Plant regulatory elements and their use

Abstract

We provide gene regulatory elements for use in plants. [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, as well as methods for using them.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Application No. 62 / 969,993, filed Feb. 4, 2020. The provisional application is hereby incorporated by reference in its entirety into this specification.

[0002] Incorporation of Sequence Listing The sequence listing contained in the file named “MONS479WO_ST25.txt” is 41.2 kilobytes (measured in Microsoft Windows®) and was created on Jan. 11, 2021. It is submitted with this specification by electronic application and is hereby incorporated by reference into this specification. (Note: There seems to be an extra closing bracket in the original Japanese text for this part. I've translated it as best as possible while keeping the structure. If it's a formatting error, the translation should be adjusted accordingly after correcting the original.)

[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 active 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 methods for using the regulatory elements to ​​​​​​The invention also provides transgenic plant cells, plants, and seeds. In one embodiment, the The regulatory element is operably ligated to a transcriptionable DNA molecule. In a particular embodiment Therefore, the transcriptionable DNA molecule may be heterogeneous with respect to the regulatory sequence. The regulatory element sequence provided by Akira, in certain embodiments, is a heterologous transcriptional DNA molecule. It can be defined as being operably connected to. The present invention also uses the adjustment element A method for doing so, and a method for producing and using recombinant DNA molecules containing the regulatory element, A transger comprising the regulatory element operably linked to a transcribable DNA molecule. Nick provides plant cells, plants, and seeds.

[0006] Therefore, in one embodiment, the present invention relates to (a) at least one of the sequence numbers 1 to 20 (b) A sequence with at least 85 percent sequence identity, or any of sequence numbers 1 to 20. (c) A sequence containing (c) any of the sequence numbers 1 to 20 having gene regulatory activity It includes a DNA sequence selected from the following group, and the sequence is made into a different type of transcriptionable DNA molecule. This provides recombinant DNA molecules that can be mobilized and linked. "Heterogeneous transcriptionable DNA molecules" refers to... For a polynucleotide sequence in which a transcriptionable DNA molecule is functionally linked, This means that, in certain embodiments, the recombinant DNA molecule is one of sequence numbers 1 to 20. For either DNA sequence, at least approximately 85 percent, at least approximately 86 percent , at least about 87 percent, at least about 88 percent, at least about 89 percent , at least about 90 percent, at least 91 percent, at least 92 percent at least 93 percent, at least 94 percent, at least 95 percent T, at least 96 percent, at least 97 percent, at least 98 percent or containing a DNA sequence having at least 99 percent sequence identity.

[0007] In another embodiment, the following is provided herein: (a) for any of Sequence IDs 1 to 20 (b) Sequences having at least approximately 85 percent sequence identity, (b) Sequence IDs 1-20 (c) A sequence containing any of the sequences in sequence numbers 1 to 20 that have gene regulatory activity. A DNA sequence selected from a group consisting of fragments, wherein the DNA sequence is a heterologous transcriptional DNA A transgenic plant cell containing a recombinant DNA molecule operably linked to molecule A In a particular embodiment, the transgenic plant cell is a monocotyledonous plant cell. In other embodiments, the transgenic plant cell is a dicotyledonous plant cell.

[0008] In yet another embodiment, the following are further provided herein: (a) any of Sequence IDs 1 to 20 (b) Sequence ID 1, which has at least approximately 85 percent sequence identity with respect to either of the other sequences. (c) Sequences containing any of ~20, and (c) Sequence IDs 1-20 having gene regulatory activity The DNA sequence comprises a sequence selected from a group consisting of any of the following fragments, wherein the sequence is transcribable to a different species. Transgenic plants containing recombinant DNA molecules that are operably linked to other DNA molecules, or that part. In a particular embodiment, the transgenic plant is the recombinant D It is a progeny plant of any generation that contains the NA molecule. Transgenic plants that are affected during growth We also offer transgenic seeds containing the recombinant DNA molecules that produce them.

[0009] In another aspect, the present invention provides a method for producing a commodity product, the method comprising obtaining a transgenic plant or a part thereof comprising a recombinant DNA molecule of the present invention and producing a commodity product therefrom. In one embodiment, the commodity product is a seed, processed seed, protein concentrate, protein isolate, starch, grain, plant part, seed oil, biomass, fine powder and coarse powder. obtaining a transgenic plant or a part thereof comprising the recombinant DNA molecule and producing a commodity product therefrom. In one embodiment, the commodity product is a seed, processed seed, protein concentrate, protein isolate, starch, grain, plant part, seed oil, biomass, fine powder and coarse powder.

[0010] In yet another aspect, the present invention provides a method for producing a transgenic plant comprising the recombinant DNA molecule of the present invention, the method 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. 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. 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. 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 promoter, P-Zm.GRMZM2G487322:2, operably linked to a leader and derived from Zea mays. SEQ ID NO: 1 is a DNA sequence of a promoter, P-Zm.GRMZM2G487322:2, operably linked to a leader and derived from Zea mays.

[0012] SEQ ID NO: 2 is a DNA sequence of a 3'UTR, T-Zm.GRMZM2G 487322:2 and derived from Zea mays

[0013] SEQ ID NO: 3 is a DNA sequence of a promoter, P-Zm.GRMZM2G339781:1, operably linked to a leader and derived from Zea mays. SEQ ID NO: 3 is a DNA sequence of a promoter, P-Zm.GRMZM2G339781:1, operably linked to a leader and derived from Zea mays.

[0014] SEQ ID NO: 4 is a DNA sequence of a 3'UTR, T-Zm.GRMZM2G 339781:1 and derived from Zea mays.

[0015] Sequence ID 5 is derived from Zea mays and is located in the intron (I-Zm.Xet:1). Operablely coupled, promoter operably coupled to the leader (P-Zm.Xe EXP-Zm.Xe is a group of regulatory expression elements or EXPs composed of t:1). This is a t:1 DNA sequence.

[0016] Sequence ID 6 is derived from Zea Mays, a promo that is operable and linked to the reader. This is the DNA sequence of the molecule P-Zm.Xet:1.

[0017] Sequence ID 7 is derived from Zea mays, an intron, I-Zm.Xet:1 D It is an NA sequence.

[0018] Sequence ID 8 is derived from Zea mays, 3'UTR, T-Zm.Xet:1 D It is an NA sequence.

[0019] Sequence ID 9 is an intron (I-Zm.Sat6:1) derived from Zea mays. A promoter (P-Zm.S) operably connected to the leader, which is operably connected to the leader. EXP is composed of at6:1, and is the DNA sequence EXP-Zm.Sat6:1. ru.

[0020] Sequence ID 10 is derived from Zea Mays, a pro that is operablely linked to the reader. This is the DNA sequence of the motor, P-Zm.Sat6:1.

[0021] Sequence ID 11 is an intron, I-Zm.Sat6:1, derived from Zea mays. This is the DNA sequence.

[0022] Sequence ID 12 is derived from Zea mays, 3'UTR, T-Zm.Sat6:1 This is the DNA sequence.

[0023] Sequence ID 13 is derived from Zea mays, an intron (I-Zm.GRMZM2 Promo Operablely connected to the reader, G049726:1) EXP is composed of (P-Zm.GRMZM2G049726:1), EX This is the DNA sequence P-Zm.GRMZM2G049726:1.

[0024] Sequence ID 14 is derived from Zea Mays, a pro that is operablely linked to the reader. This is the DNA sequence of motor P-Zm.GRMZM2G049726:1.

[0025] Sequence ID 15 is an intron, I-Zm.GRMZM2, derived from Zea mays. This is the DNA sequence G049726:1.

[0026] Sequence ID 16 is derived from Zea mays, 3'UTR, T-Zm.GRMZM2 This is the DNA sequence G049726:1.

[0027] Sequence ID 17 is derived from Zea Mays, a pro that is operable to the reader. This is the DNA sequence of the motor, P-Zm.GRMZM2G141762:1.

[0028] Sequence ID 18 is derived from Zea Mays, a pro that is operablely linked to the reader. This is the DNA sequence of the motor, P-Zm.DSUL:1.

[0029] Sequence ID 19 is derived from Zea Mays, a pro that is operablely linked to the reader. This is the DNA sequence of the motor, P-Zm.GRMZM2G512113:1.

[0030] Sequence ID 20 is derived from Zea mays, 3'UTR, T-Zm.GRMZM2 This is the DNA sequence G512113:1.

