Plant regulatory elements and their use

Abstract

This provides useful regulatory elements in the fields of plant molecular biology and plant genetic engineering. [Solution] The present invention provides recombinant DNA molecules and constructs useful for regulating gene expression in plants, as well as their nucleotide sequences. The present invention also provides transgenic plants, plant cells, plant parts, and seeds containing recombinant DNA molecules operably linked to heterologous transcriptionable DNA molecules, and also provides methods for using them.

Description

Technical Field

[0001] Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 016,904, filed Apr. 28, 2020, the entire disclosure of which is hereby incorporated by reference herein.

[0002] Incorporation of Sequence Listing The sequence listing contained in the file named “MONS481WO-sequence_listing.txt” is 13,550 bytes (measured in Microsoft Windows®) and was created on Apr. 22, 2021, and is hereby submitted with this specification via electronic submission and is incorporated by reference herein. (registered trademark)

[0003] The present invention relates to the fields of plant molecular biology and plant genetic engineering. More specifically, the present invention relates to DNA molecules useful for regulating gene expression in plants.

Background Art

[0004] Regulatory elements are genetic elements that regulate gene activity by regulating the transcription of a transcriptionally capable DNA molecule operably linked thereto. Such elements can include promoters, leaders, introns, and 3′ untranslated regions, which are useful in the fields of plant molecular biology and plant genetic engineering.

Summary of the Invention

[0005] The present invention provides gene regulatory elements for use in plants. The present invention also provides recombinant DNA molecules comprising the regulatory elements. The present invention also provides transformation vectors comprising the regulatory elements. We also provide genetically modified plant cells, plants, and seeds. In one embodiment, we regulate the The rement is operably linked to a transcribable DNA molecule. In certain embodiments In this case, the transcriptionable DNA molecule may be heterogeneous with respect to the regulatory sequence. Therefore, The regulatory element array provided by the invention, in certain embodiments, is a heterogeneous transcription It can be defined as being operablely linked to a possible DNA molecule. The present invention also provides a regulatory element. Using a ment, recombinant DNA molecules containing regulatory elements, as well as transcriptional DNA molecules Transgenic plant cells, plants, and plants containing regulatory elements operably linked to their offspring. Methods for producing and using seeds are also provided.

[0006] Therefore, in one embodiment, the present invention relates to a recombinant DNA molecule comprising (a) sequence A sequence having at least approximately 85 percent sequence identity with any of numbers 1 to 5, b) A sequence containing any of sequence numbers 1 to 5, and (c) a sequence having gene regulatory activity, The DNA sequence includes a DNA sequence selected from the group consisting of any fragment from sequence number 1 to 5, A recombinant DNA molecule in which the sequence is operably linked to a different type of transcriptionable DNA molecule. It provides "heterogeneous transcriptionable DNA molecules" means that transcriptionable DNA molecules are operational. This means that the polynucleotide sequence linked to it is heterogeneous. In certain embodiments The recombinant DNA molecule is at least approximately [number missing] for any of the DNA sequences of SEQ ID NOs: 1-5. 85 percent, at least about 86 percent, at least about 87 percent, at least Approximately 88 percent, at least approximately 89 percent, at least approximately 90 percent, and less At least 91 percent, at least 92 percent, at least 93 percent, less 94 percent, at least 95 percent, at least 96 percent, and at least 97 percent, at least 98 percent, or at least 99 percent array Contains identical DNA sequences.

[0007] In another aspect, this specification describes transgenic plant molecules containing recombinant DNA molecules. A cell, and (a) containing at least about 85 percent of the sequence of any of sequence numbers 1-5 (b) sequences having identity, (c) sequences containing any of sequence numbers 1 to 5, and (d) genetic A selection is made from the group consisting of any of the SEQ ID NOs: 1 to 5, which have regulatory activity. It includes a DNA sequence, and the DNA sequence is operably linked to a different transcriptionable DNA molecule. The present invention provides transgenic plant cells containing recombinant DNA molecules. In this embodiment, the transgenic plant cell is a monocotyledonous plant cell. Other embodiments In this context, transgenic plant cells are dicotyledonous plant cells.

[0008] In yet another aspect, this specification further describes trans... A genic plant or a part thereof, a) at least one of sequence numbers 1-5 a) A sequence having 85 percent sequence identity, b) A sequence containing any of sequence numbers 1 to 5 , and c) from any of the SEQ ID NOs: 1-5, which have gene regulatory activity The DNA sequence is selected from the group and the sequence interacts with a different type of transcribable DNA molecule. A transgenic plant or part containing a recombinant DNA molecule that is movably linked. Provides a position. In certain embodiments, the transgenic plant contains recombinant DNA molecules. It is an offspring plant of any generation. Also provided are transgenic seeds containing recombinant DNA molecules that produce such transgenic plants when grown.

[0009] In another aspect, the present invention provides a method for producing a commercial product, comprising obtaining a transgenic plant or a part thereof containing the recombinant DNA molecule of the present invention and then producing a commercial product therefrom. In one embodiment, the commercial product is seeds, processed seeds, protein concentrates, protein isolates, starch, grains, plant parts, seed oils, biomass, flours, and grits.

[0010] In yet another aspect, the present invention provides a method for producing a transgenic plant containing the recombinant DNA molecule of the present invention, comprising transforming a plant cell with the recombinant DNA molecule of the present invention to produce a transformed plant cell and regenerating a transgenic plant from the transformed plant cell.

[0011] Brief Description of the Sequences SEQ ID NO: 1 is a DNA sequence of a regulatory expression element group or EXP (EXP-ANDge.Ubq:5) derived from Andropogon gerardii, which is composed of a promoter (P-ANDge.Ubq:7) operably linked to the 5' side of a leader (L-ANDge.Ubq:2) operably linked to the 5' side of an intron (I-ANDge.Ubq:2). [[ID=3�]]

[0012] SEQ ID NO: 2 is the DNA sequence of a promoter (P-ANDge.Ubq:7) derived from Andropogon gerardii.

[0013] Sequence ID 3 is a leader or 5'UT derived from Andropogon gerardii. This is the DNA sequence of R(L-ANDge.Ubq:2).

[0014] Sequence ID 4 is an intron (I-AND) derived from Andropogon gerardii. This is the DNA sequence of ge.Ubq:2).

[0015] Sequence ID 5 is derived from Arundo donax 3'UTR(T-ARUdo.TubA This is the DNA sequence of :1).

[0016] Sequence ID 6 is the DNA distribution of EXP (EXP-CaMV.35S+Zm.DnaK:12) It is a row.

[0017] Sequence ID 7 is derived from Sorgum bicolor 3'UTR(T-Sb.Nltp4) This is the DNA sequence -1:1:2).

[0018] Sequence ID 8 is the potato light-inducible tissue-specific St-LS1 gene (Genbank A β- Planting of glucuronidase (GUS, GOI-Ec.uidA+St.LS1.nno:1) This is a synthetic coding sequence optimized for gene expression. [Modes for carrying out the invention]

[0019] The present invention provides regulatory elements having gene regulatory activity in plants. The nucleotide sequences of the regulatory elements are provided as SEQ ID NOs: 1-5. The elements influence the expression of operably linked, transcribed DNA molecules within plant tissue. This can have an effect, and therefore, in transgenic plants, operably linked transgenes The gene expression of can be regulated. Furthermore, the present invention relates to recombinant D containing the provided regulatory element. Methods for modifying, producing, and using NA molecules are also provided. Furthermore, the present invention provides recombinant forms of the present invention. Composition including transgenic plant cells containing DNA molecules, plants, plant parts, and seeds. The material, as well as methods for preparing and using it, are also provided.

[0020] The following definitions and methods better define the present invention and provide guidance to those skilled in the art for implementing the present invention. Provided for the purpose of [doing so]. Unless otherwise noted, terms are defined by those skilled in the art in the relevant field. It shall be understood according to the usage of the original text.

[0021] dna molecule As used herein, the terms “DNA” or “DNA molecule” refer to 5' (upstream). A double-stranded DNA sequence of genomic or synthetic origin, read from the ) end to the 3' (downstream) end. This refers to molecule A (i.e., a polymer of deoxyribonucleotide bases or DNA molecules). As used herein, the term "DNA sequence" refers to the nucleotide sequence of a DNA molecule. Refers to a column. The nomenclature used herein is the nomenclature of 37, Section 1.822 of the Code of Federal Regulations. This corresponds to the standards set forth in Tables 1 and 3 of Annex 2 of WIPO Standard ST.25 (1998). .

