Plant regulatory elements and uses thereof

Abstract

The present invention provides recombinant DNA polynucleotides and constructs, as well as their nucleotide sequences, useful for modulating gene expression in plants. The invention also provides transgenic plants, plant cells, plant parts, and seeds comprising the recombinant DNA polynucleotides that are operably linked to a heterologous transcribable DNA polynucleotide. Also provided are methods of using the recombinant DNA polynucleotides and constructs as well as the transgenic plants, plant cells, plant parts, and seeds comprising the recombinant DNA polynucleotides and constructs.

Description

[0001] Citation of Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 375,684, filed on September 14, 2022, which is incorporated herein by reference in its entirety.

[0003] Incorporation of Sequence Listing

[0004] 67,794 bytes contained in a file named "MONS538WO_ST26.xml" (as in Microsoft The sequence listing for the invention (measured in ) was created on August 28, 2023 and was filed simultaneously by electronic submission (using the U.S. Patent Office Patent Center) and is incorporated by reference in its entirety. Technical Field

[0005] The present invention relates to the field of plant molecular biology and plant genetic engineering. More specifically, the present invention relates to DNA polynucleotides that can be used to regulate gene expression in plants. Background Art

[0006] Regulatory elements are genetic elements that regulate gene activity by regulating the transcription of operably linked transcribable DNA polynucleotides. Such elements may include promoters, leader sequences, introns, and 3' untranslated regions, and can be used in the fields of plant molecular biology and plant genetic engineering. Summary of the invention

[0007] This section provides a general summary of the disclosure, and is not a comprehensive disclosure of its full scope or all of its features.

[0008] Provided herein are gene control elements for plants.Several embodiments relate to recombinant DNA polynucleotides comprising control elements.Also provided are transgenic plant cells, plants and seeds comprising control elements.In one embodiment, control elements are operably connected to transcribable DNA polynucleotides.In certain embodiments, transcribable DNA polynucleotides can be heterologous to the control DNA sequence.Therefore, in specific embodiments, the control element DNA sequence provided herein can be defined as being operably connected to heterologous transcribable DNA polynucleotides.Several embodiments relate to the use of control elements and the preparation and use of recombinant DNA polynucleotides comprising control elements and transgenic plant cells, plants and seeds comprising control elements operably connected to transcribable DNA polynucleotides.

[0009] 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 to any one of SEQ ID NOs: 1-23; (b) a sequence comprising any one of SEQ ID NOs: 1-23; and (c) a fragment of any one of SEQ ID NOs: 1-23, wherein the fragment comprises gene regulatory activity; wherein the DNA sequence is operably linked to a heterologous transcribable 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 to the DNA sequence of any one of SEQ ID NOs: 1-23.

[0010] In another aspect, the present invention provides a transgenic plant cell comprising a recombinant DNA polynucleotide comprising a DNA sequence selected from the group consisting of: (a) a sequence having at least about 85% sequence identity to any one of SEQ ID NO: 1-23; (b) a sequence comprising any one of SEQ ID NO: 1-23; and (c) a fragment of any one of SEQ ID NO: 1-23, wherein the fragment comprises gene regulatory activity; wherein the DNA sequence is operably linked to a heterologous transcribable DNA polynucleotide. In certain embodiments, the transgenic plant cell may be a monocotyledonous plant cell. In other embodiments, the transgenic plant cell may be a dicotyledonous plant cell.

[0011] In another aspect, the present invention further provides a transgenic plant or part thereof comprising a recombinant DNA polynucleotide, wherein the recombinant DNA polynucleotide comprises a DNA sequence selected from the group consisting of: a) a sequence having at least 85% sequence identity with any one of SEQ ID NO: 1-23; b) a sequence comprising any one of SEQ ID NO: 1-23; and c) a fragment of any one of SEQ ID NO: 1-23, wherein the fragment has gene regulatory activity; wherein the DNA sequence is operably linked to a heterologous transcribable DNA polynucleotide. In a specific embodiment, the transgenic plant can be a progeny plant of any generation comprising the recombinant DNA polynucleotide. A transgenic seed comprising the recombinant DNA polynucleotide is also provided, wherein the transgenic seed produces such a transgenic plant when grown.

[0012] Several embodiments relate to a method of producing a commercial product, comprising obtaining a transgenic plant or part thereof containing a recombinant DNA polynucleotide as described herein (such as those comprising a DNA sequence selected from SEQ ID NO: 1-23), and producing a commercial product therefrom. In one embodiment, the commercial product can be seed, processed seed, protein concentrate, protein isolate, starch, grain, plant part, seed oil, biomass, flour and / or meal.

[0013] Several embodiments relate to a method for producing a transgenic plant comprising a recombinant DNA polynucleotide, wherein the recombinant DNA polynucleotide comprises a DNA sequence selected from the group consisting of: a) a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-23; b) a sequence comprising any one of SEQ ID NOs: 1-23; and c) a fragment of any one of SEQ ID NOs: 1-23, wherein the fragment comprises gene regulatory activity, the method comprising transforming a plant cell with the recombinant DNA polynucleotide to produce a transformed plant cell and regenerating a transgenic plant from the transformed plant cell.

[0014] Sequence Description

[0015] SEQ ID NO: 1 is the DNA sequence of the promoter operably linked to the native leader sequence of the promoter, P-Gm.08G282100:2.

[0016] SEQ ID NO:2 is the DNA sequence of 3'UTR, T-Gm.08G282100:2.

[0017] SEQ ID NO:3 is the DNA sequence of the promoter operably linked to the native leader sequence of the promoter, P-Gm.02G215700:2.

[0018] SEQ ID NO:4 is the DNA sequence of 3'UTR, T-Gm.02G215700:2.

[0019] SEQ ID NO:5 is the DNA sequence of the promoter operably linked to the native leader sequence of the promoter, P-Gm.11G155000:2.

[0020] SEQ ID NO:6 is the DNA sequence of 3'UTR, T-Gm.11G155000:2.

[0021] SEQ ID NO:7 is the DNA sequence of the promoter operably linked to the native leader sequence of the promoter, P-Gm.PSII:1.

[0022] SEQ ID NO:8 is the DNA sequence of 3'UTR, T-Gm.PSII:1.

[0023] SEQ ID NO:9 is the DNA sequence of the promoter operably linked to the native leader sequence of the promoter, P-Gm.01G238800:1.

[0024] SEQ ID NO: 10 is the DNA sequence of 3'UTR, T-Gm.01G238800:1.

[0025] SEQ ID NO: 11 is the DNA sequence of the promoter of P-Gm.19G007700:1, operably linked to the native leader sequence of the promoter.

[0026] SEQ ID NO: 12 is the DNA sequence of 3'UTR, T-Gm.19G007700:1.

[0027] SEQ ID NO: 13 is the DNA sequence of the promoter operably linked to the native leader sequence of the promoter, P-Gm.05G007100:1.

[0028] SEQ ID NO: 14 is the DNA sequence of the promoter operably linked to the native leader sequence of the promoter, P-Gm.16G089000:1.

[0029] SEQ ID NO: 15 is the DNA sequence of 3'UTR, T-Gm.16G089000:1.

[0030] SEQ ID NO: 16 is the DNA sequence of the promoter operably linked to the promoter's native leader sequence P-Gm.16G089000_trunc: 1.

[0031] SEQ ID NO: 17 is the DNA sequence of the promoter operably linked to the native leader sequence of the promoter, P-Gm.15G057600:1.

[0032] SEQ ID NO: 18 is the DNA sequence of 3'UTR, T-Gm.15G057600:1.

[0033] SEQ ID NO: 19 is the DNA sequence of the promoter operably linked to the native leader sequence of the promoter, P-Gm.07G156100:1.

[0034] SEQ ID NO:20 is the DNA sequence of 3'UTR, T-Gm.07G156100:1.

[0035] SEQ ID NO:21 is the DNA sequence of the promoter operably linked to the promoter's native leader sequence P-Gm.07G156100_trunc:1.

[0036] SEQ ID NO:22 is the DNA sequence of the promoter of P-Gm.17G020600:1, operably linked to the native leader sequence of the promoter.

[0037] SEQ ID NO:23 is the DNA sequence of the promoter operably linked to the native leader sequence of the promoter, P-Gm.02G101100:1.

[0038] SEQ ID NO:24 is the DNA sequence of the synthetic 3'UTR, T-Zm.GST59.nno:1.

[0039] SEQ ID NO: 25 is a synthetic coding sequence for plant expression of β-glucuronidase (GUS) with a processable intron derived from the potato light-inducible, tissue-specific St-LS1 gene (GenBank Accession No.: X04753). DETAILED DESCRIPTION

[0040] 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 the present disclosure. Unless otherwise noted, terms should be understood according to conventional usage by those of ordinary skill in the relevant art.

[0041] Provided herein are regulatory elements with gene regulatory activity in plants. The nucleotide sequences of these regulatory elements are provided as SEQ ID NO: 1-23. These regulatory elements can affect the expression of transcribable DNA polynucleotides operably connected in plant tissues, thereby regulating the gene expression of transgenic operably connected transgenes in transgenic plants. Also provided are methods for modifying, producing and using recombinant DNA polynucleotides containing the regulatory elements provided. Also provided are compositions comprising transgenic plant cells, plants, plant parts and seeds and methods for preparing and using the compositions, wherein the transgenic plant cells, plants, plant parts and seeds contain recombinant DNA polynucleotides containing one or more regulatory elements as described herein.

