Plant regulatory elements and their use for automated excision

Abstract

Recombinant DNA molecules and constructs are provided for use in regulating gene expression in plants. One or more expression cassettes of the recombinant DNA molecule or construct can be excised from the transgenic plant after transformation by expressing a site-specific recombinase encoded by the recombinant DNA molecule or construct, through the presence of flanking site-specific recombination sites in the recombinant DNA molecule or construct. Such a recombinase system can be used to remove one or more expression cassettes from plants transformed with the recombinant DNA construct or vector. The recombinase transgene can be operatively linked to a promoter suitable for automatic excision in the transformed plant without hybridization with different transgenic lines expressing the recombinase. Methods for inducing automatic excision of one or more expression cassettes in transgenic plants are also provided, as well as plants and cells containing or transformed with the recombinant DNA molecule or construct of this disclosure.

Description

Citation of relevant applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 639,891, filed April 29, 2024, which is incorporated herein by reference in its entirety.

[0002] Merging of sequence lists The file named “MONS591WO_2_ST26.xml” contains a sequence list of 52,107 bytes (as measured in Microsoft Windows®), was created on April 14, 2025, and was submitted electronically with this document and incorporated herein by reference. Technical Field

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

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

[0005] The use of transgenic technology has provided many beneficial traits for agricultural purposes, but it has also encountered some challenges. One concern involves the presence of marker genes in transgenic crops that confer resistance to antibiotics or herbicides. Furthermore, there may be other transgenic cassettes or DNA sequences that are designed for specific purposes and present in the initial transformation, but are not needed in the final transgenic product. In the field of plant biotechnology, the removal of such marker genes and other unwanted expression cassettes and DNA sequences is highly desirable.

[0006] Many strategies have been devised to produce marker-free transgenic plants. For example, the marker gene expression cassette can be removed using a dual T-DNA transformation system or a site-specific recombinase system.

[0007] The dual T-DNA transformation system utilizes a plant binary transformation vector containing two separate T-DNAs (dual T-DNA transformation system). One T-DNA contains a marker gene expression cassette. The other T-DNA contains one or more expression cassettes designed to retain one or more genes of interest in the transgenic plant. Plant cells can be transformed using Agrobacterium (…). Agrobacterium Transformation is performed via T-DNA mediated by [a specific gene]. Each T-DNA can be integrated into a separate chromosome of the genome of the transformed plant cell. After transformation and plant regeneration, R0 plants self-pollinate to produce R1 progeny. R1 progeny plants are selected that have T-DNA containing one or more expression cassettes intended for use in the final transgenic product, but lack T-DNA containing one or more marker gene expression cassettes (see, for example, Komari, T. et al., Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by [a specific gene]). Agrobacterium tumefaciens and segregation of transformants free from selection markers, The Plant Journal 10 (1): 165-174, 1996). The double T-DNA transformation system has some drawbacks in terms of efficiency. In the double T-DNA transformation system, the transformant R0 plant can have more than one copy of one or two T-DNAs. Such plants may have to be excluded, and the percentage of selected plants with only one copy of each T-DNA may be low.

[0008] Another system for removing marker gene expression cassettes from transgenic plants relies on excision using site-specific recombinases. Various site-specific recombinases can be used, such as Cre recombinase, Flp recombinase (Lyznik, L. et al., Gene Transfer Mediated by Site-Specific Recombination Systems). Plant Molecular Biology Manual , N1: 1-26, 2000), R recombinase (Machida, C. et al., Use of the R-RS Site-Specific Recombination System in Plants, Plant Molecular Biology Manual (N2: 1-23, 2000) or Gin recombinase (Maeser S. et al., The Gin-recombinase of phage Mu can catalyze site-specific recombination in plant protoplasts, Mol Gen Genet (230: 170-176, 1991). Within a construct (such as a T-DNA insert), one or more marker gene expression cassettes are flanked by site-specific recombinase recognition sequences, such that the construct sequence between the site-specific recombinase recognition sequences can be excised by recombinase expression. One or more expression cassettes intended to be retained in the transgenic plant after excision exist within the construct outside of the site-specific recombinase recognition sequences.

[0009] Expression cassettes flanked by site-specific recombinase recognition sequences can be removed using hybridization strategies or by automatic excision. In hybridization strategies, plants preferably homozygous for the presence of the construct (e.g., R1 progeny) are hybridized with another transgenic plant line transformed using an expression cassette for expressing a site-specific recombinase. The resulting F1 progeny are then selected for the presence of the construct, and the expression cassette flanked by the site-specific recombinase recognition sequence is excised. In the case of automatic excision, additional expression cassettes encoding the site-specific recombinase are present in the construct, which has one or more other expression cassettes flanked by the site-specific recombinase recognition sequence to be excised, such that all such expression cassettes are excised by the site-specific recombinase. Predictable and robust control of site-specific recombinase expression is crucial for efficient automatic excision without reducing transformation efficiency. Typically, promoters will have a preference or specificity for driving expression in a particular type of cell or tissue. Not all promoters and expression elements are suitable for efficient automatic excision, and experiments are required to identify the correct promoters, introns, and 3' UTRs to drive recombinase expression to obtain the desired excision frequency and results.

[0010] There is a need to drive efficient automatic excision in one or more crop plants, preferably with expression elements that do not reduce transformation efficiency. This disclosure provides several expression elements identified through years of experimental testing, which can be used to drive the expression of recombinases and, after transformation, generate marker and / or recombinase transgenes and / or one or more other expression cassettes for efficient automatic excision in multiple crop species. Summary of the Invention

[0011] This disclosure provides gene regulatory elements for driving site-specific recombinases in plants, said site-specific recombinases causing efficient automatic excision of marker gene expression cassettes and expression cassettes for genome editing. This disclosure also provides recombinant DNA molecules comprising regulatory elements. This disclosure further provides recombinant DNA constructs comprising regulatory elements. In some embodiments, the regulatory element is operatively linked to a site-specific recombinase. In other embodiments, the regulatory element is contained within a construct containing at least three transgenic cassettes. This disclosure also provides methods for using the regulatory element and methods for manufacturing and using recombinant DNA molecules and constructs comprising said regulatory element. This disclosure also provides a synthetic Cre recombinase coding sequence for expression in plant cells.

[0012] Therefore, in one aspect, this disclosure provides a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: (a) a DNA sequence having at least 85% sequence identity with any one of SEQ ID NO: 1-14; (b) a DNA sequence comprising any one of SEQ ID NO: 1-14; and (c) (i) a fragment of any one of SEQ ID NO: 1-14 or (ii) a DNA sequence having at least 85% sequence identity with any one of SEQ ID NO: 1-14, wherein the fragment has gene regulatory activity, and wherein the DNA sequence is operatively ligated to a heterologous transcribed DNA sequence. In specific embodiments, the DNA sequence may have at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the DNA sequence of any one of SEQ ID NO: 1-14. In other embodiments, the recombinant DNA molecule may comprise a DNA sequence that may have gene regulatory activity, or may have promoter activity, or may have 3' UTR activity. In some embodiments, the heterologous transcribed DNA sequence may encode a site-specific recombinase. In a particular embodiment, the site-specific recombinase may be selected from the group consisting of Cre recombinase, Flp recombinase, R recombinase, and Gin recombinase; or wherein said site-specific recombinase is a Cre recombinase. In another aspect, this disclosure provides a recombinant DNA construct comprising a recombinant DNA molecule and further comprising (i) an expression cassette containing a selectively labeled transgene; and / or (ii) an expression cassette encoding a site-specific nuclease; and / or (iii) one or more expression cassettes encoding one or more guide RNAs; and / or (iv) an expression cassette containing a transgene of agronomical value. In a specific embodiment, the recombinant DNA construct may further comprise a pair of site-specific recombination site sequences flanking one or more of the following: the recombinant DNA molecule and / or the expression cassette containing a selectively labeled transgene; and / or the expression cassette containing a site-specific nuclease; and / or one or more expression cassettes encoding one or more guide RNAs, wherein the site-specific recombination site is cleavable by the site-specific recombinase.In other embodiments, the site-specific recombination sequence pairs may be arranged head-to-tail, and / or each site-specific recombination sequence pair may be selected from the group consisting of LoxP, FRT, RS, and GIX; or each site-specific recombination sequence pair may be a LoxP sequence; or each site-specific recombination sequence pair may contain SEQ ID NO:18. In certain embodiments, selectively labeled transgenes may confer resistance to herbicides or antibiotics. In other embodiments, transgenes with agronomical value may confer herbicide tolerance to plants, or may confer resistance to plant pests or diseases, or may confer increased yield or stress tolerance to plants, or may encode dsRNA, miRNA, or siRNA. In some embodiments, the guide RNA may contain a targeting sequence that targets a sequence in the genome of a eukaryotic cell or plant cell for genome editing or site-specific integration. In some embodiments, the site-specific nuclease may be an RNA-guided endonuclease; or the RNA-guided endonuclease may be selected from the group consisting of: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12a, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2 The RNA-guided endonuclease may be Cas12a. Alternatively, this disclosure provides a DNA transformation vector comprising a recombinant DNA molecule or a recombinant DNA construct. In some embodiments, the recombinant DNA molecule or recombinant DNA construct contains a heterologous transcribed DNA sequence that encodes a site-specific recombinase. In some embodiments, the DNA transformation vector may also comprise a T-DNA segment defined by a left and a right boundary. In a specific embodiment, the heterologous transcribed DNA sequence encoding the site-specific recombinase may be located between the left and right boundaries of the T-DNA segment. In other embodiments, one or more of the following may be located between the left and right boundaries of the T-DNA segment: the heterologous transcribed DNA sequence encoding the site-specific recombinase, and / or a selectively labeled transgene, and / or an expression cassette encoding the site-specific nuclease, and / or one or more expression cassettes encoding one or more guide RNAs, and / or a transgene with agronomical value.In another aspect, this disclosure provides a transgenic plant, plant part, or plant cell or bacterial cell comprising a recombinant DNA molecule or recombinant DNA construct. In some embodiments, the recombinant DNA molecule or recombinant DNA construct is stably transformed into the genome of the transgenic plant, plant part, or plant cell. In other embodiments, the transgenic plant, plant part, or plant cell may be selected from the group consisting of maize, soybean, cotton, or rapeseed plants, plant parts, or plant cells. Another aspect of this disclosure is a method for producing a transgenic plant or plant part, the method comprising the steps of: (a) transforming plant cells of an explant with a DNA molecule or DNA transformation vector comprising a recombinant DNA molecule or recombinant DNA construct; and (b) regenerating or developing a transgenic plant from the explant, wherein the transgenic plant comprises a recombinant DNA construct stably transformed into the genome of one or more cells of the transgenic plant. In some embodiments, the method may further comprise the steps of (c) isolating or harvesting plant parts from the transgenic plant; and / or (d) hybridizing one or more of the progeny plants with itself or another plant. In some embodiments, plant cells may be transformed via Agrobacterium-mediated transformation, Rhizobium-mediated transformation, microsphere-mediated transformation, or particle bombardment-mediated transformation. In other embodiments, the transgenic plant, plant part, or plant cell may be selected from the group consisting of: maize, soybean, cotton, or rapeseed plants or plant cells. Another aspect of this disclosure is a method for excising an expression cassette from the genome of a transgenic plant, the method comprising the steps of: (a) transforming explant plant cells with a recombinant DNA construct or a DNA transformation vector containing said recombinant DNA construct, wherein said recombinant DNA construct or DNA transformation vector contains a pair of site-specific recombination site sequences flanking one or more of the following: a recombinant DNA molecule and / or an expression cassette containing a selectively labeled transgene; and / or an expression cassette containing a site-specific nuclease; and / or one or more expression cassettes encoding one or more guide RNAs, wherein said site-specific recombination site is capable of being site-specifically recombined. (a) cleavage of histase, and containing a heterologous transcribed DNA sequence coding site-specific recombinase in a recombinant DNA construct or DNA transformation vector for transformation; (b) at least partially regenerating or developing or obtaining a transgenic plant from one or more stably transformed plant cells; (c) hybridizing the transgenic plant with itself or another plant; and (d) selecting one or more progeny plants in which one or more of the heterologous transcribed DNA sequence of the coding site-specific recombinase between the site-specific recombinant site sequence pairs and / or the selectively labeled transgene and / or the expression cassette encoding guide RNA are excised and are no longer present in the genome of the progeny plant.In some embodiments, plant cells may be transformed via Agrobacterium-mediated transformation, Rhizobium-mediated transformation, microsphere-mediated transformation, or particle bombardment-mediated transformation. In other embodiments, the transgenic plant or plant cells may be selected from the group consisting of maize, soybean, cotton, or rapeseed plants or plant cells. In another aspect, this disclosure provides a recombinant DNA molecule comprising a DNA sequence having at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity with SEQ ID NO:21, wherein said DNA sequence is a Cre recombinase encoding sequence.

[0013] In another aspect, this disclosure provides a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: (a) a DNA sequence having at least 85% identity with any one of SEQ ID NO: 1-14; (b) a DNA sequence comprising any one of SEQ ID NO: 1-14; and (c) (i) a fragment of any DNA sequence having at least 85% sequence identity with any one of SEQ ID NO: 1-14 or (ii) a fragment of any DNA sequence having at least 85% sequence identity with any one of SEQ ID NO: 1-14, wherein the fragment may contain gene regulatory activity, and wherein the DNA sequence is operatively linked to a heterologous transcribed DNA sequence that encodes a gene of agronomical value or that encodes dsRNA, miRNA, or siRNA. In some embodiments, the gene of agronomical value may confer herbicide tolerance to plants or may confer pest resistance to plants. Another aspect of this disclosure is a transgenic plant, plant part, plant cell, transgenic plant seed, or its progeny plant or plant part comprising a recombinant DNA molecule containing a DNA sequence selected from the group consisting of: (a) a DNA sequence having at least 85% sequence identity with any one of SEQ ID NO: 1-14; (b) a DNA sequence comprising any one of SEQ ID NO: 1-14; and (c) (i) a fragment of a DNA sequence having at least 85% sequence identity with any one of SEQ ID NO: 1-14 or (ii) a DNA sequence having at least 85% sequence identity with any one of SEQ ID NO: 1-14, wherein the fragment has gene regulatory activity, wherein the DNA sequence is operatively linked to a heterologous transcribed DNA molecule that encodes a gene of agronomical value or that encodes dsRNA, miRNA, or siRNA. In some embodiments, the gene of agronomical value may confer herbicide tolerance or pest resistance to the plant. In a specific embodiment, the transgenic plant cell may be a monocotyledonous plant cell or a dicotyledonous plant cell. On the other hand, this document provides a method for producing a commercial product, the method comprising obtaining a transgenic plant or a portion thereof. In some embodiments, the commercial product may be selected from the group consisting of: seeds, processed seeds, protein concentrates, protein isolates, starch, cereals, plant parts, seed oils, biomass, flour, and semolina. Yet another aspect of this disclosure is a method for expressing a transcribed DNA molecule, the method comprising obtaining a transgenic plant and culturing the plant, wherein the transcribed DNA molecule is expressed.

[0014] These and other features disclosed herein are set forth in this document.

[0015] Brief description of the sequence SEQ ID NO:1 is the DNA sequence of the promoter P-Gm.CALa, which contains a 5' promoter operatively linked to its natural leader sequence.

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

[0017] SEQ ID NO:3 is the DNA sequence of promoter P-Gm.Mads17, which contains a 5' promoter operatively linked to its natural leader sequence.