[0022] As used herein, "recombinant DNA molecule" means a molecule that does not naturally coexist without human intervention. This refers to DNA molecules containing combinations of DNA molecules that would not otherwise occur. For example, recombinant DNA. A molecule is composed of at least two DNA molecules that are heterogeneous to each other. A molecule, a DNA molecule containing a DNA sequence that deviates from naturally occurring DNA sequences, synthetic D DNA molecules containing NA sequences, or host cells through gene transformation or gene editing. It could also be a DNA molecule incorporated into the DNA.

[0023] References to “isolated DNA molecules” or equivalent terms or expressions in this application are DNA molecules exist alone or in combination with other compositions, but in their natural environment... It is intended to mean something that does not exist. For example, a code array, in TRON sequences, untranslatable reader sequences, promoter sequences, transcription termination sequences, etc., are biological Nucleic acid elements found naturally within the DNA of a genome are those elements that are part of the genome of an organism. As long as it is located within the genome and the element is in a naturally occurring position within the genome, it is "isolated". It is not considered to be ". However, each of these elements, and these elements The lower part is that the element is not present in the genome of an organism, and the element is found in nature. Unless located within the genome, it is considered “isolated” within the scope of this disclosure. , insecticidal proteins or any naturally occurring insecticidal variants of those proteins The nucleotide sequence to be coded is the sequence in which the nucleotide sequence codes for the protein. Unless it is found in the DNA of naturally occurring bacteria, it is considered an isolated nucleotide sequence. A synthetic nucleotide sequence that codes for the amino acid sequence of a naturally occurring insecticidal protein. For the purposes of this disclosure, it is considered to be isolated. And any transgenic nucleotide sequence, i.e., plant or bacterial cell DNA nucleotides that are inserted into the genome or present in an extrachromosomal vector The sequence is located within a plasmid or similar structure used for cell transformation. Even if present within the genome of a plant or bacterium, or if derived from a plant or bacterium, Even if present in detectable amounts in textiles, progeny, biological samples, or commodity products, isolation is possible. It is considered to be a nucleotide sequence.

[0024] As used herein, the term “sequence identity” refers to two optimal alignments. A polynucleotide sequence or two optimally aligned polypeptide sequences It refers to a certain degree of oneness. The optimal array alignment is for two arrays, for example, a reference array and one other. Manually align one sequence with another to ensure proper internal nucleotide insertions, deletions, and This is achieved by maximizing the number of nucleotide matches in a sequence alignment with gaps. This is how it is produced. In this specification, the term "reference sequence" refers to sequence numbers 1-5. This refers to the DNA sequence provided as such.

[0025] As used herein, "sequence identity percentage" or "identity percentage" The term "identity %" is the percentage of identity multiplied by 100. The "percentage of identity" for sequences that have been optimally aligned is the optimal alignment The number of nucleotide matches within the reference sequence is the total number of nucleotides in the reference sequence, for example, the full-length reference sequence. This is obtained by dividing by the total number of nucleotides. Therefore, one embodiment of the present invention is described in this specification. When optimally aligned with the reference arrays provided as sequence numbers 1-5 in the book. , at least approximately 85 percent identity with respect to the reference sequence, at least approximately 86 percent Identity, at least approximately 87 percent identity, at least approximately 88 percent identity , at least approximately 89 percent identity, at least approximately 90 percent identity, less Both have approximately 91 percent identity, at least approximately 92 percent identity, and at least approximately 9 3 percent identity, at least about 94 percent identity, at least about 95 percent Identity of the subject, at least approximately 96 percent identity, at least approximately 97 percent identity One gender, at least approximately 98 percent identity, at least approximately 99 percent identity, The present invention provides a DNA molecule containing a sequence with at least approximately 100 percent identity. .

[0026] Adjustment element Promoter, leader (also known as 5'UTR), enhancer, intron, and transcription Regulatory elements such as the termination region (or 3'UTR) affect the overall expression of genes in living cells. It plays an essential role in this. When used herein, the term "regulating element" is used The term refers to a DNA molecule that has gene regulatory activity. When used herein, "gene regulatory activity" refers to a DNA molecule that has gene regulatory activity. The term "nodal activity" refers, for example, to the transcription of operably linked transcriptionable DNA molecules and / or affect the translation of a operably linked transcriptable DNA molecule This refers to the ability to influence expression. In plants, it refers to promoters, leaders, and enhancers that function. Regulatory elements such as sensors, introns, and 3'UTRs are used in genetically engineered plant expression. It is useful for modifying the current form.

[0027] As used herein, the term "regulatory expression element group" or "EXP sequence" refers to the operation of the element. A group of modulating elements that can be linked together, for example, enhancer, promoter, leader, This can refer to a group of introns. For example, the group of regulatory expression elements may include, for example, introns. A leader that is operablely connected to the 5' side of the array, and a leader that is operablely connected to the 5' side of the array. It may consist of linked promoters. An EXP useful for carrying out the present invention is sequence number It is presented as item number 1.

[0028] The regulatory elements affect their gene expression patterns, for example, positive and / or negative effects. For example, constitutive expression or temporal, spatial, developmental, tissue, environmental, physiological, or pathological , the cell cycle, and / or the expression of chemical responses, as well as any combination thereof, in addition It can be characterized by quantitative or qualitative indicators. When used herein, "genetics" refers to genetics. "Sub-expression pattern" refers to the process by which operably linked DNA molecules are transcribed into transcription RNA molecules. It is any pattern. The transcribed RNA molecule is translated to produce a protein molecule. In some cases, antisense RNA or other regulatory RNA molecules, such as double-stranded RNA, may be involved. (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), It can also produce microRNAs (miRNAs), small interfering RNAs (siRNAs), etc. ru.

[0029] As used herein, the term "protein expression" means that a transcription RNA molecule is a protein It is any pattern that is translated into protein molecules. Protein expression is temporal and spatial. Characterized by developmental or morphological properties, in addition by quantitative or qualitative indicators. It can be attached.

[0030] A promoter is used to regulate the expression of operably linked, transcriptable DNA molecules. It is useful as a regulatory element. When used herein, the term "promoter" is used The term generally refers to RNA polymerase II and other proteins (for example) that initiate transcription. This refers to DNA molecules involved in the recognition and binding of trans-acting transcription factors. It can be initially isolated from the 5' untranslated region (5'UTR) of a genomic gene copy, or For the purposes of this disclosure, the promoters provided herein shall have 5 rights to the leader. It consists of a promoter operably connected to the side. Alternatively, the promoter is The DNA molecule may be synthetically produced or manipulated. Furthermore, the promoter may be a chimeric one. It may also be the case that a chimeric promoter is produced by the fusion of two or more heterologous DNA molecules. A promoter useful for carrying out the present invention is presented as Sequence ID No. 2. or a fragment or variant thereof. In certain embodiments of the present invention , the DNA molecules of the claims and any variant thereof as described herein or Derivatives are further defined as those containing promoter activity, i.e., transgenic It can act as a promoter within host cells such as those of nicks. Further specific implementations are also possible. Morphologically, the fragment is a promo possessed by the initiation promoter molecule from which it originates. Sometimes defined as something that exhibits teratogenic activity, fragments are basal-level transcriptions. This leads to the RNA polymerase II complex recognizing and binding to it in order to initiate transcription. Includes a "minimal promoter" composed of a TATA box or equivalent DNA sequence. In some cases, it may be the case.

[0031] In one embodiment, a fragment of the promoter sequence disclosed herein is provided The promoter fragment may contain the promoter activity described above, and , alone, or with other promoters (for example, when constructing a chimeric promoter) In combination with promoter fragments, or with other expression elements and expression elements It may be useful in combination with the fragments of the following: In certain embodiments, at least about 5 0, at least about 75, at least about 95, at least about 100, at least about 125, At least approximately 150, at least approximately 175, at least approximately 200, at least approximately 225, At least approximately 250, at least approximately 275, at least approximately 300, at least approximately 500, At least about 600, at least about 700, at least about 750, at least about 800, At least about 900, or at least about 1000 consecutive nucleotides, or The above includes a DNA molecule having the promoter activity disclosed herein, - A fragment is provided. Such a fragment is produced from the start promoter molecule. The methods for producing these products are well known in the relevant technical field.

[0032] A set derived from the promoter element of sequence number 2 (e.g., internal or 5' deletion). The product, for example, has either a positive or negative effect on expression in order to improve or modify its expression. Removal of elements that affect, duplication of elements that have a positive or negative effect on expression, and / or duplication of elements that have tissue-specific or cell-specific effects on expression or It can be produced using methods known in the art, including removal. Promoter of Sequence ID No. 2 - Elements (TATA box elements or equivalent arrays and downstream arrays are removed) Using a composition derived from the 3' deletion that is present, for example, enhance - Elements can be created. Further deletions can be performed to create tissue-specific, detailed elements with positive or negative expression. Cell-specific or time-specific (e.g., circadian rhythm) effects Any element affecting it can be removed. Promo provided as Sequence ID 2 Using a ter element, and fragments or enhancers derived therefrom This allows for the creation of chimeric transcription regulatory element compositions.