[0031] Sequence ID 21 is the potato light-inducible tissue-specific St-LS1 gene (Genbank). Accession: X04753) has a β- with a processingable intron. Synthetic coding sequence optimized for plant expression of glucuronidase (GUS,GOI-Ec.u idA+St.LS1.nno:1) [Modes for carrying out the invention]

[0032] The present invention provides regulatory elements that have gene regulatory activity in plants. The nucleotide sequences of the regulatory elements are shown as SEQ ID NOs: 1-20. The elements influence the expression of operably linked, transcribed DNA molecules within plant tissue. It is possible to exert an effect, and consequently, to enable operable linkage within transgenic plants. It is possible to regulate the gene expression of the introduced gene. Furthermore, the present invention makes it possible to regulate the gene expression of the introduced gene. Methods for modifying, producing, and using recombinant DNA molecules containing regulatory elements are also provided. Furthermore, the present invention relates to transgenic plant cells, plants, and plants containing the recombinant DNA molecule of the present invention. The present invention also provides compositions containing plant parts and seeds, as well as methods for preparing and using them.

[0033] 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 [unclear]. Unless otherwise stated, terms are defined by those skilled in the art in the relevant field. It shall be understood according to its intended use.

[0034] dna molecule As used herein, the terms “DNA” or “DNA molecule” refer to the 5' (upstream) A double-stranded DNA molecule of genomic or synthetic origin, read from the terminal to the 3' (downstream) terminal. This refers to a polymer of deoxyribonucleotide bases or a DNA molecule. In this context, the term "DNA sequence" refers to the nucleotide sequence of a DNA molecule. The nomenclature used in the specification corresponds to the nomenclature in 37, Section 1.822 of the Code of Federal Regulations, W This is defined in Tables 1 and 3 of Annex 2, IPO Standard ST.25 (1998).

[0035] As used herein, "recombinant DNA molecules" refers to molecules that do not naturally coexist without human intervention. This refers to DNA molecules that contain combinations of DNA molecules that do not result in the formation of a recombinant DNA molecule. A DNA molecule is composed of at least two DNA molecules that are heterogeneous to each other, naturally DNA molecules containing DNA sequences that deviate from existing DNA sequences, and synthetic DNA sequences. DNA molecules, or DNA molecules incorporated into the host cell's DNA through gene transformation or gene editing. It may also be embedded DNA molecules.

[0036] References to “isolated DNA molecules” or equivalent terms or expressions in this application are The DNA molecule exists alone or in combination with other compositions, but in its natural environment This means that it is a DNA molecule that does not exist inside. For example, a coding sequence, i Chromon sequences, untranslated reader sequences, promoter sequences, transcription termination sequences, etc., Nucleic acid elements found naturally within the DNA of an organism's genome are those elements that contribute to the organism's genome. As long as it is located within the genome and the element is in a naturally occurring position within the genome, "isolation" It is not considered that each of these elements, and these elements The lower part of the ment refers to the fact that the element is not present in the genome of an organism, and that the element is naturally occurring. Unless found at a genomic location, a substance is considered "isolated" within the scope of this disclosure. In addition, insecticidal proteins or any naturally occurring insecticidal variants of those proteins The nucleotide sequence to be coded is the nucleotide sequence that codes for the protein. Unless the sequence is found in the DNA of naturally occurring bacteria, it is considered an isolated nucleotide sequence. The synthetic nucleotide sequence that codes for the amino acid sequence of naturally occurring insecticidal proteins is, For the purposes of this disclosure, any traceable is deemed to be isolated. For the purposes of this disclosure, any traceable is deemed to be isolated. A nucleotide sequence, i.e., an insertion into the genome of a plant or bacterial cell. The nucleotide sequence of the DNA present in the extrachromosomal vector is, It is present in plasmids or similar structures used for cell transformation, or in plants or It is present in the genome of bacteria, or in tissues, progeny, and biological systems derived from plants or bacteria. Whether present in detectable amounts in the target sample or commercial product, isolated nucleotides It is considered to be a rheotide sequence.

[0037] As used herein, the term “sequence identity” refers to two optimally aligned points. The more identical the dinucleotide sequence or the two optimally aligned polypeptide sequences are This refers to the degree. The optimal array alignment is between two arrays, for example, a reference array and another array. Manually align the array with appropriate internal nucleotide insertions, deletions, or gaps. It is created by maximizing the number of nucleotide matches within the column alignment. As used in this document, the term "reference sequence" refers to the DNA sequence shown as sequence numbers 1-20. It refers to an array.

[0038] As used herein, “sequence identity percentage” or “identity percentage” The term "identity%" is obtained by multiplying the identity fraction by 100. The "fraction of identity" for an optimally aligned array is the number of elements within the optimal alignment. The number of cleotide matches is the total number of nucleotides in the reference sequence, for example, the total number of nucleotides in 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 herein. When optimally aligned with the reference sequences indicated as sequence numbers 1 to 20, 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 DNA molecules containing sequences that have at least approximately 100 percent identity.

[0039] 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. The term “regulating element” as used herein This refers to DNA molecules that have gene regulatory activity. The term "transmission" is used, for example, to describe the transcription of operably linked transcriptable DNA molecules and / or Alternatively, by influencing translation, the expression of operably linked, transcriptable DNA molecules. This refers to the ability to influence something. In plants, it functions as a promoter, leader, enhancer, or other agent. Regulatory elements such as trantrons and 3'UTRs are used to modify plant phenotypes through genetic engineering. It is useful for that purpose.

[0040] As used herein, the “regulatory expression element group” or “EXP” sequence refers to an operable sequence. Modulation elements linked to the function, such as enhancers, promoters, leaders, and This can refer to a group of introns. For example, a group of regulatory expression elements could be, for example, an intron sequence. A leader array operably linked to 5' for a leader array operably linked to 5' for a leader array It can be composed of a promoter. An EXP useful for carrying out the present invention is Sequence ID No. 5 Examples include 9 and 13.

[0041] 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, pathological , the cell cycle, and / or the expression of chemical responses, as well as any combination thereof, It may be characterized by quantitative or qualitative indicators. The term "genetics" as used herein refers to genetics. "Sub-expression pattern" refers to the expression pattern of a operably linked DNA molecule to a transcribed RNA molecule. This is any pattern of transcription. The transcribed RNA molecule is translated to produce a protein molecule. In some cases, antisense RNA or other regulatory RNA molecules may be present, such as double-stranded R. NA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA) ), in cases where microRNA (miRNA), small interfering RNA (siRNA), etc. There are also others.

[0042] As used herein, the term "protein expression" refers to the transcribed RNA molecule. Protein expression is an arbitrary pattern of translation into protein molecules. By their specific, developmental, or morphological properties, and also by quantitative or qualitative indicators, It can be marked.

[0043] A promoter is used to regulate the expression of operably linked, transcriptable DNA molecules. Useful as a regulatory element. The term "promoter" as used herein. Generally, RNA polymerase II and other proteins are needed to initiate transcription, for example. This refers to DNA molecules involved in the recognition and binding of trans-acting transcription factors. The promoter is First, it can be isolated from the 5' untranslated region (5'UTR) of the genome copy of the gene, For the purposes of this disclosure, the promoters provided herein are operable to the leader at 5'. It consists of a promoter linked to it. Alternatively, the promoter is produced synthetically. In some cases, it may be a manipulated DNA molecule. Also, the promoter is a chimera. That's fine. Chimeric promoters are produced by the fusion of two or more different DNA molecules. Useful promoters for carrying out the present invention include SEQ ID NOs: 1, 3, 5, 6, 9, 1 Promoter elements included in any of 0, 13, 14, 17, 18, and 19, Or fragments or variants thereof. In a particular embodiment of the present invention, The DNA molecules and any variants or derivatives thereof described in the claims of the specification are Furthermore, it is defined as containing promoter activity, i.e., transgenic plants It can act as a promoter within host cells such as these. Further specific implementations In this state, the fragment exhibits the promoter activity of the initiation promoter molecule from which it originates. Sometimes defined as a whole, a fragment brings about a basic level of transcription and initiates transcription. The TATA box is used for recognition and binding of the RNA polymerase II complex. Alternatively, it may include a "minimal promoter" composed of equivalent DNA sequences.

