RU2774709C2 - Plant regulatory elements and their application options - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to the field of biochemistry, in particular to a recombinant DNA molecule for the expression of a heterologous transcribed polynucleotide molecule. A transgene plant cell, a transgene plant, a part of a transgene plant, and a transgene seed containing the specified DNA molecule are also disclosed. A method for the expression of the specified DNA molecule, and a method for the production of the specified cell of a transgene plant are disclosed.

EFFECT: invention allows for effective expression a heterologous transcribed polynucleotide molecule.

18 cl, 2 tbl, 4 ex

Description

LINK TO RELATED APPLICATION

[0001] This application claims priority from U.S. Provisional Application No. 62/340,656, filed March 24, 2016, the contents of which are hereby incorporated by reference in their entirety.

INCLUDING A SEQUENCE LIST

[0002] The sequence listing, which is contained in a file called "MONS419WO-sequence_listing", which is 14.1 kB in size (measured on Microsoft Windows®) and was created on March 22, 2017, is filed electronically with this application and incorporated into the present description by reference.

FIELD OF TECHNOLOGY

[0003] The present invention relates to the fields of plant molecular biology and plant genetic engineering. In particular, the invention relates to DNA molecules suitable for modulating gene expression in plants.

BACKGROUND OF THE INVENTION

[0004] Regulatory elements are genetic elements that regulate the operation of genes by modulating the transcription of an operably linked transcribed DNA molecule. Such elements may include promoters, leader sequences (leaders), introns, and 3' untranslated regions, and may be used in the fields of plant molecular biology and plant genetic engineering.

BRIEF DESCRIPTION OF THE INVENTION

[0005] The invention provides novel regulatory gene elements for use in plants. The invention also provides DNA constructs containing regulatory elements. The present invention also provides transgenic plant cells, plants and seeds containing regulatory elements. In one embodiment, the regulatory elements are operably linked to the transcribed DNA molecule. In some embodiments, the transcribed DNA molecule may be heterologous with respect to the regulatory sequence. Thus, the proposed according to the invention the sequence of regulatory elements in specific implementations can be defined as operably linked to a heterologous transcribed DNA molecule. The present invention also provides methods for the production and use of regulatory elements, DNA constructs containing regulatory elements, and transgenic plant cells, plants and seeds containing regulatory elements operably linked to a transcribed DNA molecule.

[0006] Thus, in one aspect of the invention, there is provided a recombinant DNA molecule comprising a DNA sequence that is selected from the group consisting of: (a) a sequence having at least about 85 percent sequence identity with any one of SEQ ID NO: 1 -5 or 7; (b) a sequence containing any of SEQ ID NOs: 1-5 or 7; and (c) a fragment of any of SEQ ID NOs: 1-5 or 7, wherein the fragment has gene regulatory activity; wherein said sequence is operably linked to a heterologous transcribed DNA molecule. By "heterologous transcribed DNA molecule" is meant that the transcribed DNA molecule is heterologous with respect to the polynucleotide sequence to which it is operably linked. In certain embodiments, the recombinant DNA molecule contains a DNA sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the DNA sequence of any of SEQ ID NOs: 1-5. In certain embodiments, the DNA sequence contains a regulatory element. In some embodiments, the regulatory element comprises a promoter. In other embodiments, the heterologous transcribed DNA molecule contains a gene of interest from an agronomic point of view, such as a gene having the ability to confer plant resistance to herbicides or a gene having the ability to confer plant pest resistance. In other embodiments of the invention, a construct is provided that contains a recombinant DNA molecule as provided herein.

[0007] In another aspect, provided herein are transgenic plant cells that comprise a recombinant DNA molecule comprising a DNA sequence that is selected from the group consisting of: (a) a sequence having at least 85 percent sequence identity with any of SEQ ID NO: 1-5 or 7; (b) a sequence containing any of SEQ ID NOs: 1-5 or 7; and (c) a fragment of any of SEQ ID NOs: 1-5 or 7, wherein the fragment has gene regulatory activity; wherein the DNA sequence is operably linked to a heterologous transcribed DNA molecule. In certain embodiments, the transgenic plant cell is a monocot cell. In other embodiments, the transgenic plant cell is a monocotyledonous plant cell or a dicotyledonous plant cell.

[0008] In yet another aspect, the present document further provides a transgenic plant, or part thereof, which(s) contains a recombinant DNA molecule containing a DNA sequence that is selected from the group consisting of: (a) a sequence characterized by at least 85- percent sequence identity with any of SEQ ID NOs: 1-5 or 7; (b) a sequence containing any of SEQ ID NOs: 1-5 or 7; and (c) a fragment of any of SEQ ID NOs: 1-5 or 7, wherein the fragment has gene regulatory activity; wherein the DNA sequence is operably linked to a heterologous transcribed DNA molecule. In specific embodiments, the transgenic plant is a progeny of any generation that contains the recombinant DNA molecule. The present document also provides a transgenic seed containing a recombinant DNA molecule from which such a transgenic plant grows.