[0033] According to the present invention, the promoter or promoter fragment is a known promoter - Elements, namely TATA boxes and other known transcription factor binding site motifs The presence of certain DNA sequence characteristics may be analyzed. Such known promoters - The identification information of the element is such that, as a person skilled in the art, it has the same expression pattern as the original promoter. It can be used to design variants of promoters.

[0034] As used herein, the term "leader" refers to the untranslated 5' region (5') of a gene. Isolated from UTR, between the transcription start site (TSS) and the protein coding sequence start site. It refers to a DNA molecule, generally defined as a nucleotide segment. Alternatively, The leader may be a synthetically produced or manipulated DNA element. 5' regulatory element for regulating the expression of operably linked transcriptable DNA molecules It can be used as a leader molecule. The leader molecule is a heterologous promoter or their native It may be used in conjunction with a promoter. A useful reader for carrying out the present invention is sequence number It is presented as item 3, or a fragment or variant thereof. In one embodiment, such a DNA sequence is used in a host cell (e.g., transgenic It can be defined as being able to act as a leader within (including plant cells). In one embodiment, Therefore, such sequences are decoded as containing leader activity.

[0035] The leader sequence of sequence number 3 (also called 5'UTR) is composed of regulatory elements. In some cases, this may involve the transcription or translation of a operably linked, transcribed DNA molecule. In some cases, a secondary structure that can have an effect on the translation is adopted. The leader sequence of sequence number 3 is used in the original Use according to the instructions to cast shadow on the transcription or translation of operably linked transcribed DNA molecules. It is possible to create chimeric modulating elements that impart resonance.

[0036] As used herein, the term "intron" means an intron isolated or identified from a gene. Therefore, splicing is removed during pre-translational messenger RNA (mRNA) processing. It can be generally defined as a region of a DNA molecule that is synthesized. Alternatively, an intron is a compound. They may be chemically generated or manipulated DNA elements. Introns are operable. It may also include an enhancer element that brings about the transcription of the linked gene. Introduction The regulator is for adjusting the expression of operably linked transcriptable DNA molecules. It can be used as a ment. The structure may include an intron, which is a transformation. The DNA molecules that can be copied may or may not be heterogeneous. Examples of introns include the actin intron in rice and the HSP70 in maize. Introns are one example.

[0037] In plants, if a gene construct includes several introns, constructs lacking introns... Compared to this, the accumulation of mRNA and protein increases. This effect is due to the "in" gene expression. This is called "intron-mediated enhancement" (IME). It is known to stimulate expression in plants. The tron ​​is a maize gene (e.g., tubA1, Adh1, Sh1, and Ubi 1) Rice genes (e.g., tpi), as well as petunia genes (e.g., rbcS), Ja Derived from *Cornus thaliana* (e.g., st-ls1) and *Arabidopsis thaliana*. Identified in dicotyledonous plant genes such as those derived from (for example, ubq3 and pat1) It is possible that gene expression is reduced due to deletions or mutations within the intron splice site. This indicates that splicing may be necessary for IMEs. However, in dicotyledonous plants, the IME is the pat1 gene of A. thaliana. This is indicated by point mutations within the splice site. The same in one plant Multiple uses of TRON have been shown to present disadvantages. In such cases, appropriate To construct recombinant DNA elements, it is essential to assemble basic regulatory elements. This is essential. An exemplary intron useful for carrying out the present invention is presented as Sequence ID No. 4. It is being done.

[0038] As used herein, the terms "3' transcription termination molecule," "3' untranslated region," or "3'" The term "UTR" is used during transcription of the 3' portion of an mRNA molecule into the untranslated region. This refers to NA molecules. The 3' untranslated region of mRNA molecules undergoes specific cleavage and 3' polyadenylation. It can be produced by (also known as poly-A tail). The 3'UTR is a transcriptable DNA It may be operably linked to a molecule and located downstream of it, involved in transcription, mRNA processing, or Includes polyadenylation signals and other regulatory signals that can affect gene expression. But that's fine. The poly(A) tail is thought to function in mRNA stability and translation initiation. An example of a 3' transcription termination molecule in the relevant technical field is the nopaline synthase 3' region. Wheat hsp17 3' region, endowed visco subunit 3' region, cotton E6 This is the 3' region and the coixin 3'UTR.

[0039] Typically, the 3'UTR is useful for the recombinant expression of specific DNA molecules. This is acknowledged. A weak 3' UTR has the potential to result in a lead-through, and this lead-through This could potentially affect the expression of DNA molecules within adjacent expression cassettes. By appropriately controlling transcription termination, downstream localized DNA sequences (for example, other expression cassettes) can be controlled. This prevents read-through to (T), and furthermore, efficiently recycles RNA polymerase. This can improve gene expression. Efficient termination of transcription (RNA from DNA) The release of polymerase II is a necessary condition for resuming transcription, and therefore the overall transcription level It directly affects the cell. Following transcription termination, mature mRNA is released from the synthesis site, and the template is released from the cell. It is transported to the atmosphere. Eukaryotic mRNA accumulates in vivo as poly(A) form. Therefore, it becomes difficult to detect the transcription termination site using conventional methods. Functional and efficient 3'UTR prediction using the Matics method makes it easy to obtain a valid 3'UTR. The difficulty lies in the fact that there are no conserved DNA sequences that would allow for prediction.

[0040] From a practical standpoint, typically the 3'UTR used in expression cassettes has the following characteristics: Having it is beneficial. Firstly, the 3'UTR efficiently and effectively transcribes the transgene. It should be possible to terminate the process, and multiple expressions present within a single transmutation DNA (T-DNA) should be possible. Any adjacent DNA sequences that can be composed of another expression cassette, as in the case of a cassette, Alternatively, it can prevent the read-through of transcripts to adjacent chromosomal DNA into which the T-DNA is inserted. It should be done. Next, the 3'UTR is used to promote the expression of DNA molecules. Motors, leaders, enhancers, and introns cause a reduction in transcriptional activity. It should not be rubbed. Finally, in plant biotechnology, 3'UTR is a transmutation. It is often used for priming the amplification reaction of reverse-transcribed RNA extracted from succinous plants. Then, (1) evaluate the transcriptional activity or expression of the expression cassette once it has been incorporated into the plant chromosome. (2) Evaluate the copy number of insertions in plant DNA, and (3) After breeding It is used to evaluate the conjugation ability of the obtained seeds. Also, 3'UTR is inserted To characterize the integrity of the set, an amplification reaction of DNA extracted from transgenic plants was performed. It is also used. A 3'UTR useful for carrying out the present invention is presented as Sequence ID No. 5. It is being done.

[0041] As used herein, the terms "enhancer" or "enhancer element" are used to refer to the same term. The term refers to a cis-action regulatory element (also known as a cis element), which is operable. One aspect of the overall expression pattern of linked transcriptionable DNA molecules (however, usually, alone) It imparts (insufficient to drive transcription). Unlike promoters, enhancers are typically different. The element contains a transcription start site (TSS), a TATA box, or an equivalent DN. The A sequence is not included. The promoter or promoter fragment is naturally produced. One or more enhancer elements that affect the transcription of a movably linked DNA sequence. It may also include. Furthermore, the enhancer element may be fused with the promoter to enable gene development. It would be good to create a chimeric promoter system element that provides one aspect of the current overall adjustment. stomach.

[0042] Many promoter-enhancer elements bind to DNA-binding proteins, and / or affect the DNA topology, and the RNA polymerase into the DNA template Local three-dimensional structures that selectively allow or restrict access, or duplication at the transcription initiation site. It is thought to generate local three-dimensional structures that promote the selective release of the helix. - Elements may function to bind to transcription factors that regulate transcription. Some elements The Hanser element binds to two or more transcription factors, and the transcription factors bind to two or more transcription factors with different affinities. It can interact with the enhancer domain above. Identifying the enhancer element is possible through deletion resolution. Analysis (i.e., one or more nucleotides from the 5' end or inside of the promoter) (deleting the protein), or DNA-binding protein analysis using the DNaseI footprint method, Methylation interference, electrophoretic mobility shift assay, ligation-mediated polymerase chain reaction In vivo genome footprinting methods using PCR, and other conventional methods This can be done using multiple techniques, including Sei, or by using conventional DNA sequence ratios such as BLAST. A known cis-element motif or a target sequence or target motif by comparison. This can be done by DNA sequence similarity analysis using enhancer elements. The ultrastructure of the enhancer domain involves mutagenesis (or substitution) of one or more nucleotides. This may be further studied by or by other conventional methods known in the art. Enhancer elements are produced by chemical synthesis, or by modulating elements containing such elements. These can be obtained by isolation from the ment, and these facilitate the manipulation of subsequences. It can be synthesized with additional adjacent nucleotides containing useful restriction sites. Therefore, Disclosed herein for regulating the expression of movably linked, transcribed DNA molecules The design, construction, and use of enhancer elements in accordance with the method described herein are included in this invention. The enhancer may originate from the promoter presented as Sequence ID No. 2.