[0044] In one embodiment, a fragment of a promoter sequence disclosed herein is provided. The promoter fragment may contain the promoter activity described above, and may also be used alone or For example, other promoters and promoter fragments when constructing a chimeric promoter. In combination with, or in combination with other expression elements and fragments of expression elements It may be useful in certain embodiments. In certain embodiments, at least about 50, at least about 75, less At least about 95, at least about 100, at least about 125, at least about 150, less At least about 175, at least about 200, at least about 225, at least about 250, and less At least about 275, at least about 300, at least about 500, at least about 600, and at least At least about 700, at least about 750, at least about 800, at least about 900, and This includes at least about 1000 consecutive nucleotides, or more, as disclosed herein. This provides a promoter fragment containing a DNA molecule having promoter activity. Methods for producing such fragments from motor molecules are well known in this field.

[0045] For example, sequences 1, 3, 5, 6, 9, 10, 13, 14, such as internal or 5' deletions. Derived from any of the promoter elements included in 17, 18, and 19 The composition is a known method in the art for improving or modifying expression, i.e., expression To remove elements that have a positive or negative effect on expression This involves duplicating elements that have a negative effect, and / or making the expression tissue-specific or This involves using methods such as duplicating or removing elements that have a cell-specific effect. It can be produced by TATA box elements or their equivalent arrays and downstream arrays. Sequence IDs 1, 3, 5, 6, 9, 10, 13, 14, which consist of the removed 3' deletion, Derived from any of the promoter elements included in 17, 18, and 19 Using such a composition, for example, an enhancer element can be fabricated. By performing deletions, we can determine whether they have a positive or negative effect on expression, tissue-specific effects, or cell-specific effects. It exerts a resonance or timing-specific influence (such as, but not limited to, circadian rhythms). You may remove any element. Sequence numbers 1, 3, 5, 6, 9, 10, 13, 1 Promoter elements shown as being included in any of 4, 17, 18, and 19 Using either fragments or enhancers derived therefrom, chimeric transcriptional regulatory agents A rement composition can be prepared.

[0046] According to the present invention, a promoter or promoter fragment is a known promoter element. DNA binding sites, such as the TATA box and other known transcription factor binding site motifs. The existence of sequence characteristics can be analyzed. Identification information of such known promoter elements. Those skilled in the art can determine a variant of a promoter having an expression pattern similar to that of the original promoter. It can be used to design an Ant.

[0047] As used herein, the term "leader" refers to the untranslated 5' region (5'U) of a gene. Isolated from TR, between the transcription start site (TSS) and the protein coding sequence start site This refers to DNA molecules, generally defined as nucleotide segments. Alternatively, a leader This can be either a synthetically produced DNA element or a manipulated DNA element. The commander is a 5' regulator for controlling the expression of operable, transcribed DNA molecules. It can be used as an element. Leader molecules are heterologous promoters or their natural forms. It can be used in conjunction with the promoter. A useful leader for carrying out the present invention is: Includes any of the sequence numbers 1, 3, 5, 6, 9, 10, 13, 14, 17, 18, and 19 Examples include leader elements, or fragments or variants thereof. In this embodiment, such DNA sequence is used, for example, in transgenic plant cells. It can be defined as being able to act as a leader within the principal cell. In one embodiment, The sequence is decoded to include leader activity.

[0048] In any of the sequence numbers 1, 3, 5, 6, 9, 10, 13, 14, 17, 18, and 19 The included leader sequence (also called 5'UTR) may also constitute a regulatory element. If present, it may affect the transcription or translation of a operably linked, transcribed DNA molecule. In some cases, a secondary structure is adopted. Sequence numbers 1, 3, 5, 6, 9, 10, 13, 14, 1 A leader array included in any of 7, 18, and 19 is used in accordance with the present invention and operated Chimeric regulatory elements that affect the transcription or translation of potentially linked transcriptionable DNA molecules It is possible to create a ment.

[0049] As used herein, the term “intron” refers to a term used to describe a gene that has been isolated or identified from a gene. It is possible that, generally, the processing of pretranslational messenger RNA (mRNA) This refers to DNA molecules that can be defined as regions to be spliced ​​and removed in between. Alternatively, NTPs are either synthetically produced DNA elements or manipulated DNA elements. Good. Introns are enhancer elements that result in the transcription of functionally linked genes. It may contain introns. Introns are used to express transcribed DNA molecules that are operably linked. It may be used as a regulating element to adjust. The structure includes an intron. However, the intron is often heterogeneous or non-heterogeneous with respect to the transcriptionable DNA molecule. This is also good. Examples of introns in this technology include the actin intron of rice and One example is the corn HSP70 intron.

[0050] In plants, if a gene construct includes some introns, then if it does not include those introns, Compared to the construct, the accumulation of mRNA and protein increases. This effect is due to gene expression This is called "intron-mediated enhancement" (IME). It is known to stimulate expression in plants. The introns are found in maize genes (e.g., tubA1, Adh1, Sh1) , and Ubi1), rice genes (e.g., tpi), and dicotyledonous plant genes, e.g. , genes derived from petunias (e.g., rbcS), genes derived from potatoes (e.g., s t-ls1), and genes derived from Arabidopsis thaliana (for example, Identified in ubq3 and pat1). Deletion within the splice site of an intron or It is known that mutations reduce gene expression, and splicing is necessary for IMEs. This indicates that it may be necessary. However, in dicotyledonous plants, A. thalia An IME caused by a point mutation in the splice site of the na pat1 gene has been shown. It has been shown that using the same intron multiple times in plants is detrimental. In addition, basic regulatory elements are collected to construct appropriate recombinant DNA elements. It is necessary. Examples of introns useful in carrying out the present invention are Sequence IDs 7, 11, and It is shown as 15.

[0051] As used herein, the terms "3' transcription termination molecule," "3' untranslated region," or "3'UT" are used to refer to the same terms. The term "R" is used in the process of transcription to the untranslated region of the 3' portion of the mRNA molecule. This refers to NA molecules. The 3' untranslated region of mRNA molecules undergoes specific cleavage and poly-A tail formation. It can also be caused by 3' polyadenylation, also known as the 3'UTR. The 3'UTR is a transcriptional DNA molecule. It may be operably connected to and located downstream thereof, and the polyadenylation signal and transcription, mRNA processing or other regulatory signals that can affect gene expression It may also include. The poly(A) tail functions in mRNA stability and translation initiation. It is thought that an example of a 3' transcription termination molecule in this field is nopalin synthase 3'. Region, wheat hsp17 3' region, pea melbisco small subunit 3' region, wa This is the E6 3' region and the coixin 3'UTR.

[0052] The 3'UTR is typically found to have beneficial applications in the recombinant expression of specific DNA molecules. The 3'UTR can cause read-through, which can lead to the expression within adjacent expression cassettes. This may affect the expression of DNA molecules located in that region. Proper control of transcription termination is crucial. This prevents read-through to downstream localized DNA sequences (e.g., other expression cassettes). It can be stopped, and furthermore, the efficient use of RNA polymerase to improve gene expression This enables recycling. Efficient transcription termination (RNA polymerization from DNA) The release of -ase II is a prerequisite for transcription restart, and therefore directly affects the overall transcription level. This has an effect. Following transcription termination, mature mRNA is released from the synthesis site, and the template is imported into the cytoplasm. It is sent. Eukaryotic mRNA accumulates in vivo as poly(A) type, Detecting transcription termination sites using conventional methods is difficult. However, bioinformatics Predicting 3'UTR functionally and efficiently using the Matics method is effective for predicting 3'UTR. This is difficult because there are no conserved DNA sequences that would allow for easy prediction.

[0053] From a practical standpoint, the 3'UTR typically used in expression cassettes has the following characteristics: This is beneficial. Firstly, the 3'UTR efficiently and effectively terminates the transcription of the transgene. It should be possible to do so, and it should be possible to do so in a single transfer DNA (T-DNA). In the case of multiple expression cassettes, any adjacent expression cassette that can be composed of another expression cassette Reads of a DNA sequence, or a transcript of a T-DNA inserted into adjacent chromosomal DNA. It should be possible to prevent this. Secondly, the 3'UTR is the expression of DNA molecules. The promoter, leader, enhancer, and intron used to drive the Therefore, this should not cause a reduction in the confederated transcriptional activity. Finally, plant biotechnology In logy, the 3'UTR is often the reverse transcription RNA extracted from transformed plants. It is used for priming the amplification reaction, and (1) once incorporated into plant chromosomes To evaluate the transcriptional activity or expression of the expression cassette, (2) copy the insertions in the plant DNA. - Used to evaluate the number and (3) to evaluate the conjugation ability of seeds obtained after breeding. 3'UTR also characterizes the intactness of the inserted cassette, It is also used in the amplification reaction of DNA extracted from conversion plants. 3'UTR is indicated as sequence numbers 2, 4, 8, 12, 16, and 20.