[0042] DNA polynucleotide

[0043] As used herein, the term "DNA" or "DNA polynucleotide" refers to a double-stranded DNA polynucleotide of genomic or synthetic origin read from the 5' (upstream) end to the 3' (downstream) end, that is, a polymer of deoxyribonucleotide bases or a DNA polynucleotide. As used herein, the term "DNA sequence" refers to the nucleotide sequence of a DNA polynucleotide. The nomenclature used herein corresponds to Title 37 of the U.S. Federal Regulations §1.822 and is set forth in WIPO Standard ST.26 (2021), Annex I, Tables 1 and 3.

[0044] As used herein, a "recombinant DNA polynucleotide" is a DNA polynucleotide comprising a combination of DNA polynucleotides that would not naturally exist together 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 comprising a DNA sequence that deviates from a DNA sequence that exists in nature, or a DNA polynucleotide comprising a synthetic DNA sequence, or a DNA polynucleotide that has been incorporated into the DNA of a host cell by genetic transformation or gene editing.

[0045] As used herein, a "synthetic nucleotide sequence" or "artificial nucleotide sequence" is a nucleotide sequence that is known not to exist or does not naturally occur in nature. Preferably, the synthetic nucleotide sequence shares little or no extended homology with the natural sequence. In this context, extended homology generally refers to 100% sequence identity extending over about 25 nucleotides of the contiguous sequence. An example of a synthetic nucleotide sequence is 3'UTR, T-Zm.GST59.nno:1 (SEQ ID NO:24).

[0046] Reference to "isolated DNA polynucleotides" or equivalent terms or phrases in this application is intended to mean that the DNA polynucleotide is a DNA polynucleotide that exists alone or in combination with other compositions, but is not in its natural environment. For example, as long as nucleic acid elements naturally present in the DNA of the genome of an organism (such as coding sequences, intron sequences, non-translated leader sequences, promoter sequences, transcription termination sequences, etc.) are located within the genome of the organism and at the location within the genome where they naturally occur, the elements are not considered to be "isolated". However, as long as each of these elements and sub-portions of these elements are not within the genome of the organism and are not at the location within the genome where they naturally occur, the elements are "isolated" within the scope of the present disclosure. Similarly, as long as the nucleotide sequence encoding an insecticidal protein or any naturally occurring insecticidal variant of the protein is not in the DNA of the bacteria where the sequence encoding the protein naturally occurs, the nucleotide sequence is an isolated nucleotide sequence. 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 of DNA inserted into the genome of a plant or bacterial cell or present in an extrachromosomal vector, will be considered an isolated nucleotide sequence, whether it is present in a plasmid or similar construct used to transform cells, in the genome of a plant or bacteria, or in a detectable amount in a tissue, progeny, biological sample, or commercial product derived from a plant or bacteria.

[0047] As used herein, the term "sequence identity" refers to the degree to which two optimally aligned polynucleotide sequences or two optimally aligned polypeptide sequences are identical. An optimal sequence alignment is generated 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 sequences provided as SEQ ID NOs: 1-23 are used as reference sequences.

[0048] As used herein, the term "percent sequence identity" or "percent identity" or "% identity" is the identity score multiplied by 100. The "identity score" of a sequence optimally aligned to a reference sequence is the number of nucleotide matches in the optimal alignment, divided by the total number of nucleotides in the reference sequence, e.g., the total number of nucleotides over the entire length of the reference sequence. Thus, several embodiments relate to a DNA polynucleotide comprising a sequence that, when optimally aligned with a reference sequence provided herein as SEQ ID NOs: 1-23, has at least about 85% identity, at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, or at least about 100% identity with the reference sequence. The sequences disclosed herein may have the activity of any one of the reference sequences from which they were derived, e.g., SEQ ID NOs: 1-23.

[0049] Regulatory elements

[0050] Regulatory elements, such as promoters, leader sequences (also known as 5'UTRs), enhancers, introns, and transcription termination regions (or 3'UTRs), play an integral role in the overall expression of genes in living cells. As used herein, the term "regulatory element" (or "expression element") refers to a DNA polynucleotide having gene regulatory activity. As used herein, the term "gene regulatory activity" refers to the ability to affect the expression of an operably linked transcribable DNA polynucleotide, for example, by affecting the transcription and / or translation of an operably linked transcribable DNA polynucleotide. Regulatory elements that function in plants, such as promoters, leader sequences, enhancers, introns, and 3'UTRs, can be used to modify plant phenotypes by genetic engineering.

[0051] As used herein, "regulatory expression element group" or "EXP" sequence can refer to a group of operably linked regulatory elements, such as enhancers, promoters, leader sequences and introns. For example, the regulatory expression element group can include, for example, a promoter operably linked to the 5' of the leader sequence and operably linked to the 5' of the intron sequence.

[0052] Regulatory elements can be characterized by their gene expression patterns, for example, positive and / or negative effects such as constitutive expression or time, space, development, organization, environment, physiology, pathology, cell cycle and / or chemical response expression, and any combination thereof, and characterized by quantitative or qualitative indication. As used herein, "gene expression pattern" is any pattern of transcribed RNA that is operably connected DNA polynucleotides. Transcribed RNA can be translated to produce protein or can provide antisense or other regulatory RNA, such as double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), small interfering RNA (siRNA) etc.

[0053] As used herein, the term "protein expression" is any pattern of translation of transcribed RNA into protein. Protein expression can be characterized by its temporal, spatial, developmental or morphological properties, and by quantitative or qualitative indications.

[0054] Promoter can be used as a regulatory element for regulating the expression of operably connected transcribable DNA polynucleotides. As used herein, the term "promoter" generally refers to a DNA polynucleotide that is involved in identifying and binding RNA polymerase (e.g., RNA polymerase II) and other proteins (such as trans-acting transcription factors) to initiate transcription. The promoter can be initially separated from the 5' untranslated region (5'UTR) of the genomic copy of the gene. In some embodiments, the promoter is operably connected to the 5' of the leader sequence. The promoter can be a DNA polynucleotide synthetically produced or manipulated. The promoter can also be chimeric. Chimeric promoters are produced by the fusion of two or more heterologous DNA polynucleotides. In some embodiments, the promoter is present as SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 14, 16, 17, 19, 21, 22 and 23 or its fragment or variant. In some embodiments, the DNA polynucleotides claimed as described herein and any fragment or variant thereof are further defined as comprising promoter activity, i.e., can act as a promoter in a host cell (such as in a transgenic plant cell). In other specific embodiments, the fragment may be defined as exhibiting the promoter activity possessed by the initiator promoter molecule from which it is extended, or the fragment may comprise a "minimal promoter" that provides a basal level of transcription and comprises a TATA box or equivalent DNA sequence for recognizing and binding the RNA polymerase II complex to initiate transcription.

[0055] In one embodiment, a fragment of a promoter sequence disclosed herein is provided. The promoter fragment may include promoter activity as described above, and may be used alone or in combination with other promoters and promoter fragments, such as when constructing a chimeric promoter, or in combination with other expression elements and expression element fragments. In some embodiments, a fragment of a promoter 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 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900, or at least about 1000 continuous nucleotides or longer of a DNA polynucleotide with promoter activity as disclosed herein. In some embodiments, a fragment of a promoter 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 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900, at least about 1000, at least about 1050, at least about 1100, or at least about 1150 consecutive nucleotides of a DNA sequence comprising a TATA box and having promoter activity as disclosed herein. NO: 1, 3, 5, 7, 9, 11, 13, 14, 16, 17, 19, 21, 22 and 23 have at least about 85% identity, at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, or at least about 100% identity. The fragments disclosed herein can have the activity of the reference sequence from which it is derived, such as the activity of any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 14, 16, 17, 19, 21, 22 and 23.

[0056] Methods for generating such fragments from a starting promoter polynucleotide are well known in the art.

[0057] Compositions derived from promoter elements of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 14, 16, 17, 19, 21, 22 and 23 (such as internal or 5' deletions) can be generated, for example, using methods known in the art to improve or alter expression, including by removing elements that have a positive or negative effect on expression; duplicating elements that have a positive or negative effect on expression; and / or duplicating or removing elements that have a tissue or cell-specific effect on expression. Compositions derived from promoter elements of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 14, 16, 17, 19, 21, 22 and 23 can be used, for example, to prepare enhancer elements, wherein the promoter element comprises a 3' deletion in which the TATA box element or its equivalent sequence and downstream sequences are removed. Further deletions can be made to remove any element that has a positive or negative; tissue-specific; cell-specific; or time-specific (such as but not limited to circadian) effect on expression. Any promoter element provided by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 14, 16, 17, 19, 21, 22 and 23, as well as fragments or enhancers derived therefrom, can be used to prepare chimeric transcriptional regulatory element compositions.

[0058] Promoters or promoter fragments as described herein can be analyzed for the presence of known promoter elements, e.g., DNA sequence characteristics, such as the TATA box and other known transcription factor binding site motifs. One skilled in the art can use the identification of such known promoter elements to design variants of promoters that have similar expression patterns to the original promoter.