[0043] As used herein, the term “chimera” means a combination of a first DNA molecule and a second DNA molecule. This refers to a single DNA molecule produced by fusing with other molecules, and the first DNA molecule is also The second DNA molecule is also not usually found in this configuration (i.e., the configuration fused with the other). Therefore, chimeric DNA molecules are novel and not typically found in nature by other means. It is a DNA molecule. As used herein, the term "chimeric promoter" means this This refers to promoters produced by manipulating DNA molecules, such as chimeric promoters. A promoter combines two or more DNA fragments (for example, a promoter enhancer). - It can be fused with an element. Therefore, it can be operably bound and transferable. Chimeric promolars according to the methods disclosed herein for modulating the expression of DNA molecules The design, construction, and use of the device are included in this invention.

[0044] Chimeric regulatory elements can be modified by various methods known in the art, such as restriction enzyme digestion and Ligation, ligation-independent cloning, and modularization of PCR products during amplification. Direct chemical synthesis of radiator assemblies or regulating elements, and known in the art Designed to include various component elements that can be operably connected by other means. It is possible. The various chimeric modifier elements obtained may be the same constituent elements or the same constituent elements. It may consist of variants of elements, but the components must be operably connected. The DNA sequences (multiple) containing the linking DNA sequences (multiple) that enable this are different. The DNA sequences provided as Sequence IDs 1-5 provide regulatory element reference sequences. In some cases, the constituent elements that make up the reference array are those known in the relevant art. They may be linked by law, and one or more may occur naturally in the transformation of bacteria and plant cells. This may include substitutions, deletions, and / or insertions of the nucleotides listed above, or mutations.

[0045] As used herein, the term "variant" refers to a variant whose composition is similar to that of the first DNA molecule. This refers to a second DNA molecule that is similar but not identical, such as a regulatory element, and in this case The second DNA molecule, for example, has transcriptional activity roughly equivalent to that of the first DNA molecule. The general functionality of that DNA molecule, i.e., the same or similar expression pattern, remains unchanged. It maintains. A variant is a shorter or abbreviated version of the first DNA molecule, This is a modified version of the sequence of the first DNA molecule, for example, with different restriction enzyme sites, and / or It may be a version with internal deletions, substitutions, or insertions. Also, "Varian "T" is a nucleotide that includes the substitution, deletion, or insertion of one or more nucleotides in a reference sequence. It may also include regulatory elements having a 3D array, in which case the derivative regulatory element corresponds It is larger, smaller, or has equivalent transcriptional or translational activity to the parent regulatory molecule. Furthermore, regulatory elements, or "variants," naturally occur in the transformation of bacterial and plant cells. This also includes variants resulting from mutations. In the present invention, provided as SEQ ID NOs: 1 to 5 Using a polynucleotide sequence, the DNA sequence and composition are similar to those of the original regulatory element. However, they are not identical, while the general functionality of the original adjustment element, i.e., the same or It is possible to create variants that still maintain a similar expression pattern. The production of riant is well within the scope of the skills of those skilled in the art in light of this disclosure and within the scope of the present invention. It is included in.

[0046] Modifications, duplications, and The effects of deletions are described in the study examples herein to verify the results. The efficacy may also be tested experimentally in stable and transient plant assays. This can vary depending on the modifications made to the starting DNA molecule and the purpose of those modifications.

[0047] Structures As used herein, the term “construction” means a construct in which at least one DNA molecule is distinct It is functionally ligated to the DNA molecule in a manner that allows it to operate, that is, it is ligated in an operable manner. A DNA molecule containing a genomically integrated or self-replicating DNA molecule, originating from any source. Rasmids, cosmids, viruses, phages, or linear or circular DNA molecules or This refers to any recombinant DNA molecule, such as an RNA molecule. When used herein, "vector" refers to any recombinant DNA molecule. The term "transformation" refers to the introduction of heterologous DNA or RNA into a host cell. It means any structure that can be used for the purpose of doing so. A structure is typically one or more Includes an expression cassette. As used herein, “expression cassette” means one or more regulatory The element is typically operably coupled to at least a promoter and 3'UTR. This refers to a recombinant DNA molecule containing at least one transcriptable DNA molecule.

[0048] As used herein, the term “operably linked” refers to a second DNA molecule. This refers to the first DNA molecule linked to the first DN, where the first and second DNA molecules are the first DN Molecule A is positioned to influence the function of the second DNA molecule. The two DNA molecules are single It may or may not be part of a continuous DNA molecule, and it may also be adjacent to it. In some cases, this is not the case. For example, a promoter may cause the transcription of a target transcribable DNA molecule within a cell. When regulated, this promoter is operably linked to a transcriptionable DNA molecule. For example, if a leader can influence the transcription or translation of a DNA sequence, D It is operablely linked to the NA sequence.

[0049] In one embodiment, the construct of the present invention is possessed by A. tumefaciens cells Along with transposition molecules, T-DNA enables integration of T-DNA into the plant cell genome. Includes Ti+ isolated from Agrobacterium tumefaciens The right-hand boundary (RB or AGRtu.RB) and left-hand boundary (LB or AGRtu.RB) of the mid. It may be provided as a dual tumor induction (Ti) plasmid boundary construct having an LB) region (e.g. For example, see U.S. Patent No. 6,603,061). Also, the construct is found in multiple bacterial cells. Plasmid backbone DNA segments that contribute to functional and antibiotic selection, e.g., ori3 Escherichia coli replication start points such as 22, oriV or oriRi Which broad-host-range replication origin, and for spectinomycin or streptomycin Tn7 aminoglycoside adenyltransferase (aadA) that confers resistance Selective markers such as Spec / Strp, or gentamicin (Gm, Gen) t) The coding region for the selection marker gene may also be included. For plant transformations The host bacterial strain is often A. tumefaciens ABI, C58, or LB. A4404, however, other strains known to those skilled in the art in the field of plant transformation are also included. It can function in the invention.

[0050] A transcriptionable DNA molecule is translated into a functional mRNA molecule that is expressed as a protein. A method for assembling a construct and introducing it into a cell in a manner that allows for transcription is, in the field of the said technology. This is publicly known. In carrying out the present invention, the conventional method for preparing and using constructs and host cells Conventional compositions and methods are well known to those skilled in the art. Useful for nucleic acid expression in higher plants. Typical vectors are well known in the art, and include Agrobacteri Vectors and pCaMVCN derived from the Ti plasmid of um tumefaciens It includes an introduction control vector.

[0051] Various adjustment elements, including any adjustment elements provided herein, can be used in a structure. It may include. Any such adjustment element may be provided in combination with other adjustment elements. Such combinations may be designed or modified to produce the desired regulatory function. Obtain. In one embodiment, the construct of the present invention is operably connected to 3'UTR It comprises a transcriptionable DNA molecule and at least one regulatory element operably linked to it. .

[0052] The constructs of the present invention are provided herein or any known in the art. It may include a promoter or leader. For example, the promoter of the present invention may include heterogeneous Operable with non-translated 5' readers (e.g., those derived from heat shock protein genes). It may be linked to a different promoter, for example, Cali. Alternatively, the leader of the present invention may be a different promoter, for example, Cali. It may be operably linked to the Flower Mosaic Virus 35S transcript promoter.

[0053] Expression cassettes also contain operablely linked proteins, particularly in chloroplasts, leucomas, and other areas. or other plastid organelles, mitochondria, peroxisomes, vacuoles, or extracellular spaces Transport peptides that encode peptides useful for targeting at or below the cellular level relative to location. The docode sequence may also be included. Many chloroplast-localized proteins are precursors derived from nuclear genes. It is expressed and targeted to chloroplasts by chloroplast transport peptides (CTPs). Examples of isolated chloroplast proteins include, but are not limited to, ribulose- 1,5-Bisphosphate carboxylase small subunit (SSU), ferredoxin, Eredoxin oxidoreductase, light-harvesting complex protein I and protein II, thio Regarding redoxin F and enolpyruvir shikimate phosphate synthase (EPSPS) Related examples include chloroplast transport peptides, for example, U.S. 7,193,13 This is explained in issue 3. Non-chloroplast proteins are introduced genes that encode non-chloroplast proteins. By expressing heterologous CTPs operably bound to genes, chloroplasts can be targeted. This has been proven.