[0054] As used herein, the terms "enhancer" or "enhancer element" are used in a manner that is not applicable to this specification. The term refers to a cis-acting regulatory element, also known as a cis element, which is operably linked. It certifies one aspect of the overall expression pattern of the linked transcriptionable DNA molecules, but usually, it is simple. In Germany, it is insufficient to drive transcription. Unlike promoters, enhancer elements The entry typically contains a transcription start site (TSS), a TATA box, or an equivalent DNA sequence. It does not include. The promoter or promoter fragment is a activatably ligated DNA compound. It may naturally contain one or more enhancer elements that affect the transcription of the column. By fusing the enhancer element with the promoter, one aspect of the overall regulation of gene expression is achieved. A chimeric promoter cis element that confers may be produced.

[0055] Many promoter-enhancer elements bind to DNA-binding proteins, and / or affect the DNA topology, and act on the DNA template for RNA polymerase Local conformations that selectively allow or restrict Seth, or double helix at the transcription initiation site It is thought to produce a local conformation that promotes the selective release of enhancer elements. It can function by binding to transcription factors that regulate transcription. Some enhancers Rement binds to multiple transcription factors, and these transcription factors then bind to multiple enhancers with different affinities. It can interact with the domain. Enhancer elements are deletion analysis, i.e., one or more Deleting the nucleotide from the 5' end or inside to the promoter, DNase DNA-binding protein analysis using I-footprinting, methylation interference, and electrophoresis. Mobility shift assay, ligation-mediated polymerase chain reaction (PCR) Several technologies, including vivo genome footprinting and other conventional assays. In some cases, the target sequence may be identified by conventional DNA sequence comparison methods such as BLAST. Alternatively, a known cis-element motif or enhancer element as a target motif. It may also be identified by DNA sequence similarity analysis using the enhancer domain. The ultrastructure of this can be altered by mutagenesis (or substitution) of one or more nucleotides, or by this technique. The enhancer element can be further studied using other conventional methods known in the field. , obtained by chemical synthesis or by isolation from regulatory elements containing such elements These can be used to add restriction enzyme sites that are useful for manipulating partial sequences. It can be synthesized together with adjacent nucleotides. Thus, a operably linked, transcribed Enhancer element for modulating DNA molecule expression according to the method disclosed herein The design, construction, and use of the enhancer are included in this invention. The enhancer is represented by Sequence IDs 1, 3, Any of the promoters included in 5, 6, 9, 10, 13, 14, 17, 18, and 19 It may originate from crabs.

[0056] As used herein, the term “chimera” means a molecule in which a first DNA molecule is divided into two DNA molecules. This refers to a single DNA molecule produced by fusing with the offspring, and this first DNA molecule is also The second DNA molecule is also usually found in its configuration, that is, fused with the other. No. Therefore, chimeric DNA molecules are novel DNA molecules that are not normally found in nature. The term "chimeric promoter" as used herein refers to a DNA molecule that This refers to a promoter produced by manipulation. A chimeric promoter is made from two or more DNA molecules. The fragments can be combined, for example, by fusing the promoter with the enhancer element. This may be used to regulate the expression of operably linked transcriptable DNA molecules. The design, construction, and use of a chimeric promoter according to the methods disclosed herein constitute the present invention. It is included.

[0057] Chimeric regulatory elements can be modified by various methods known in the art, such as restriction enzyme digestion and Ligation, ligation-independent cloning, and the module of PCR products during amplification. Direct chemical synthesis of assemblies or regulating elements, and known in the art. Designed to include various component elements that can be operably connected by other means. Obtain. The various chimera modifier elements obtained are the same constituent element or its variant. It can be composed of multiple ligatures, but the ligatures are linked together by a series of ligatures (multiple) The DNA sequences (multiple sequences are possible) containing the (possibly) are different. In this invention, these are shown as Sequence IDs 1 to 20. The DNA sequence may provide a reference sequence for a regulatory element, and may include such reference sequence. The constituent elements can be connected by methods known in the art, and bacteria and Substitutions, deletions, and / or nucleotide substitutions that occur naturally in plant cell transformations. This may include insertions or mutations.

[0058] As used herein, the term “variant” refers to a molecule whose composition is similar to that of the first DNA molecule. This refers to a second DNA molecule that is identical but not identical, such as a regulatory element, where the The second DNA molecule has the same or similar general function as the first DNA molecule. The expression pattern is maintained, for example, by roughly equivalent transcriptional activity. The variant is , a shortened or cleaved form of the first DNA molecule, or a modified form of the sequence of the first DNA molecule For example, having different restriction enzyme sites and / or internal deletions, substitutions, or insertions. It can be of a certain type. Also, a "variant" is a substitution of one or more nucleotides in the reference sequence. Regulatory elements having nucleotide sequences including deletions or insertions can also be included. Here, the derivative regulatory element performs transcription or transfer that is approximately equivalent to that of the corresponding parent regulatory molecule. It has translational activity. In addition, the "variant" of the regulatory element is shaped like a bacterial or plant cell. This also includes variants arising from naturally occurring mutations during qualitative transformation. In the present invention, the sequence number Using the polynucleotide sequence shown as 1-20, the DNA of the original regulatory element The arrangement and composition are similar but not identical, while the general function of the original regulatory element is similar. Therefore, variants that still maintain the same or similar expression patterns may be created. The production of such variants of the present invention is sufficiently within the scope of the skills of those skilled in the art in light of this disclosure. This is included within the scope of the present invention.

[0059] The modifications, duplications, or deletions described herein to the desired expression aspects of a particular transgene The effectiveness of the assay is stable and transient, as described in the examples of the assay described herein. Experimental testing was conducted in S.A., and the results varied depending on the modifications made to the initial DNA molecule and the purpose of those modifications. You may verify the results you obtain.

[0060] Structures As used herein, the term “construction” is derived from any source and incorporates genomes. They are capable of collaborative or autonomous replication, and at least one DNA molecule functions with another DNA molecule. Containing DNA molecules linked in a way that is operable, i.e., operablely linked, Any recombinant DNA molecule, e.g., plasmid, cosmid, virus, phage, or This refers to linear or circular DNA or RNA molecules. In this specification, "B" means a linear or circular DNA or RNA molecule. The term "transformer" refers to the process of introducing heterologous DNA or RNA into a host cell. This refers to any construct that can be used for the purpose of inputting. A construct is typically one or more expressions. Includes cassette. As used herein, “expression cassette” means one or more modulatory elements. The nt, typically operably connected to at least the promoter and 3'UTR, This also refers to DNA molecules that contain transcribed DNA molecules.

[0061] As used herein, the term “operably linked” means that the first DNA molecule It is linked to a second DNA molecule, and the first DNA molecule influences the function of the second DNA molecule. This refers to the arrangement of the first DNA molecule and the second DNA molecule in a certain manner. The two DNA molecules may or may not be part of a single continuous DNA molecule. They may or may not be adjacent. For example, a promoter in a cell may perform the desired transcription. When regulating the transcription of possible DNA molecules, the promoter acts on the transcribable DNA molecules. They are linked in a possible way. For example, a leader can influence the transcription or translation of a DNA sequence. If possible, it is operably linked to the DNA sequence.

[0062] In one embodiment, the construct of the present invention is Agrobacterium tumefa Along with the transfer molecules provided by ciens cells, the T-DNA in plant cell genomes A T-DNA isolated from A. tumefaciens that enables integration into the body. The right-hand boundary (RB or AGRtu.RB) and the left-hand boundary (LB or It is proposed as a dual tumor induction (Ti) plasmid boundary construct having the AGRtu.LB region. It may be provided (see, for example, U.S. Patent No. 6,603,061). In addition, the structure may contain bacteria. Plasmid backbone DNA segments that contribute to replication function and antibiotic selection in cells, e.g. For example, Escherichia coli replication start points such as ori322, oriV or Broad-host replication origins such as oriRi, and spectinomycin or streptomycin Tn7 aminoglycoside adenyltransferase (aa) confers resistance to A selection marker such as Spec / Strp that codes for dA), or gentamicin (Gm This may also include coding regions for selection marker genes (Gent). Therefore, the host bacterial strain is often A. tumefaciens ABI, C58, and This is LBA4404, but other strains known to those skilled in the art in the field of plant transformation However, this may also function in the present invention.