[0059] As used herein, the term "leader sequence" refers to a DNA polynucleotide separated from the non-translated 5' region (5'UTR) of a gene and is generally defined as a nucleotide segment between a transcription start site (TSS) and a protein coding sequence start site. Alternatively, the leader sequence can be a synthetically produced or manipulated DNA element. The leader sequence can be used as a 5' regulatory element for regulating the expression of an operably connected transcribable DNA polynucleotide. The leader polynucleotide can be used with a heterologous promoter or with its native promoter. Several embodiments relate to leader sequences or fragments or variants thereof present in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 14, 16, 17, 19, 21, 22 and 23. In a specific embodiment, such DNA sequences can be defined as being able to serve as leader sequences in host cells (including, for example, transgenic plant cells). In one embodiment, such sequences are decoded as comprising leader sequence activity. In some embodiments, a fragment of a leader sequence is provided, which comprises at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, or at least about 95 consecutive nucleotides of a DNA sequence having a leader sequence activity as disclosed herein. The leader sequences contained in NO: 1, 3, 5, 7, 9, 11, 13, 14, 16, 17, 19, 21, 22 and 23 have at least about 85% identity, at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, or at least about 100% identity. The leader sequences disclosed herein can have the activity of the reference sequence from which it is derived, such as the activity of any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 14, 16, 17, 19, 21, 22 and 23.

[0060] The leader sequence (also referred to as 5'UTR) contained in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 14, 16, 17, 19, 21, 22 and 23 may contain a regulatory element, or may adopt a secondary structure that may have an effect on the transcription or translation of an operably linked transcribable DNA polynucleotide. The leader sequence contained in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 14, 16, 17, 19, 21, 22 and 23, or a fragment or variant thereof, may be used to prepare a chimeric regulatory element that affects the transcription or translation of an operably linked transcribable DNA polynucleotide.

[0061] As used herein, the term "intron" refers to a DNA polynucleotide that can be isolated or identified from a gene and can be generally defined as a region spliced ​​during messenger RNA (mRNA) processing prior to translation. Alternatively, an intron can be a synthetically produced or manipulated DNA element. An intron can contain an enhancer element that enables transcription of an operably linked gene. An intron can be used as a regulatory element for regulating the expression of an operably linked transcribable DNA polynucleotide. A construct can include an intron, and the intron may be heterologous or may not be heterologous relative to the transcribable DNA polynucleotide. Examples of introns include rice actin introns and corn HSP70 introns.

[0062] In plants, relative to the construct lacking introns, some introns are included in the gene construct to cause mRNA and protein accumulation to increase. This effect is referred to as "intron-mediated enhancement" (IME) of gene expression. Introns known to stimulate expression in plants have been identified in maize genes (e.g., tubA1, Adh1, Sh1 and Ubil), rice genes (e.g., tpi) and dicot genes (e.g., rbcS), such as those from petunia (petunia) (e.g., rbcS), potato (e.g., st-ls1) and Arabidopsis thaliana (Arabidopsis thaliana) (e.g., ubq3 and pat1). Studies have shown that deletions or mutations in intron splicing sites reduce gene expression, indicating that IME may require splicing. However, IME in dicots has been confirmed by point mutations in Arabidopsis thaliana pat1 gene splicing sites. Repeated use of the same intron in a plant has been shown to exhibit shortcomings. In those cases, it is necessary to collect basic control elements to construct appropriate recombinant DNA elements.

[0063] As used herein, the term "3' transcription termination polynucleotide", "3' untranslated region" or "3'UTR" refers to a DNA polynucleotide used during transcription of the untranslated region of the 3' portion of an mRNA. The 3' untranslated region of an mRNA (also known as the polyA tail) can be generated by specific cleavage and 3' polyadenylation. The 3'UTR can be operably linked to and downstream of a transcribable DNA polynucleotide, and can include a polyadenylation signal and other regulatory signals capable of affecting transcription, mRNA processing or gene expression. The polyA tail is believed to play a role in mRNA stability and translation initiation. Examples of 3' transcription termination polynucleotides are the nopaline synthase 3' region, the wheat hsp17 3' region, the pea rubisco small subunit 3' region, the cotton E6 3' region, and the coixin 3'UTR.

[0064] 3'UTR is generally beneficial to the recombinant expression of specific DNA polynucleotides. Weak 3'UTR is likely to generate read-through, which may affect the expression of DNA polynucleotides located in adjacent expression cassettes. Proper control of transcription termination can prevent read-through of DNA sequences located downstream (e.g., other expression cassettes), and can further allow effective recycling of RNA polymerase to improve gene expression. Effective termination of transcription (releasing RNA polymerase II from DNA) is a prerequisite for restarting transcription, which directly affects the overall transcription level. After transcription termination, mature mRNA is released from the synthesis site and the template is transported to the cytoplasm. Eukaryotic mRNA accumulates in the form of poly (A) in vivo, making it difficult to detect transcription termination sites by conventional methods. However, it is difficult to predict functional and efficient 3'UTRs by bioinformatics methods because there is no conserved DNA sequence that allows easy prediction of effective 3'UTRs.

[0065] From a practical point of view, it is generally beneficial for the 3'UTR used in the expression cassette to have the following properties. First, the 3'UTR should be able to efficiently and effectively terminate the transcription of the transcribable DNA polynucleotide (e.g., a transgene) and prevent the transcript from reading through any adjacent DNA sequence, which may contain another expression cassette, such as when multiple expression cassettes are present on one transfer DNA (T-DNA) or on adjacent chromosomal DNA that has been inserted into the T-DNA during plant transformation. Second, the 3'UTR should not result in a reduction in the transcriptional activity conferred by the promoter, leader sequence, enhancer, and intron used to drive the expression of the transcribable DNA polynucleotide. Finally, in plant biotechnology, the 3'UTR is often used to initiate amplification reactions of reverse transcribed RNA extracted from transformed plants and is used to: (1) evaluate the transcriptional activity or expression after the expression cassette is integrated into the plant chromosome; (2) evaluate the copy number of the insertion within the plant DNA; and (3) evaluate the zygosity of the resulting seeds after breeding. The 3'UTR is also used in amplification reactions of DNA extracted from transformed plants to characterize the integrity of the insertion cassette. The 3'UTR used in conjunction with regulatory elements (e.g., regulatory elements present as SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 14, 16, 17, 19, 21, 22, and 23) is present as SEQ ID NOs: 2, 4, 6, 8, 10, 12, 15, 18, 20, and 24.

[0066] As used herein, the term "enhancer" or "enhancer element" refers to a cis-acting regulatory element, also referred to as a cis-element, which confers aspects of the overall expression pattern, but is generally insufficient to drive transcription of an operably linked transcribable DNA polynucleotide alone. Unlike a promoter, an enhancer element generally does not include a transcription start site (TSS) or a TATA box or equivalent DNA sequence. A promoter or promoter fragment may naturally contain one or more enhancer elements that affect transcription of an operably linked DNA sequence. An enhancer element may also be fused to a promoter to produce a chimeric promoter cis-element that confers aspects of the overall regulation of gene expression.

[0067] It is believed that many promoter enhancer elements bind DNA binding proteins and / or affect DNA topology, producing a local conformation that selectively allows or restricts RNA polymerase to approach the DNA template or promotes the selective opening of the double helix at the transcription start site. Enhancer elements can play a role in binding to transcription factors that regulate transcription. Some enhancer elements bind more than one transcription factor, and transcription factors can interact with more than one enhancer domain with different affinities. Enhancer elements can be identified by a variety of techniques, including deletion analysis, i.e., deleting one or more nucleotides from the 5' end or inside of the promoter; DNA binding protein analysis using DNase I footprinting, methylation interference, electro-shift cutting assay, in vivo genomic footprinting and other conventional assays performed by ligation-mediated polymerase chain reaction (PCR), or by using known cis-element motifs or enhancer elements as target sequences or target motifs, using conventional DNA sequence comparison methods (such as BLAST) to perform DNA sequence similarity analysis. The fine structure of the enhancer domain can be further studied by mutagenesis (or substitution) of one or more nucleotides or by other conventional methods known in the art. Enhancer elements can be obtained by chemical synthesis or by separation from regulatory elements comprising such elements, and they can be synthesized together with additional flanking nucleotides containing useful restriction enzyme sites to facilitate subsequent manipulations. Therefore, the design, construction and use of enhancer elements for regulating the expression of operably linked transcribable DNA polynucleotides according to the methods disclosed herein are contemplated herein. Enhancers can be derived from promoters present as SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 14, 16, 17, 19, 21, 22 and 23.

[0068] As used herein, the term "chimeric" refers to a single DNA polynucleotide produced by fusing a first DNA polynucleotide to a second DNA polynucleotide, wherein the first DNA polynucleotide and the second DNA polynucleotide generally do not exist in this configuration (i.e., fused to each other). Therefore, a chimeric DNA polynucleotide is a new DNA polynucleotide that does not normally exist in nature. As used herein, the term "chimeric promoter" refers to a promoter produced by such manipulation of a DNA polynucleotide. A chimeric promoter can combine two or more DNA fragments, such as a fusion of a promoter and an enhancer element. Therefore, the design, construction and use of a chimeric promoter for regulating the expression of an operably linked transcribable DNA polynucleotide according to the methods disclosed herein are contemplated herein.