[0054] Transcriptional DNA molecules As used herein, the term “transcribeable DNA molecule” means “transcribeable RNA molecule.” This refers to any DNA molecule that can be converted into a protein-coding sequence, which may contain a protein-coding sequence. substances, substances that encode guide RNA, and RNA molecules that have sequences useful for gene repression. This includes, but is not limited to, those that produce DNA. The types of DNA molecules include the same plant DNA molecules from one plant, DNA molecules from another plant, DNA molecules from a different organism, or Synthetic DNA molecules, for example, DNA molecules containing antisense messages for genes, or derivatives It contains DNA molecules that encode artificial, synthetic, or altered versions of the input gene. However, it is not limited to these. Examples of transferable D for incorporation into the constructs of the present invention. Examples of NA molecules include DNA molecules from species other than the one incorporating the DNA molecule, or Genes, or originating from the same species, or present in the same species, but using classical breeding techniques One example is a gene that is incorporated into recipient cells through genetic engineering methods.

[0055] An "introduced gene" is a gene that differs from the host cell in terms of its location within the host cell genome, at least. A species of transcribed DNA molecule, and / or the current or any past generation of a cell. This refers to a transcriptionable DNA molecule that has been artificially incorporated into the genome of a host cell.

[0056] The regulatory elements of the present invention, such as promoters, are transcription elements that are heterogeneous to the regulatory elements. It may be operably ligated to any DNA molecule. When used herein, "heterogeneous" and The term refers to a combination of two or more DNA molecules in which such a combination is not typically found in nature. This refers to cases where it is not released. For example, the two DNA molecules may originate from different species, and / Alternatively, the two DNA molecules may originate from different genes (for example, different genes from the same species). Genes, or the same genes from different species. Therefore, regulatory elements are operably linked. If such a combination is not normally found in nature for a transcribed DNA molecule That is, a transcribable DNA molecule is operably linked to a regulatory element and occurs naturally. If they are not, they are different species.

[0057] A transcriptable DNA molecule is, in general, any DNA molecule whose transcript expression is desired. To obtain. The expression of such transcripts leads to the translation of the resulting mRNA molecule, therefore Protein expression can be brought about by this. Alternatively, for example, a transcriptionable DNA molecule can be used. It can be designed to ultimately cause a decrease in the expression of a gene or protein. In this embodiment, it uses a transferable DNA molecule oriented in the antisense direction. This can be achieved by [doing something]. Those skilled in the art are familiar with the use of such antisense techniques. In this manner, any gene may be negatively regulated, and in one embodiment, a transcriptional DN A molecule expresses specific genes through the expression of dsRNA, siRNA, or miRNA molecules. It may be designed to suppress it.

[0058] Therefore, one embodiment of the present invention is that the construct is transgenic plant cell geno When integrated into the molecule, the transcription of the transcribable DNA molecule is performed at a desired level or in a desired pattern. The sequence number is operably linked to heterologous transcriptionable DNA molecules to allow for adjustment. Recombinant DNA molecules containing the regulatory elements of the present invention, such as those provided as 1-5. In one embodiment, a transcriptionable DNA molecule transcribed the protein-coding region of a gene. In another embodiment, the transcriptionable DNA molecule includes the antisense region of a gene. .

[0059] Genes for agricultural purposes Transcriptional DNA molecules may be genes for agricultural purposes. When used herein, The term "gene for agricultural purposes" refers to the expression of a specific plant tissue, cell, or cell type. This refers to a transcribable DNA molecule that confers a desired characteristic when transcribed. (Agricultural gene) The products include plant morphology, physiology, growth, development, yield, grain composition, nutritional profile, and diseases. Alternatively, it may produce effects on pest resistance and / or environmental or chemical tolerance. In some cases, it acts within the plant, while in other cases, it acts as an insecticide by being a food source for pests that regularly feed on plants. In some cases, this adjustment is performed. In one embodiment of the present invention, the adjustment element of the present invention adjusts The element operably ligates to a transcribable DNA molecule, which is a gene for agricultural purposes. The sea urchin is incorporated into the structure. Transgenic plants containing such structures Therefore, the expression of genes for agricultural purposes can confer beneficial agricultural traits. For example, though not limited to, herbicide resistance, insect control, modified yield, Disease resistance, pathogen resistance, modified plant growth and development, modified starch content, modified Modified oil content, modified fatty acid content, modified protein content, modified fruit content Maturation, nutritional enhancement for animals and humans, biopolymer production, environmental stress resistance, pharmaceutical peptides, Improvement of processing quality, improvement of flavor, usefulness of hybrid seed production, improvement of fiber production, carbon sequestration This can be seen as an enhancement of biofuels, as well as the production of desirable biofuels.

[0060] Examples of genes for agricultural purposes that are publicly known in the relevant technical field include herbicide resistance (US Patent). No. 6,803,501; No. 6,448,476; No. 6,248,876; No. 6,2 No. 25,114; No. 6,107,549; No. 5,866,775; No. 5,804,4 (Patents No. 25; No. 5,633,435; and No. 5,463,175), increased yield (US Patent) USRE No. 38,446; No. 6,716,474; No. 6,663,906; No. 6, No. 476,295; No. 6,441,277; No. 6,423,828; No. 6,399, No. 330; No. 6,372,211; No. 6,235,971; No. 6,222,098 ; and No. 5,716,837), insect control (US Patent No. 6,809,078; No. 6, No. 713,063; No. 6,686,452; No. 6,657,046; No. 6,645, No. 497; No. 6,642,030; No. 6,639,054; No. 6,620,988 ; No. 6,593,293; No. 6,555,655; No. 6,538,109; No. 6, No. 537,756; No. 6,521,442; No. 6,501,009; No. 6,468, No. 523; No. 6,326,351; No. 6,313,378; No. 6,284,949 ; No. 6,281,016; No. 6,248,536; No. 6,242,241; No. 6, No. 221,649; No. 6,177,615; No. 6,156,573; No. 6,153, No. 814; No. 6,110,464; No. 6,093,695; No. 6,063,756 ;No.6,063,597;No.6,023,013;No.5,959,091;No.5, No. 942,664; No. 5,942,658; No. 5,880,275; No. 5,763,2 (Patent No. 45; and No. 5,763,241), fungal disease resistance (U.S. Patent No. 6,653,28) No. 0; No. 6,573,361; No. 6,506,962; No. 6,316,407; No. No. 6,215,048; No. 5,516,671; No. 5,773,696; No. 6,12 (Nos. 1,436; 6,316,407; and 6,506,962), virus resistance Sex (U.S. Patent No. 6,617,496; No. 6,608,241; No. 6,015,940) (Nos. 6,013,864; Nos. 5,850,023; and Nos. 5,304,730) Nematode resistance (U.S. Patent No. 6,228,992), bacterial disease resistance (U.S. Patent No. 5,5 (Patent No. 16,671), Plant Growth and Development (Patent Nos. 6,723,897 and 6,518) (Patent No. 488), starch production (U.S. Patent No. 6,538,181; No. 6,538,179) (Numbers 6,538,178; 5,750,876; and 6,476,295) , modified oil production (U.S. Patent No. 6,444,876; No. 6,426,447; No. 6 (Patents No. 380, 462), high oil production (US Patent No. 6,495, 739; No. 5,608, (No. 149; No. 6,483,008; No. 6,476,295), Modified fatty acid content (US Patent Nos. 6,828,475; 6,822,141; 6,770,465) ; No. 6,706,950; No. 6,660,849; No. 6,596,538; No. 6, No. 589,767; No. 6,537,750; No. 6,489,461; No. 6,459, (No. 018), high protein production (US No. 6,380,466), fruit ripening (US (Patent No. 5,512,466), Nutritional fortification of animals and humans (U.S. Patent No. 6,723,836) No. 7; No. 6,653,530; No. 6,5412,59; No. 5,985,605; No. (Patent No. 6,171,640), Biopolymers (USRE No. 37,543; No. 6,22) (Nos. 8,623; 5,958,745, and 6,946,588), environmental stress Resistance (US Patent No. 6,072,103), pharmaceutical peptides and secretory peptides (US Patent No. 6,072,103) Licenses No. 6,812,379; No. 6,774,283; No. 6,140,075; and (Patent No. 6,080,560), Improvement of processing traits (Patent No. 6,476,295) , improved digestibility (US Patent No. 6,531,648), low raffinose (US Patent No. 6 (Patents No. 166, 292), Industrial enzyme production (US Patent No. 5,543, 576), Flavor modification Good (U.S. Patent No. 6,011,199), Nitrogen fixation (U.S. Patent No. 5,229,114) , hybrid seed production (US Patent No. 5,689,041), fiber production (US Patent No. 6 Nos. 576,818; Nos. 6,271,443; Nos. 5,981,834; and Nos. 5,8 Examples include patents 69,720, and biofuel production (US Patent No. 5,998,700). It is possible.