[0009] According to another aspect of the invention, a method for producing a commercial product is provided, including obtaining a transgenic plant or part thereof containing (s) a recombinant DNA molecule according to the present invention, and obtaining from it (her) said commercial product. In one embodiment, the marketable product is treated seeds, grains, plant parts, oils, and meal.

[0010] According to another aspect of the invention, there is provided a method for producing a transgenic plant containing a recombinant DNA molecule according to the present invention, which includes transforming a plant cell with a recombinant DNA molecule according to the present invention to obtain a transformed plant cell and regenerating a transgenic plant from this transformed plant cell.

[0011] According to some aspects of the invention, methods for producing a transgenic plant cell are provided, comprising introducing into the plant cell a recombinant DNA molecule as provided herein. In certain embodiments, introducing said recombinant DNA molecule into said plant cell comprises transforming or regenerating a transgenic plant from said plant cell. In further embodiments, introducing said recombinant DNA molecule into said plant cell comprises crossing a transgenic plant as provided herein with another plant to produce a progeny plant containing said plant cell.

[0012] In a further aspect of the invention, methods for producing a transgenic plant are provided, comprising transforming a plant cell with a recombinant DNA molecule as provided herein. In certain embodiments, the methods of the invention further comprise regenerating a transgenic plant from a plant cell that has been transformed with a DNA molecule as provided herein.

BRIEF DESCRIPTION OF SEQUENCES

[0013] SEQ ID NO: 1 is the DNA sequence of the Expression Regulatory Elements (EXP) group that contains a promoter derived from a putative ubiquitin gene from Buteloua gracilis operably linked at the 5' end to its leader, which is operably linked at the 5' end with its native first intron.

[0014] SEQ ID NO: 2 is a promoter sequence derived from a putative ubiquitin gene from the graceful butelua ( Bouteloua gracilis ).

[0015] SEQ ID NO: 3 is a leader derived from a putative ubiquitin gene from the graceful boutelua ( Bouteloua gracilis ).

[0016] SEQ ID NO: 4 is an intron sequence derived from a putative ubiquitin gene from the graceful butelua ( Bouteloua gracilis ).

[0017] SEQ ID NO: 5 is a 3' untranslated region (UTR) derived from a putative ubiquitin gene from the dainty butelua ( Bouteloua gracilis ).

[0018] SEQ ID NO: 6 is a synthetic coding sequence encoding β-glucuronidase for expression in a plant cell.

[0019] SEQ ID NO: 7 is a promoter enhancer derived from the putative Bouteloua gracilis ubiquitin gene.

[0020] SEQ ID NO: 8 is a 3' untranslated region (UTR) derived from a gene for a lipid transfer protein (LTP) like protein of Oryza sativa .

[0021] SEQ ID NO: 9 is an enhancer-enhanced promoter and leader sequence derived from cauliflower mosaic virus.

[0022] SEQ ID NO: 10 is the 3' untranslated region (UTR) derived from the nopaline synthase gene of Agrobacterium tumefaciens .

DETAILED DESCRIPTION OF THE INVENTION

[0023] The invention provides DNA molecules having gene regulatory activity in plants. The nucleotide sequences of such DNA molecules are provided as SEQ ID NOs: 1-5 or 7. These DNA molecules have the ability to influence the expression of an operably linked transcribed DNA molecule in plant tissues and therefore regulate the expression of an operably linked transgene in transgenic plants. The invention also provides methods for modifying, preparing and using such molecules. The invention also provides compositions that contain transgenic plant cells, plants, plant parts and seeds containing recombinant DNA molecules according to the present invention, as well as methods for their preparation and use.

[0024] The following definitions and methods are provided in order to better define the present invention and provide guidance to those of ordinary skill in the art in carrying out the present invention. Unless otherwise indicated, the terms are to be understood in accordance with their standard usage by those of ordinary skill in the relevant art.

DNA molecules

[0025] As used herein, the term “DNA” or “DNA molecule” refers to a double-stranded DNA molecule of genomic or synthetic origin, i.e., a polymer of deoxyribose bases or a DND molecule read from the 5' end (reverse direction) to 3' end (forward direction). As used herein, the term "DNA sequence" refers to the nucleotide sequence of a DNA molecule. The nomenclature used in this document is that of Title 37 of the United States Code of Federal Regulations 1.822 and is tabulated in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.