[0069] Chimeric regulatory elements can be designed to include various components that can be operably connected by various methods known in the art, such as restriction enzyme digestion and connection, modular assembly of PCR products during cloning, amplification, or direct chemical synthesis of regulatory elements and other methods known in the art. The various chimeric regulatory elements obtained can include the same components or variants thereof, but are different in terms of one or more DNA sequences of the connection DNA sequences that allow one or more components to be operably connected. In some embodiments, the DNA sequence provided as SEQ ID NO: 1-23 can provide a regulatory element reference sequence, wherein the component comprising the reference sequence can be connected by methods known in the art and can include substitution, deletion, and / or insertion of one or more nucleotides or naturally occurring mutations in bacteria and plant cell transformation.

[0070] As used herein, the term "variant" refers to a second DNA polynucleotide (such as a regulatory element) that is similar in composition to but not identical to a first DNA polynucleotide, and wherein the second DNA polynucleotide still maintains the general functionality of the first DNA polynucleotide, for example, the same or similar expression pattern, for example, by more or less equivalent transcriptional activity. The variant can be a shortened or truncated version of the first DNA polynucleotide or an altered version 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" can also encompass a regulatory element having a nucleotide sequence that comprises a substitution, deletion or insertion of one or more nucleotides of a reference sequence, wherein the derived regulatory element has more or less or equal transcription or translation activity compared to the corresponding parent regulatory polynucleotide. The regulatory element "variant" will also encompass variants produced by naturally occurring mutations in bacterial and plant cell transformation. In some embodiments, the polynucleotide sequence provided as SEQ ID NO: 1-23 can be used to produce a variant that is similar but not identical to the DNA sequence composition of the original regulatory element, while still maintaining the general functionality of the original regulatory element, for example, the same or similar expression pattern. The generation of such variations is well within the ordinary skill in the art in view of the present disclosure and is contemplated herein.

[0071] The efficacy of the modifications, duplications or deletions described herein on the desired expression of a particular transgene can be tested empirically in stable and transient plant assays (such as those described in the working examples herein) to verify the results, which may vary depending on the changes made in the starting DNA polynucleotide and the target of the changes.

[0072] Constructs

[0073] As used herein, the term "construct" means any recombinant DNA polynucleotide, such as a plasmid, a cosmid, a virus, a phage, or a linear or circular DNA or RNA polynucleotide, which is derived from any source, capable of genome integration or autonomous replication, comprising a DNA polynucleotide, wherein at least one DNA polynucleotide has been connected (i.e., operably connected) to another DNA polynucleotide in a functionally operational manner. As used herein, the term "vector" means any construct that can be used for transformation purposes (i.e., introducing heterologous DNA or RNA into a host cell). Constructs typically include one or more expression cassettes. As used herein, "expression cassette" refers to a recombinant DNA polynucleotide comprising at least a transcribable DNA polynucleotide operably connected to one or more regulatory elements (usually at least a promoter and 3'UTR).

[0074] As used herein, the term "operably connected" refers to a first DNA polynucleotide being connected to a second DNA polynucleotide, wherein the first and second DNA polynucleotides are arranged so that the first DNA polynucleotide affects the function of the second DNA polynucleotide. Two DNA polynucleotides may or may not be part of a single continuous DNA polynucleotide, and may or may not be adjacent. For example, if a promoter regulates the transcription of a transcribable DNA polynucleotide of interest in a cell, the promoter is operably connected to the transcribable DNA polynucleotide. For example, when a leader sequence is capable of affecting the transcription or translation of a DNA sequence, it is operably connected to a DNA sequence.

[0075] In some embodiments, one or more regulatory elements operably linked to a transcribable DNA polynucleotide as described herein are provided in a double tumor-inducing (Ti) plasmid border construct having a right border (RB or AGRtu.RB) and left border (LB or AGRtu.LB) region of a Ti plasmid isolated from Agrobacterium tumefaciens containing T-DNA, wherein the one or more regulatory elements, together with a transport molecule provided by the Agrobacterium tumefaciens cell, allow the T-DNA to be integrated into the plant cell genome (see, e.g., U.S. Pat. No. 6,603,061). The construct may also contain plasmid backbone DNA segments that provide replication function and antibiotic selection in bacterial cells, for example, an Escherichia coli replication origin, such as ori322, a broad host range replication origin, such as oriV or oriRi, and a coding region for a selective marker, such as Spec / Strp encoding Tn7 aminoglycoside adenylyltransferase (aadA) that confers resistance to spectinomycin or streptomycin or gentamicin (Gm, Gent) selective marker genes. For plant transformation, the host bacterial strain is typically A. tumefaciens ABI, C58 or LBA4404, although other strains known to those skilled in the art of plant transformation may also work.

[0076] Methods for assembling and introducing constructs into cells in a manner that allows transcription of transcribable DNA polynucleotides into functional mRNA polynucleotides that are 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 vectors derived from the Ti plasmid of Agrobacterium tumefaciens and the pCaMVCN transport control vector.

[0077] Various regulatory elements may be included in the construct, 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 features. In one embodiment, the construct may include at least one regulatory element operably connected to a transcribable DNA polynucleotide, which is operably connected to a 3'UTR.

[0078] In some embodiments, constructs may include any promoter or leader sequence provided herein or known in the art. For example, a promoter (e.g., SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 14, 16, 17, 19, 21, 22, and 23 or a fragment or variant thereof) may be operably linked to a heterologous non-translated 5' leader sequence, such as a leader sequence derived from a heat shock protein gene. Alternatively, a leader sequence (e.g., comprising or present in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 14, 16, 17, 19, 21, 22, and 23 or a fragment or variant thereof) may be operably linked to a heterologous promoter, such as a cauliflower mosaic virus 35S transcript promoter.

[0079] The expression cassette can also include a transit peptide coding sequence, which encodes a peptide for subcellular targeting of an operably connected protein, particularly subcellular targeting to chloroplasts, leucoplasts or other plastid organelles; mitochondria; peroxisomes; vacuoles; or extracellular locations. Many chloroplast localization proteins are expressed by nuclear genes as precursors and are targeted to chloroplasts by chloroplast transit peptides (CTPs). Examples of such isolated chloroplast proteins include, but are not limited to, those associated with: small subunits (SSUs) of ribulose-1,5,-bisphosphate carboxylase, ferredoxin, ferredoxin oxidoreductase, light-harvesting complex proteins I and II, thioredoxin F, and enolpyruvate shikimate phosphate synthase (EPSPS). Chloroplast transit peptides are described in, for example, U.S. Patent No. 7,193,133. It has been demonstrated that non-chloroplast proteins can be targeted to chloroplasts by expression of heterologous CTPs operably linked to transgenics encoding non-chloroplast proteins.

[0080] Transcriptable DNA polynucleotide

[0081] As used herein, the term "transcribable DNA polynucleotide" refers to any DNA polynucleotide that can be transcribed into RNA, including but not limited to those having protein coding sequences (e.g., mRNA), those encoding guide RNA (gRNA), and those producing RNA having sequences that can be used for gene inhibition (e.g., siRNA, miRNA, and dsRNA). The type of DNA polynucleotide may include but is 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 transgenics. Examples of transcribable DNA polynucleotides for incorporation into constructs as described herein include, for example, DNA polynucleotides or genes from 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 by genetic engineering methods rather than classical breeding techniques.

[0082] As used herein, the term "heterologous transcribable DNA polynucleotide" refers to a transcribable DNA polynucleotide that is heterologous with respect to one or more regulatory elements to which it is operably linked.

[0083] "Transgene" refers to a transcribable DNA polynucleotide that is heterologous to a host cell, at least with respect to its location in the host cell genome and / or that has been artificially incorporated into the host cell genome in the current or any prior generation of cells.

[0084] A regulatory element, such as a promoter (e.g., SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 14, 16, 17, 19, 21, 22, and 23, or a fragment or variant thereof), can be operably linked to a transcribable 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), which combination does not typically exist 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, e.g., different genes from the same species or the same gene from different species. Therefore, if such a combination does not typically exist in nature, i.e., the transcribable DNA polynucleotide is not naturally operably linked to the regulatory sequence, then the regulatory element is heterologous to the transcribable DNA polynucleotide that is operably linked.

[0085] The transcribable DNA polynucleotide can be any DNA polynucleotide of the expression of the desired transcript in general. This expression of the transcript can lead to the translation of the resulting mRNA, thereby leading to protein expression. Or, for example, the transcribable DNA polynucleotide can be designed to ultimately lead to a reduction in the expression of a specific gene or protein. In one embodiment, this can be achieved by using a transcribable DNA polynucleotide oriented in the antisense direction. Those of ordinary skill in the art are familiar with the use of this antisense technology. Any gene can be negatively regulated in this way, and in one embodiment, the transcribable DNA polynucleotide can be designed to suppress a specific gene by the expression of dsRNA, siRNA or miRNA.

[0086] Thus, one embodiment provides a recombinant DNA polynucleotide comprising a regulatory element (such as those provided as SEQ ID NOs: 1-23) operably linked to a heterologous transcribable DNA polynucleotide such that when the construct is integrated into the genome of a plant cell or a transgenic plant cell, transcription of the transcribable DNA polynucleotide is regulated at a desired level or in a desired pattern. In one embodiment, the transcribable DNA polynucleotide comprises a protein coding region of a gene, and in another embodiment, the transcribable DNA polynucleotide comprises an antisense region of a gene.