[0061] Alternatively, genes for agricultural purposes can lead to targeted regulation of gene expression of endogenous genes. By encoding the RNA molecule that triggers it, for example, antisense (for example, US patent) See Patent No. 5,107,065), inhibitory RNA ("RNAi"; e.g., U.S. Patent Publication No. 2 Patent Nos. 006 / 0200878 and 2008 / 0066206, and U.S. Patent Applications As explained in issue 11 / 974,469, miRNA, siRNA, trans This includes the regulation of gene expression through mechanisms mediated by active siRNA and phase sRNA. The above-mentioned plant characteristics or phenotypes are affected by (or by a mechanism mediated by co-inhibition). This can have an effect. Furthermore, RNA can be manipulated to cleave the desired endogenous mRNA product. Catalytic RNA molecules (e.g., ribozymes or riboswitches; e.g., US No. 2006 / 0) (See Issue 200878) This may also be the case. A transcriptionable DNA molecule suppresses genes. To construct a construct and introduce it into cells in a manner that is transcribed into molecules that can cause control The method is publicly known in the relevant technical field.

[0062] Selection Marker Selective marker-transfer genes may also be used in conjunction with the regulatory elements of this invention. When used in a specification, the term "selection marker transgene" is a transgenic term. The expression, or lack of expression, in plants, tissues, or cells is screened in any way. Or it refers to any transcribable DNA molecule that can be scored. Selectable marker genes for use, as well as their related selection and screening. The ning technique is known in the relevant field and includes β-glucuronidase (GUS) , green fluorescent protein (GFP), antibiotic resistance-constituting proteins, and herbicide resistance It includes, but is not limited to, transcriptionable DNA molecules that encode proteins that confer resistance. Not determined. An example of a selective marker-introduced gene is provided as SEQ ID NO: 8.

[0063] Genome editing Some embodiments include site-directed genome modification enzymes for performing genome modification and / or It is operablely ligated to heterologous DNA sequences encoding any one related protein(s). Furthermore, a sequence having at least approximately 85% sequence identity with any of sequence numbers 1 to 5. This relates to recombinant DNA constructs that include expression cassettes (or more) containing the fragments. The nuclease expression cassette(s) are donor templates for template editing and Within the same molecule or vector (cis), or on a separate molecule or vector (tra). (Enzymes) may exist. Various sequence-specific genome modification enzymes that modify genomic DNA ( There are several editing methods involving a complex of proteins and / or guide RNA. This is known in the art. In some embodiments, site-directed genome modification enzymes are used as desired. Inducing double-strand breaks (DSBs) or nicks at genomic sites or gene loci Further genome modification. In some embodiments, DSBs introduced by genome-modifying enzymes Alternatively, during the process of repairing the nick, the donor template DNA may be at the DSB site or It can be incorporated into the genome at a nick site. In some embodiments, by genome modifying enzymes During the process of repairing the introduced DSB or nick, an insertion mutation or deletion mutation (in Dell may be introduced into the genome. In some embodiments, site-directed genome modification enzymes It contains cytidine deaminase. In some embodiments, the site-directed genome modification enzyme is It contains adenine deaminase. In this disclosure, site-directed genome modification enzymes include E Endonuclease, recombinase, transposase, deaminase, helicase, Examples include birth transcriptases and any combination thereof.

[0064] Some embodiments encode one or more components of a genome editing system, heterogeneous Gene regulatory elements described herein, operably linked to a transcriptionable DNA molecule Regarding genome editing systems, one or more insertions, deletions, or substitutions are made in the genome of a host cell. It can be used to introduce base modifications, translocations, or inversions. In some embodiments, this The gene regulatory elements described in the specification are CRISPR-Cas effector proteins. Sequence-specific proteins such as zinc finger proteins or transcription-activating (TAL) proteins. It encodes a DNA-binding domain and is operably ligated to a heterologous, transcriptional DNA molecule. In some embodiments, the sequence-specific DNA-binding domain may be a fusion protein. In some embodiments, the gene regulatory element described herein is CRISPR-Cas. It is operablely linked to a heterologous, transcribable DNA molecule that encodes a fector protein. In some embodiments, the CRISPR-Cas effector protein is Type Type I CRISPR-Cas system, Type II CRISPR-Cas system, Type III CRISPR-Cas system, Type IV CRISPR-C as system, Type V CRISPR-Cas system, or Type VI Selected from CRISPR-Cas systems. In some embodiments, as described herein. Gene regulatory elements are created in heterologous, transcriptional DNA molecules that encode guide RNA. Movable ligation. As used herein, “guide RNA” or “gRNA” is It recognizes the target DNA sequence and directs the CRISPR effector protein to the target DNA sequence. It refers to RNA that is complementary to the target DNA. The region (called crRNA) and the region that binds to the CRISPR effector protein ( It is composed of a single RNA molecule (called tracrRNA). Guide RNA is a single RNA molecule (s This may be gRNA or two separate RNA molecules (two gRNAs). Some embodiments So, gRNA is an RNA template (peg) for reverse transcriptionase. It may also contain RNA.

[0065] Some embodiments involve a CRISPR-Cas effector protein and a guide RN. A encodes one or more components of the CRISPR-Cas genome editing system, including A. , gene regulatory agents as described herein, operably linked to heterologous transcriptionable DNA molecules Regarding rement. Examples of CRISPR-Cas effector proteins include limited... It is not something that can be done, but Cas9, C2c1, C2c3, C2c4, C2c5, C2c8, C2c9, C2c10, Cas12a (also called Cpf1), Cas12b, Cas 12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, C as13a, Cas13b, Cas13c, Cas13d, Casl, CaslB, Ca s2, Cas3, Cas3´, Cas3”, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Cs yl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Cs n2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cm r4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Cs f2, Csf3, Csf4(dinG), Csf5, Cas14a, Cas14b, and Examples include Cas14c effector proteins. In some embodiments, the following is described herein. The gene regulatory elements listed are nuclease active sites (for example, RuvC, HNH, etc.) For example, the RuvC site of the Cas12a nuclease domain; for instance, Cas9 nuclease CRISPR-Cas containing mutations in the RuvC site and / or HNH site of the domain It is operablely linked to the effector protein. It has a mutation in the nuclease active site, Therefore, CRISPR-Cas effector tanno no longer contains nuclease activity. The protein is generally referred to as "dead," for example, dCas. In some embodiments, CRISPR-Cas effector protein with mutations in the nuclease active site The protein or polypeptide is the same CRISPR-Cas effector protein without mutations. Compared to its quality, its activity may be impaired or reduced. In embodiments, the gene regulatory element described herein is mutated at the nuclease active site. Operatably linked to a CRISPR-Cas effector protein having a river It generates nickas activity that is operably linked to the transscriptase enzyme.

[0066] Cell transformation The present invention also includes one or more regulatory elements operably linked to a transcriptionable DNA molecule. It is also directed towards methods for producing transformed cells and plants, including [specific components].

[0067] The term "transformation" refers to the introduction of DNA molecules into a recipient host. When used in this context, the term "host" means a bacterium, fungus, or plant, and this includes, This includes cells, tissues, organs, or progeny of bacteria, fungi, or plants, especially for specific purposes. The plant tissues and cells include protoplasts, callus, roots, tubers, seeds, stems, leaves, seedlings, It contains the embryo and pollen.

[0068] As used herein, the term "transformed" refers to a foreign DNA molecule (e.g., This refers to a cell, tissue, organ, or organism into which a construct has been introduced. The introduced D is incorporated into the genomic DNA of recipient cells, tissues, organs, or organisms. The NA molecule may be passed on to the next generation. "Transgenic" or " "Transformed" cells or organisms also have progeny, and such transformations occur in the offspring of cells or organisms and in mating. Produced from a breeding program using lancegenic organisms as parents, and containing foreign DNA molecules This may also include offspring that exhibit altered phenotypes resulting from the presence of the gene. Furthermore, it may include the introduced DNA. The molecule is transiently introduced into recipient cells, and the introduced DNA molecule is then inherited by the next generation. It is acceptable to prevent it from being inherited. The term "transgenic" refers to one or more different species. This refers to bacteria, fungi, or plants that contain DNA molecules.