[0018] SEQ ID NO:4 is the DNA sequence of 3' UTR, T-Gm.Mads17.

[0019] SEQ ID NO:5 is the DNA sequence of promoter P-Gm.AP1, which contains a 5' promoter operatively linked to its natural leader sequence.

[0020] SEQ ID NO:6 is the DNA sequence of 3' UTR, T-Gm.AP1.

[0021] SEQ ID NO:7 is the DNA sequence of promoter P-Gm.10G071400, which contains a 5' promoter operatively linked to its natural leader sequence.

[0022] SEQ ID NO:8 is the DNA sequence of 3' UTR, T-Gm.10G071400.

[0023] SEQ ID NO:9 is the DNA sequence of promoter P-Gm.16G200800, which contains a 5' promoter operatively linked to its natural leader sequence.

[0024] SEQ ID NO:10 is a 3' UTR, T-Gm.16G200800 DNA sequence.

[0025] SEQ ID NO:11 is the DNA sequence of promoter P-Gm.11G080000, which contains a 5' promoter operatively linked to its natural leader sequence.

[0026] SEQ ID NO:12 is the DNA sequence of 3' UTR, T-Gm.11G080000.

[0027] SEQ ID NO:13 is the DNA sequence of promoter P-Gm.02G121600, which contains a 5' promoter operatively linked to its natural leader sequence.

[0028] SEQ ID NO:14 is a 3' UTR, T-Gm.02G121600 DNA sequence.

[0029] SEQ ID NO:15 is the DNA sequence of promoter P-At.Erl1, which contains a 5' promoter operatively linked to its natural leader sequence.

[0030] SEQ ID NO:16 is the DNA sequence of 3' UTR, T-Mt.AC140914v20.

[0031] SEQ ID NO:17 is a DNA sequence encoding a synthetic sequence for plant expression of Cre recombinase (Cre) containing a processable intron derived from the potato light-inducible tissue-specific St-LS1 gene (GenBank accession number: X04753).

[0032] SEQ ID NO:18 is the DNA sequence of the Cre recombinase recognition sequence LoxP.

[0033] SEQ ID NO:19 is a DNA sequence encoding a synthetic coding sequence of aadA, a selective marker used to select transformed plant cells using spectinomycin selection.

[0034] SEQ ID NO:20 is a synthetic coding sequence for plant expression of β-glucuronidase (GUS) containing a processable intron derived from the potato light-inducible tissue-specific St-LS1 gene (GenBank accession number: X04753).

[0035] SEQ ID NO:21 is a DNA sequence encoding a synthetic sequence for plant expression of Cre recombinase (Cre-2) containing a processable intron derived from the potato light-inducible tissue-specific St-LS1 gene (GenBank accession number: X04753). Detailed Implementation

[0036] This disclosure provides gene regulatory elements for driving the expression of site-specific recombinases in plants, the expression of which leads to efficient automatic excision of marker gene expression cassettes. This disclosure also provides recombinant DNA molecules comprising regulatory elements, DNA constructs, and DNA transformation vectors. The nucleotide sequences of these gene regulatory elements are provided as SEQ ID NO:1-16 or variants or fragments thereof. These gene regulatory elements are capable of efficiently regulating the automatic excision of marker gene expression cassettes. This disclosure also provides a method for automatically excising at least two transgenic expression cassettes from the genome of a transgenic plant using a construct comprising a transgenic cassette, wherein the gene regulatory elements described herein are operatively linked to site-specific recombinase genes.

[0037] The following definitions are provided for certain terms and phrases used herein to define and clarify their meaning with reference to relevant embodiments of this disclosure as used herein, and to guide those skilled in the art in understanding this disclosure. Unless otherwise defined in this disclosure, the terms and phrases used herein should be understood according to their conventional meaning in the relevant fields, particularly in molecular biology and plant transformation. 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, 8th edition, Oxford University Press: New York , 2014; and Lewin, Genes IX, Oxford University Press: New York , 2007. (e.g., 37 CFR) The nomenclature of DNA bases as described in 1.822 is adopted and is set forth in Tables 1 and 3 of Annex I of WIPO Standard ST.26 (2021).

[0038] When describing elements of this disclosure or its embodiments, the articles “a”, “an”, “the”, and “said” are intended to mean the presence of one or more elements.

[0039] When used in a list of two or more items, the term “and / or” means any one of the items, any combination of items, or all items related to the term.

[0040] The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that additional elements may be present in addition to those listed. For example, any method that “comprising,” “having,” or “including” one or more steps is not limited to having only those steps and may also cover other steps not listed. Similarly, any composition or apparatus that “comprising,” “having,” or “including” one or more features is not limited to having only those features and may also cover other features not listed.

[0041] Site-specific recombinases and DNA segment excision As used herein, a “site-specific recombinase” is an enzyme that binds to a specific DNA recognition sequence and catalyzes DNA cleavage, DNA strand exchange, and DNA rejoining between two site-specific recombinase site sequences. “Site-specific recombination,” “site-specific recombinase system,” “site-specific recombinase technology,” “site-directed recombination,” “site-directed recombinase system,” or “site-directed recombinase technology” describe various specialized recombination processes involving the mutual exchange between defined DNA sites. As used herein, the term “flanking” means that two or more sequences (such as site-specific recombinant site sequences) are located on either side of one or more specific loci, genes, sequences, transgenes, or expression cassettes. Site-specific recombinant site sequences can be cloned in a recombinant DNA construct relative to the 5' and 3' of a DNA segment containing one or more expression cassettes from which recombination will occur (i.e., flanking said DNA segment). Depending on the initial arrangement of the parental site-specific recombinant sites, site-specific recombination has one of three possible outcomes: integration (insertion of a foreign DNA segment), excision (removal of a DNA segment), or inversion (rotation of the DNA segment 180 degrees followed by rejoining of the two end fragments). Integration is caused by recombination between sites on different DNA molecules (provided that at least one of the parent chromosomes is circular).

[0042] For recombination sites located on the same DNA molecule or chromosome, the results can be determined by their relative orientation. Inversions of DNA segments can be caused by exchanges between inverted (head-to-head) sites, while excisions can be caused by recombination between sites in a head-to-tail orientation (Grindley ND et al., Mechanisms of Site-Specific Recombination). Annu. Rev. Biochem, 75: 567-605, 2006). Various site-specific recombinases can be used to excise DNA between two site-specific recombinase recognition sites, such as Cre recombinase recognizing the Lox site and Flp recombinase recognizing the FRT site (see, for example, Lyznik L. et al., Gene Transfer Mediated by Site-Specific Recombination Systems, 75: 567-605, 2006). Plant Molecular Biology Manual (N1: 1-26, 2000), R recombinases that recognize RS sites (see, for example, Machida C. et al., Use of the R-RS Site-Specific Recombination System in Plants, N1: 1-26, 2000), Plant Molecular Biology Manual (N2: 1-23, 2000) or Gin recombinases that recognize the GIX site (see, for example, Maeser S. et al., The Gin recombinase of phage Mu can catalyze site-specific recombination in plant protoplasts, Mol Gen Genet (230: 170-176, 1991). Each of the above site-specific recombinase systems has been shown to function in plants. The Cre / Lox site-specific recombinase system is a highly dependent marker-excision system in plant biotechnology.

[0043] Site-specific recombinases are used in plant biotechnology to remove marker gene expression cassettes, as well as other expression cassettes and DNA segments, from transgenic plants. Typically, plants are transformed using recombinant DNA constructs or vectors containing multiple expression cassettes. Expression cassettes can be used to express transgenes that provide advantageous traits to the plant, as well as transgenes used as markers for selecting transformed plant cells, such as antibiotic resistance genes, herbicide tolerance genes, or other transgenes useful in the selection process. The marker gene cassette is flanked by a pair of site-specific recombinase recognition sites. After transformation and selection, regenerated transformed plants grow. The marker gene can then be excised using various hybridization strategies, either by hybridization with a site-specific recombinase-expressing plant line or by automatic excision.

[0044] Hybridization using site-specific recombinase-expressing plant lines is typically performed as follows: Plants allowed to transform to R0 (e.g., plants transformed with a recombinant DNA construct containing one or more desired expression cassettes, wherein one or more marker gene expression cassettes may be laterally linked with a site-specific recombinase recognition sequence) are self-crossed. R1 progeny plants are then selected for the presence of the recombinant DNA construct. The selected R1 progeny plants are then self-crossed, and R2 progeny plants homozygous for the insertion of the recombinant DNA construct are selected. The homozygous R2 progeny plants are then hybridized with another line expressing the recombinase. This hybridization results in the recombinase excising one or more marker gene expression cassettes laterally linked with a site-specific recombinase recognition sequence, thereby producing F1 progeny plants containing one or more desired expression cassettes, but with the one or more marker gene expression cassettes excised from the genome; that is, the one or more marker gene expression cassettes are no longer present in the F1 progeny genome, only the one or more desired expression cassettes are present. The resulting F1 progeny are then self-crossed, and F2 progeny plants lacking the recombinase but homozygous for the insertion of the now-modified recombinant DNA construct are selected.

[0045] Another strategy for removing one or more marker gene expression cassettes is through automatic excision. Similar to the excision methods described above, the expressed recombinase is used to excise one or more marker gene expression cassettes, but instead of hybridizing the transformed plant with another line expressing the recombinase, the recombinase gene expression cassette is positioned within the same recombinant DNA construct used for transformation and lateralized with a site-specific recombinase site sequence and one or more marker gene expression cassettes. One or more expression cassettes intended to be retained in the transgenic plant after automatic excision exist within the recombinant DNA construct outside of the site-specific recombinase site sequence, i.e., without lateralization of the site-specific recombinase site sequence. After transformation and plant regeneration, R0 plants containing the recombinant DNA construct are produced. These R0 plants can then be self-pollinated, and the resulting R1 progeny plants can be selected for the presence of a modified recombinant DNA construct, in which one or more marker gene expression cassettes and the recombinase expression cassette have been excised. The advantage of the automatic excision system is that it removes one or more marker gene expression cassettes in fewer generations compared to using a site-specific recombinase excision system (which requires hybridization with another line expressing the site-specific recombinase).

[0046] The complexity of automatic excision lies in identifying gene expression elements (also known as "expression elements," "regulatory elements," or "gene regulatory elements") that provide expression of site-specific recombinases at the correct developmental stage and in the correct tissue to enable automatic excision and produce label-free R1 progeny plants. Not all expression elements will provide a successful outcome where automatic excision occurs effectively. Furthermore, expression elements may only provide effective automatic excision in specific crop species (such as maize, soybean, or cotton, but not all three). Therefore, numerous experiments have been conducted to identify the expression elements disclosed herein.

[0047] DNA molecules As used herein, the terms “DNA,” “DNA molecule,” “DNA polynucleotide,” and “nucleic acid molecule” refer to a double-stranded DNA molecule of genomic or synthetic origin, i.e., a polymer of deoxyribonucleotide bases. DNA consists of two polynucleotide chains. As used herein, the term “DNA sequence” refers to the nucleotide sequence of a DNA molecule read from the 5' (upstream) end to the 3' (downstream) end, i.e., a continuous nucleotide sequence within a DNA molecule. As used herein with respect to DNA sequences or nucleotides of DNA molecules, the terms “continuous” and “adjacent” are interchangeable and synonymous, and refer to DNA polynucleotides or nucleotides linked together in a DNA sequence, strand, or molecule without any gaps or breaks between them.

[0048] As used herein, a “recombinant DNA molecule” or a “recombinant DNA construct” is a DNA molecule or construct comprising a combination of DNA sequences that would not naturally coexist without human intervention. For example, a recombinant DNA molecule may comprise at least two DNA sequences that are heterologous to each other, DNA sequences that deviate from DNA sequences found in nature, synthetic DNA sequences, and / or DNA sequences that have been incorporated into the genomic DNA of a host cell, for example, through genetic transformation, genome editing, or site-specific integration.

[0049] As used herein, a “synthetic nucleotide sequence” or “artificial nucleotide sequence” or “synthetic coding sequence” is a nucleotide sequence that is known not to exist in nature or is not naturally occurring. An example of a synthetic coding sequence is shown in SEQ ID NO:17.

[0050] The use of the term "isolated DNA molecule" or equivalent in this application is intended to mean a DNA molecule that exists alone or in combination with other compositions but is not in its natural environment. For example, a nucleic acid element (such as a coding sequence, intron sequence, untranslated sequence, leader sequence, promoter sequence, transcription termination sequence, etc.) that is naturally present in the genome of an organism is not considered "isolated" as long as it is natural with respect to the genome of the organism and is located in its naturally occurring genome. However, an element is considered "isolated" within the scope of this disclosure as long as each of these elements and a sub-part of these elements is not present in its naturally occurring genome and / or not located in its naturally occurring genome. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA inserted into the genome of a plant or bacterial cell or present in an extrachromosomal vector, will be considered an isolated nucleotide sequence, whether it is present in a plasmid or similar vector used for transforming cells, in the genome of a plant or bacteria, or in a detectable amount in tissues, progeny, biological samples, or commercial products derived from plants or bacteria.

[0051] As used herein, the term "sequence identity" refers to the degree to which two optimally aligned polynucleotide sequences or two optimally aligned polypeptide sequences are identical. Optimal sequence alignment of two sequences (e.g., a reference sequence and another sequence) is achieved by aligning the two sequences to maximize the number of nucleotide matches with appropriate internal nucleotide insertions, deletions, or gaps in the sequence alignment. As used herein, the term "reference sequence" may refer to a DNA sequence comprising one or more of SEQ ID NO:1-16.

[0052] As used herein, the terms “sequence identity percentage” or “sequence identity %”, “identity percentage”, and “identity %” refer to the identity score of two best-aligned sequences multiplied by 100. The “identity score” of a best-aligned sequence to a reference sequence is the number of nucleotide matches in the best alignment divided by the total number of nucleotides in the reference sequence (i.e., the total number of nucleotides in the entire length of the reference sequence). Therefore, some embodiments of this disclosure provide recombinant DNA molecules comprising a DNA sequence that, upon optimal alignment with a reference sequence (such as one of SEQ ID NO: 1-16), has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 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%, at least 99%, or 100% identity with the reference sequence. According to embodiments of this disclosure, the DNA sequence can be operatively ligated to a heterologous transcribed DNA sequence that encodes a site-specific recombinase.

[0053] Control element Regulatory elements (also known as “expression elements” or “gene expression elements”) such as promoters, leader sequences, enhancers, introns, and transcription termination regions (also known as 3' UTRs) play an essential role in the overall expression of genes in living cells. As used herein, the term “regulatory element” or “expression element” refers to a DNA molecule or sequence or segment of DNA that has gene regulatory activity. As used herein, the term “gene regulatory activity” refers to the ability to influence the expression of operably linked transcribed DNA molecules, for example, by affecting the transcription and / or translation of operably linked transcribed DNA molecules. Regulatory elements that function in plants, such as promoters, leader sequences, enhancers, introns, and 3' UTRs, can be used to modify plant phenotypes through genetic engineering.