[0063] A transcriptionable DNA molecule is translated into a functional mRNA molecule that is expressed as a protein. Methods for assembling constructs and introducing them into cells so that they can be transcribed are already available in this field. It is knowledge. With regard to the implementation of the present invention, conventional methods for preparing and using constructs and host cells The composition and method are well known to those skilled in the art. A typical example useful for nucleic acid expression in higher plants. The bacterium is well known in this field, and it includes Agrobacterium tum Vectors and pCaMVCN transfers derived from efaciens' Ti plasmid —Includes control vectors.

[0064] Various adjustment elements, including any of the adjustment elements provided herein, can be used in structures. It may be included in. Any such adjustment element is provided in combination with other adjustment elements. In some cases, such combinations may be designed or modified to produce the desired adjustment function. It is possible. In one embodiment, the construct of the present invention is operably connected to the 3'UTR It comprises at least one regulatory element operably linked to a transcriptionable DNA molecule. .

[0065] The constructs of the present invention are any promoters provided herein or known in the art. or may include a leader. For example, the promoter of the present invention may include a heterogeneous non-translating 5' leader. —For example, it can be operably linked to something derived from a heat shock protein gene. Alternatively, the leader of this invention is a heterologous promoter, for example, cauliflower mosaic virus. It can be operably connected to the S35S transcript promoter.

[0066] Expression cassettes activatably link proteins, particularly in chloroplasts, leucomas, or Other plastid organelles, mitochondria, peroxisomes, vacuoles, or extracellular locations In contrast, transport peptides encoding peptides useful for targeting at or below the cellular level This may also include code sequences. Many chloroplast-localized proteins are expressed as precursors from nuclear genes. It is then targeted to chloroplasts by chloroplast transport peptides (CTPs). An example of a chloroplast protein is ribulose-1,5-bisphosphate carboxylase. Subunit (SSU), ferredoxin, ferredoxin oxidoreductase, photocatalytic Harvesting complex protein I and protein II, thioredoxin F, and enolpyrubin Examples include those related to lucicic acid phosphate synthase (EPSPS), but these are not limited to these. Not specified. Chloroplast transport peptides are, for example, described in U.S. Patent No. 7,193,133. Non-chloroplast proteins are activated by transgenes that encode non-chloroplast proteins. It has been demonstrated that the expression of heterologous CTP linked to brain activity can target chloroplasts.

[0067] Transcriptional DNA molecules As used herein, the term “transcribeable DNA molecule” refers to a DNA molecule that can be transcribed into an RNA molecule. This refers to any DNA molecule that can perform protein coding, and this includes those that have a protein-coding sequence. , produce RNA molecules that encode guide RNA and RNA molecules with sequences useful for gene repression. This includes, but is not limited to, living organisms. The types of DNA molecules can also be derived from the same plant. DNA molecules from one plant, DNA molecules from another organism, or synthesized DNA molecules, for example, DNA molecules containing antisense messages for genes, or introduced genes This may include DNA molecules that encode artificial, synthetic, or otherwise modified versions of the gene. Examples of transcribable DNA molecules for incorporation into the constructs of the present invention include: For example, DNA molecules or genes originating from a species other than the species into which the DNA molecule is incorporated, Alternatively, they originate from the same species or exist within the same species, but are genetically inherited rather than through classical breeding techniques. It contains genes that are incorporated into recipient cells by genetic engineering.

[0068] An "introduced gene" is a gene that is different from the host cell, at least in terms of its location within the host cell genome. A transcribable DNA molecule that is the current generation or any past generation of a cell. This refers to a transcriptional DNA molecule that has been artificially incorporated into the genome of a host cell.

[0069] The regulatory element, for example, the promoter of the present invention, is heterogeneous with respect to the regulatory element. It can be operably ligated to a transcribable DNA molecule. The term "heterogeneous" as used herein and The term refers to a case where a combination of two or more DNA molecules is not normally found in nature. This refers to a combination. For example, the two DNA molecules may originate from different species, and The two DNA molecules may originate from different genes, for example, from the same species. It can be different genes that originate from different species, or the same gene that originates from different species. Therefore, the regulatory element The nt is a operably linked, transcribable DNA molecule, and such combinations are natural. In cases not typically found in the world, i.e., when a transcribed DNA molecule is naturally present in a regulatory element If it does not occur when operably connected to the terminal, it is a different type.

[0070] A transcriptable DNA molecule is, in general, any DNA molecule whose transcript expression is desired. To obtain. The expression of the transcript leads to the translation of the resulting mRNA molecule, and consequently, to the translation of the transcript. Protein expression can result. Alternatively, for example, a transcriptionable DNA molecule can lead to the expression of a specific gene. It may be designed to ultimately cause a decrease in gene or protein expression. Morphologically, this uses a transcribable DNA molecule oriented in the antisense direction. This can be carried out by [method]. Those skilled in the art are familiar with the use of such antisense techniques. Any gene may be negatively regulated by this method, and in one embodiment, a transcriptional DNA molecule This suppresses specific genes through the expression of dsRNA, siRNA, or miRNA molecules. It may be designed to do so.

[0071] Therefore, one embodiment of the present invention relates to a construct that is incorporated into the genome of a transgenic plant cell. When integrated, the transcription of transcriptionable DNA molecules can be performed at the desired level or in the desired pattern. Sequence IDs 1-20 are operably linked to heterologous transcriptionable DNA molecules to regulate them. These are recombinant DNA molecules containing the regulatory elements of the present invention, such as those shown as. In this embodiment, the transcriptionable DNA molecule includes a protein-coding region of a gene, and another In this embodiment, the transcriptionable DNA molecule includes the antisense region of a gene.

[0072] Genes for agricultural purposes Transcriptional DNA molecules can be genes for agricultural purposes. The term "gene for agricultural purposes" refers to a gene expressed in a specific plant tissue, cell, or cell type. In general, it refers to a transcribable DNA molecule that confers a desired characteristic. The product of a gene for agricultural purposes is , plant morphology, physiology, growth, development, yield, grain composition, nutritional profile, diseases or Plants to produce effects against pests and / or environmental or chemical resistance. It can act within the substance itself, or it can act as an insecticide by being a food source for plant-eating pests. Yes. In one embodiment of the present invention, the adjustment element of the present invention is such that the adjustment element is Within the construct, it is operably linked to a transcribable DNA molecule that is a gene for agricultural purposes. It is incorporated into the transgenic plant containing such a structure, for agricultural purposes The expression of genes can confer beneficial agricultural traits. Beneficial agricultural traits include, for example, limited While not definitively established, herbicide resistance, insect control, modified yield, disease resistance, pathogens Resistance, modified plant growth and development, modified starch content, modified oil content, modified Modified fatty acid content, modified protein content, modified fruit ripening, animal and human Nutritional enhancement, biopolymer production, environmental stress resistance, pharmaceutical peptides, improvement of processing quality, Improvement of flavor, usefulness of hybrid seed production, improvement of fiber production, and desired biofuels Production can be cited as an example.