[0026] As used herein, the term "recombinant DNA molecule" means a DNA molecule containing a combination of DNA molecules that do not naturally occur together without human intervention. For example, a recombinant DNA molecule may be a DNA molecule that contains at least two DNA molecules that are heterologous to each other, a DNA molecule that contains a DNA sequence that is different from DNA sequences that naturally exist, or a DNA molecule that has been inserted into Host cell DNA through genetic transformation or genome editing.

[0027] Where reference is made herein to "an isolated DNA molecule" or an appropriate term or phrase, this should mean that the DNA molecule is present alone or in combination with other compositions, but in its natural environment. For example, nucleic acid elements, such as a coding sequence, an intron sequence, an untranslated leader sequence, a promoter sequence, a transcription termination sequence, and the like, that are naturally found in the DNA of an organism's genome are not considered "isolated" as long as the element is in the organism's genome. and in the part of the genome in which it occurs naturally. However, each of such elements and constituent parts of such elements will be "isolated" within the scope of this description, if the specified element is not found in the genome of the organism and in the region of the genome in which it occurs naturally. Similarly, a nucleotide sequence encoding an insecticidal protein, or any naturally occurring variant of such a protein, would be an isolated nucleotide sequence, provided that the nucleotide sequence is not in the DNA of the bacterium in which the protein-coding sequence occurs naturally. For the purposes of this specification, a synthetic nucleotide sequence encoding the amino acid sequence of a naturally occurring insecticidal protein will be considered isolated. For the purposes of this description, any transgenic nucleotide sequence, i.e. a DNA nucleotide sequence inserted into the genome of a plant or bacterial cell, or present in an extrachromosomal vector, will be considered an isolated nucleotide sequence, whether it is present in a plasmid or similar structure used for cell transformation, in the genome of a plant or bacterium, or is present in detectable amounts in tissues, progeny, biological samples or commercial products derived from a plant or bacterium.

[0028] As used herein, the term "sequence identity" refers to the degree to which two optimally aligned polynucleotide sequences or two optimally aligned polypeptide sequences are identical. Optimal sequence alignment is achieved by manually aligning two sequences, such as a reference sequence and another sequence, to maximize the number of nucleotide matches in the aligned sequence with corresponding internal nucleotide insertions, deletions, or breaks. As used herein, the term "reference sequence" refers to a DNA sequence provided as SEQ ID NO: 1-5 or 7.

[0029] As used herein, the term "percent sequence identity" or "percent identity" or "% identity" is the proportion of identity multiplied by 100. The "proportion of identity" of a sequence optimally aligned with a reference sequence is the number of nucleotide matches at optimal alignment divided by the total number of nucleotides in the reference sequence, for example , the total number of nucleotides in the full-length reference sequence. Thus, according to one embodiment of the invention, a DNA molecule is provided that contains a sequence that, when optimally aligned with the reference sequence described herein as SEQ ID NOs: 1-5 and 7, has at least about 85 percent identity, at least about 86 percent identity, at least about 87 percent identity, at least about 88 percent identity, at least about 89 percent identity, at least about 90 percent identity, at least about 91 percent identity, at least about 92 percent identity, at least 93 percent identity, at least about 94 percent identity, at least about 95 percent identity, at least about 96 percent identity, at least about 97 percent identity, at least about 98 percent identity, at least about 99 percent close identity or at least about 100 percent identity with the reference sequence. In certain embodiments, provided herein are sequences having some percent identity with any of SEQ ID NOs: 1-5 or 7 and having full-length sequence activity.

Regulatory elements

[0030] Regulatory elements such as promoters, leaders (also known as 5' UTRs), enhancers, introns, and transcription termination sites (also known as 3' UTRs) play an integral role in overall gene expression in living cells. As used herein, the term "regulatory element" refers to a DNA molecule having gene regulation activity. As used herein, the term "gene regulatory activity" refers to the ability to influence the expression of an operably linked transcribed DNA molecule, for example by influencing transcription and/or translation of an operably linked transcribed DNA molecule. Thus, regulatory elements such as promoters, leaders, enhancers, introns and 3' UTRs that function in plants are suitable for modifying plant phenotypes through genetic engineering.

[0031] As used herein, the term "expression regulatory element groups" or "EXP" sequence may refer to a group of operably related regulatory elements such as enhancers, promoters, leaders, and introns. Thus, a group of expression control elements may contain, for example, a promoter operably linked to the 5' end of the leader sequence. EXP that can be used to implement the present invention are presented as SEQ ID no: 1.