[0054] According to embodiments of this disclosure, the regulating element is a promoter having a sequence comprising SEQ ID NO:1, 3, 5, 7, 9, 11, 13 or 15, or having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 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%, at least 99%, or 100% identity with SEQ ID NO:1, 3, 5, 7, 9, 11, 13 or 15, or any of the aforementioned sequences, including a promoter operatively connected to its natural leader sequence. According to embodiments of this disclosure, the regulatory element is a leader sequence having a sequence included in SEQ ID NO:1, 3, 5, 7, 9, 11, 13 or 15, or a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 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%, at least 99%, or 100% identity with the leader sequence included in SEQ ID NO:1, 3, 5, 7, 9, 11, 13 or 15, or any of the aforementioned sequences, a functional fragment or portion that can influence the expression of an operably linked transcribed DNA sequence.

[0055] As used herein, a “fragment” of a regulatory element comprises a fragment or portion of a regulatory element, and a “functional fragment” of a regulatory element comprises a fragment or portion of a regulatory element that influences, regulates, or drives the expression of an operably linked transcribed DNA sequence, i.e., has gene regulatory activity. According to some embodiments, the “functional fragment” of a regulatory element influences, regulates, or drives the expression of an operably linked transcribed DNA sequence in a manner similar to that of the corresponding regulatory element. According to another embodiment, a fragment of any one of SEQ ID NO:1-16 is provided, which may have gene regulatory activity. In other embodiments, a fragment of any one of SEQ ID NO:1-16 is provided, which may exhibit gene regulatory activity similar to that of its corresponding regulatory element. In yet another embodiment, a fragment of any one of SEQ ID NO:1-16 is provided, which may contain a reference sequence from which it is derived, i.e., has the same or similar gene regulatory activity as any one of SEQ ID NO:1-16.

[0056] Regulatory elements can be characterized by their associated gene expression patterns in plants, plant tissues, and plant cells, for example, by their positive and / or negative effects on expression, such as constitutive expression or specific expression patterns, such as temporal, spatial, developmental, tissue, environmental, physiological, pathological, or cell cycle expression and / or chemical responses or inducible expression, and any combination thereof, as well as by quantitative or qualitative indications or expression patterns. As used herein, a “gene expression pattern” is any pattern in which operatively linked DNA molecules are transcribed into transcribed RNA molecules, resulting in the relative levels and abundance of transcribed RNA molecules in various plant tissues and cells during development. Regulatory elements may include enhancers, promoters, leader sequences, introns, and / or 3' UTRs.

[0057] As used herein, the term "promoter" generally refers to a DNA molecule, segment, or sequence that participates in recognizing and binding RNA polymerase II and other proteins (such as trans-acting transcription factors) to initiate or regulate transcription. A promoter may initially separate from the upstream of a copy of the gene's genome or from the 5' untranslated region (5' UTR). Alternatively, a promoter may be a synthetically produced or engineered DNA molecule. Promoters can also be chimeric. Chimeric promoters are produced by the fusion of two or more heterologous DNA molecules. Promoters that can be used to practice embodiments of the present disclosure may include promoter elements comprising sequences of SEQ ID NO:1, 3, 5, 7, 9, 11, 13 or 15 or sequences having at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity. In specific embodiments, DNA molecules as described herein, and any variants, fragments, portions, or derivatives thereof, are further defined as containing promoter activity, i.e., capable of acting as a promoter in a host cell (such as in transgenic plants). In other specific embodiments, fragments of promoter sequences may be defined as exhibiting the promoter activity of the initiator promoter molecule from which they originate, or fragments may contain a “minimal promoter” that provides basal levels of transcription and contains a TATA box or equivalent DNA sequence for recognizing and binding the RNA polymerase II complex to initiate transcription.

[0058] In one embodiment, a fragment of the promoter sequence disclosed herein is provided. The promoter fragment may contain the promoter activity or gene regulatory activity as described above, and may be used alone or in combination with other promoters and / or promoter fragments, such as in the construction of chimeric promoters, or in combination with other expression or regulatory elements and expression or regulatory element fragments. In specific 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, or at least about 1500 consecutive nucleotides or longer from a reference sequence from which it is derived, namely any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15 (such as promoters, promoter sequences, or DNA molecules having promoter or gene regulatory activity disclosed herein). Fragments of any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 may have the activity of the base sequence, such as the promoter activity of the base sequence.

[0059] A recombinant DNA molecule or construct comprising any promoter element provided in SEQ ID NO:1, 3, 5, 7, 9, 11, 13 or 15, or any sequence from any of SEQ ID NO:1, 3, 5, 7, 9, 11, 13 or 15, such as an internal or truncated sequence or a sequence with a 5' deletion, may be modified or altered for expression using methods known in the art, such as by removing one or more elements or portions of one or more elements or one or more nonfunctional spacer sequences that may have a positive or negative effect on expression; replicating elements that have a positive or negative effect on expression; inserting elements that have a positive or negative effect on expression; and / or replicating or removing elements that have tissue-specific, developmental or cell-specific effects on expression. Any recombinant DNA construct or molecule containing a promoter or regulatory element can be used, for example, to prepare enhancer elements, wherein the promoter or regulatory element is derived from any promoter element provided as in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, or 15, and contains a 3' deletion, wherein the TATA box element or its equivalent sequence and downstream sequence are removed. Further deletions can be made to remove any elements that have a positive or negative effect on expression; tissue-specific; cell-specific; or time-specific (such as, but not limited to, circadian rhythms or developmental timing). Any promoter element provided as in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, or 15, and fragments or enhancers derived therefrom, can be used to prepare chimeric transcriptional regulatory element compositions.

[0060] According to this disclosure, promoters or promoter fragments can be analyzed against known promoter elements, i.e., DNA sequence features, such as the presence of TATA boxes and other known transcription factor binding site motifs. Those skilled in the art can use the identification of such known promoter elements to design promoter variants that have similar expression patterns to the original promoter.

[0061] As used herein, the term "leader sequence" refers to a DNA molecule that can be initially separated from the upstream or 5' untranslated region (5' UTR) of a gene's genomic copy, and is generally defined as a nucleotide segment between the transcription start site (TSS) and the protein-coding sequence start site. Alternatively, a leader sequence can be a synthetically produced or engineered DNA element. Leader sequences can be used as 5' regulatory elements for regulating the expression of operatively linked, transcribed DNA sequences. Leader sequences can be used with heterologous promoters or with their native promoters. The leading sequence that can be used to practice the embodiments of this disclosure may include the leading sequence contained in SEQ ID NO:1, 3, 5, 7, 9, 11, 13 or 15, or a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 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%, at least 99% or 100% identity with the leading sequence contained in SEQ ID NO:1, 3, 5, 7, 9, 11, 13 or 15, or a fragment or functional fragment or portion of any of the foregoing sequences. In specific embodiments, such DNA sequences can be defined as capable of serving as leader sequences in host cells, including, for example, transgenic plant cells. In one embodiment, such sequences are defined as containing leader sequence activity.

[0062] The leader sequence included in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, or 15 may contain a regulatory element and / or may employ a secondary structure that influences the transcription or translation of an operably linked transcribed DNA sequence. The leader sequence included in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, or 15, or any fragment thereof, may be used according to this disclosure to prepare a chimeric regulatory element that influences the transcription or translation of an operably linked transcribed DNA sequence.

[0063] As used herein, the term "intron" refers to a DNA molecule or sequence that can be isolated or identified from a gene and can be generally defined as a region spliced ​​during pre-translational messenger RNA (mRNA) processing. Alternatively, an intron can be a synthetically produced or engineered DNA element. Introns may contain enhancer elements that influence the transcription of operably linked gene or transcribed DNA sequences. Introns can serve as regulatory elements for regulating the expression of operably linked transcribed DNA sequences. Constructs may contain introns, and introns may or may not be heterologous relative to transcribed DNA sequences. Examples of introns in the art include the rice actin intron and the maize HSP70 intron.

[0064] In plants, the presence of introns in gene constructs, relative to those lacking introns, leads to increased accumulation of mRNA and protein. This effect is known as "intron-mediated enhancement" (IME) of gene expression. Introns known in the art to stimulate expression in plants have been identified in maize genes (e.g., tubA1, Adh1, Sh1, and Ubi1), rice genes (e.g., tpi), and dicotyledonous genes such as those from petunia (e.g., rbcS), potato (e.g., st-ls1), and Arabidopsis (e.g., rbcS). Arabidopsis thaliana Those (e.g., ubq3 and pat1). It is known in the art that deletions or mutations within the splice site of an intron reduce gene expression, indicating that the IME of gene expression may require splicing. However, the IME of gene expression in dicotyledonous plants has been demonstrated by point mutations within the splice site of the pat1 gene from Arabidopsis thaliana. Multiple uses of the same intron in a single plant are known to be disadvantageous. In those cases, a set of introns is needed to construct a suitable recombinant DNA element.

[0065] As used herein, the terms “3’ transcription termination sequence,” “3’ untranslated region,” or “3’ UTR” refer to a DNA sequence transcribed into the untranslated region within the 3’ portion of an mRNA molecule, as commonly understood in the art. The 3’ untranslated region of an mRNA molecule can be generated through specific cleavage and 3’ polyadenylation, also known as the formation of a polyA tail. A 3’ UTR can be operatively linked to and downstream of the RNA or protein-coding portion of a transcribed DNA sequence and can include polyadenylation signals as well as other regulatory elements or signals capable of influencing transcription, mRNA processing, and / or gene expression. Polyadenylated tails are thought to play a role in mRNA stability and translation initiation. Examples of 3’ transcription termination molecules in the art are the 3’ region of carmine synthase, the 3’ region of wheat hsp17, the 3’ region of the small subunit of pea ribulose diphosphate carboxylase (rubisco), the 3’ region of cotton E6, and the 3’ UTR of coixin.

[0066] 3' UTRs are generally beneficial for the recombination expression of specific DNA molecules. Weak 3' UTRs can generate readthroughs, which may affect the expression of DNA molecules located in adjacent expression cassettes. Proper control of transcription termination can prevent readthroughs of downstream DNA sequences (e.g., other expression cassettes) and can further allow efficient recycling of RNA polymerase to improve gene expression. Efficient termination of transcription (release of RNA polymerase II from DNA) is a prerequisite for restarting transcription, thus directly affecting the overall transcriptional level. After transcription termination, mature mRNA is released from the site of synthesis and the template is transported to the cytoplasm. Eukaryotic mRNA accumulates in vivo in poly(A) form, making it difficult to detect transcription termination sites using conventional methods. However, it is difficult to predict functional and efficient 3' UTRs using bioinformatics methods because there are no conserved DNA sequences that allow for easy prediction of efficient 3' UTRs.

[0067] Regulating gene function through 3' UTRs is a relatively new field, as only recent sequencing technologies have enabled a deeper understanding of 3' UTRs across species and cell types. Prior to the availability of sequencing technologies, only a few model 3' UTRs were studied in detail for their functions and mechanisms. While these model 3' UTRs have made significant contributions to the overall understanding of 3' UTR biology, conclusions regarding their regulatory functions are limited and largely focused on mRNA stability. (Mayr, C., Regulation by 3'-Untranslated Regions, Annual Review of Genetics (Xie X. et al., 51: 171-194, 2017) Whole-genome computer simulation analysis revealed that the motifs in the 3' UTR are mainly conserved on one strand, consistent with 3' UTRs used to regulate gene expression at the posttranscriptional level (Xie X. et al., Systematic discovery of regulatory motifs in human promoters and 3' UTRs by comparison of several mammals, 51: 171-194, 2017). Nature 434: 338-345, 2005). 3' UTR determines protein levels by regulating mRNA stability and translation primarily mediated by AU-rich elements and miRNAs. 3' UTR also enables local translation by regulating mRNA localization. 3' UTR length can be regulated by selective cleavage and polyadenylation. 3' UTR mediates protein-protein interactions (PPIs), which have a wide range of influences on protein complex formation, protein localization, and protein function. 3' UTR regulates gene expression by binding to RNA-binding proteins (RBPs). RBPs bind to cis-elements of 3' UTRs and mediate 3' UTR function by recruiting effector proteins. RBPs synergize with other bound RBPs to achieve in vivo function specificity. The composition of RBPs bound to 3' UTRs at a given time is dynamic and can vary depending on the local environment, such as through the addition of post-translational modifications, local expression of other RBPs, and interactions with membranes and cytoskeletal filaments. RBP binding is also influenced by the formation of secondary and tertiary RNA structures that regulate 3' UTR accessibility (Mayr, C., Regulation by 3'-UntranslatedRegions). Annual Review of Genetics , 51: 171-194, 2017).

[0068] The poly(A) tail is generated by adding a series of adenosine bases to the 3' end of the RNA molecule. This provides a binding site for a class of regulatory factors called poly(A)-binding proteins (PABPs) that play a role in the regulation of gene expression, including mRNA export, stability and decay, and translation. The 5' cap structure of mRNA and the poly-A tail work synergistically to control mRNA translation. The association of PABPs with the poly(A) tail promotes the interaction with eIF4F bound to the 5' cap structure, leading to mRNA circularization, thereby promoting translation initiation and ensuring ribosome recycling and efficient translation. This interaction also allows for the inhibition of translation by repressor proteins that bind to the 3' UTR (Barret LW et al., Regulation of eukaryotic gene expression by the untranslated regions and other non-coding elements). Cell. Mol. Life Sci. 69: 3613-3634, 2012).

[0069] From a practical perspective, the following characteristics are generally beneficial for the 3' UTR used in expression cassettes. First, the 3' UTR should be able to efficiently and effectively terminate the transcription of the transgene, and it should be able to prevent the transcript from reading through to any adjacent DNA sequence that may contain another expression cassette, such as when multiple expression cassettes are located in a DNA construct or exist in adjacent chromosomal DNA that has been inserted into the construct. Second, the 3' UTR should not result in a reduction of the transcriptional activity conferred by the promoter, leader sequence, enhancer, and introns that drive the expression of the DNA sequence. Finally, in plant biotechnology, the 3' UTR is commonly used to initiate the amplification reaction of reverse-transcribed RNA extracted from transformed plants and is used for: (1) assessing transcriptional activity or expression after the expression cassette is integrated into the plant chromosome; (2) assessing the copy number of the insertion within the plant DNA; and (3) assessing the conjugation of the resulting seeds after breeding. The 3' UTR is also used for the amplification reaction of DNA extracted from transformed plants to characterize the integrity of the insertion cassette. The 3' UTR that can be used to practice the teachings of this disclosure is represented as SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16.

[0070] As used herein, the term "chimerism" refers to a single DNA molecule produced by fusing a first DNA molecule to a second DNA molecule, wherein the first and second DNA molecules would not normally exist in this configuration (i.e., fused together). Therefore, a chimeric DNA molecule is a novel DNA molecule that does not normally exist in nature. As used herein, the term "chimeric promoter" refers to a promoter produced by this manipulation of a DNA molecule. Chimeric promoters can combine two or more DNA fragments, such as the fusion of a promoter with an enhancer element. Therefore, this disclosure covers the design, construction, and use of chimeric promoters according to the methods disclosed herein for regulating the expression of operatively linked, transcribed DNA molecules.

[0071] Chimeric regulatory elements can be designed to comprise various constituent elements that can be operatively linked by various methods known in the art, such as restriction enzyme digestion and ligation, ligation-independent cloning, modular assembly of PCR products during amplification, or direct chemical synthesis of the regulatory element, as well as other methods known in the art. The resulting chimeric regulatory elements may comprise the same constituent elements or variations thereof, but differ in that they contain one or more ligation DNA sequences that allow for the operative linking of the constituent elements. In this disclosure, DNA sequences provided as SEQ ID NO:1-16 provide reference sequences for regulatory elements, wherein the constituent elements containing the reference sequences can be linked by methods known in the art and may contain substitutions, deletions, and / or insertions of one or more nucleotides or mutations naturally present in bacterial and plant cell transformations.