[0073] Examples of genes known in this field for agricultural purposes include herbicide resistance (US 6, No. 803,501, No. 6,448,476, No. 6,248,876, No. 6,225, No. 114, No. 6,107,549, No. 5,866,775, No. 5,804,425 (Patents No. 5,633,435, and No. 5,463,175), increased yield (USA) RE38,446, 6,716,474, 6,663,906, 6,476 ,295, No.6,441,277, No.6,423,828, No.6,399,330 Nos. 6,372,211, 6,235,971, 6,222,098, and (Patents No. 5,716,837), Insect Control (US Patent No. 6,809,078, No. 6,713) ,063, No. 6,686,452, No. 6,657,046, No. 6,645,497 No. 6,642,030, No. 6,639,054, No. 6,620,988, No. 6 , No. 593,293, No. 6,555,655, No. 6,538,109, No. 6,537 ,756, No.6,521,442, No.6,501,009, No.6,468,523 No. 6,326,351, No. 6,313,378, No. 6,284,949, No. 6 , No. 281,016, No. 6,248,536, No. 6,242,241, No. 6,221 ,649, No.6,177,615, No.6,156,573, No.6,153,814 No. 6,110,464, No. 6,093,695, No. 6,063,756, No. 6 ,063,597, No.6,023,013, No.5,959,091, No.5,942 ,664, No.5,942,658, No.5,880,275, No.5,763,245 (Patent No. 5,763,241), fungal disease resistance (Patent No. 6,653,280) , No. 6,573,361, No. 6,506,962, No. 6,316,407, No. 6, No. 215,048, No. 5,516,671, No. 5,773,696, No. 6,121, (Nos. 436, 6,316,407, and 6,506,962), virus resistance ( U.S. Patent Nos. 6,617,496, 6,608,241, 6,015,940, (Nos. 6,013,864, 5,850,023, and 5,304,730), line Insect resistance (U.S. Patent No. 6,228,992), bacterial disease resistance (U.S. Patent No. 5,516) (Patent No. 671), Plant Growth and Development (Patent Nos. 6,723,897 and 6,518, U.S. Patent Nos. 6,723,897 and 6,518) (No. 488), starch production (U.S. Patent No. 6,538,181, No. 6,538,179) (Nos. 6,538,178, 5,750,876, 6,476,295), modified Oil production (U.S. Patent No. 6,444,876, No. 6,426,447, and No. 6, (Patents No. 380,462), high oil production (US Patent No. 6,495,739, No. 5,608,1) (Nos. 49, 6,483,008, and 6,476,295), containing modified fatty acids Quantity (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 Nos. 589,767, 6,537,750, 6,489,461, and 6,4 (Patent No. 59,018), high protein production (Patent No. 6,380,466), fruit ripening Nutritional Fortification (U.S. Patent No. 5,512,466), Animal and Human Nutritional Fortification (U.S. Patent No. 6,7266) No. 3,837, No. 6,653,530, No. 6,5412,59, No. 5,985,60 (Patents No. 5, and No. 6,171,640), Biopolymers (USRE No. 37,543) Nos. 6,228,623, 5,958,745, and 6,946,588 ), environmental stress resistance (U.S. Patent No. 6,072,103), pharmaceutical peptides and secretions Peptides (U.S. Patent Nos. 6,812,379, 6,774,283, 6,140, (Patents No. 075, and No. 6,080,560), Improvement of processing traits (U.S. Patent No. 6,4) (Patent No. 76,295), improved digestibility (Patent No. 6,531,648), lower raffinol (US Patent No. 6,166,292), Industrial Enzyme Production (US Patent No. 5,543,57) (No. 6), Flavor improvement (U.S. Patent No. 6,011,199), Nitrogen fixation (U.S. Patent No. 5,2 (Patent No. 29,114), hybrid seed production (U.S. Patent No. 5,689,041), fiber production (US Patent Nos. 6,576,818, 6,271,443, 5,981,834) Patent No. 5,869,720, and Biofuel Production (U.S. Patent No. 5,998, Examples include those related to issue 700.

[0074] Alternatively, genes for agricultural purposes can trigger targeted regulation of gene expression of endogenous genes. By encoding the RNA molecule that triggers it, for example, antisense (for example, US patent) No. 5,107,065), inhibitory RNA (“RNAi”, e.g. published application US .2006 / 0200878 and US2008 / 0066206, and U.S. Patent As described in application No. 11 / 974,469, miRNA, siRNA, trans-active (Including regulation of gene expression by mechanisms mediated by siRNA and phase sRNA) The above-mentioned plant characteristics or phenotypes are affected by mechanisms mediated by co-inhibition. Furthermore, RNA is used as a catalyst RN, which is engineered to cleave the desired endogenous mRNA product. Molecule A (e.g., ribozyme or riboswitch, e.g., US2006 / 0200) (See 878) This may be the case. A transcriptionable DNA molecule can become a molecule that can cause gene repression. Methods for constructing and introducing constructs into cells in a manner that allows for transcription are in this field. It is already known.

[0075] Selection Marker Selective marker-transfer genes can also be used in conjunction with the regulatory elements of the present invention. The term "selection marker transgene" used in the document refers to transgenic plants. The expression or lack of expression in tissues or cells is screened in some way. Or refers to any transcribable DNA molecule that can be scored. Selective marker genes for this purpose, as well as related selection and screening techniques, This is known in the art, and includes β-glucuronidase (GUS) and green fluorescent protein. Proteins that confer antibiotic resistance (GFP), proteins that confer antibiotic resistance, and proteins that confer herbicide resistance This includes, but is not limited to, transcriptionable DNA molecules that code for proteins. An example of a maker-introduced gene is shown as Sequence ID No. 21.

[0076] Genome editing Some embodiments are site-directed genome modification enzymes and / or genome modification for It is operablely linked to heterologous DNA sequences encoding any one related protein(s) of the specified protein(s). At least approximately 85 percent of any one or a fragment of sequence numbers 1-20 were included. Regarding recombinant DNA constructs containing expression cassettes (multiple cassettes are possible) that have sequence identity: These nuclease expression cassettes (or multiple cassettes) are the same as the donor templates used for template editing. They may exist in a molecule or vector (cis), or they may exist in another molecule or vector. In some cases, it may be trans. Different sequence-specific genome modifying enzymes modify genomic DNA. Some editing methods include a protein complex and / or guide RNA. This is known in the art. In some embodiments, the site-directed genome modification enzyme is two Inducing strand breaks (DSBs) or creating breaks at desired genomic locations or gene loci. The genome is modified by induction. In some embodiments, the genome-modifying enzyme In the process of introducing DSBs or repairing breaks, the donor template DNA is the DSB Alternatively, the genome can be integrated at the site of the break. In some embodiments, the genome modification DSBs are introduced by enzymes, or the process of repairing breaks involves insertion or deletion mutations. (indels) can be introduced into the genome. In some embodiments, site-directed genome modification is performed. The mutating enzymes include cytidine deaminase. In some embodiments, site-directed genome modification is performed. The mutating enzymes include adenine deaminase. In this disclosure, site-directed genome-modifying enzymes include: Endonucleases, recombinases, transposases, deaminases, helicases, This includes reverse transcriptase and any combination thereof.

[0077] Some embodiments encode heterogeneous components of a genome editing system. Gene regulatory elements described herein, operably linked to a transcriptionable DNA molecule. Regarding the use of genome editing systems to insert or delete one or more elements into the genome of a host cell. Substitutions, base modifications, translocations, or inversions may be introduced. In some embodiments, this specification The gene regulatory elements described in the book are CRISPR-Cas effector proteins, and Sequence-specific proteins such as thrombocytopenia (TAL) proteins or activators of transcription (TAL) proteins. It is operably ligated to a heterologous, transcriptional DNA molecule that encodes a DNA-binding domain. In some embodiments, the sequence-specific DNA-binding domain may be a fusion protein. In some embodiments, the gene regulatory elements described herein are CRISPR -Makes it operable to heterologous transcriptional DNA molecules encoding Cas effector proteins They are linked. In some embodiments, the CRISPR-Cas effector protein These are Type I CRISPR-Cas systems, Type II CRISPR-Cas systems, and Type III CRISPR-Cas systems. Type IV CRISPR-Cas system, Type V CRISPR-Cas system The system can be selected from either the PR-Cas system or the VI-type CRISPR-Cas system. In some embodiments, the gene regulatory element described herein controls the guide RNA. It is operably ligated to a different type of transcribable DNA molecule. Guide RNA (or gRNA) recognizes the target DNA sequence and triggers CRISPR effects. This refers to RNA that directs or "guides" a protein towards a target DNA sequence. The doRNA contains a region complementary to the target DNA (called crRNA) and CRISPR. It consists of a region that binds to the receptor protein (called tracrRNA). DRNA can be a single RNA molecule (sgRNA) or two separate RNA molecules. (In some cases, it is a two-piece gRNA.) In some embodiments, the gRNA is reverse transcriptase It may further include an RNA template (pegRNA) for this purpose.

[0078] Some embodiments involve a CRISPR-Cas effector protein and a guide RN. A gene encoding one or more components of the CRISPR-Cas genome editing system, including A. Gene regulatory elements described herein, operably linked to a species' transcribable DNA molecule. Regarding the phenotype. An example of a CRISPR-Cas effector protein is Cas9. C2c1, C2c3, C2c4, C2c5, C2c8, C2c9, C2c10, Cas1 2a (also called Cpf1), Cas12b, Cas12c, Cas12d, Cas1 2e, Cas12h, Cas12i, Cas12g, Cas13a, Cas13b, Ca s13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', C as3”, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (Csn1 and Also known as 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, C sx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4(dinG) Csf5, Cas14a, Cas14b, and Cas14c effector proteins Quality is one example, but is not limited to these. In some embodiments, as described herein Gene regulatory elements are their nuclease active sites (for example, RuvC, HNH, etc.) For example, the RuvC site of the Cas12a nuclease domain, for instance, Cas9 nuclease CRISPR-Cas variants containing mutations in the RuvC and / or HNH sites of the domain It is operably linked to the effector protein. It has a mutation in the nuclease active site, Furthermore, CRISPR-Cas effector proteins no longer possess nuclease activity. This is generally called "dead," for example, dCas. In some embodiments, CRISPR-Cas effector protein with mutations in the nuclease active site The in or polypeptide is the same CRISPR-Cas effector that does not contain the mutation. Compared to protein, its activity may be impaired or reduced. In some embodiments, the gene regulatory element described herein is its nuclea It can be activated in CRISPR-Cas effector proteins that have mutations in the enzyme active site. It is linked and produces nickasase activity that is operably linked to reverse transcriptase.