[0087] Genes with agronomic value

[0088] Transcribable DNA polynucleotide can comprise the gene with agronomic value.As used herein, term " gene with agronomic value " refers to the transcribable DNA polynucleotide that gives the desired characteristic when expressed in specific plant tissue, cell or cell type.The product of the gene with agronomic value can play a role in plant, so that plant morphology, physiology, growth, development, yield, grain composition, nutritional status, disease or pest resistance and / or environment or chemical tolerance are affected, or can serve as the pesticide in the diet of the insect that feeds on plants.In one embodiment, regulatory element (such as those provided as SEQ ID NO:1-23) is incorporated into construct so that regulatory element is operably connected to the transcribable DNA polynucleotide as the gene with agronomic value.In the transgenic plant containing this construct, the expression of the gene with agronomic value can give useful agronomic traits. Beneficial agronomic traits may include, for example, 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 maturity, 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 desirable biofuel production.

[0089] Examples of genes with agronomic value known in the art include genes for herbicide resistance (U.S. Pat. Nos. 6,803,501; 6,448,476; 6,248,876; 6,225,114; 6,107,549; 5,866,775; 5,804,425; 5,633,435; and 5,463,175), increased yield (U.S. Pat. Nos. USRE38,446; 6,716,474; 6,663,906; 6,476,295; 6,441,277; 6,423,828; 6,399,330; 6,372,211; 6,235,971; 6,222,098; and 5,7 16,837), insect control (U.S. Patent Nos. 6,809,078; 6,713,063; 6,686,452; 6,657,046; 6,645,497; 6,642,030; 6,639,054; 6,620,988; 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; 6,177,61 5; 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 (U.S. Pat. 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 6,506,96 2), virus resistance (U.S. Patent Nos. 6,617,496; 6,608,241; 6,015,940; 6,013,864; 5,850,023; and 5,304,730), nematode resistance (U.S. Patent 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. Pat. Nos. 6,495,739; 5,608,149; 6,483,008; and 6,476,295), improved fatty acid content (U.S. Pat. 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 (U.S. Patent No. 6,380,466), fruit ripening (U.S. Patent No. 5,512,466), enhanced animal and human nutrition (U.S. Patent Nos. 6,723,837; 6,653,530; 6,5412,59; 5,985,605; and 6,171,640), biopolymers (U.S. Patent Nos. USRE37,543; 6,228,623; and 5,9 58,745 and 6,946,588), environmental stress resistance (U.S. Pat. No. 6,072,103), pharmaceutical peptides and secretable peptides (U.S. Pat. Nos. 6,812,379; 6,774,283; 6,140,075; and 6,080,560), improved processing properties (U.S. Pat. No. 6,476,295), improved digestibility (U.S. Pat. No. 6,531,648), low raffinose (U.S. Pat. No. 6,166,292) , industrial enzyme production (U.S. Patent No. 5,543,576), improved flavor (U.S. Patent No. 6,011,199), nitrogen fixation (U.S. Patent No. 5,229,114), hybrid seed production (U.S. Patent No. 5,689,041), fiber production (U.S. Patent Nos. 6,576,818; 6,271,443; 5,981,834; and 5,869,720), and biofuel production (U.S. Patent No. 5,998,700).

[0090] Alternatively, a gene of agronomic value can affect the above-mentioned plant characteristics or phenotypes by encoding an RNA that causes targeted regulation of gene expression of an endogenous gene, such as by antisense RNA (see, e.g., U.S. Pat. No. 5,107,065); inhibitory RNA ("RNAi"), including regulation of gene expression by miRNA, siRNA, trans-acting siRNA, and phased sRNA-mediated mechanisms, such as those described in published applications US2006 / 0200878 and US2008 / 0066206 and U.S. Patent Application No. 11 / 974,469); or co-suppression-mediated mechanisms. The RNA can also be a catalytic RNA (e.g., a ribozyme or riboswitch; see, e.g., US2006 / 0200878) that is engineered to cleave a desired endogenous mRNA product. Methods for constructing a construct and introducing it into a cell in a manner that allows transcription into a molecule capable of causing gene inhibition are known in the art.

[0091] Selective markers

[0092] The transcribable DNA polynucleotides encoding selective markers can also be used with regulatory elements (such as those provided as SEQ ID NO: 1-23). ​​As used herein, the term "selective marker" refers to any transcribable DNA polynucleotides whose expression or lack of expression in transgenic plants, tissues or cells can be screened or scored in some ways. Selectable markers (also referred to as reporter genes) and their related selection and screening techniques are known in the art, and include but are not limited to, transcribable DNA polynucleotides encoding beta-glucuronidase (GUS), green fluorescent protein (GFP), proteins that confer antibiotic resistance, and proteins that confer herbicide tolerance. The example of a reporter transgenic is provided as SEQ ID NO: 25.

[0093] It is well known in the art to use reporter gene assays (or reporter transgenic assays) to determine the gene regulatory activity (or expression profile) of regulatory elements (e.g., Clarke et al., Unit 4, Chapter 21, 3rd Edition, Academic Press, Elsevier Inc., 2019). As used herein, the term "reporter gene assay" refers to a method in which a reporter gene (such as a transgenic encoding β-glucuronidase (GUS) protein) is first used as a heterologous transcribable DNA polynucleotide operably linked to a specific regulatory element to determine the gene regulatory activity (or expression profile) of the latter (e.g., a promoter or 3'UTR). In subsequent reporter gene assays, qualitative and quantitative GUS analysis can be used to evaluate the gene regulatory activity (or expression profile) of the regulatory element in the selected plant organs and / or tissues of the transformed plants. It should be understood that the gene regulatory activity (or expression profile) of the regulatory element (e.g., promoter or 3'UTR) determined by using a reporter gene assay (e.g., GUS assay) is the same or substantially the same or substantially similar to other transcribable DNA molecules operably linked to GUS. In one embodiment, the other operably linked transcribable DNA molecule may be a gene of agronomic value, including but not limited to those described herein.

[0094] Genome editing

[0095] Several embodiments relate to a recombinant DNA construct comprising an expression cassette comprising a sequence having at least about 85% sequence identity, 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 sequence identity with SEQ ID NO: 1-23 or a fragment thereof operably linked to a heterologous DNA sequence encoding a site-specific genome modification enzyme and / or any associated protein for genome modification. These one or more site-specific genome modification enzyme expression cassettes may be present in the same molecule or vector (cis) with a donor template for template editing, or in a separate molecule or vector (trans). Several editing methods are known in the art, which involve different sequence-specific genome modification enzymes (or complexes of proteins and / or guide RNAs) for modifying genomic DNA. In some embodiments, the site-specific genome modification enzyme modifies the genome by inducing double-strand breaks (DSB) or nicks at the desired genome site or locus. In some embodiments, during the process of repairing the DSB or nicks introduced by the genome modification enzyme, the donor template DNA can be integrated into the genome at the site of the DSB or nicks. In some embodiments, during the process of repairing the DSB or nicks introduced by the genome modification enzyme, insertion or deletion mutations (indels) can be introduced into the genome. In some embodiments, the site-specific genome modification enzyme comprises a cytidine deaminase. In some embodiments, the site-specific genome modification enzyme comprises adenine deaminase. In the present disclosure, the site-specific genome modification enzyme includes endonucleases, recombinases, transposases, deaminases, helicases, reverse transcriptases and any combination thereof.

[0096] Several embodiments relate to a gene regulatory element as described herein, which is operably connected to a heterologous transcribable DNA molecule encoding one or more components of a genome editing system. The genome editing system 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, a gene regulatory element as described herein is operably connected to a heterologous transcribable DNA polynucleotide encoding a sequence-specific DNA binding domain (such as a CRISPR-Cas effector protein, a zinc finger protein, or a transcription activator (TAL) protein). In some embodiments, a sequence-specific DNA binding domain can be a fusion protein. In some embodiments, a gene regulatory element as described herein is operably connected to a heterologous transcribable DNA polynucleotide encoding a CRISPR-Cas effector protein. In some embodiments, a CRISPR-Cas effector protein is selected from a type I CRISPR-Cas system, a type II CRISPR-Cas system, a type III CRISPR-Cas system, a type IV CRISPR-Cas system, a type V CRISPR-Cas system, or a type VI CRISPR-Cas system. In some embodiments, a gene regulatory element as described herein is operably linked to a heterologous transcribable DNA polynucleotide encoding a guide RNA. As used herein, "guide RNA" or "gRNA" refers to an RNA that recognizes a target DNA sequence and guides or "guides" a CRISPR effector protein to a target DNA sequence. The guide RNA includes a region complementary to the target DNA (referred to as crRNA) and a region (referred to as tracrRNA) that binds to the CRISPR effector protein. The guide RNA can be a single RNA molecule (sgRNA) or two separate RNA molecules (2-piece gRNA). In some embodiments, the gRNA may further include an RNA template (pegRNA) for a reverse transcriptase.