[0072] As used herein, the term "variant" refers to a second DNA molecule (such as a regulatory element) that is similar to but not identical to a first DNA molecule, and wherein the second DNA molecule retains the general functionality of the first DNA molecule, i.e., the same or similar expression pattern, for example, through more or less equivalent transcriptional activity. A variant can be a shortened or truncated form of the first DNA molecule or a modified form of the sequence of the first DNA molecule, such as DNA molecules with different restriction enzyme sites and / or internal deletions, substitutions, or insertions. "Variant" may also cover regulatory elements having a nucleotide sequence comprising substitutions, deletions, or insertions of one or more nucleotides of a reference sequence, wherein the derived regulatory element has more or less or equivalent transcriptional or translational activity compared to the corresponding parental regulatory molecule. Regulatory element "variants" will also cover variants resulting from mutations naturally occurring in bacterial and plant cell transformations. In this disclosure, the polynucleotide sequences provided as SEQ ID NO:1-16 can be used to generate variants that are similar to but not identical to the DNA sequence of the original regulatory element, while retaining the general functionality of the original regulatory element, i.e., the same or similar expression pattern. In view of this disclosure, the generation of such variations of this disclosure is entirely within the scope of ordinary skill in the art, and this is taken into consideration herein.

[0073] As used herein, a “transcribed DNA sequence” is any DNA sequence that can be transcribed into RNA when operatively linked to a regulatory element or promoter. Transcribed RNA molecules encoded by transcribed DNA sequences operatively linked to one or more regulatory elements provided herein can be translated to produce protein molecules, or can provide antisense or other functional or regulatory RNA molecules, such as double-stranded hairpin RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), small interfering RNA (siRNA), crRNA, gRNA, etc.

[0074] As used herein, the term "protein expression" is any pattern in which transcribed RNA molecules are translated into protein molecules. Protein expression can be characterized by its temporal, spatial, developmental, or morphological properties, as well as by its quantitative or qualitative indicators or expression patterns.

[0075] The efficacy of the modifications, duplications, or deletions described herein in achieving the desired expression of a specific transgene can be empirically tested in stable and transient plant assays (such as those described in the working examples herein) to validate the results, which may vary depending on the alterations made in the starting DNA molecule and the target of those alterations.

[0076] Construct As used herein, the term "construct" means any DNA molecule or vector, or segment or portion of a DNA molecule, vector, or chromosome, derived from one or more sources and capable of transfection or genome integration, comprising at least two DNA sequences functionally linked to each other. For example, a construct may comprise two operatively linked sequences, such as regulatory elements or promoters operatively linked to coding or transcribed DNA sequences. A construct may be a recombinant DNA construct. An example of a construct as a linear recombinant DNA segment is T-DNA. As used herein, "vector" means a DNA molecule that may contain or include constructs of this disclosure, such as plasmids, granules, viruses, bacteriophages, or other linear or circular DNA molecules, and "DNA transformation vector" means any DNA molecule or vector containing a recombinant DNA construct or recombinant DNA molecule that can be used for transformation purposes, i.e., for introducing a recombinant DNA molecule or construct into a host cell, such as a plant cell. According to some embodiments, a DNA transformation vector may comprise a T-DNA segment defined by a left boundary sequence and / or a right boundary sequence, which can be used for bacterial-mediated transformation, such as Rhizobium-mediated or Agrobacterium-mediated transformation. A construct typically contains one or more expression cassettes operatively linked to one or more expression elements (such as promoters), one or more gene coding sequences, or transcribed DNA sequences. As used herein, an "expression cassette" is a DNA sequence that contains at least a transcribed DNA sequence operatively linked to one or more regulatory elements (typically at least a promoter and a 3' UTR).

[0077] As used herein, the term "operably linked" refers to a functional relationship between two or more physically linked DNA sequences in a DNA molecule, construct, vector, or chromosome, comprising first and second DNA sequences arranged such that the first DNA sequence influences the function or expression of the second DNA sequence. The two DNA sequences may or may not be part of a single, continuous DNA molecule, and may or may not be adjacent. For example, if a promoter regulates the transcription of a transcribed DNA sequence of interest in a cell, the promoter is operably linked to the transcribed DNA sequence. For example, a leader sequence is operably linked to a transcribed DNA sequence when it is capable of influencing transcription or translation of the DNA sequence.

[0078] In one embodiment, the construct of this disclosure may be provided as a dual tumor-inducing (Ti) plasmid boundary construct having a right boundary (RB or AGRtu.RB) and a left boundary (LB or AGRtu.LB) region of a Ti or Ri plasmid isolated from an Agrobacterium genus containing T-DNA (e.g., Agrobacterium tumefaciens or Agrobacterium rhizogenes), the T-DNA, together with a delivery molecule provided by the Agrobacterium cell, allowing the T-DNA to integrate into the genome of the plant cell (see, for example, U.S. Patent 6,603,061). The construct may also contain a plasmid backbone DNA segment that provides replication function and antibiotic selection in bacterial cells, such as Escherichia coli (E. coli). Escherichia coli Replication origins, such as ori322; broad host-range replication origins, such as oriV or oriRi (see, for example, Ye et al., Transgenic Research 20(4): 773-86, 2011); and selectively labeled coding regions, such as Tn7 aminoglycoside adenylate transferases that confer resistance to spectinomycin or streptomycin ( aadA The Spec / Strp or gentamicin (Gm, Gent) selective marker gene. For plant transformation, the host bacterial strain is often Agrobacterium tumefaciens ABI, C58, or LBA4404; however, other strains known to those skilled in the art of plant transformation may function in accordance with the teachings of this disclosure.

[0079] Methods for assembling and introducing constructs into cells in a manner that causes transcribed DNA molecules to be transcribed into functional mRNA molecules that are translated and expressed as proteins are known in the art. Conventional compositions and methods for preparing and using constructs and host cells are well known to those skilled in the art in practice for the purpose of practicing this disclosure. Typical vectors that can be used to express nucleic acids in higher plants are well known in the art and include vectors derived from the Ti plasmid of Agrobacterium tumefaciens and the pCaMVCN delivery control vector.

[0080] The construct may contain a variety of regulatory elements, including any of those provided herein. Any such regulatory element may be provided in combination with other regulatory elements. Such combinations may be designed or modified to produce desired regulatory characteristics. In one embodiment, the construct of this disclosure includes at least one regulatory element operatively linked to a transcribed DNA molecule operatively linked to a 3' UTR.

[0081] The constructs disclosed herein may include any promoter or leader sequence provided herein or known in the art. For example, the promoters of this disclosure may be operatively linked to a heterologous untranslated 5' leader sequence, such as a 5' leader sequence derived from a heat shock protein gene. Alternatively, the leader sequence of this disclosure may be operatively linked to a heterologous promoter, such as the cauliflower mosaic virus 35S transcript promoter.

[0082] The expression cassette may also include a transport peptide encoding a sequence that encodes a peptide for subcellular targeting of a protein that can be operatively linked, particularly to chloroplasts, leucosomal or other plastid organelles; mitochondria; peroxisomes; vacuoles; or extracellular locations. Many chloroplast-localizing proteins are expressed as precursors by nuclear genes and target chloroplasts via chloroplast transport peptides (CTPs). Examples of such isolated chloroplast proteins include, but are not limited to, those associated with: the small subunit (SSU) of ribulose-1,5,2-bisphosphate carboxylase, ferricredoxin, ferricredoxin oxidoreductase, light-harvesting complex proteins I and II, thioredoxin F, and enolpyruvate shikimate phosphate synthase (EPSPS). Chloroplast transport peptides are described, for example, in U.S. Patent No. 7,193,133. It has been demonstrated that non-chloroplast proteins can target chloroplasts via expression of a heterologous CTP operatively linked to a transgene encoding a non-chloroplast protein.

[0083] Transcribed DNA sequences As used herein, the term "transcribed DNA sequence" refers to any DNA sequence capable of being transcribed into RNA molecules, including, but not limited to, those having protein-coding sequences and those producing RNA molecules with sequences that can be used for gene repression. Types of DNA sequences may include, but are not limited to, DNA sequences from the same plant, DNA sequences from another plant, DNA sequences from different organisms, or synthetic DNA sequences (such as DNA sequences containing antisense information of a gene) or DNA sequences encoding artificial, synthetic, or otherwise modified transgenic forms. Exemplary transcribed DNA sequences used to be incorporated into constructs of this disclosure include, for example, DNA sequences or genes from species different from the species into which the DNA sequence is incorporated, or genes derived from or present in the same species but incorporated into recipient cells through genetic engineering methods rather than classical breeding techniques.

[0084] "Transgenic" refers to a heterologous transcribed DNA sequence relative to the host cell genome at least with respect to its position in the host cell genome and / or an transcribed DNA sequence artificially incorporated into the host cell genome in the current or any previous generation of cells.

[0085] Regulatory elements, such as the promoters of this disclosure, can be operatively linked to a heterologous transcribed DNA sequence relative to the regulatory element. As used herein, the term "heterologous" refers to a combination of two or more DNA sequences when such a combination is not normally present in nature. For example, the two DNA sequences may originate from different species and / or the two DNA sequences may originate from different genes, for example, different genes from the same species or the same gene from different species. Therefore, if such a combination is not normally present in nature, i.e., the transcribed DNA sequence is not naturally operatively linked to the regulatory element, then the regulatory element is heterologous relative to the operatively linked transcribed DNA sequence. A "heterologous transcribed DNA sequence" refers to a transcribed DNA sequence that is heterologous to the DNA sequence to which it is operatively linked. In one embodiment, the heterologous transcribed DNA sequence comprises a gene encoding a site-specific recombinase.

[0086] Transcribed DNA sequences can generally be any DNA sequence for which expression of a transcript is desired. Such expression of the transcript leads to the translation of the resulting mRNA molecule, which in turn leads to protein expression. Alternatively, for example, a transcribed DNA sequence can be programmed to ultimately result in reduced expression of a specific gene or protein. In one embodiment, this can be achieved by using a transcribed DNA sequence oriented in an antisense direction. Those skilled in the art are familiar with the use of such antisense techniques. Any gene can be negatively regulated in this manner, and in one embodiment, the transcribed DNA sequence can be programmed to repress a specific gene via the expression of dsRNA, siRNA, or miRNA molecules.

[0087] Therefore, one embodiment of this disclosure is a recombinant DNA molecule comprising the regulatory elements of this disclosure, such as those provided as SEQ ID NO:1-16 or fragments thereof, or sequences having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 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%, at least 99%, or 100% identity with any of SEQ ID NO:1-16 or fragments thereof, said regulatory elements being operatively linked to a heterologous transcribed DNA sequence to regulate transcription of the transcribed DNA sequence at a desired level or in a desired pattern when the construct is integrated into the genome of a transgenic plant cell. In one embodiment, the transcribed DNA sequence comprises a protein-coding region of a gene, and in another embodiment, the transcribed DNA sequence comprises an antisense region of a gene or any other transcribed DNA sequence that causes repression of one or more specific target genes. In one embodiment, the transcribed DNA sequence may comprise a gene encoding a site-specific recombinase. In another embodiment, the transcribed DNA sequence may comprise a gene encoding a Cre recombinase. Examples of Cre recombinases are provided as SEQ ID NO:17 (Cre) or SEQ ID NO:21 (Cre-2).

[0088] Genes with agronomical value Transcribed DNA sequences can be genes of agronomic value. As used herein, the terms "gene of agronomic value" or "transgenic gene of agronomic value" refer to a transcribed DNA sequence that, when expressed in a particular plant tissue, cell, or cell type, confers a desired characteristic or trait. The product of a gene of agronomic value or a transgenic gene can function within a plant to influence plant morphology, physiology, growth, development, yield, cereal composition, nutritional status, disease or pest resistance, and / or environmental or chemical tolerance, or can act as an insecticide in the diet of plant-feeding pests. In one embodiment of this disclosure, the regulatory elements of this disclosure are incorporated into a construct such that the regulatory elements are operatively linked to a transcribed DNA sequence that is a gene of agronomic value or a transgenic gene. In transgenic plants containing such a construct, expression of a gene of agronomic value can confer beneficial agronomic traits. Beneficial agronomic traits may include, but are not limited to, herbicide tolerance, insect control, improved or increased 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 tolerance or resistance, pharmaceutical peptides, improved processing quality, improved flavor, hybrid seed production utility, improved fiber production, and desired biofuel production.

[0089] Non-limiting examples of genes (or transgenes) known in the art to have agronomical value include the following genes (or transgenes): herbicide resistance (US Patent Nos. 6,803,501; 6,448,476; 6,248,876; 6,225,114; 6,107,549; 5,866,775; 5,804,425; 5,633,435; and 5,463,175), increased yield (US Patent Nos. USRE 38,446; 6,716,474; 6,663,906; 6,476,295; 6,441,277; 6,423,828; 6,399,330; 6,372,211; 6,235,971; 6, 222,098; and 5,716,837), insect control (US Patent Nos. 6,809,078; 6,713,063; 6,686,452; 6,657,046; 6,645,497; 6,642,030; 6,639,054; 6,620,988; 6,593,293; 6,555,655; 6,538,109; 6,537,756; 6,521,442; 6,501,009; 6,468,523; 6,326,351; 6,313,378; 6,284,949; 6,281,016; 6,248,536; 6,242,241; 6,221,649; 6,177,615; 6,156,573; 6,153,814; 6,110,464; 6,093,695; 6,063,756; 6,063,597; 6,023,013; 5,959,091; 5,942,664; 5,942,658; 5,880,275; 5,763,245; and 5,763,241), fungal disease resistance (US Patent Nos. 6,653,280; 6,573,361; 6,506,962; 6,316,407; 6,215,048; 5,516,671; 5,773,696; 6,121,436; 6,316,407; and 6,50 6,962), virus resistance (US Patent Nos. 6,617,496; 6,608,241; 6,015,940; 6,013,864; 5,850,023; and 5,304,730), nematode resistance (US Patent No. 6,228,992), bacterial disease resistance (US Patent No. 5,516,671), plant growth and development (US Patent Nos. 6,723,897 and 6,518,488), starch production (US Patent Nos. 6,538,181; 6,538,179; 6,538,178; 5,750,876; 6,476,295), and improved oil production (US Patent Nos. 6,444,876; 6,426,447).And 6,380,462), high oil production (US Patent Nos. 6,495,739; 5,608,149; 6,483,008; and 6,476,295), improved fatty acid content (US Patent Nos. 6,828,475; 6,822,141; 6,770,465; 6,706,950; 6,660,849; 6,596,538; 6,589,767; 6,537,750; 6,489 High protein production (US Patent No. 6,380,466), fruit ripening (US Patent No. 5,512,466), enhanced animal and human nutrition (US Patent Nos. 6,723,837; 6,653,530; 6,5412,59; 5,985,605; and 6,171,640), biopolymers (US Patent Nos. USRE37,543; 6,228,623; and 5,985,605). 58,745 and 6,946,588), environmental stress resistance (US Patent No. 6,072,103), pharmaceutical peptides and secretible peptides (US Patent Nos. 6,812,379; 6,774,283; 6,140,075; and 6,080,560), improved processing properties (US Patent No. 6,476,295), improved digestibility (US Patent No. 6,531,648), low raffinose (US Patent No. 6,166,292). Industrial enzyme production (US Patent No. 5,543,576), improved flavor (US Patent No. 6,011,199), nitrogen fixation (US Patent No. 5,229,114), hybrid seed production (US Patent No. 5,689,041), fiber production (US Patent Nos. 6,576,818; 6,271,443; 5,981,834; and 5,869,720), and biofuel production (US Patent No. 5,998,700).