[0079] Cell transformation The present invention also includes one or more regulatory elements operably linked to a transcriptionable DNA molecule. The present invention relates to a method for producing transformed cells and plants, including [specific components].

[0080] The term "transformation" refers to the introduction of DNA molecules into a recipient host. The term "host" as used here includes any cell, tissue, or organ, including bacteria, fungi, etc. This refers to plants, or the bacteria, fungi, or plant progeny. Particularly important plant tissues and fine The spores include protoplasts, callus, roots, tubers, seeds, stems, leaves, seedlings, embryos, and pollen. It can be listed.

[0081] As used herein, the term "transformed" refers to a foreign DNA molecule, for example, This refers to a cell, tissue, organ, or organism into which a construct has been introduced. DNA molecules that are integrated into and introduced into the genomic DNA of cells, tissues, organs, or organisms. It may be possible to pass it on to future generations. "Transgenic" or "transformed" The cells or organisms that have been "transformed" also have progeny, and such transgender cells or organisms can be transgendered through mating. It is produced from a breeding program using phenic organisms as parents, and the presence of foreign DNA molecules This may also include offspring that exhibit the altered phenotype. Furthermore, the introduced DNA molecule may then To prevent it from being passed down to future generations, the introduced DNA molecule is transiently introduced into the recipient cell. It may be included. The term "transgenic" refers to a substance containing one or more heterogeneous DNA molecules. This refers to bacteria, fungi, or plants.

[0082] There are many methods well known to those skilled in the art for introducing DNA molecules into plant cells. Generally, the process involves selecting suitable host cells and transforming them with a vector. The invention includes the steps of performing a certain procedure and obtaining transformed host cells. A method for transforming plant cells by introducing plant constructs into the plant genome, and The materials may include any of the well-known and proven methods. Preferred methods are limited to: It is not something that is done, but among them, bacterial infections (for example, Agrobacterium), NariBAC vectors, direct delivery of DNA (e.g., PEG-mediated transformation, desiccation / inhibition media) DNA incorporation via intermediary, electroporation, stirring with silicon carbide fibers, and DNA co Examples include acceleration of pharmacokinetic particles and gene editing (e.g., the CRISPR-Cas system). It is possible.

[0083] The host cell is any cell or organism, for example, plant cells, algal cells, algae, fungal cells, These may be fungal, bacterial, or insect cells. In certain embodiments, the host cells and transforming cells may be used. The cells may include cells from crop plants.

[0084] The transgenic plants are then regenerated from the transgenic plant cells of the present invention. It is possible to breed seeds from this transgenic plant using conventional breeding techniques or self-pollination. It may produce offspring. Such seeds, and the progeny resulting from growth from such seeds. The substance becomes transgenic because it contains the recombinant DNA molecule of the present invention.

[0085] The transgenic plant of the present invention is self-pollinated (homozygous to the recombinant DNA molecule). The present invention may also provide seeds of homozygous transgenic plants, By crossing non-transgenic plants or different transgenic plants, (recombinant D Seeds of the heterozygous transgenic plant of the present invention (heterozygous to NA molecules) They can also be provided. Such homozygous and heterozygous transgenic plants Both are referred to as “progeny” in this specification. Progeny are the original transgenic plants. This is a transgenic plant derived from a plant lineage and containing the recombinant DNA molecule of the present invention. The seeds produced using the transgenic plant of the invention are harvested and used in this A transgenic plant that contains the construct of the invention and expresses genes for agricultural purposes, that is, In addition, the generations of the progeny plants of the present invention can be grown. Descriptions of breeding methods commonly used for different crops can be found in one of several reference books. For example see Allard, Principles of Plant Breeding, Joh n Wiley & Sons, NY, U. of CA, Davis, CA, 50 - 98 (1960), Simmonds, Principles of Crop Impro vement, Longman, Inc., NY, 369 - 399(1979), Sne ep and Hendriksen, Plant breeding Perspec tives, Wageningen(ed), Center for Agricult ural Publishing and Documentation(1979), Fehr, Soybeans: Improvement, Production and Uses, 2nd Edition, Monograph, 16:249(1987) Fehr, Principles of Variety Development, Theory and Technique, (Vol.1) and Crop Speci es Soybean(Vol.2), Iowa State Univ., Macmi llan Pub. Co., NY, 360 - 376(1987). Please refer to these references.

[0086] Transgenic plants can be analyzed for the presence of the target gene(s), as well as the expression level and / or profile conferred by the regulatory elements of the present invention. Those skilled in the art will recognize numerous methods available for analyzing transgenic plants. For example, for plant analysis see a number of methods available for the analysis of transgenic plants. For example, for plant analysis Methods include Southern blotting or Northern blotting, PCR-based approaches, These include biochemical analysis, phenotypic screening, field evaluation, and immunodiagnostic assays. However, it is not limited to these. The expression of transcriptionable DNA molecules is controlled by TaqMan®. Reagents and The method described by the manufacturer and the TaqMan® Testing Mat It can be measured using the PCR cycle time determined using ix. Alternatively, I nvader (registered trademark) (Third Wave Technologies, Mad Using the reagents from ison,WI) and the method described by the manufacturer, the expression of the transgene is performed It can be evaluated.

[0087] The present invention also provides plant parts of the present invention. Plant parts include leaves, stems, roots, tubers, Examples include, but are not limited to, seeds, endosperm, ovules, and pollen. This includes being viable, not viable, regenerative, and / or not regenerative. In some cases, the present invention also includes transformed plant cells containing the DNA molecule of the present invention, and this Provided. The transformed or transgenic plant cells of the present invention are regenerative and / or non-renewable plant cells are included.

[0088] 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 sequence numbers 1 to 20. It contains a detectable amount of DNA containing a DNA sequence selected from the group. "Commercial products" refers to transgenic plants containing the recombinant DNA molecule of the present invention, seeds, etc. Any composition or product consisting of material derived from children, plant cells, or plant parts This refers to commercial products, which include, but are not limited to, processed seeds, grains, plant parts, and coarse flours. Not determined. The commercial product of the present invention contains a detectable amount corresponding to the recombinant DNA molecule of the present invention. Contains DNA. Using the detection of one or more of this DNA in the sample, the commercial product The content or source may be identified. Any standard method, including the detection method disclosed herein. Various DNA molecule detection methods may be used.

[0089] The present invention may be more easily understood by referring to the following embodiments. Unless otherwise specified, these embodiments are provided for illustrative purposes only and do not limit the present invention. This is not intended. Those skilled in the art will see that the techniques disclosed in the following embodiments are useful in carrying out the present invention. The fact that it functions well is representative of the technology discovered by the inventors. Please understand. However, those skilled in the art will understand that in light of this disclosure, the specific implementations disclosed Many modifications may be made to the form, and even then, it will not deviate from the spirit and scope of the present invention. It should be understood that similar or identical results can be obtained, and therefore, explanation All content presented or shown is illustrative and not limited to any specific meaning. It should not be interpreted as a taste. [Examples]

[0090] Example 1 Identification and cloning of regulatory elements In this embodiment, we identify, synthesize, and classify regulatory expression elements derived from Zea mays. This section describes roaning.

[0091] Genes preferentially expressed in pollen were identified using public and proprietary transcriptome data. Each locus was investigated bioinformaticslly to identify the corresponding gene promoter, leader, intron, and 3’UTR. The identified EXPs, promoters / leaders, introns, and 3’UTRs are shown in Table 1 below.

Table 1

[0092] The identified EXPs, promoters / leaders, and 3’UTRs were synthesized using methods known in the art and cloned into a binary plant transformation vector construct in an expression cassette used to drive β-glucuronidase (GUS) expression as described in Example 2 below, and their activities were evaluated in stably transformed maize plants.

[0093] Example 2 Analysis of Regulatory Elements Driving GUS Expression in Stably Transformed Maize Plants Maize plants were transformed with a vector, specifically a plant expression vector containing a test regulatory element that drives the expression of the β-glucuronidase (GUS) transgene. The resulting plants were analyzed for GUS protein expression to evaluate the effect exerted by the selected regulatory element on expression.