[0097] Several embodiments relate to a gene regulatory element as described herein, operably linked to a heterologous transcribable DNA polynucleotide encoding one or more components of a CRISPR-Cas genome editing system comprising a CRISPR-Cas effector protein. Examples of CRISPR-Cas effector proteins include, but are not limited to, Cas9, C2c1, 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, 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, Cs aX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), Csf5, Cas14a, Cas14b and Cas14c effector proteins. In some embodiments, the gene regulatory element as described herein is operably connected to a CRISPR-Cas effector protein containing a mutation in its nuclease active site (e.g., RuvC, HNH and / or NUC domain). CRISPR-Cas effector proteins having mutations in their nuclease active sites and therefore no longer containing nuclease activity are generally referred to as "dead", such as dCas. In some embodiments, a CRISPR-Cas effector protein domain or polypeptide having a mutation in its nuclease active site may have impaired activity or reduced activity compared to the same CRISPR-Cas effector protein without a mutation. In some embodiments, a gene regulatory element as described herein is operably connected to a CRISPR-Cas effector protein having a mutation in its nuclease active site to generate a nickase activity operably connected to a reverse transcriptase.

[0098] Cell transformation

[0099] Also provided are methods of producing transformed cells and plants comprising one or more regulatory elements operably linked to a transcribable DNA polynucleotide, such as those provided as SEQ ID NOs: 1-23.

[0100] The term "transformation" refers to the introduction of a DNA polynucleotide 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, rice seedlings, embryos and pollen.

[0101] As used herein, the term "transformation" refers to a cell, tissue, organ or organism into which an exogenous DNA polynucleotide (such as a construct as described herein) has been introduced. The introduced DNA polynucleotide can be integrated into the genomic DNA of the recipient cell, tissue, organ or organism so that the introduced DNA polynucleotide is inherited by subsequent offspring. "Transgenic" or "transformed" cells or organisms also include offspring of cells or organisms and offspring produced in a breeding program that uses such transgenic plants as parents in hybridization and exhibits a changed phenotype caused by the presence of external DNA molecules. The introduced DNA polynucleotide can also be transiently introduced into the recipient cell so that the introduced DNA polynucleotide is not inherited by subsequent offspring. The term "transgenic" refers to bacteria, fungi or plants containing one or more heterologous DNA polynucleotides.

[0102] There are many well-known methods for introducing DNA polynucleotides into plant cells by those skilled in the art. The method generally includes selecting a suitable host cell, transforming the host cell with a vector, and obtaining the steps of transformed host cells. The methods and materials for transforming plant cells by introducing plant constructs into the plant genome may include any well-known and proven methods. Suitable methods include, but are not limited to, bacterial infection (e.g., Agrobacterium), binary BAC vectors, direct delivery of DNA (e.g., by PEG-mediated conversion, drying / suppression-mediated DNA absorption, electroporation, stirring using silicon carbide fibers and acceleration of DNA-coated particles), gene editing (e.g., CRISPR-Cas systems), etc.

[0103] The host cell can be any cell or organism, such as a plant cell, an algae cell, an algae, a fungal cell, a fungus, a bacterial cell or an insect cell. In a specific embodiment, the host cell and the transformed cell can include a cell from a crop plant. In a further specific embodiment, the host cell and the transformed cell can include a cell from a soybean plant.

[0104] The transgenic plant can then be regenerated from the transgenic plant cell as described herein. Seeds can be produced from such transgenic plants using conventional breeding techniques or self-pollination. Such seeds and resulting offspring plants grown from such seeds will contain the recombinant DNA polynucleotides as described herein (such as a recombinant DNA molecule comprising a sequence selected from SEQ ID NO: 1-23), and will therefore be transgenic.

[0105] Transgenic plants can be self-pollinated to provide seeds of homozygous transgenic plants (homozygous for recombinant DNA molecules as described herein), or hybridized with non-transgenic plants or different transgenic plants to provide seeds of heterozygous transgenic plants (heterozygous for recombinant DNA molecules as described herein). Such homozygous and heterozygous transgenic plants are referred to as "progeny plants" herein. Progeny plants are transgenic plants that originate from the original transgenic plants and contain recombinant DNA polynucleotides as described herein. Seeds produced using transgenic plants can be harvested and used to cultivate generations of transgenic plants, i.e., progeny plants that contain recombinant DNA molecules as described herein and express genes with agronomic 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 (eds.), Center for Agricultural Publishing and Documentation (1979); Fehr, Soybeans: Improvement, Production and Uses, 2nd ed., 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).

[0106] The transformed plants can be analyzed for the presence of one or more genes of interest and the expression levels and / or profiles conferred by regulatory elements (such as those provided as SEQ ID NOs: 1-23). ​​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 transcribable DNA polynucleotides can be detected using (Applied Biosystems, Foster City, CA) reagents and measurements were performed as described by the manufacturer and using Test matrix to determine PCR cycle time. Alternatively, use Transgene expression was assessed using the ELISA kit (Third Wave Technologies, Madison, WI) and methods described by the manufacturer.

[0107] Also provided are parts of plants as described herein. Plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperms, ovules, and pollen. Plant parts can be viable, non-viable, regenerative, and / or non-regenerative. Also provided are transformed plant cells comprising DNA polynucleotides as described herein, such as those provided as SEQ ID NOs: 1-23. Transformed or transgenic plant cells include regenerative and / or non-regenerative plant cells.

[0108] Commercial products produced by transgenic plants or parts thereof containing recombinant DNA polynucleotides as described herein (such as those provided as SEQ ID NOs: 1-23). ​​In some embodiments, commercial products contain detectable amounts of DNA comprising a DNA sequence selected from the group consisting of SEQ ID NOs: 1-23 or its fragments or variants. As used herein, "commercial products" refers to any composition or product comprising materials derived from transgenic plants, seeds, plant cells, or plant parts, which contain recombinant DNA polynucleotides as described herein, such as those provided as SEQ ID NOs: 1-23. Commercial products include, but are not limited to, processed seeds, grains, plant parts, and meal. Commercial products containing detectable amounts of DNA corresponding to recombinant DNA molecules as described herein, such as those provided as SEQ ID NOs: 1-23, are contemplated. One or more detections of such DNA in a sample can be used to determine the content or source of a commercial product. Any standard method for detecting DNA polynucleotides can be used, including detection methods disclosed herein.

[0109] The definitions and methods provided define the present disclosure and guide those of ordinary skill in the art to practice the present disclosure. Unless otherwise stated, the terms should be understood according to the conventional usage of those of ordinary skill in the relevant field. The definitions of commonly used terms and methods in molecular biology can also be found in Clark et al., Molecular Biology, 3rd edition, Academic Press, Elsevier Inc., 2019; Alberts et al., Molecular Biology of The Cell, 5th edition, Garland Science Publishing, Inc.: New York, 2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; King et al., A Dictionary of Genetics, 6th edition, Oxford University Press: New York, 15 2247; and Lewin, Genes IX, Oxford University Press: New York, 2007.

[0110] Implementation

[0111] For further illustration, additional exemplary non-limiting embodiments of the present disclosure are set forth below.

[0112] Embodiment 1 relates to a recombinant DNA polynucleotide comprising a DNA sequence selected from the group consisting of: a) a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-23; b) a sequence comprising any one of SEQ ID NOs: 1-23; and c) a fragment of any one of SEQ ID NOs: 1-23, wherein the fragment has gene regulatory activity; wherein the sequence is operably linked to a heterologous transcribable DNA polynucleotide.

[0113] Embodiment 2 relates to the recombinant DNA polynucleotide of embodiment 1, wherein the sequence has at least 90% sequence identity with the DNA sequence of any one of SEQ ID NOs: 1-23.

[0114] Embodiment 3 relates to the recombinant DNA polynucleotide of embodiment 1, wherein the sequence has at least 95% sequence identity with the DNA sequence of any one of SEQ ID NOs: 1-23.

[0115] Embodiment 4 relates to the recombinant DNA polynucleotide of embodiment 1, wherein the DNA sequence comprises gene regulatory activity.

[0116] Embodiment 5 relates to the recombinant DNA polynucleotide of embodiment 1, wherein the heterologous transcribable DNA polynucleotide comprises a gene of agronomic value.

[0117] Embodiment 6 relates to the recombinant DNA polynucleotide of embodiment 5, wherein the gene of agronomic value confers herbicide tolerance to the plant.

[0118] Embodiment 7 relates to the recombinant DNA polynucleotide of embodiment 5, wherein the gene of agronomic value confers resistance to plant pests.

[0119] Embodiment 8 relates to the recombinant DNA polynucleotide of embodiment 1, wherein the heterologous transcribable DNA polynucleotide encodes dsRNA, miRNA or siRNA.

[0120] Embodiment 9 relates to a transgenic plant cell comprising a recombinant DNA polynucleotide, wherein the recombinant DNA polynucleotide is selected from the group consisting of: a) a sequence having at least 85% sequence identity with any one of SEQ ID NOs: 1-23; b) a sequence comprising any one of SEQ ID NOs: 1-23; and c) a fragment of any one of SEQ ID NOs: 1-23, wherein the fragment has gene regulatory activity; wherein the sequence is operably linked to a heterologous transcribable DNA polynucleotide.

[0121] Embodiment 10 relates to the transgenic plant cell of embodiment 9, wherein the transgenic plant cell is a monocotyledonous plant cell.

[0122] Embodiment 11 relates to the transgenic plant cell of embodiment 9, wherein the transgenic plant cell is a dicotyledonous plant cell.

[0123] Embodiment 12 relates to a transgenic plant or part thereof, which comprises the recombinant DNA polynucleotide as described in embodiment 1.

[0124] Embodiment 13 relates to a progeny plant or a part thereof of the transgenic plant of embodiment 12, wherein the progeny plant or a part thereof comprises the recombinant DNA polynucleotide.