[0090] Alternatively, agronomically valuable genes or transgenes can influence the aforementioned plant characteristics or phenotypes by encoding RNA molecules that target and regulate the expression of endogenous genes, for example, through antisense (see, for example, U.S. Patent 5,107,065); repressive RNA (“RNAi”, including mechanisms that regulate gene expression through miRNA, siRNA, trans-acting siRNA, and phased sRNA, for example, as described in published applications US 2006 / 0200878 and US 2008 / 0066206 and U.S. Patent Application 11 / 974,469); or co-repressive mechanisms. The RNA can also be a catalytic RNA molecule engineered to cleave the desired endogenous mRNA product (e.g., a ribozyme or riboswitch; see, for example, US 2006 / 0200878). Methods for constructing DNA constructs and introducing them into cells in a manner that causes transcribed DNA sequences to be transcribed into RNA molecules capable of inducing gene repression are known in the art.

[0091] Selective marking Selective marker transgenes may also be used in conjunction with the regulatory elements of this disclosure. As used herein, the term “selective marker transgene” refers to any transcribed DNA sequence whose expression or absence in a transgenic plant, tissue, or cell is selected or scored in some manner. Selective marker genes used in practices of this disclosure and their associated selection and screening techniques are known in the art and include, but are not limited to, transcribed DNA sequences encoding β-glucuronidase (GUS), green fluorescent protein (GFP), proteins conferring antibiotic resistance, and proteins conferring herbicide tolerance. An example of a selective marker transgene is provided as aadA (SEQ ID NO:19), used for selecting transformed plant cells by spectinomycin selection. An example of a reporter transgene is provided as SEQ ID NO:20 (GUS).

[0092] The use of reporter gene assays (or reporter transgene assays) to determine the gene regulatory activity (or expression profile) of regulatory elements is well known in the art; see, for example, Clark et al., Molecular Biology, 3rd Edition, Unit 4, Chapter 21. Academic Press, Elsevier Inc , 2019. As used herein, the term “reporter assay” refers to a method of determining the gene regulatory activity (or expression profile) of a reporter gene (such as a transgene encoding a β-glucuronidase (GUS) protein) as a heterologous transcribed DNA sequence operably linked to a specific regulatory element (e.g., a promoter or 3' UTR) using a reporter gene assay. In a subsequent reporter gene assay, qualitative and quantitative GUS analyses can be used to evaluate the gene regulatory activity (or expression profile) of the regulatory element in selected plant organs and / or tissues in transformed plants. It should be understood that the gene regulatory activity (or expression profile) of a regulatory element (e.g., a promoter or 3' UTR) determined by using a reporter gene assay (e.g., a GUS assay) is the same as, substantially the same as, or substantially similar to one or more other operably linked transcribed DNA molecules other than GUS. In some embodiments, such other operably linked transcribed DNA molecules may be genes of agronomical value, including but not limited to those described herein. In other embodiments, such other operably linked transcribed DNA molecules may be DNA sequences encoding site-specific recombinases. In some implementations, the site-specific recombinase may be a Cre recombinase.

[0093] Site-specific nucleases As used herein, the term "genome editing" refers to the modification of a DNA molecule or gene sequence at a target site in the genome or chromosome of an organism or cell (such as the genome of an agricultural crop plant) by deleting, substituting, and / or inserting a DNA sequence at or near a target site, which can be produced using site-specific nucleases. "Site-specific integration" or "site-specific integration" are terms used to refer to the insertion of a DNA sequence or construct at a target site into the genome or chromosome of an organism or cell.

[0094] As used in this article, the term "site-specific nuclease" refers to a DNA-cutting nuclease that produces double-strand breaks or nicks at or near a specific target site or location in a DNA molecule, chromosome, or genome.

[0095] As used herein, a “target site” for genome editing refers to the location of a polynucleotide sequence within the plant genome that is bound and cleaved by a site-specific nuclease, thereby introducing a double-strand break (or single-strand nick) into the nucleic acid backbone of the polynucleotide sequence and / or its complementary DNA strand. Following the break or cleavage, cellular DNA repair mechanisms can recognize and repair the break or nick via non-homologous end joining (NHEJ) or homologous targeted repair, and may introduce mutations and / or insertions at the target site, as understood in the art.

[0096] The site-specific nucleases provided in this article can be selected from the group consisting of: zinc finger nucleases (ZFNs), broad-spectrum nucleases, RNA-guided endonucleases such as CRISPR-associated nucleases, TALE-endonucleases (TALENs), recombinases, transposases, or any other possible endonucleases. See, for example, Khandagale K. et al., Genome editing for targeted improvement in plants, Plant Biotechnol Rep 10: 327-343, 2016; and Gaj T. et al., ZFN, TALEN and CRISPR / Cas-based methods for genome engineering, Trends Biotechnol. The contents and publication of the cited document 31(7): 397-405, 2013 are incorporated herein by reference. The expression cassettes provided herein encode site-specific nucleases. Such expression cassettes may contain a transcribed DNA sequence operatively linked to a plant-expressible promoter encoding a site-specific nuclease. Alternatively, the recombinant DNA constructs provided herein may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten expression cassettes encoding one or more site-specific nucleases.

[0097] According to embodiments of this disclosure, the recombinase may be a serine recombinase linked to a DNA recognition motif, a tyrosine recombinase linked to a DNA recognition motif, or other recombinases known in the art. The recombinase or transposase may be a DNA transposase or recombinase linked to a DNA-binding domain. The tyrosine recombinase linked to the DNA recognition motif may be selected from the group consisting of Cre recombinase, Flp recombinase, and Tnp 1 recombinase. According to some embodiments, the Cre recombinase or Gin recombinase provided herein is tethered to a zinc finger DNA-binding domain. In another embodiment, the serine recombinase linked to a DNA recognition motif provided herein is selected from the group consisting of PhiC31 integrase, R4 integrase, and TP-901 integrase. In yet another embodiment, the DNA transposase linked to a DNA-binding domain provided herein is selected from the group consisting of TALE-piggyBac and TALE- mutants.

[0098] According to the embodiments of this disclosure, the RNA-guided endonuclease or CRISPR-related nuclease may be selected from the group consisting of: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cas12a (also known as Cpf1), CasX, CasY, and their homologs or modified forms, Argonaute (non-limiting examples of Argonaute proteins include thermophilic bacteria) Thermus thermophilus Argonaute (TtAgo), *Streptococcus pyogenes* ( Pyrococcus furiosus Argonaute (PfAgo), Halophilic bacillus griseus ( Natronobacterium gregoryi (Argonaute (NgAgo)) and its homologs or modified forms. According to some embodiments, the RNA-guided endonuclease can be a Cas9 or Cas12a enzyme. In some embodiments, the site-specific nucleases provided herein are selected from the group consisting of Cas9 or Cas12a enzymes.

[0099] For RNA-guided endonucleases or CRISPR-associated nucleases, guide RNA (gRNA) molecules may be required to direct the endonuclease to a target site in a plant's DNA molecule, chromosome, or genome via base pairing or hybridization to induce a DSB or nick at or near the target site. gRNA can be transformed or introduced into plant cells or tissues in the form of a recombinant DNA construct (possibly along with the nuclease or a DNA construct encoding the nuclease), the recombinant DNA construct containing a transcribed DNA sequence operatively linked to a plant-expressible promoter encoding guide RNA. As understood in the art, "guide RNA" can include, for example, CRISPR RNA (crRNA), single-stranded guide RNA (sgRNA), or any other RNA molecule that can guide or direct an endonuclease to a specific target site in the genome. A "single-stranded guide RNA" (or "sgRNA") is an RNA molecule containing crRNA covalently linked to tracrRNA by an adapter sequence, which can be expressed as a single RNA transcript or molecule. Guide RNA contains a guide or targeting sequence that is the same as or complementary to the target site within the DNA molecule, chromosome, or plant genome. As is known in the art, the protospacer adjacent motif (PAM) can be located in the genome adjacent to the 5' end of the genomic target site sequence complementary to the target sequence of the guide RNA and upstream of it—that is, downstream (3') of the sense (+) strand of the genomic target site (relative to the target sequence of the guide RNA). See, for example, Wu X. et al., Target specificity of the CRISPR-Cas9 system. Quant Biol. 2(2): 59-70, 2014, the contents and publication of which are incorporated herein by reference. The genomic PAM sequence (relative to the target sequence of the guide RNA) on the sense (+) strand adjacent to the target site may contain 5'-NGG-3'. However, the corresponding sequence of the guide RNA (i.e., immediately downstream (3') of the target sequence of the guide RNA) may not typically be complementary to the genomic PAM sequence. The guide RNA may typically be a non-coding RNA molecule that does not encode a protein. The guide sequence of the guide RNA may be at least 10 nucleotides long, such as 12-40 nucleotides, 12-30 nucleotides, 12-20 nucleotides, 12-35 nucleotides, 13-30 nucleotides, 15-30 nucleotides, 17-30 nucleotides, or 17-25 nucleotides, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides long. The guide sequence may be identical or complementary to at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more consecutive nucleotides at the target site, representing at least 95%, 96%, 97%, 99%, or 100% of these sequences. The expression cassettes provided herein encode guide RNA. Such expression cassettes may contain a transcribed DNA sequence encoding guide RNA operably linked to a plant-expressible promoter. Alternatively, the recombinant DNA constructs provided herein may contain at least one, two, three, four, five, six, seven, eight, nine, or ten expression cassettes encoding one or more guide RNAs.

[0100] Zinc finger nucleases (ZFNs) are synthetic proteins composed of engineered zinc finger DNA-binding domains fused to a cleavage domain (or cleavage half domain). This cleavage domain may be derived from restriction endonucleases (e.g., Fok1 The DNA-binding domain can be canonical (C2H2) or non-canonical (e.g., C3H or C4). Depending on the target site, the DNA-binding domain may contain one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more zinc fingers). Multiple zinc fingers in the DNA-binding domain can be separated by one or more adapter sequences. ZFNs can be designed to cleave virtually any segment of double-stranded DNA by modification of the zinc finger DNA-binding domain. ZFNs originate from non-specific DNA cleavage domains fused to the DNA-binding domain (e.g., derived from...). FokI The monomers of a ZFN (a type of nuclease) form a dimer, and the DNA-binding domain comprises an array of zinc fingers engineered to bind to a target site DNA sequence. The DNA-binding domain of a ZFN typically consists of 3-4 (or more) zinc fingers. The amino acids at positions -1, +2, +3, and +6 relative to the initiation of the α-helix of the zinc fingers can be altered and customized to suit a specific target sequence, and these amino acids contribute to site-specific binding to the target site. Other amino acids can form a common backbone to produce ZFNs with different sequence specificities. Methods and rules for designing ZFNs to target and bind to specific target sequences are known in the art. See, for example, U.S. Patent Applications Nos. 2005 / 0064474, 2009 / 0117617, and 2012 / 0142062, the contents and disclosures of which are incorporated herein by reference. Fok1 Nuclease domains may require dimerization to cleave DNA and therefore require two ZFNs with their C-terminal regions to bind to opposite strands of DNA (separated by 5-7 bp) at the cleavage site. If the dual ZF binding site is palindromic, the ZFN monomer can cleave the target site. As used herein, ZFN is broad and includes monomeric ZFNs that can cleave double-stranded DNA without the assistance of another ZFN. The term ZFN can also be used to refer to one or both members of a pair of ZFNs engineered to work together to cleave DNA at the same site.

[0101] Unrestricted by any scientific theory, custom ZFNs can theoretically be constructed to target virtually any target sequence (e.g., at or near genes in the plant genome) because the DNA-binding specificity of zinc finger domains can be reengineered using one of several methods. Publicly available methods for engineering zinc finger domains include background-dependent assembly (CoDA), oligomer library engineering (OPEN), and modular assembly. In some embodiments, the methods and / or compositions provided herein comprise one or more, two or more, three or more, four or more, or five or more ZFNs. In other embodiments, the ZFNs provided herein are capable of generating targeted DSBs or nicks. In some embodiments, cells are provided with a vector containing a DNA sequence encoding one or more, three or more, four or more, or five or more ZFNs via transformation methods known in the art (e.g., but not limited to viral transfection, particle bombardment, PEG-mediated protoplast transfection, or Agrobacterium-mediated transformation). ZFN can be introduced in the form of a ZFN protein, in the form of a polynucleotide encoding a ZFN protein, and / or in the form of a combination of a protein and a polynucleotide encoding a protein.

[0102] Widespread nucleases, typically identified in microorganisms, such as the LAGLIDADG family of homing endonucleases, are unique enzymes with high activity and long recognition sequences (>14 bp), resulting in site-specific digestion of target DNA. Engineered forms of naturally occurring widespread nucleases often have extended DNA recognition sequences (e.g., 14 to 40 bp). According to some embodiments, widespread nucleases may comprise a scaffold or basease selected from the group consisting of: I-CreI、I-CeuI、I- MsoI、I-SeeI、I-AniI and I-DmoI The engineering of large-scale nucleases can be more challenging than that of ZFNs and TALENs because their DNA recognition and cleavage functions are intertwined within a single domain. Specialized mutagenesis and high-throughput screening methods have been used to generate novel large-scale nuclease variants that recognize unique sequences and possess modified nuclease activity. Therefore, large-scale nucleases can be selected or engineered to bind to genomic target sequences in plants. In one aspect, the methods and / or compositions provided herein comprise one or more, two or more, three or more, four or more, or five or more large-scale nucleases. In another aspect, large-scale nucleases can produce targeted cleavage or breakage.

[0103] Zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs) are chimeric enzymes that combine nucleases with DNA-binding domains. TALENs are sequence-specific nucleases that can be used to generate double-strand breaks at specific target sequences in the genome of plants or other organisms. TALENs work by fusing transcription activator-like effector (TALE) DNA-binding domains into nuclease domains (e.g., ...). FokI Artificial restriction enzymes are produced by binding to DNA sites flanking the target site. FokI Monomerization occurs and induces double-strand DNA breaks at the target site. (Except for wild-type) FokI Outside the cleavage domain, structures with mutations have been designed. FokI Variations of the cleavage domain are used to improve cleavage specificity and cleavage activity. FokI The domain functions as a dimer, thus requiring a construct of two unique DNA-binding domains with appropriate orientation and spacing, each targeting a site in the target genome. The TALEN DNA-binding domain and... FokI Both the number of amino acid residues between the cleavage domains and the number of bases between two individual TALEN binding sites are parameters used to achieve high activity levels. TALENs are artificial restriction enzymes produced by fusing transcription activator-like effector (TALE) DNA-binding domains into nuclease domains. In some respects, nucleases are selected from the following groups: PvuII、MutH、TevI、FokI、 AlwI、MlyI、SbfI、SdaI、StsI、CleDORF、Clo051 and Pept071 When each member of the TALEN pair binds to a DNA site flanking the target site, FokI Monomerization occurs and causes double-stranded DNA breaks at the target site. As used herein, the term TALEN is broad and includes monomeric TALENs that can cleave double-stranded DNA without the assistance of another TALEN. The term TALEN also refers to one or both members of a pair of TALENs that work together to cut DNA at the same site.