[0069] There are many methods well known to those skilled in the art for introducing DNA molecules into plant cells. The process generally involves the steps of selecting suitable host cells and then transforming the host cells with a vector. The method includes the steps of replacing and obtaining transformed host cells. Method and material for transforming plant cells by introducing a material construct into the plant genome. The materials may include any of the well-known and proven methods. A preferred method is... This is not limited to bacterial infections (for example, Agrobacterium ), binary BAC vectors, direct delivery of DNA (e.g., PEG-mediated transformation, dry Drying / inhibition-mediated DNA incorporation, electroporation, stirring with silicon carbide fibers, and (Acceleration of DNA-coated particles), gene editing (e.g., CRISPR-Cas system (Mu) is one example.

[0070] The host cell is any cell or organism, for example, plant cells, algal cells, algae, fungal cells, It may be a fungal, bacterial, or insect cell. In certain embodiments, the host cell and Transformed cells may include cells from crop plants.

[0071] The transgenic plants are then regenerated from the transgenic plant cells of the present invention. It is possible to obtain seeds from this transgenic plant using conventional breeding techniques or self-pollination. Such seeds and the resulting offspring plants can be produced. This invention contains the recombinant DNA molecule of the present invention and is therefore transgenic.

[0072] By self-pollinating the transgenic plant of the present invention, the homozygous transgenic plant of the present invention Can we obtain seeds of nicked plants (homozygous for recombinant DNA molecules), and This involves crossbreeding non-transgenic plants or different transgenic plants to develop the true form. A heterozygous transgenic plant (heterozygous with recombinant DNA molecules) Seeds can be obtained. Such homozygous and heterozygous transgenic Both plants are referred to as “progenitor plants” in this specification. Progenitor plants are the original transgenic plants. This is a transgenic plant derived from a plant species and containing the recombinant DNA molecule of the present invention. Seeds produced using the transgenic plants of the present invention were harvested, and the recombinant DNA of the present invention was used. A transgenic plant that contains offspring and expresses genes for agricultural purposes, i.e., the present invention. It can be used to grow subsequent generations of plants. It is commonly used in breeding for various crops. An explanation of this method can be found in one of several references. For example, Allard, Principles of Plant Breeding, John Wiley & Sons,NY,U.of CA,Davis,CA,50-98( 1960);Simmonds,Principles of Crop Improv ement,Longman,Inc.,NY,369-399(1979);Snee p and Hendriksen,Plant breeding Perspective ives,Wageningen(ed),Center for Agricultu ral Publishing and Documentation(1979);F ehr,Soybeans: Improvement,Production and Uses, 2nd Edition, Monograph, 16:249(1987) ;Fehr,Principles of Variety Development, Theory and Technique,(Vol.1)and Crop Spe cies Soybean(Vol.2),Iowa State Univ.,Mac See Millan Pub.Co., NY, 360-376 (1987).

[0073] Transgenic plants exhibit the presence of the target gene(s), as well as the regulatory elements of the present invention. The expression levels and / or profiles conferred can be analyzed. Those skilled in the art will know how to transform I am aware of numerous methods that can be used for the analysis of hybrid plants. For example, as a method of plant analysis, While not limited to these, Southern blot or Northern blot, PCR-based Approach, biochemical analysis, phenotypic screening methods, field evaluation, and immunodiagnostic assays. Examples include: The expression of transcriptionable DNA molecules is controlled by TaqMan(registered trademark) (Appli Reagents and manufacturers of ed Biosystems (Foster City, CA) The method of explanation, and using the TaqMan® Testing Matrix It can be measured using the PCR cycle time determined by [method / method]. Alternatively, Invader (registration Trademark)(Third Wave Technologies(Madison,WI)) The expression of the transgene can be evaluated using the reagents and methods described by the manufacturer. ru.

[0074] The present invention also provides plant parts of the present invention. The plant parts are not limited to the following: However, examples include leaves, stems, roots, tubers, seeds, endosperm, ovules, and pollen. The parts of an object may be growing, not growing, regenerating, and / or not regenerating. Furthermore, the present invention also includes transformed plant cells containing the DNA molecule of the present invention, and provides the same. The transformed or transgenic plant cells of the present invention are renewable and / or It contains plant cells that cannot be regenerated.

[0075] The present invention also relates to transgenic plants or parts thereof containing the recombinant DNA molecule of the present invention. The present invention also provides commodity products produced from the position. The commodity products of the present invention are from sequence numbers 1 to 5. The DNA used herein contains a detectable amount of DNA, which includes a DNA sequence selected from the group. In this case, "commercial product" refers to a transgenic plant containing the recombinant DNA molecule of the present invention. Any composition or raw material consisting of seeds, plant cells, or materials derived from plant parts It refers to a product. While not limited to commodity products, it can include processed seeds, grains, etc. Examples include plant parts and coarse powders. The commercial products of the present invention are recombinant DNA molecules of the present invention. This will include a detectable amount of DNA corresponding to the product. To determine this, detection of one or more of these DNAs in the sample may be used. You may use any standard DNA molecule detection method, including the detection methods disclosed in this document. stomach.

[0076] The present invention can be more easily understood by referring to the following embodiments, and their implementations Examples, unless otherwise specified, are provided for illustrative purposes only and are not intended to limit the present invention. One of ordinary skill in the art will understand that techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the implementation of the present invention. However, one of ordinary skill in the art, mindful of this disclosure, will understand that many modifications can be made to the specific embodiments disclosed without departing from the spirit and scope of the present invention, and still obtain an equivalent or similar result. Accordingly, all matters set forth in the attached drawings are to be interpreted in an illustrative, rather than a limiting, sense. It is not intended. One of ordinary skill in the art should understand that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the implementation of the present invention. However, one of ordinary skill in the art should understand that, in light of this disclosure, many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the present invention, and still obtain an equivalent or similar result. Therefore, all matters described or shown in the accompanying drawings are to be construed in an illustrative, rather than a limiting, sense.

Examples

[0077] Identification and Cloning of Regulatory Elements This example describes the identification, synthesis, and cloning of regulatory expression elements from Andropogon gerardii (Big blue stem ) and Arundo donax (Giant reed). A novel ubiquitin regulatory element was identified and isolated from genomic DNA of the monocotyledonous plant species Andropogon gerardii (Big blue stem ). Primers were designed to amplify the corresponding regulatory elements of the identified ubiquitin gene, which included a promoter, a leader (5´UTR), and a first intron, operably linked using the respective 5´ untranslated regions (5´UTRs) of each ubiquitin 1 transcript. The primers were constructed into GenomeWalker (trademark) (Cl

[0078] ) according to the manufacturer's protocol. ​​​​​​​ontech Laboratories, Inc. (Mountain View, CA) )) Use with the library to clone the 5' region of the corresponding genomic DNA sequence. did.

[0079] In addition, the ge of Arundo donax (Giant reed), a monocotyledonous plant species. A novel 3'UTR was identified and isolated from nom DNA. Andropogon geran dii's identified EXP and its corresponding promoter, leader, and introduction Table 1 shows the 3'UTR of Arundo donax. [Table 1]

[0080] In the expression cassette used to drive the expression of β-glucuronidase (GUS) Then, the identified EXP and 3'UTR are converted to binary using methods known in the art. Synthesized and cloned into plant transformation vector constructs as described in Examples 2 and 3. Thus, their activity was evaluated in stably transformed maize plants.

[0081] Example 2 EXP-AN drives GUS expression in stably transformed maize plants. Analysis of Dge.Ubq:5 Maize plants are used as vectors, specifically through the introduction of β-glucuronidase (GUS) into the genetic model. Plant expression vector containing EXP (EXP-ANDge.Ubq:5) that drives the expression of offspring The plants were transformed. The resulting plants were analyzed for GUS protein expression, and the relationship between expression and The effects of the regulatory element group (EXP) were evaluated and compared with the standard control EXP.

[0082] Maize plants were transformed with a plant GUS expression construct. Using standard methods known in the art, EXP (EXP-ANDge.Ubq:5, SEQ ID NO: 1) was cloned into a plant expression vector. The resulting plant expression vector contains the left border region of Agrobacterium tumefaciens (B-AGRtu.left border), a first transgene selection cassette for selection of transformed plant cells conferring resistance to the herbicide glyphosate, and a second transgene cassette for evaluating the activity of EXP-ANDge.Ubq:5, the second transgene cassette comprising a GUS coding sequence composed of a processing intron (SEQ ID NO: 8) operably linked to the 3´UTR (T-Sb.Nltp4-1:1:2, SEQ ID NO: 7), and operably linked to the 5´ side of the GUS coding sequence, the second transgene cassette composed of a transgene cassette containing EXP-ANDge.Ubq:5 (SEQ ID NO: 1), and the right border region of Agrobacterium tumefaciens (B-AGRtu.right border). GUS expression driven by EXP-ANDge.Ubq:5 was compared to the expression of a similar control construct containing a GUS transgene expression cassette composed of EXP (EXP-CaMV.35S+Zm.DnaK:12, SEQ ID NO: 6) operably linked to the 5´ side of the GUS coding sequence operably linked to the 3´UTR (T-Sb.Nltp4-1:1:2). <000097,2> Maize plant cells were, as is well known in the art, Agrobacte By rium-mediated transformation, the above binary transformation vector construct can be used to acquire traits. The transformation occurred. The resulting transformed plant cells were then induced to form a whole maize plant. .