[0125] Embodiment 14 relates to a transgenic seed, wherein the seed comprises the recombinant DNA polynucleotide of embodiment 1.

[0126] Embodiment 15 relates to a method for producing a commercial product, which comprises obtaining the transgenic plant or part thereof according to embodiment 12 and producing the commercial product therefrom.

[0127] Embodiment 16 relates to the method of embodiment 15, wherein the commercial product is seed, processed seed, protein concentrate, protein isolate, starch, grain, plant part, seed oil, biomass, flour and meal.

[0128] Embodiment 17 relates to a method for expressing a transcribable DNA polynucleotide, the method comprising obtaining the transgenic plant of embodiment 12 and cultivating the plant, wherein the transcribable DNA is expressed.

[0129] Embodiment 18 is a recombinant DNA polynucleotide comprising a DNA sequence selected from the group consisting of:

[0130] a) a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-23;

[0131] b) a sequence comprising any one of SEQ ID NOs: 1-23; and

[0132] c) a fragment of any one of SEQ ID NOs: 1-23, wherein the fragment comprises gene regulatory activity;

[0133] Wherein the DNA sequence is operably linked to a heterologous transcribable DNA polynucleotide.

[0134] Embodiment 19 is the recombinant DNA polynucleotide of embodiment 18, wherein the DNA sequence has at least 90% sequence identity with the DNA sequence of any one of SEQ ID NOs: 1-23.

[0135] Embodiment 20 is a recombinant DNA polynucleotide as described in any one of embodiments 18 or 19, wherein the DNA sequence has at least 95% sequence identity with the DNA sequence of any one of SEQ ID NOs: 1-23.

[0136] Embodiment 21 is a recombinant DNA polynucleotide as described in any one of embodiments 18 to 20, wherein the DNA sequence comprises gene regulatory activity.

[0137] Embodiment 22 is the recombinant DNA polynucleotide of any one of embodiments 18 to 21, wherein the heterologous transcribable DNA polynucleotide comprises a gene of agronomic value.

[0138] Embodiment 23 is the recombinant DNA polynucleotide of embodiment 22, wherein the gene of agronomic value confers herbicide tolerance to the plant.

[0139] Embodiment 24 is the recombinant DNA polynucleotide of embodiment 22, wherein the gene of agronomic value confers resistance to plant pests.

[0140] Embodiment 25 is a recombinant DNA polynucleotide as described in any one of embodiments 18 to 21, wherein the heterologous transcribable DNA polynucleotide encodes a dsRNA, a miRNA, or a siRNA.

[0141] Embodiment 26 is a transgenic plant cell comprising a recombinant DNA polynucleotide comprising a DNA sequence selected from the group consisting of:

[0142] a) a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-23;

[0143] b) a sequence comprising any one of SEQ ID NOs: 1-23; and

[0144] c) a fragment of any one of SEQ ID NOs: 1-23, wherein the fragment comprises gene regulatory activity;

[0145] Wherein the DNA sequence is operably linked to a heterologous transcribable DNA polynucleotide.

[0146] Embodiment 27 is the transgenic plant cell of embodiment 26, wherein the transgenic plant cell is a monocotyledonous plant cell.

[0147] Embodiment 28 is the transgenic plant cell of embodiment 26, wherein the transgenic plant cell is a dicotyledonous plant cell.

[0148] Embodiment 29 is a transgenic plant or part thereof comprising the recombinant DNA polynucleotide of any one of embodiments 18 to 25.

[0149] Embodiment 30 is a progeny plant or part thereof of the transgenic plant of embodiment 29, wherein the progeny plant or part thereof comprises the recombinant DNA polynucleotide of any one of embodiments 18 to 25.

[0150] Embodiment 31 is a transgenic seed, wherein the seed comprises the recombinant DNA polynucleotide of any one of embodiments 18 to 25.

[0151] Embodiment 32 is a method for producing a commercial product, comprising obtaining the transgenic plant or part thereof of embodiment 29 or the progeny plant or part thereof of embodiment 30 and producing the commercial product therefrom.

[0152] Embodiment 33 is a method as described in embodiment 32, wherein the commercial product is selected from the group consisting of: seeds, processed seeds, protein concentrates, protein isolates, starches, grains, plant parts, seed oils, biomass, flours and meals.

[0153] Embodiment 34 is a method for expressing a transcribable DNA polynucleotide, comprising obtaining the transgenic plant of embodiment 29 or the progeny plant of embodiment 30 or a part thereof and cultivating the plant, wherein the transcribable DNA polynucleotide is expressed.

[0154] Embodiment 35 is an isolated recombinant DNA molecule characterized by comprising a DNA sequence of any one of SEQ ID NOs: 1-23, wherein the DNA sequence is operably linked to a heterologous transcribable polynucleotide molecule.

[0155] Embodiment 36 is an isolated recombinant DNA molecule as described in embodiment 35, characterized in that the heterologous transcribable DNA polynucleotide molecule comprises a gene with agronomic value.

[0156] Embodiment 37 is an isolated recombinant DNA molecule according to embodiment 36, characterized in that the gene of agronomic value confers herbicide tolerance to the plant.

[0157] Embodiment 38 is an isolated recombinant DNA molecule as described in embodiment 36, characterized in that the gene with agronomic value confers resistance to plant pests.

[0158] Embodiment 39 is a method for producing a transgenic plant, characterized in that, in addition to the plant obtained by the method, it comprises:

[0159] a. transforming a plant cell with the isolated recombinant DNA molecule as described in embodiment 35 to produce a transformed plant cell; and

[0160] b. Regenerating transgenic plants from the transformed plant cells.

[0161] Embodiment 40 is a construct characterized by comprising the isolated recombinant DNA molecule described in embodiment 35.

[0162] The embodiments described herein can be more easily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting unless specified. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work better in practicing the present invention. However, in view of this disclosure, it is understood by those skilled in the art that many changes can be made in the disclosed specific embodiments, and still obtain the same or similar results without departing from the spirit and scope of the present invention, and therefore, all matters proposed or shown in the accompanying drawings should be understood to be exemplary, without limiting.

[0163] Example

[0164] Example 1

[0165] Identification and cloning of regulatory elements

[0166] This example describes the identification, synthesis, and cloning of regulatory expression elements derived from Glycine max (Soybean).

[0167] The expression elements of SEQ ID NO:1-23 are selected based on the expression patterns observed in the transcriptome of soybean variety A3555. Excavated the proprietary soybean A3555 gene expression atlas to identify the expression elements that drive the gene expression in three leaf tissues and aboveground tissues, and hardly expressed or not expressed in root and pollen. Identify those genes with suitable expression profiles and use Williams 82 genome assembly to identify the corresponding expression element sequences of those genes. Promoter is selected using a sequence about 2 kilobases (kb) upstream of the start methionine start codon and comprises both natural promoter and leader sequence (5 'UTR). Transcription termination / polyadenylation sequence (3 'UTR) is identified as about 500 base pairs (bp) after the endogenous gene stop codon. Short potential open reading frames were identified in the upstream regions of P-Gm.16G089000:1 (SEQ ID NO:14) and P-Gm.07G156100:1 (SEQ ID NO:19), leading to the design of 5' truncated P-Gm.16G089000_trunc:1 (SEQ ID NO:16) and 5' truncated P-Gm.07G156100_trunc:1 (SEQ ID NO:21).

[0168] The expression elements identified from soybean are presented in Table 1 .

[0169] Table 1. Groups of regulatory expression elements, promoters, leader sequences and introns.

[0170]

[0171]

[0172] The identified expression elements were synthesized using methods known in the art and cloned into an expression cassette for driving β-glucuronidase (GUS) expression in a binary plant transformation vector construct to evaluate the activity of the expression elements in stably transformed soybean plants, as described in Example 2.

[0173] Example 2

[0174] Analysis of soybean expression elements driving GUS expression in stably transformed soybean plants

[0175] Soybean plants were transformed with a plant binary expression vector construct containing expression elements driving expression of a β-glucuronidase (GUS) transgene as SEQ ID NOs: 1-8. The resulting plants were analyzed for GUS protein expression to assess the effect of the regulatory elements on expression.

[0176] Soybean plants were transformed with a GUS expression construct comprising the expression elements shown in Table 2 below.

[0177] Table 2. Plant GUS expression constructs used for stable transformation of soybean plants.

[0178]

[0179] The resulting plant expression vector contains: a left border from Agrobacterium tumefaciens (B-AGRtu.leftborder), a first transgenic selection cassette for selecting transformed plant cells that confer resistance to the antibiotic spectinomycin; a second expression cassette for evaluating the activity of the expression elements present in Table 2, the second expression cassette comprising a promoter (promoter and leader sequence), the promoter being 5'operably linked to a GUS coding sequence, the GUS coding sequence comprising a processable intron (SEQID NO:25), the processable intron being 5'operably linked to a 3'UTR; and a right border from Agrobacterium tumefaciens (B-AGRtu.right border).

[0180] As is well known in the art, soybean plant cells are transformed using the above binary transformation vector constructs via Agrobacterium-mediated transformation. The resulting transformed plant cells are induced to form complete soybean plants.

