction was stopped by adding 350 μL of 200 mM sodium bicarbonate solution. The reaction product, 4-methylumbelliferone (4-MU), exhibited maximum fluorescence at high pH, ​​where the hydroxyl groups were ionized. The assay was stopped by adding alkaline sodium carbonate solution, and the pH was adjusted to quantify the fluorescent product 4-MU. The amount of 4-MU formed was estimated by measuring fluorescence using a FLUOstar Omega microplate reader (BMG LABTECH) (excitation at 355 nm, emission at 460 nm). GUS activity values ​​were provided as nanomoles of 4-MU / h / mg total protein. Right now GUS expression).

[0104] The following tissues were sampled for GUS expression in the R0 generation: V5 stage saucer, primary leaf, and root; R1 stage flower, petiole, primary leaf, pollen, and root; R3 stage pod and immature seed; R5 primary leaf; and R8 seed cotyledon and embryo. The nutritional and reproductive stages of soybean are well known to those skilled in the art, and numerous publications describing these stages are available on the World Wide Web and elsewhere, such as North Dakota State University Publication A-1174, June 1999, which was reviewed and reprinted in August 2004. Table 1 shows the range and mean of GUS expression for each construct.

[0105] Table 1. Effects of the 3' UTR regulatory element on the quantitative expression of GUS in stably transformed R0 soybean plants.

[0106] As shown in Table 1 above, the effect of synthesized 3' UTR T-Zm.GST314 (SEQ ID NO:1) on GUS transgene expression differs from that of 3' UTR T-Gb.FbL2 (SEQ ID NO:4). Compared to events containing T-Gb.FbL2 (SEQ ID NO:4), transformations using constructs containing T-Zm.GST314 (SEQ ID NO:1) resulted in lower expression in most tissues, except for V5, R1 and R5 source leaves and R3 pods, where expression of both 3' UTRs was similar. When expressing transgenes, excessive expression in transgenic plants can have detrimental effects, thus requiring reduction in expression. Compared to the control T-Gb.FbL2 (SEQ ID NO:4), expression in R1 pollen was reduced by more than five-fold when using T-Zm.GST314 (SEQ ID NO:1). When expressing transgenes such as insecticidal toxin genes, reducing pollen expression is particularly important because the transgene may be effective against non-target pests that feed on pollen. Analysis of the GUS transcript containing T-Zm.GST314 (SEQ ID NO:1) demonstrated that transcription and polyadenylation were correctly and efficiently terminated (data not shown). Therefore, the 3' UTR T-Zm.GST314 (SEQ ID NO:1) behaves similarly to the naturally occurring 3' UTR, meaning it can influence transgene expression and correctly terminate the transcription of the transgene operatively linked to it.

[0107] Having described and illustrated the principles of the invention, it will be apparent to those skilled in the art that modifications may be made to the arrangement and details without departing from such principles. All publications and published patent documents cited herein are hereby incorporated by reference as if explicitly and individually indicated to be incorporated by reference.

Claims

1. A recombinant DNA polynucleotide comprising a DNA sequence selected from the group consisting of: a) A sequence that has at least 85% sequence identity with SEQ ID NO:1; b) A sequence containing SEQ ID NO:1; and c) The fragment of SEQ ID NO:1, wherein the fragment contains gene regulatory activity; The DNA sequence therein is operatively linked to a heterologous transcribed DNA polynucleotide.

2. The recombinant DNA polynucleotide of claim 1, wherein the DNA sequence has at least 90% sequence identity with the DNA sequence of SEQ ID NO:

1.

3. The recombinant DNA polynucleotide of claim 1, wherein the DNA sequence has at least 95% sequence identity with the DNA sequence of SEQ ID NO:

1.

4. The recombinant DNA polynucleotide of claim 1, wherein the DNA sequence contains gene regulatory activity.

5. The recombinant DNA polynucleotide of claim 1, wherein the heterotranscribed DNA polynucleotide comprises a gene of agronomic value.

6. The recombinant DNA polynucleotide of claim 5, wherein the gene with agronomical value confers herbicide tolerance to the plant.

7. The recombinant DNA polynucleotide of claim 5, wherein the gene with agronomical value confers plant pest resistance.

8. The recombinant DNA polynucleotide of claim 1, wherein the heterotranscribed DNA polynucleotide encodes dsRNA, miRNA or siRNA.

9. A transgenic plant cell comprising a recombinant DNA polynucleotide, said recombinant DNA polynucleotide comprising a DNA sequence selected from the group consisting of: a) A sequence that has at least 85% sequence identity with SEQ ID NO:1; b) A sequence containing SEQ ID NO:1; and c) The fragment of SEQ ID NO:1, wherein the fragment contains gene regulatory activity; The DNA sequence therein is operatively linked to a heterologous transcribed DNA polynucleotide.

10. The transgenic plant cell of claim 9, wherein the transgenic plant cell is a monocotyledonous plant cell.

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

12. A transgenic plant or a portion thereof comprising the recombinant DNA polynucleotide of claim 1.

13. A progeny plant or a portion thereof of the transgenic plant as claimed in claim 12, wherein the progeny plant or a portion thereof comprises the recombinant DNA polynucleotide as claimed in claim 1.

14. A transgenic seed, wherein the seed comprises the recombinant DNA polynucleotide of claim 1.

15. A method of producing a commercial product, comprising obtaining the genetically modified plant of claim 12 or a portion thereof and producing the commercial product therefrom.

16. The method of claim 15, wherein the commercial product is selected from the group consisting of: seeds, processed seeds, protein concentrates, protein isolates, starch, cereals, plant parts, seed oils, biomass, flour, and coarse flour.

17. A method for producing the transgenic plant of claim 12, comprising: a) Transforming plant cells with the recombinant DNA polynucleotide of claim 1 to produce transformed plant cells, and b) Regenerating transgenic plants from the transformed plant cells.

18. A method for expressing a transcribed DNA polynucleotide, comprising obtaining the transgenic plant of claim 12 and culturing the plant, wherein the transcribed DNA polynucleotide is expressed.

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