[0084] Qualitative and quantitative GUS analysis was used to analyze selected plant organs and tissues of transformed plants. The activity of the expressed element was evaluated. Qualitative analysis of GUS expression by histochemical staining was performed. Therefore, whole-mount tissue or sectioned tissue should be treated with 1 mg / mL of X-Gluc( GUS staining solution containing 5-bromo-4-chloro-3-indolyl-β-glucuronide The samples were incubated together at 37°C for 5 hours and destained with 35% EtOH and 50% acetic acid. US expression is the blue coloration of selected plant organs or tissues under a dissecting microscope or combined microscope. The assessment was made qualitatively through visual inspection.

[0085] For quantitative analysis of GUS expression using enzyme assays, transformed maize Total proteins were extracted from selected plant tissues. 1-2 micrograms of total protein. The quality is achieved by adding 4-methylunion, a fluorescence-generating substrate, at a concentration of 1 mM in a total reaction volume of 50 microliters. It was incubated with beriferyl-β-D-glucuronide (MUG). 1 After incubation for the specified time, add 350 microliters of 200 mM sodium bicarbonate solution. The reaction was stopped by adding 4-methylumbelliferone(4- MU) exhibits maximum fluorescence at high pH, ​​at which point the hydroxyl group is ionized. The assay is stopped by adding a sodium carbonate solution, and at the same time, the fluorescence product 4 - Adjust pH for MU quantification. FLUOstar Omega Micropl ate Reader (BMG LABTECH) (Excitation: 355nm, Emission: 460nm) Using m), the fluorescence of the generated 4-MU was measured and its amount was estimated. The GUS activity value was: It is supplied in nanomolar units of 4-MU / hour / mg of total protein.

[0086] The following tissues were sampled for GUS expression in the R0 generation: leaves at stage V4 and Roots; leaves and roots at stage V7; leaves, flowers / anthers, and pollen at stage VT; rachis / hairs at stage R1; pollination Seed embryo and seed endosperm at stage R3, 21 days after (DAP). Table 2 shows the tissue samples collected. It shows average quantitative GUS expression. [Table 2]

[0087] As can be seen from Table 2, EXP(EXP-ANDge.Ubq:5, Sequence ID 1) Therefore, the control (EXP-CaMV.35S+Zm.DnaK:12, SEQ ID NO: 6) A completely different expression profile was obtained in the anthers and pollen of VT, The expression level driven by P-ANDge.Ubq:5 is EXP-CaMV.35S+ Higher expression levels than those driven by Zm.DnaK:12. EXP-CaMV.35S+ Expression in leaves driven by Zm.DnaK:12 increased from stage V4 to V7. Next, it appeared to decrease up to the VT stage, while EXP-ANDge.Ubq: Expression in leaves driven by 5 remained constant throughout all three stages. In hair, expression driven by EXP-ANDge.Ubq:5 was compared to the control. It was common. EXP-ANDge.Ubq:5 (SEQ ID NO: 1) exhibits unique expression characteristics. This provides a novel tool for the expression of selected transgenes.

[0088] Example 3 3'UTR(T-ARUdo.Tu) in stably transformed maize plants bA:1) Regulation of GUS expression Maize plants are used as vectors, specifically through the introduction of β-glucuronidase (GUS) into the genetic model. Plant expression vector containing 3'UTR(T-ARUdo.TubA:1) that regulates offspring expression The plants were transformed using the following method. The resulting plants were analyzed for GUS protein expression, and the expression was determined to be related to... The effect of the 3'UTR adjustment element was evaluated.

[0089] Maize plants were transformed with plant GUS expression constructs and control constructs. In Example 2, a similar structure was used as described above. Both structures were processed by Prosessin. For a GUS code sequence consisting of a ligable intron (sequence number 8), the 5' side is made Movably linked EXP(EXP-CaMV.35S+Zm.DnaK:12, sequence) It included number 6). The control structure was as described above in Example 2. The GUS code sequence is operablely linked to 3'UTR(T-Sb.Nltp4-1:1:2). It was assumed that it was done. The other structure is 3'UTR(T-ARUdo.TubA:1, Transcription of the GUS transgene was terminated using Sequence ID No. 5).

[0090] As described in Example 2, maize plants were transformed and GUS expression was assayed. The following tissues were sampled for GUS expression in the R0 generation: leaves at stage V4 Roots; leaves and roots at stage V7; leaves, flowers / anthers, and pollen at stage VT; rachis / hairs at stage R1; receiving Seed embryos and seed endosperm at stage R3, 21 days after powder (DAP). Table 3 shows the tissue samples taken. Average quantitative GUS expression and increased GUS induced by T-ARUdo.TubA:1 It shows a high magnification. [Table 3]

[0091] As can be seen from Table 3, 3'UTR(T-ARUdo.TubA:1, Sequence ID 5 ) was compared with 3'UTR(T-Sb.Nltp4-1:1:2) when assayed. The expression level of the GUS transgene was enhanced in all tissues. Transcriptional analysis revealed that GUS m Appropriate RNA termination and polyadenylation were demonstrated. In some cases, the expression level of the transgene was shown. The enhancement was considerably large. For example, the expression level of VT in leaves was significantly higher than that of the control. It was 176.8 times more abundant. The expression level of V7 in the roots was 93 times higher than that of the control. It was 7 times more. Therefore, 3'UTR(T-ARUdo.TubA:1, sequence number 5) This enhances the expression of the introduced gene compared to the control.

[0092] The principles of the present invention have been illustrated and explained, but without departing from these principles, the arrangement of the present invention may be described. It should be obvious to those skilled in the art that the details can be modified. (Inventors) This patent claims all modifications to the intent and scope of the claims. All publications and published patent documents are treated as if they were individual publications or patent applications. To the same extent that each is clearly and individually indicated to be referenced, This is incorporated herein by reference.

Claims

1. Recombinant DNA molecules, a) A sequence having at least 85 percent sequence identity with sequence number 5, b) Sequences including sequence number 5, and c) A fragment of Sequence ID No. 5 having gene regulatory activity, It includes a DNA sequence selected from the group consisting of, The recombinant DNA molecule wherein the sequence is operably linked to a different type of transcriptionable DNA molecule.

2. The recombinant DNA molecule according to claim 1, wherein the sequence has at least 90 percent sequence identity with respect to the DNA sequence of sequence number 5.

3. The recombinant DNA molecule according to claim 1, wherein the sequence has at least 95 percent sequence identity with respect to the DNA sequence of sequence number 5.

4. The recombinant DNA molecule according to claim 1, wherein the DNA sequence contains gene regulatory activity.

5. The recombinant DNA molecule according to claim 1, wherein the heterogeneous transcriptionable DNA molecule contains a gene for an agricultural purpose.

6. The recombinant DNA molecule according to claim 5, wherein the gene for the aforementioned agricultural purpose confers herbicide resistance in plants.

7. The recombinant DNA molecule according to claim 5, wherein the gene for the aforementioned agricultural purpose confers pest resistance in plants.

8. The recombinant DNA molecule according to claim 1, wherein the heterogeneous transcriptionable DNA molecule encodes dsRNA, miRNA, or siRNA.

9. Transgenic plant cells, a) A sequence having at least 85 percent sequence identity with sequence number 5, b) Sequences including sequence number 5, and c) A fragment of Sequence ID No. 5 having gene regulatory activity, It includes a recombinant DNA molecule containing a sequence selected from the group consisting of, The transgenic plant cell wherein the sequence is operably linked to a heterologous transcriptionable DNA molecule.

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

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

12. A transgenic plant or a part thereof comprising the recombinant DNA molecule described in claim 1.

13. A progeny of a transgenic plant according to claim 12, or a part thereof, wherein the progeny or part thereof contains the recombinant DNA molecule.

14. A transgenic seed, wherein the seed contains the recombinant DNA molecule described in claim 1.

15. A method for producing a commodity product, comprising obtaining a transgenic plant or a part thereof as described in claim 12, and producing a commodity product therefrom.

16. The method according to claim 15, wherein the product is selected from the group consisting of seeds, processed seeds, protein concentrates, protein isolates, starch, grains, plant parts, seed oil, biomass, fine powders, and coarse powders.

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