[0104] Transcription activator-like effectors (TALEs) can be engineered to bind virtually any DNA sequence, such as those at or near genomic loci of genes in plants. A TALE has a central DNA-binding domain consisting of 13 to 28 repeating monomers of 33 to 34 amino acids each. Except for hypervariable amino acid residues at positions 12 and 13, the amino acids in each monomer are highly conserved. The DNA-binding domain of a TALE effector may contain repeating sequences of 33-35 amino acids, including repeating variable di-residues (RVDs) at residues 12 and 13, which determine their specificity in DNA binding. Each repeating sequence binds a specific nucleotide, which facilitates the engineering of specific DNA-binding domains by selecting combinations of repeating sequence segments containing appropriate RVDs. The number of repeating sequences of the RVD sequence determines the length and sequence of the target sequence to be identified (e.g., Podevin N. et al.). Trends in Biotechnology 31(6): 375-383, 2013). The two variable amino acids are called repeat variable di-residues (RVDs). The amino acid pairs of RVDs preferentially recognize certain nucleotide bases, and the regulation of RVDs can recognize consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA-binding domains by selecting combinations of repeat sequence segments containing appropriate RVDs.

[0105] Except for wild type FokI Outside the cleavage domain, structures with mutations have been designed. FokI Variations of the cleavage domain are used to improve cleavage specificity and cleavage activity. FokI The domain functions as a dimer, thus requiring a construct of two unique DNA-binding domains with appropriate orientation and spacing, each targeting a site in the target genome. The TALEN DNA-binding domain and... FokI Both the number of amino acid residues between cleavage domains and the number of bases between two individual TALEN binding sites are parameters used to achieve high activity levels. PvuII、MutH and TevI The cleavage domain is intended for use with TALE. FokI and FokI Useful alternatives to the variant. When coupled with TALE, PvuII It functions as a highly specific cleavage domain (see, for example, Yanik M et al., TALE-PvuII Fusion Proteins – Novel Tools for GeneTargeting). PLoS One, 8(12): e82539, 2013). MutH Capable of introducing strand-specific nicks into DNA (see, for example, Gabsalilow L. et al.) , Nucleic Acids Research, 41(7):e83, 2013). Tevl Introducing double-strand breaks at target sites in DNA (see, for example, Beurdeley M. et al., Compact designer TALENs for efficient genome engineering). Nat Commun 4: 1762, 2013). The relationship between amino acid sequences and DNA recognition of TALE-binding domains allows for the designability of proteins. TALE constructs can be designed using software programs such as DNA Works. Other methods for designing TALE constructs are known to those skilled in the art. See, for example, Doyle E.L. et al., TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction. Nucleic Acids Research , 40(W1): W117–W122, 2012; Cermak T. et al., Nucleic Acids Research , 39: e82, 2011; and / or tale-nt.cac.comell.edu / about. In one aspect, the recombinant DNA constructs provided herein may contain one or more, two or more, three or more, four or more, or five or more TALENs. In another aspect, the TALENs provided herein are capable of producing targeted cleavage or breakage at the target site.

[0106] Zinc finger nucleases (ZFNs) comprise a zinc finger DNA-binding domain and a double-break-inducible domain. Recognition site specificity is conferred by the zinc finger domain, which may contain two, three, or four zinc fingers, for example, a C2H2 structure, although other zinc finger structures are also known and engineered. The zinc finger domain can be used to design peptides that specifically bind to selected polynucleotide recognition sequences. ZFNs consist of engineered DNA-binding zinc finger domains linked to non-specific endonuclease domains, such as nuclease domains from type IIs endonucleases, like FokI. Additional functions can be fused to the zinc finger binding domain, including transcription activator domains, transcription repressor domains, and methyltransferases. In some instances, dimerization of the nuclease domain is necessary for cleavage activity. Each zinc finger recognizes three consecutive base pairs in the target DNA. For example, a three-finger domain recognizes a nine-nucleotide sequence, while two sets of zinc finger triplets are used to bind an eighteen-nucleotide recognition sequence due to the nuclease dimerization requirement (Gaj T. et al.). Trends Biotechnology , 31(7): 397-405, 2013; and Urnov FD et al., Nature Reviews Genetics ,11: 636-646, 2010).

[0107] Cell transformation This disclosure also relates to methods for producing transformed cells and plants comprising one or more regulatory elements operatively linked to a transcribed DNA molecule.

[0108] The term "transformation" refers to the introduction of DNA molecules into a recipient host. As used herein, the term "host" refers to bacteria, fungi, or plants, including any cell, tissue, organ, or progeny of bacteria, fungi, or plants. Plant tissues and cells of particular interest include protoplasts, callus, roots, tubers, seeds, stems, leaves, seedlings, embryos, and pollen.

[0109] As used herein, the term "transformed" refers to a cell, tissue, organ, or organism into which a foreign DNA molecule (such as a construct) has been introduced. The introduced DNA molecule may be integrated into the genomic DNA of the recipient cell, tissue, organ, or organism, such that the introduced DNA molecule is inherited by subsequent offspring. "Transgenic" or "transformed" cells or organisms may also include offspring of the cell or organism, as well as offspring produced in breeding programs that use such a transgenic organism as a parent in hybridization and exhibit altered phenotypes due to the presence of the foreign DNA molecule. The introduced DNA molecule may also be transiently introduced into the recipient cell, such that the introduced DNA molecule is not inherited by subsequent offspring. The term "transgenic" refers to bacteria, fungi, or plants containing one or more heterologous DNA molecules.

[0110] Numerous methods well-known to those skilled in the art exist for introducing DNA molecules into plant cells and transforming plant cells. These methods typically include the steps of selecting a suitable host cell or explant, transforming the cell or explant with a molecule or vector, and obtaining transformed cells. In the practice of this disclosure, methods and materials for transforming plant cells by introducing plant constructs into the plant genome can include any well-known and proven methods. Suitable methods include, but are not limited to, bacterial infection (e.g., Agrobacterium), binary BAC vectors, direct DNA delivery (e.g., transformation via PEG, drying / inhibition-mediated DNA uptake, electroporation, acceleration using stirring and DNA-coated particles with silicon carbide fibers), gene editing (e.g., CRISPR-Cas systems), etc. According to certain embodiments, transformation methods include Agrobacterium or Rhizobium-mediated transformation or particle bombardment or microsphere-mediated transformation.

[0111] The host cell can be any cell or organism, such as plant cells, algal cells, fungal cells, bacterial cells, or insect cells. In a specific embodiment, the host cell and the transformed cell may include cells derived from a crop plant.

[0112] The transformation of plant material is practiced in tissue cultures on nutrient media (e.g., mixtures of nutrients that allow cells to grow in vitro). Targets of recipient cells include, but are not limited to, meristematic cells, shoot tips, hypocotyls, callus, immature or mature embryos, and gamete cells such as microspores and pollen. Callus may originate from tissue sources including, but not limited to, immature or mature embryos, hypocotyls, seedling apical meristems, microspores, etc. The growth of cells containing a transgenic nucleus into a transgenic plant is also called an R0 plant. As used herein, “R0 plant” refers to the initial regeneration transformant. As used herein, “R1 seed” refers to a seed produced by self-pollination of an R0 plant. As used herein, “R1 plant” refers to a plant grown from an R1 seed. As used herein, “R2 seed” refers to a seed produced by self-pollination of an R1 plant. As used herein, “R2 plant” refers to a plant grown from an R2 seed.

[0113] Transgenic plants can be regenerated from the transgenic plant cells disclosed herein. Seeds can be produced from such transgenic plants using conventional breeding techniques or self-pollination. Such seeds, and the offspring plants obtained from such seeds, will contain the recombinant DNA molecules disclosed herein, and will therefore be transgenic.

[0114] The transgenic plants disclosed herein can be self-pollinated to provide seeds of homozygous transgenic plants (homozygous for the recombinant DNA molecule) of this disclosure, or hybridized with non-transgenic plants or different transgenic plants to provide seeds of heterozygous transgenic plants (heterozygous for the recombinant DNA molecule). Such homozygous and heterozygous transgenic plants are referred to herein as “progeny plants.” Progeny plants are transgenic plants originating from the original transgenic plant and containing the recombinant DNA molecule of this disclosure. Seeds produced using the transgenic plants of this disclosure can be harvested and used to grow generations of the transgenic plants of this disclosure, i.e., progeny plants, which contain the constructs of this disclosure and express genes of agronomical value or genes encoding site-specific recombinases. Descriptions of breeding methods commonly used for different crops can be found in one of several reference books, see, for example, Allard, Principles of Plant Breeding , John Wiley&Sons, NY, U. of CA, Davis,CA, 50-98,1960; Simmonds, Principles of Crop Improvement , Longman, Inc., NY, 369-399, 1979; Sneep and Hendriksen, Plant breeding Perspectives , Wageningen (ed.), Center for Agricultural Publishing and Documentation, 1979; Fehr, Soybeans: Improvement, Production and Uses (2nd edition, Monograph, 16:249, 1987); Fehr, Principles of Variety Development , Theory and Technique, (Volume 1) and Crop Species Soybean (Volume 2), Iowa State Univ., Macmillan Pub. Co., NY, 360-376, 1987.

[0115] The presence of one or more genes of interest for analysis of transformed plants, as well as the expression levels and / or expression profiles conferred by the regulatory elements of this disclosure, can be assessed. Those skilled in the art will recognize a variety of methods that can be used to analyze transformed plants. For example, methods for plant analysis include, but are not limited to, DNA blotting or RNA blotting, PCR-based methods, biochemical analysis, phenotypic screening methods, field assessment, and immunodiagnostic assays. Expression of transcribed DNA sequences can be measured using TaqMan® (Applied Biosystems, Foster City, CA) reagents and as described by the manufacturer, and PCR cycle times can be determined using a TaqMan® test matrix. Alternatively, transgenic expression can be evaluated using Invader® (Third Wave Technologies, Madison, WI) reagents and as described by the manufacturer.

[0116] This disclosure also provides plant parts of the present disclosure. "Plant part" can refer to any organ or entire tissue of a plant, and includes, but is not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovules, and pollen, or any part thereof. Plant parts of the present disclosure can be viable, non-viable, renewable, and / or non-renewable. This disclosure also includes and provides transformed plant cells containing the DNA molecules of the present disclosure. Transformed or transgenic plant cells of the present disclosure contain renewable and / or non-renewable plant cells.

[0117] As used in this article, the “vegetative stage” of plant development is the growth period between germination and flowering. The stages of the soybean vegetative stage are as follows: VE (emergence), VC (cotyledon stage), V1 (first ternate leaflet), V2 (second ternate leaflet), V3 (third ternate leaflet), V(n) (nth ternate leaflet), and V6 (approaching flowering). As used in this article, the “reproductive stage” of plant development is the period between flowering and the end of harvest. The stages of the soybean reproductive stage are as follows: R1 (beginning of flowering, first flower); R2 (full bloom, flowers in the top two nodes); R3 (beginning of pod formation, 3 / 16-inch pods in the top four nodes); R4 (complete pod, 3 / 4-inch pods in the top four nodes); R5 (1 / 8-inch seeds in the top four nodes); R6 (full-sized seeds in the top four nodes); R7 (beginning of maturity, one mature pod); and R8 (fully mature, 95% of the pods on the plant have reached mature color). The nutritional and reproductive stages of soybean are well known to those skilled in the art, and numerous publications describing these stages are available on the World Wide Web and elsewhere, such as North Dakota State University Publication A-1174, June 1999, which was reviewed and reprinted in August 2004.

[0118] This disclosure also provides commercial products derived from transgenic plants or portions thereof containing recombinant DNA molecules of this disclosure. The commercial products of this disclosure contain detectable amounts of DNA comprising a DNA sequence selected from the group consisting of SEQ ID NO:1-16. As used herein, “commercial product” means any composition or product comprising material derived from transgenic plants, seeds, plant cells, or plant parts containing recombinant DNA molecules of this disclosure. Commercial products include, but are not limited to, processed seeds, cereals, plant parts, and flours. The commercial products of this disclosure may contain detectable amounts of DNA corresponding to the recombinant DNA molecules of this disclosure. One or more detections of such DNA in a sample can be used to determine the content or source of the commercial product. Any standard method for detecting DNA molecules, including the detection methods disclosed herein, can be used.

[0119] Unless otherwise stated herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. Any and all instances or instance-like language (e.g., “such as”) provided with respect to certain embodiments herein are intended only to illustrate this disclosure and do not limit the scope of this disclosure unless otherwise claimed.

[0120] This disclosure may be more readily understood by reference to the following embodiments, which are provided for illustration and are not intended to limit the disclosure unless otherwise specified. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques that the inventors have found to work well in practicing the teachings of this disclosure. However, given this disclosure, those skilled in the art will understand that many variations can be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the principles of this disclosure; therefore, all matters presented should be understood as illustrative and not restrictive.

[0121] Example Example 1 Identification of regulatory elements capable of driving automatic excision in crop plants This embodiment provides a regulatory element that, identified in years of experiments, can drive efficient automatic excision in genetically modified soybeans.

[0122] First, regulatory elements with the potential to drive efficient autoexcision in transgenic crops were identified through a combination of literature searches and searches of public and proprietary databases. Efficient autoexcision of over thirty soybean binary transformation vector constructs containing different regulatory elements and combinations has been determined using the Cre / Lox recombinase system. From these studies, a small number of regulatory elements capable of providing efficient autoexcision were identified. Table 1 below presents the regulatory elements (“bp” refers to base pairs) identified for autoexcision assays.

[0123] Table 1. Regulatory elements for automatic excision assays in soybean plants.

[0124] Example 2 Soybean expression elements P-Gm.CALa, P-Gm.Mads17, and P-Gm.AP1 can drive automatic excision in stably transformed soybean plants. Soybean plants were transformed with recombinant DNA constructs containing different regulatory elements that drive Cre recombinase expression, particularly plant binary transformation constructs, to evaluate the ability and efficiency of Cre recombinase expressed under different regulatory elements in driving Cre recombinase expression cassettes and the automatic excision of multiple expression cassettes.

[0125] Soybean plants were transformed using plant binary transformation constructs (further referred to as “constructs” or “DNA constructs”) as shown in Table 2 below. Each plant binary transformation construct contains five transgene expression cassettes: a first Cre recombinase expression cassette, a second selective marker gene expression cassette (further referred to as a “marker gene expression cassette”), a third expression cassette for expressing Cas12a, and a fourth expression cassette for expressing guide RNA (gRNA). All expression cassettes are flanked by two LoxP sites; and a fifth expression cassette located outside the LoxP sites for expressing an agronomically valuable gene (GOI-HT). The Cre recombinase expression cassette was used to assay different expression elements to test their ability to drive efficient automatic excision of the Cre recombinase expression cassette, marker gene expression cassette, Cas12a expression cassette, and gRNA expression cassette located between LoxP sites. The Cre recombinase expression cassette includes a promoter (SEQ ID NO: 1, 3, 5, 7, or 9) 5' of which is operatively linked to a synthetic coding sequence (e.g., a codon redesigned for expression in plant cells) encoding the Cre recombinase (Cre, SEQ ID NO: 17), the synthetic coding sequence containing a processable intron derived from the potato light-inducible tissue-specific ST-LS1 gene (GenBank accession number: X04753), the synthetic coding sequence 5' being operatively linked to a 3' UTR (SEQ ID NO: 2, 4, 6, 8, or 10). Each plant transformation construct includes a marker gene expression cassette for expressing a conjugated spectinomycin resistance (aadA) transgene driven by a constitutive promoter and for selecting transformed plant cells using spectinomycin selection. Two additional expression cassettes are cloned near the Cre and spectinomycin expression cassettes, one for expressing Cas12a driven by a constitutive promoter and the other for expressing gRNA driven by a polymerase III promoter. In total, five plant binary transformation constructs were generated using techniques known in the art, and are presented in Table 2 below (constructs-1 to constructs-5) along with their respective promoters and 3' UTRs.