[0181] Use qualitative and quantitative GUS analysis to evaluate the expression element activity in the selected plant organs and tissues in the transformed plants. In order to qualitatively analyze 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-b-glucuronide) at 37°C for 5 hours and decolorized with 35% EtOH and 50% acetic acid. The expression of GUS was qualitatively determined by visually inspecting the selected plant organs or tissues with blue staining under a dissecting microscope or compound microscope.

[0182] In order to quantitatively analyze GUS expression by enzyme assay, total protein was extracted from the selected tissues of the transformed corn plants. One to two micrograms of total protein were incubated with the fluorescent substrate 4-methylumbelliferyl-β-D-glucuronide (MUG) at a concentration of 1 mM in a total reaction volume of 50 microliters. After incubation at 37°C for 1 hour, the reaction was stopped by adding 350 microliters of 200 mM sodium bicarbonate solution. The reaction product 4-methylumbelliferyl (4-MU) emits maximum fluorescence at high pH, ​​wherein the hydroxyl group is ionized. Alkaline sodium carbonate solution was added to stop the assay at the same time, and 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 and emission at 460 nm). GUS activity values ​​were provided in nanomoles of 4-MU / hour / mg total protein.

[0183] The following tissues were sampled for GUS expression in the R0 generation: V5 stage sink leaves, source leaves and roots; R1 stage flowers, petioles, source leaves, pollen and roots; R3 stage pods and immature seeds; R5 source leaves; and R8 seed cotyledons and embryos. Soybean vegetative and reproductive stages are well known to those skilled in the art, and many publications describing these stages can be found 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. Tables 3-6 show the range and average GUS expression of each sampled tissue transformed with each construct (Construct-1 to Construct-4).

[0184] Table 3. Average and range of GUS expression driven by P-Gm.08G282100:2 (SEQ ID NO: 1) and T-Gm.08G282100:2 (SEQ ID NO: 2) in stable soybean transformed plants.

[0185]

[0186]

[0187] Table 4. Average and range of GUS expression driven by P-Gm.02G215700:2 (SEQ ID NO:3) and T-Gm.02G215700:2 (SEQ ID NO:4) in stable soybean transformed plants.

[0188]

[0189] Table 5. Average and range of GUS expression driven by P-Gm.11G155000:2 (SEQ ID NO:5) and T-Gm.11G155000:2 (SEQ ID NO:6) in stable soybean transformed plants.

[0190]

[0191]

[0192] Table 6. Average and range of GUS expression driven by P-Gm.PSII:1 (SEQ ID NO:7) and T-Gm.PSII:1 (SEQ ID NO:8) in stable soybean transformed plants.

[0193]

[0194] As can be seen from Tables 3 to 6, the expression elements derived from each soybean gene have unique expression patterns. Qualitative expression analysis of selected tissues demonstrated that each combination of expression elements had different expression patterns. For example, for R1 source leaves, GUS expression was mainly observed in the mesophyll in soybean plants transformed with construct-1 (P-Gm.08G282100:2 / T-Gm.08G282100:2). Soybean plants transformed with construct-2 (P-Gm.02G215700:2 / T-Gm.02G215700:2) showed R1 source leaf expression mainly in the vascular bundle, while soybean plants transformed with construct-4 (P-Gm.PSII:1 / T-Gm.PSII:1) showed expression in the phloem, stomata, mesophyll, and vascular bundles.

[0195] Each combination of expression elements was able to drive gene expression in stably transformed soybean plants.

[0196] Example 3

[0197] Analysis of soybean expression elements driving GUS expression in stably transformed soybean plants

[0198] Soybean plants were transformed with a plant binary expression vector construct containing expression elements driving expression of a β-glucuronidase (GUS) transgene as SEQ ID NOs: 9-24. The resulting plants were analyzed for GUS protein expression to assess the effect of the regulatory elements on expression.

[0199] Soybean plants were transformed with a GUS expression construct comprising the expression elements shown in Table 7 below.

[0200] Table 7. Plant GUS expression constructs for stable transformation of soybean plants.

[0201]

[0202]

[0203] The resulting plant expression vector contains: a left border from Agrobacterium tumefaciens (B-AGRtu.leftborder), a first transgenic selection cassette for selecting transformed plant cells that confer resistance to the antibiotic spectinomycin; a second expression cassette for evaluating the activity of the expression elements present in Table 7, the second expression cassette comprising a promoter (promoter and leader sequence), the promoter being 5'operably linked to a GUS coding sequence, the GUS coding sequence comprising a processable intron (SEQID NO:25), the processable intron being 5'operably linked to a 3'UTR; and a right border from Agrobacterium tumefaciens (B-AGRtu.right border).

[0204] As is well known in the art, soybean plant cells are transformed using the above binary transformation vector constructs via Agrobacterium-mediated transformation. The resulting transformed plant cells are induced to form complete soybean plants.

[0205] Qualitative and quantitative expression assays were as described previously in Example 2. The following tissues were sampled for GUS expression in the R0 generation: V5 stage sink leaves, source leaves, and roots; R1 stage flowers, petioles, source leaves, pollen, and roots; R3 stage pods and immature seeds; and R5 source leaves; and R8 seed cotyledons and seed embryos. Tables 8-17 show the range and average GUS expression for each sampled tissue transformed with each construct.

[0206] Table 8. Average and range of GUS expression driven by P-Gm.01G238800:1 and T-Gm.01G238800:1 in stable soybean transformed plants.

[0207]

[0208]

[0209] Table 9. Average and range of GUS expression driven by P-Gm.19G007700:1 and T-Gm.19G007700:1 in stable soybean transformed plants.

[0210]

[0211] Table 10. Average and range of GUS expression driven by P-Gm.05G007100:1 and T-Zm.GST59.nno:1 in stable soybean transformed plants.

[0212]

[0213]

[0214] Table 11. Average and range of GUS expression driven by P-Gm.16G089000:1 and T-Gm.16G089000:1 in stable soybean transformed plants.

[0215]

[0216] Table 12. Average and range of GUS expression driven by P-Gm.16G089000_trunc:1 and T-Gm.16G089000:1 in stable soybean transformed plants.

[0217]

[0218]

[0219] Table 13. Average and range of GUS expression driven by P-Gm.15G057600:1 and T-Gm.15G057600:1 in stable soybean transformed plants.

[0220]

[0221] Table 14. Average and range of GUS expression driven by P-Gm.07G156100:1 and T-Gm.07G156100:1 in stable soybean transformed plants.

[0222]

[0223]

[0224] Table 15. Average and range of GUS expression driven by P-Gm.07G156100_trunc:1 and T-Gm.07G156100:1 in stable soybean transformed plants.

[0225]

[0226] Table 16. Average and range of GUS expression driven by P-Gm.17G020600:1 and T-Zm.GST59.nno:1 in stable soybean transformed plants.

[0227]

[0228]

[0229] Table 17. Average and range of GUS expression driven by P-Gm.02G101100:1 and T-Zm.GST59.nno:1 in stable soybean transformed plants.

[0230]

[0231] As can be seen from Table 8 to Table 17, the expression elements derived from each soybean gene have unique expression patterns. Compared with V5 and R1 roots, the expression elements in constructs-6 to constructs-9 show higher levels of average GUS expression in V5 and R1 source leaves. For example, compared with roots at the same growth stage, the expression element of construct-6 drives the average GUS expression in V5 and R1 source leaves ten times higher. The expression element of construct-7 can drive very high average GUS expression in V5 source leaves, R1 petioles and source leaves, while the average GUS expression in V5 and R1 roots is about twenty times lower than that in source leaves.

[0232] Having shown and described the principles of the invention, it will be apparent to one skilled in the art that the invention may be modified in arrangement and detail without departing from such principles. We claim protection for all modifications within the spirit and scope of the claims. All publications herein are incorporated herein by reference to the same extent as if each individual publication or patent application was expressly and individually indicated to be incorporated herein by reference.

Claims

1. A recombinant DNA polynucleotide comprising a DNA sequence selected from the group consisting of: a) a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-23; b) a sequence comprising any one of SEQ ID NOs: 1-23; and c) a fragment of any one of SEQ ID NOs: 1-23, wherein the fragment comprises gene regulatory activity; Wherein the DNA sequence is operably linked to a heterologous transcribable 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 any one of SEQ ID NOs: 1-23.

3. The recombinant DNA polynucleotide of claim 2, wherein the DNA sequence has at least 95% sequence identity with the DNA sequence of any one of SEQ ID NOs: 1-23.

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

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

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

7. The recombinant DNA polynucleotide of claim 5, wherein the gene of agronomic value confers resistance to plant pests.

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

9. A transgenic plant cell comprising a recombinant DNA polynucleotide comprising a DNA sequence selected from the group consisting of: a) a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-23; b) a sequence comprising any one of SEQ ID NOs: 1-23; and c) a fragment of any one of SEQ ID NOs: 1-23, wherein the fragment comprises gene regulatory activity; Wherein the DNA sequence is operably linked to a heterologous transcribable 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 part thereof, comprising the recombinant DNA polynucleotide of claim 1.

13. A progeny plant or part thereof of the transgenic plant of claim 12, wherein the progeny plant or part thereof comprises the recombinant DNA polynucleotide of claim 1.

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

15. A method for producing a commercial product, comprising obtaining the transgenic plant or part thereof according to claim 12 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, starches, grains, plant parts, seed oils, biomass, flours, and meals.

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

Images