[0094] [[ID=*]]Maize plants were transformed with a plant GUS expression construct. Using standard methods known in the art, the regulatory element was cloned into a base plant expression vector. The resulting plant expression vector was from Agrobacterium tumefaciens ​​Left border area (B-AGRtu.left border), against the herbicide glyphosate The first transgene selection cassette used to select transformed plant cells that confer resistance It consists of a promoter and a leader, and optionally, in a particular vector design, G is a processing intron that is operably connected to Ron at 5'. Adjustable element operably connected to the US code sequence and operably connected to 3'UTR A second transgene cassette (SEQ ID NO: 21) for evaluating the activity of the Agro The right-hand boundary region from bacterium tumefaciens (B-AGRtu). It included the right border.

[0095] Table 2 shows the composition of the expression elements in the GUS expression cassette for each construct. Constructs 1- Item 5 is a native promoter and leader (indicated by "P-") that are operably connected. or operably linked native promoters, leaders, and introns ("E Includes a GUS expression cassette consisting of constructs 1 and 2 (indicated as "XP-"). The S expression cassette does not contain introns. Constructs 1-5 and 8 are the same gene. Promoter / Leader of the Intron or Promoter / Leader / Intron Native Includes 3'UTR. In constructs 6 and 7, native promoter and leader (Indicated by "P-") is an intron that is not part of the native gene but can be expressed in plants. and operably linked to the 3'UTR. Construct 8 is also expressible in plants, nativity It contains introns that are not part of the gene. [Table 2]

[0096] Maize plant cells are transmitted via Agrobacterium, as is well known in this field. The cells were transformed using the above-mentioned binary transformation vector construct. The transformed plant cells were induced to form a whole maize plant.

[0097] 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, the whole or sectioned tissue is treated with 1 mg / mL of X-Gluc(5-bromo-4-chloro Inoculate with GUS staining solution containing -3-indolyl-β-glucuronide at 37°C for 5 hours. The samples were incubated and decolorized with 35% EtOH and 50% acetic acid. Examination was performed using a dissecting microscope or a combined microscope. Below, visual inspection of the blue coloration of selected plant organs or tissues indicates the development of GUS. The current situation was qualitatively identified.

[0098] 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 determined by a 1 mM concentration of the fluorescent substrate 4-methylwheat in a total reaction volume of 50 microliters. Methyleumbelliferyl-β-D-glucuronide Incubated with MUG. After incubation at 37°C for 1 hour, 350 The reaction can be stopped by adding a microliter of 200 mM sodium bicarbonate solution. The reaction product was 4-methylumbellifolone (methlyumbellifolone). erone)(4-MU) exhibits maximum fluorescence at high pH, ​​and the hydroxyl group is ionic. It is converted. The assay is stopped by adding a basic sodium carbonate solution. Next, adjust the pH to quantify the fluorescent product 4-MU. The amount of 4-MU produced is measured using FLU. Ostar Omega Microplate Reader (BMG LABTECH) (Excitation 3) It was estimated by measuring its fluorescence using 55 nm (emission at 460 nm). S activity values ​​are expressed in nanomolar units of 4-MU / hour / mg of total protein.

[0099] The following tissues were sampled for GUS expression in the R0 generation: V4 stage Leaves and roots of the plant, leaves and roots of the V7 stage, leaves of the VT stage, flowers / anthers and pollen, R1 stage The cob rachis / hairs of the seed, as well as the R3 stage seed embryo and seed 21 days after pollination (DAP). endosperm.

[0100] Tables 3 and 4 show the average quantitative GUS expression in the sampled tissues, where "b "dl" indicates that the level is below the detection level, and "NA" indicates that the assay was not performed. This shows the GU for the flowers / anthers and pollen of the VT stage, and the rachis / hairs of the R1 stage. The range of S expression is also shown in Table 4. [Table 3] [Table 4]

[0101] As shown in Tables 3 and 4, many of the GUS expression cassettes in each of these constructs are With the exception of constructs 6 and 7, higher expression was observed in VT flowers / anthers and / or pollen. Regarding construct 6, high expression was quantitatively observed in the anthers of VT and in the embryos and endosperm of 21DAP. Measured. In the case of construct 7, high expression of GUS was quantitatively observed in the anthers of VT and the rachis / hairs of R1. Measured. In the case of construct 8, the expression of VT anthers was lower compared to constructs 6 and 7. The GUS expression of VT pollen was quantitatively determined for constructs 6, 7, and 8. Not measured. Constructs 1 and 2 showed high GUS expression with VT pollen, and other From the tissue, GUS expression was high in the flowers / anthers of VT.

[0102] Furthermore, selected tissues from these transformation events were observed under a microscope, and GUS staining of the tissue was performed. The observed qualitative expression aspects were identified. Table 5 below summarizes the observations made in these tissues. To summarize. [Table 5]

[0103] As shown in Table 5, GUS expression in constructs 1 to 5 was mainly observed in VT pollen. Therefore, when measured quantitatively, higher expression was observed in VT flowers / anthers. Thus, construct 1 ~The expression element in the GUS expression cassette of construct 5 is the "pollen-preferential" expression element. Regarding GUS expression from constructs 6 and 7, GUS expression is observed in VT flowers. It was observed not only in the powder but also in other tissues such as the leaf and root cells of V7. Interestingly, GUS expression from structure 7 was also observed in the hairs of R1. In the case of structure 8, staining of VT pollen. Furthermore, staining of the root tip of V4 and the embryo and endosperm tissue of R3 with 21DAP was clearly visible. It was done.

[0104] Therefore, the expression elements contained in the GUS expression cassettes of constructs 1 to 5 are pollen This shows the preferred expression pattern. It is included in the GUS expression cassettes of constructs 6, 7, and 8. The expressed element is expressed in pollen while being stably transformed corn. It is also expressed in other plant tissues.

[0105] The principles of the present invention have been illustrated and explained, and those skilled in the art will not deviate from these principles. It should be clear that the arrangement and details of the present invention may be modified. All modifications within the spirit and scope of the claims are claimed. All publications and published patent documents used are, each of the individual publications or patent applications. To the same extent as when it is specifically and individually indicated that something is incorporated by reference, This specification is more fully incorporated here.

Claims

1. (a) At least 85 percent sequence identity for any of sequence numbers 1 to 20 The array that has (b) Sequences containing any of sequence numbers 1 to 20, and (c) Any fragment of Sequence ID No. 1 to 20 having gene regulatory activity A recombinant DNA molecule comprising a DNA sequence selected from the group consisting of, The sequence is operably linked to a different type of transcriptionable DNA molecule, the recombinant DNA molecule child.

2. The sequence is at least 90 percent of any of the DNA sequences of sequence numbers 1 to 20. A recombinant DNA molecule according to claim 1, having sequence identity.

3. The sequence is at least 95 percent of any of the DNA sequences of sequence numbers 1 to 20. A recombinant DNA molecule according to claim 1, having sequence identity.

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

5. The set according to claim 1, wherein the heterogeneous transcriptionable DNA molecules include genes for agricultural purposes. Replacement DNA molecule.

6. The recombinant D according to claim 5, wherein the gene for the agricultural purpose confers herbicide resistance to the plant. NA molecule.

7. The recombinant D according to claim 5, wherein the gene for the aforementioned agricultural purpose confers pest resistance to the plant. NA molecule.

8. The aforementioned heterogeneous transcriptionable DNA molecules are used to convert dsRNA, miRNA, or siRNA. A recombinant DNA molecule according to claim 1.

9. (a) At least 85 percent sequence identity for any of sequence numbers 1 to 20 The array that has (b) Sequences containing any of sequence numbers 1 to 20, and (c) Any fragment of Sequence ID No. 1 to 20 having gene regulatory activity Transgenic plant molecules containing recombinant DNA molecules containing sequences selected from the group consisting of the following: It is a cell, The aforementioned sequence is operably linked to a different type of transgenic DNA molecule. plant cells.

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

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

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

13. Progeny of the transgenic plant according to claim 12, comprising the recombinant DNA molecule or that part.

14. A transgenic seed containing the recombinant DNA molecule described in claim 1.

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

16. The aforementioned commercial products include seeds, processed seeds, protein concentrates, protein isolates, and starch. The method according to claim 15, which is grain, plant parts, seed oil, biomass, fine powder, and coarse powder. Law.

17. A method for expressing a transcriptionable DNA molecule, the transgenicity described in claim 12. This includes obtaining a buck plant and cultivating the plant, thereby the transferable DN The method by which A is expressed.

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