[0126] Table 2. Constructs used for transforming soybean plants.

[0127] Each of the five constructs contains four expression cassettes: the marker gene expression cassette, the Cre recombinase expression cassette, the Cas12a expression cassette, and the gRNA expression cassette, with two LoxPCre recombinase recognition sequences (SEQ ID NO: 18) side-mounted head-to-tail. If autoexcision is effective, expression of Cre recombinase in transformed plant cells is expected to result in the excision of all four expression cassettes. The agronomically valuable gene (GOI-HT) cassette (i.e., the fifth expression cassette mentioned above) was cloned outside the LoxPCre recombinase recognition sequences and expressed using a constitutive promoter.

[0128] Five promoters (SEQ ID NO: 1, 3, 5, 7, or 9) derived from soybean genes were operatively linked to a transcribed DNA sequence encoding a Cre recombinase (Cre, SEQ ID NO: 17), and their ability to drive automatic excision in stably transformed soybean plants was determined. Each promoter driving Cre contains a promoter operatively linked to its native leader sequence. Each Cre expression cassette also contains a corresponding 3' UTR (SEQ ID NO: 2, 4, 6, 8, or 10), the 3' UTR being derived from the same gene from which the promoter originates, and the 3' of the UTR being operatively linked to the Cre recombinase coding sequence within the expression cassette.

[0129] Soybean plant cells were transformed using the above-described plant binary transformation constructs (Table 2) via Agrobacterium-mediated transformation. Agrobacterium-mediated transformation methods are well-known in the art. The resulting transformed plant cells were then regenerated into soybean plants under spectinomycin selection.

[0130] Single-copy and two-copy R0 plants were selected and self-pollinated. The presence of Cre, aadA, and the agronomically valuable gene (GOI-HT) in the resulting R1 plants was then analyzed using TaqMan® assays. The conjugation of the R1 plants of the integrated construct was also determined using TaqMan® assays of the agronomically valuable gene (GOI-HT). The deletion of Cre and aadA was inferred as evidence of automatic excision.

[0131] Table 3 below shows the total number of homozygous and hemizygous marker-free R1 plants from the five constructs. As shown in Table 3, the expression elements driving Cre recombinase expression in constructs-1,-2, and-3 exhibited higher efficiency in driving automatic excision than those in constructs-4 and-5. Constructs-1,-2, and-3 provided a larger percentage of homozygous and hemizygous marker-free R1 plants (“Homozygous / Hemizygous MF Percentage” in Table 3), indicating that Cre recombinase expression occurred at the correct developmental stage in the relevant cell types to provide marker-free progeny. In contrast, the event involving construct-4 had very few marker-free R1 plants, likely due to Cre recombinase expression not being in the relevant tissues at the correct developmental stage. The event involving construct-5 was less efficient in providing marker-free plants compared to constructs-1 through-3, suggesting that the timing and expression of Cre recombinase were not optimal.

[0132] Table 3. Total number and percentage of homozygous and hemizygous marker-free (MF) R1 plants from five different constructs.

[0133] Example 3 Determination of autoexcision activity in stably transformed soybean plants Soybean plants were transformed with a plant binary expression construct containing an expression element that drives Cre recombinase expression as shown in SEQ ID NO:11-16, and its ability to effectively drive automatic excision was determined.

[0134] Soybean plants were transformed using recombinant DNA constructs, particularly those similar to those described in Example 2 above, which contain regulatory elements driving the expression of the Cre recombinase coding sequence (SEQ ID NO: 17) as presented in Table 4 below, to evaluate the ability and efficiency of the expressed Cre recombinase in driving the Cre recombinase expression cassette and the automatic excision of multiple expression cassettes. The plant transformation constructs presented in Table 4 are similar in design to those described in Example 2 above.

[0135] Table 4. Constructs for stable transformation of soybean plants.

[0136] Single-copy and two-copy R0 plants were selected and self-pollinated. The presence of Cre, aadA, and the agronomically valuable gene (GOI-HT) in the resulting R1 plants was then analyzed using TaqMan® assays. The conjugation of the R1 plants of the integrated construct was also determined using TaqMan® assays of the agronomically valuable gene (GOI-HT). The deletion of Cre and aadA was inferred as evidence of automatic excision. Table 5 below shows the total number of homozygous and hemizygous marker-free R1 plants from the three constructs.

[0137] Table 5. Total number and percentage of homozygous and hemizygous marker-free (MF) R1 plants from three different constructs.

[0138] As shown in Table 5 above, no marker-free plants were obtained using construct-6, while constructs-7 and-8 produced a low percentage of homozygous marker-free plants. Similarly, when compared with constructs-1, construct-2, and construct-3 from Example 2 above, constructs-7 and-8 produced a lower percentage of hemizygous marker-free plants, indicating that the timing and expression of Cre recombinase were not optimal in these constructs.

[0139] Example 4 Determination of expression element activity in stably transformed soybean plants Soybean plants were transformed using a plant binary expression construct (also known as a "plant binary expression vector" or "plant binary transformation vector") containing expression elements that drive the expression of the β-glucuronidase (GUS) transgene, as shown in SEQ ID NO:1-16. The expression of the GUS protein in the resulting plants was analyzed to assess the effect of the regulatory elements on the expression of the GUS transgene.

[0140] The plant binary expression vector for plant transformation contains a left boundary region (B-AGRtu. left boundary) from Agrobacterium tumefaciens; a first selective marker gene expression cassette for selecting transformed plant cells conferring resistance to the antibiotic spectinomycin, which expresses a spectinomycin-resistant transgene (aadA) driven by a constitutive promoter; a second expression cassette for evaluating the activity of expression elements presented as SEQ ID NO:1, 3, 5, 7, 9, 11, 13 or 15, the second expression cassette containing a promoter (promoter and leader sequence) 5' operably linked to a coding sequence of GUS (SEQ ID NO:20), the coding sequence of GUS containing a processable intron 5' operably linked to a 3' UTR presented as SEQ ID NO:2, 4, 6, 8, 10, 12, 14 or 16; and a right boundary region (B-AGRtu. right boundary) from Agrobacterium tumefaciens.

[0141] As is well known in the art, soybean plant cells were transformed using the aforementioned plant binary transformation vector via Agrobacterium-mediated transformation. The resulting transformed plant cells were induced to form complete soybean plants. The following tissues were sampled for GUS expression in generation R0: V5 stage saucer, progenitor leaf, and root; R1 stage flower, petiole, progenitor leaf, pollen, and root; R3 stage pod and immature seed; R5 progenitor leaf; and R8 seed cotyledon and seed embryo.

[0142] Qualitative and quantitative GUS analysis was used to evaluate expression element activity, specifically the effect of regulatory elements on GUS transgene expression in selected plant organs and tissues of transformed plants. For qualitative analysis of GUS expression via histochemical staining, whole or sectioned tissues were incubated with a GUS staining solution containing 1 mg / mL X-Gluc (5-bromo-4-chloro-3-indolyl-β-glucuronide) at 37°C for 5 hours, followed by destaining with 35% EtOH and 50% acetic acid. GUS expression was qualitatively determined by visual examination of blue staining in selected plant organs or tissues under a dissecting microscope or a combination microscope.

[0143] To quantify GUS expression via enzymatic assay, total protein was extracted from selected tissues of transformed soybean plants. One to two micrograms of total protein were incubated with 1 mM of the fluorescent substrate 4-methylumbelliferyl-β-D-glucuronide (MUG) in a total reaction volume of 50 μL. After incubation at 37 °C for 1 hour, the reaction was stopped by adding 350 μL of 200 mM sodium bicarbonate solution. The reaction product, 4-methylumbelliferone (4-MU), exhibited maximum fluorescence at high pH, ​​where the hydroxyl group was ionized. The assay was stopped by adding alkaline sodium carbonate solution, and the pH was adjusted to quantify the fluorescent product 4-MU. The amount of 4-MU formed was estimated by measuring fluorescence using a FLUOstar Omega microplate reader (BMG LABTECH) (excitation at 355 nm, emission at 460 nm). GUS activity values ​​were provided as nanomoles of 4-MU / hour / mg total protein.

[0144] Example 5 Determination of expression element activity in stably transformed soybean plants Soybean plants were transformed using a plant binary expression construct containing expression elements as presented in SEQ ID NO: 1-6 that drive the expression of the β-glucuronidase (GUS) transgene. GUS protein expression in the resulting plants was analyzed to assess the effect of the regulatory elements on expression.

[0145] Soybean plants were transformed using plant binary expression constructs similar to those described in Example 4 above. Qualitative and quantitative GUS expression was also determined as described in Example 4. The following tissues from vegetative and reproductive plant developmental stages were sampled for GUS expression in generation R0: V5 stage saucer, progenitor leaf, and root; R1 stage flower, petiole, progenitor leaf, pollen, and root; R3 stage pod and immature seed; and R5 progenitor leaf; and R8 seed cotyledon and seed embryo. In addition to the above tissues, GUS expression was examined microscopically by histochemical staining of axillary floral meristems and apical meristems of vegetative branches from selected plants. Tables 6 and 7 below show the range and mean of quantitative GUS expression for each combination of expression elements, including standard error (Std Err).

[0146] Table 6. Range, mean, and standard error of GUS expression in stably transformed soybean plants.

[0147] Table 7. Range, mean, and standard error of GUS expression in stably transformed soybean plants.

[0148] As can be seen in Tables 6 and 7 above, all three promoters, P-Gm.CALa (SEQ ID NO:1), P-Gm.Mads17 (SEQ ID NO:3), and P-Gm.AP1 (SEQ ID NO:5), drive constitutive expression of the GUS transgene. Quantitatively, promoters P-Gm.CALa (SEQ ID NO:1) and P-Gm.Mads17 (SEQ ID NO:3) drive expression at similar levels in all sampled tissues. In contrast, histochemical staining analysis of GUS expression (as described above, determined by visual examination of the blue staining of selected plant organs or tissues; data not shown) revealed qualitative differences, particularly in R1 flowers. GUS expression driven by P-Gm.CALa (SEQ ID NO:1) was observed in nectaries and petals, while GUS expression driven by P-Gm.Mads17 (SEQ ID NO:3) was observed in nectaries and anthers. Furthermore, GUS staining of R1-origin leaves driven by P-Gm.CALa (SEQ ID NO:1) demonstrated expression in the phloem, epidermis, stomata, mesophyll, and vascular bundles, while expression driven by P-Gm.Mads17 (SEQ ID NO:3) was observed in the phloem, mesophyll, and vascular bundles. Microscopic examination of axillary flower meristems and vegetative branch apical meristems revealed that GUS expression driven by P-Gm.CALa (SEQ ID NO:1) and P-Gm.Mads17 (SEQ ID NO:3) was present only in axillary flower meristems and not in vegetative branch apical meristems. Compared with P-Gm.CALa (SEQ ID NO:1) and P-Gm.Mads17 (SEQ ID NO:3), GUS expression driven by P-Gm.AP1 (SEQ ID NO:5) was higher in flowers and pods. Histochemical staining analysis of GUS expression (as described above, determined by visual examination of the blue staining of selected plant organs or tissues; data not shown) revealed that GUS expression in R1 flowers was primarily found in the nectaries and anthers. Expression was also observed in axillary meristems.

[0149] Example 6 Autoexcision efficiency was determined in stable-transformed soybean plants using a redesigned Cre recombinase synthesis coding sequence. Soybean plants were transformed with a plant binary expression construct containing expression elements as shown in SEQ ID NO:1-4, which drive the expression of a codon-redesigned coding sequence (SEQ ID NO:21) encoding the Cre recombinase Cre-2. The ability of the resulting plants to effectively drive automatic excision was determined.

[0150] Soybean plants were transformed with a plant binary expression construct containing a redesigned coding sequence (SEQ ID NO:21) encoding the codon for Cre-2. The Cre-2 (SEQ ID NO:21) and Cre (SEQ ID NO:17) coding sequences presented in Example 2 both contain the same processable intron derived from the potato light-inducible tissue-specific St-LS1 gene (GenBank accession number: X04753), but are located differently relative to the two DNA fragments encoding the Cre recombinase. The Cre (SEQ ID NO:17) coding sequence contains a processable intron between nucleotides 431-621 of its 1221 bp synthetic coding sequence. Cre-2 (SEQ ID NO:21) has an intron located between nucleotides 208-396 of its corresponding 1221 bp synthetic coding sequence. Alignment of the two linked coding fragments of Cre and Cre-2 showed that they shared 74% sequence identity (based on alignments created using the Clustal W computational alignment method). Two constructs, similar in design to those described in Example 2 and presented in Table 8 below, were used to evaluate the ability and efficiency of the redesigned synthetic Cre-2 coding sequence (SEQ ID NO:21) to drive automatic excision, as described in Example 2 above.

[0151] Table 8. Constructs containing the redesigned synthetic Cre-2 coding sequence SEQ ID NO:21 for transforming soybean plants.

[0152] The efficiency of using the Cre Cre recombinase synthesis coding sequence (SEQ ID NO:17) presented in Example 2 above for construct-1 (containing P-Gm.CALa, SEQ ID NO:1) to drive automatic excision and produce homozygous and hemizygous marker-free plants was compared with the efficiency of using the Cre-2 Cre recombinase synthesis coding sequence (SEQ ID NO:21) for construct-A. Similarly, the efficiency of automatic excision of construct-2 (containing P-Gm.Mads17, SEQ ID NO:3) presented in Example 2 above was compared with that of construct-B to determine whether the newly designed Cre-2 Cre recombinase synthesis coding sequence (SEQ ID NO:21) produces an improved automatic excision frequency compared to the Cre Cre recombinase synthesis coding sequence (SEQ ID NO:17). As shown in Table 9 below, constructs 1 and 2 (both containing Cre, SEQ ID NO: 17) provided a higher percentage of homozygous and hemizygous marker-free plants when compared to constructs-A and-B (both containing Cre-2, SEQ ID NO: 21). This indicates that the Cre recombinase synthesis coding sequence presented in SEQ ID NO: 17 (Cre) is more efficient in providing marker-free plants when compared to the Cre recombinase synthesis coding sequence presented in SEQ ID NO: 21 (Cre-2).

[0153] Table 9. Comparison of the coding sequences for synthetic Cre recombinases in providing marker-free plants.

[0154] Implementation Plan To further illustrate this, additional non-restrictive embodiments of this disclosure are described below.

[0155] Implementation scheme 1 is a recombinant DNA molecule containing a DNA sequence selected from the group consisting of: a. A DNA sequence having at least 85% identity with any one of SEQ ID NO:1-14; b. A sequence containing any one of SEQ ID NO: 1-14; and c. (i) any one of SEQ ID NO:1-14 or (ii) a fragment of any DNA sequence having at least 85% sequence identity with any one of SEQ ID NO:1-14, wherein the fragment has gene regulatory activity; wherein the DNA sequence is operatively ligated to a heterologous transcribed DNA sequence.

[0156] Implementation scheme 2 is a recombinant DNA molecule as described in implementation scheme 1, wherein the DNA sequence has at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity with the DNA sequence of any one of SEQ ID NO:1-14.

[0157] Implementation scheme 3 is a recombinant DNA molecule as described in implementation scheme 1 or 2, wherein the DNA sequence has regulatory activity, or wherein the DNA sequence has promoter activity.

[0158] Implementation scheme 4 is a recombinant DNA molecule as described in any one of implementation schemes 1-3, wherein the heterologous transcribed DNA sequence coding site-specific recombinase is described.

[0159] Embodiment 5 is a recombinant DNA molecule as described in Embodiment 4, wherein the site-specific recombinase is selected from the group consisting of Cre recombinase, Flp recombinase, R recombinase and Gin recombinase; or wherein the site-specific recombinase is Cre recombinase.

[0160] Implementation Scheme 6 is a recombinant DNA construct comprising a recombinant DNA molecule as described in any one of Implementation Schemes 1-5 and including: i. Expression cassettes containing selectively marker-driven transgenes; and / or ii. Expression cassettes encoding site-specific nucleases; and / or iii. One or more expression cassettes encoding one or more guide RNAs; and / or iv. Expression cassettes containing transgenic materials with agronomical value.

[0161] Implementation 7 is a recombinant DNA construct as described in Implementation 6, further comprising a pair of site-specific recombination site sequences flanking one or more of the following: the recombinant DNA molecule and / or the expression cassette containing the selectively labeled transgene; and / or the expression cassette containing the site-specific nuclease; and / or the one or more expression cassettes encoding the one or more guide RNAs; The site-specific recombination sites thereon can be cleaved by site-specific recombinases.

[0162] Implementation scheme 8 is a recombinant DNA construct as described in implementation scheme 7, wherein: i. The site-specific recombination site sequence pairs are arranged head-to-tail; and / or ii. Each of the site-specific recombination sequence pairs is selected from the group consisting of: LoxP, FRT, RS, and GIX; or each of the site-specific recombination sequence pairs is a LoxP sequence; or each of the site-specific recombination sequence pairs contains SEQ ID NO:18.

[0163] Implementation scheme 9 is a recombinant DNA construct as described in any one of implementation schemes 6-8, wherein: i. The selectively labeled transgene confers resistance to herbicides or antibiotics; and / or ii. The aforementioned transgenic material with agronomical value confers herbicide tolerance, or pest or disease resistance, or increased yield or stress tolerance, or encodes dsRNA, miRNA, or siRNA.

[0164] Implementation scheme 10 is a recombinant DNA construct as described in any one of implementation schemes 6-9, wherein: i. The guide RNA contains a targeting sequence that targets a sequence in the genome of a eukaryotic or plant cell for genome editing or site-specific integration; and / or ii. The site-specific nuclease is an RNA-guided endonuclease; or iii. The RNA-guided endonuclease is selected from the group consisting of: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12a, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, CasX, and CasY; or iv. The RNA-guided endonuclease is Cas12a.

[0165] Implementation scheme 11 is a DNA transformation vector, which comprises: i. A recombinant DNA molecule as described in any one of embodiments 1 to 5, or a recombinant DNA construct as described in any one of embodiments 6 to 10; or ii. A recombinant DNA molecule as described in any one of embodiments 1 to 5, or a recombinant DNA construct as described in any one of embodiments 6 to 10, and a T-DNA segment defined by a left boundary and a right boundary.

[0166] Embodiment 12 is a DNA transformation vector as described in Embodiment 11, comprising a site-specific recombinase for the heterologous transcribed DNA sequence coding site in the recombinant DNA molecule and located between the left and right boundaries of the T-DNA segment.

[0167] Embodiment 13 is a DNA transformation vector as described in Embodiment 12, wherein the selectively labeled transgene, and / or the expression cassette encoding the site-specific nuclease, and / or the one or more expression cassettes encoding the one or more guide RNAs, and / or the agronomically valuable transgene is located between the left and right boundaries of the T-DNA segment.

[0168] Embodiment 14 is a transgenic plant, plant part or plant cell or bacterial cell containing a recombinant DNA molecule as described in any one of Embodiments 1-5 or a recombinant DNA construct as described in any one of Embodiments 6-10.

[0169] Embodiment 15 is a transgenic plant, plant part or plant cell as described in Embodiment 14, wherein a recombinant DNA molecule as described in any one of Embodiments 1-5 or a recombinant DNA construct as described in any one of Embodiments 6-10 is stably transformed into the genome of the transgenic plant, plant part or plant cell; and / or the transgenic plant, plant part or plant cell is selected from the group consisting of: maize, soybean, cotton or rapeseed plants, plant parts or plant cells.

[0170] Implementation scheme 16 is a method for producing transgenic plants or plant parts, the method comprising: a. Transforming explant cells with a recombinant DNA molecule as described in any one of embodiments 1-5, or a recombinant DNA construct as described in any one of embodiments 6-10, or a DNA transformation vector as described in any one of embodiments 11-13; and b. Regenerating or developing a transgenic plant from the explant, wherein the transgenic plant contains the recombinant DNA molecule or recombinant DNA construct stably transformed into the genome of one or more cells of the transgenic plant.

[0171] Implementation scheme 17 is the method as described in implementation scheme 16, the method further comprising: c. Isolating or harvesting plant parts from the genetically modified plant; and / or d. To hybridize one or more of the offspring plants with itself or another plant.

[0172] Implementation Scheme 18 is a method for excising expression cassettes from the genome of a transgenic plant, the method comprising: a. Transform explant cells using the following methods: i. A recombinant DNA construct as described in any one of embodiments 7-10, comprising a site-specific recombinase for the heterologous transcribed DNA sequence coding site in the recombinant DNA molecule, or ii. A DNA transformation vector as described in any one of embodiments 11-13, comprising a site-specific recombinase for the heterologous transcribed DNA sequence coding site in the recombinant DNA molecule; b. Regeneration or development of or acquisition of transgenic plants, at least in part, from one or more of the stably transformed plant cells; c. To hybridize the genetically modified plant with itself or another plant; and d. Select one or more progeny plants in which one or more of the heterologous transcribed DNA sequence encoding the site-specific recombinase and / or the selectively labeled transgene and / or the expression cassette encoding the site-specific nuclease and / or the expression cassette encoding the guide RNA between the site-specific recombination site sequence pairs of the recombinant DNA construct are excised and are no longer present in the genome of the progeny plant.

[0173] Implementation scheme 19 is the method as described in any one of implementation schemes 16-18, wherein: i. The plant cells are transformed via Agrobacterium-mediated transformation, Rhizobium-mediated transformation, Microsphere-mediated transformation, or Particle bombardment-mediated transformation; and / or ii. The transgenic plant or plant cell is selected from the group consisting of: corn, soybean, cotton or rapeseed plants or plant cells.

[0174] Embodiment 20 is a recombinant DNA molecule comprising a DNA sequence having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity with SEQ ID NO:21, wherein said DNA sequence is a Cre recombinase encoding sequence.

[0175] Implementation scheme 21 is a recombinant DNA molecule as described in any one of implementation schemes 1-3, wherein: i. The heterologous transcribed DNA molecule contains a gene of agronomical value; or ii. The heterologous transcribed DNA molecule encodes dsRNA, miRNA, or siRNA.

[0176] Implementation scheme 22 is a DNA molecule as described in implementation scheme 21, wherein: i. The agronomically valuable genes confer herbicide tolerance to plants; or ii. The agronomically valuable genes confer plant pest resistance.

[0177] Implementation scheme 23 is a transgenic plant cell containing recombinant DNA molecules as described in implementation scheme 21 or 22.

[0178] Implementation scheme 24 is a transgenic plant cell as described in implementation scheme 23, wherein the transgenic plant cell is a monocotyledonous plant cell or a dicotyledonous plant cell.

[0179] Implementation scheme 25 is a transgenic plant or plant part, transgenic plant seed, or its progeny plant or plant part, which contains a recombinant DNA molecule as described in implementation scheme 21 or 22.

[0180] Implementation scheme 26 is a method for producing a commercial product, the method comprising obtaining a genetically modified plant or a portion thereof according to implementation scheme 25.

[0181] Implementation scheme 27 is the method as described in implementation scheme 26, wherein the commercial product is selected from the group consisting of: seeds, processed seeds, protein concentrates, protein isolates, starch, grains, plant parts, seed oils, biomass, flour and coarse flour.

[0182] Embodiment 28 is a method for expressing a transcribed DNA molecule, comprising obtaining a transgenic plant according to Embodiment 25 and culturing the plant, wherein the transcribed DNA molecule is expressed.

Claims

1. A recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: a) A DNA sequence having at least 85% sequence identity with any one of SEQ ID NO:1-14; b) A DNA sequence containing any one of SEQ ID NO:1-14; and c) (i) any one of SEQ ID NO:1-14 or (ii) a fragment of a DNA sequence having at least 85% sequence identity with any one of SEQ ID NO:1-14, wherein the fragment has gene regulatory activity; The DNA sequence therein is operatively linked to a heterologous transcribed DNA sequence.

2. The recombinant DNA molecule of claim 1, wherein the DNA sequence has at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity with the DNA sequence of any one of SEQ ID NO:1-14.

3. The recombinant DNA molecule of claim 1, wherein the DNA sequence has gene regulatory activity, or wherein the DNA sequence has promoter activity, or wherein the DNA sequence has 3'UTR activity.

4. The recombinant DNA molecule of claim 1, wherein the heterologous transcribed DNA sequence coding site-specific recombinase.

5. The recombinant DNA molecule of claim 4, wherein the site-specific recombinase is selected from the group consisting of Cre recombinase, Flp recombinase, R recombinase and Gin recombinase; or wherein the site-specific recombinase is Cre recombinase.

6. A recombinant DNA construct comprising the recombinant DNA molecule as claimed in claim 1 and further comprising: (i) Expression cassettes containing selectively marker-driven transgenes; and / or (ii) Expression cassettes for coding site-specific nucleases; and / or (iii) One or more expression cassettes encoding one or more guide RNAs; and / or (iv) Expression cassettes containing transgenic materials with agronomical value.

7. The recombinant DNA construct of claim 6, further comprising a pair of site-specific recombination site sequences flanking one or more of the following: the recombinant DNA molecule and / or the expression cassette containing the selectively labeled transgene; and / or the expression cassette containing the site-specific nuclease; and / or the one or more expression cassettes encoding the one or more guide RNAs; The site-specific recombination sites thereon can be cleaved by site-specific recombinases.

8. The recombinant DNA construct of claim 7, wherein: (i) The site-specific recombination site sequence pairs are arranged head-to-tail; and / or (ii) Each of the site-specific recombination site sequence pairs is selected from the group consisting of LoxP, FRT, RS and GIX; or each of the site-specific recombination site sequence pairs is a LoxP sequence; or each of the site-specific recombination site sequence pairs contains SEQ ID NO:

18.

9. The recombinant DNA construct of claim 6, wherein... (i) The selectively labeled transgene confers resistance to herbicides or antibiotics; and / or (ii) The agronomically valuable transgenic material confers herbicide tolerance, or pest or disease resistance, or increased yield or stress tolerance, or encodes dsRNA, miRNA or siRNA.

10. The recombinant DNA construct of claim 6, wherein: (i) The guide RNA contains a targeting sequence that targets a sequence in the genome of a eukaryotic or plant cell for genome editing or site-specific integration; and / or (ii) The site-specific nuclease is an RNA-guided endonuclease; or (iii) The site-specific nuclease is selected from the group consisting of: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12a, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, CasX and CasY; or (iv) The site-specific nuclease is Cas12a.

11. A DNA transformation vector comprising: (i) the recombinant DNA molecule as claimed in claim 1, or the recombinant DNA construct as claimed in claim 6; or (ii) The recombinant DNA molecule as claimed in claim 1, or the recombinant DNA construct as claimed in claim 6, and the T-DNA segment defined by the left and right boundaries.

12. The DNA transformation vector of claim 11, comprising a site-specific recombinase for the heterologous transcribed DNA sequence in the recombinant DNA molecule and located between the left and right boundaries of the T-DNA segment.

13. The DNA transformation vector of claim 12, comprising the expression cassette for selectively labeling the transgene, and / or the expression cassette encoding the site-specific nuclease, and / or the one or more expression cassettes encoding the one or more guide RNAs, and / or the agronomically valuable transgene located between the left and right boundaries of the T-DNA segment.

14. A transgenic plant, plant part or plant cell comprising a recombinant DNA molecule as claimed in claim 1 or a recombinant DNA construct as claimed in claim 6.

15. The transgenic plant, plant part, or plant cell of claim 14, wherein the recombinant DNA molecule of claim 1 or the recombinant DNA construct of claim 6 is stably transformed into the genome of the transgenic plant, plant part, or plant cell; and / or the transgenic plant, plant part, or plant cell is selected from the group consisting of: maize, soybean, cotton, or rapeseed plants, plant parts, or plant cells.

16. A method for producing a transgenic plant or plant part, the method comprising: a) Transforming explant cells with the recombinant DNA molecule as described in claim 1, or the recombinant DNA construct as described in claim 6, or the DNA transformation vector as described in claim 11; and b) Regenerating or developing a transgenic plant from the explant, wherein the transgenic plant contains the recombinant DNA molecule or recombinant DNA construct stably transformed into the genome of one or more cells of the transgenic plant.

17. The method of claim 16, further comprising: c) Isolating or harvesting plant parts from the genetically modified plant; and / or d) To hybridize one or more of the offspring plants with itself or another plant.

18. A method for excising an expression cassette from the genome of a transgenic plant, the method comprising: a) Transform the plant cells of the explants using the following methods: (i) The recombinant DNA construct of claim 7, comprising a site-specific recombinase for the heterologous transcribed DNA sequence coding site in the recombinant DNA molecule, or (ii) The DNA transformation vector of claim 11, comprising a site-specific recombinase for the heterologous transcribed DNA sequence coding site in the recombinant DNA molecule; b) Regeneration or development or acquisition of transgenic plants, at least in part, from one or more of the stably transformed plant cells; c) Hybridizing the genetically modified plant with itself or another plant; and d) Select one or more progeny plants in which one or more of the heterologous transcribed DNA sequence encoding the site-specific recombinase and / or the selectively labeled transgene and / or the expression cassette encoding the site-specific nuclease and / or the expression cassette encoding the guide RNA between the site-specific recombinant site sequence pairs of the recombinant DNA construct are excised and are no longer present in the genome of the progeny plant.

19. The method of claim 16, wherein: (i) The plant cells are transformed via Agrobacterium-mediated transformation, Rhizobium-mediated transformation, Microsphere-mediated transformation, or Particle bombardment-mediated transformation; and / or (ii) The transgenic plant or plant part or plant cell is selected from the group consisting of: corn, soybean, cotton or rapeseed, plant part or plant cell.

20. The method of claim 18, wherein: (i) The plant cells are transformed via Agrobacterium-mediated transformation, Rhizobium-mediated transformation, Microsphere-mediated transformation, or Particle bombardment-mediated transformation; and / or (ii) The transgenic plant or plant part or plant cell is selected from the group consisting of: corn, soybean, cotton or rapeseed, plant part or plant cell.

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