[0032] Regulatory elements can be characterized by their gene expression pattern, e.g. , positive and/or negative effects such as permanent or temporal expression, spatial, physiological, pathological, developmental, tissue, environmental, cell cycle, and /or chemically sensitive expression, and any combination thereof, as well as quantitatively or qualitatively. As used herein, the term "gene expression pattern" is any transcription pattern of an operably linked DNA molecule into a transcribed RNA molecule. The transcribed RNA molecule can be translated into a protein molecule, or an antisense or other regulatory RNA molecule can be generated, such as double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), miRNA, and the like.

[0033] As used herein, the term "protein expression" refers to any pattern of translation of a transcribed RNA molecule into a protein molecule. The expression of a protein can be described by its temporal, spatial, developmental or morphological qualities, as well as quantitative or qualitative indicators.

[0034] A promoter can be used as a regulatory element to modulate the expression of an operably linked transcribed DNA molecule. As used herein, the term "promoter" generally refers to a DNA molecule that is involved in the recognition and binding of RNA polymerase II and other proteins, such as trans-acting transcription factors, to initiate transcription. The promoter can be initially isolated from the 5'-untranslated region (5'-UTR) of the genomic copy of the gene. Alternatively, promoters may be synthetically produced or processed DNA molecules. Promoters can also be chimeric. Chimeric promoters are produced by hybridization of two or more heterologous DNA molecules. A promoter that can be used to implement the present invention includes the promoter elements contained in SEQ ID NO: 2, or fragments or variants thereof. In specific embodiments of the invention, the DNA molecules specified in the claims and any of their variants or derivatives described herein are further defined as having promoter activity, i.e. capable of acting as a promoter in a host cell, for example, in a transgenic plant. In certain other embodiments, a fragment may be defined as exhibiting the promoter activity that the original promoter molecule from which it was derived has, or the fragment may contain a "minimal promoter" that provides a basic level of transcription and consists of a TATA box, another known site motif. binding transcription factors, or an equivalent DNA sequence for recognizing and binding an RNA polymerase II complex to initiate transcription.

[0035] In one embodiment, fragments of the promoter sequence described herein are provided. Promoter fragments may have promoter activity as described above and may be used alone or in combination with other promoters and promoter fragments, such as when creating chimeric promoters, or in combination with other expression elements and expression element fragments. In certain embodiments, promoter fragments are provided comprising at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175 at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at at least about 750, at least about 800, at least about 900, or at least about 1000 or more contiguous nucleotides of a DNA molecule having promoter activity as described herein. In certain embodiments, provided herein are fragments of any of SEQ ID NOs: 1-5 and 7 having full-length sequence activity. Methods for obtaining such fragments from the original promoter molecule are well known in the art.

[0036] Compositions derived from any of the promoter elements contained in SEQ ID NO: 2, such as internal or 5' deletions, for example, can be obtained using methods known in the art to improve or alter expression, including removing elements that have a positive or negative effect on expression; duplication of elements that have a positive or negative effect on expression; and/or duplication or deletion of elements that have a tissue-specific or cell-specific effect on expression. Compositions that are derived from any of the promoter elements contained in SEQ ID NO: 2 containing 3' deletions in which the TATA box element or equivalent sequence is removed and the sequence located in the forward reading direction can be used, for example, to create enhancer elements. Additional deletions can be introduced to remove any elements that are positive or negative; tissue-specific; cell-specific; or time-specific (including but not limited to circadian rhythm) effects on expression. Any of the promoter elements contained in SEQ ID NO: 2 and fragments or enhancers derived from them can be used to create compositions of chimeric transcriptional regulatory elements.

[0037] In accordance with the present invention, a promoter or promoter fragment can be analyzed to determine the presence of known promoter elements, i.e. DNA sequence characteristics such as the TATA box and other known transcription factor binding site motifs. One of skill in the art can use the identification of such known promoter elements to design promoter variants that have a similar expression pattern to the original promoter.

[0038] As used herein, the term "leader" refers to a DNA molecule isolated from the untranslated 5' region (5'-UTR) of a gene and is generally defined as the nucleotide segment between the transcriptional start site (TSS) and the start site of the coding sequence squirrel. Alternatively, the leaders may be synthetically produced or processed DNA elements. Leaders can be used as a 5' regulatory element to modulate the expression of an operably linked transcribed DNA molecule. The leaders can be used with a heterologous promoter or with their native promoter. The leaders that can be used to implement the present invention are represented as SEQ ID NO: 3 or any of the leader elements contained in SEQ ID NO: 3 or fragments and variants thereof. In certain embodiments, such DNA sequences can be defined as having the ability to act as a leader in a host cell, including, for example, a transgenic plant cell. In one embodiment, such sequences are decoded as having leader activity.

[0039] Leader sequences (also referred to as 5' UTRs) shown as SEQ ID NO: 3, or any of the leader elements included in SEQ ID NO: 3, may contain regulatory elements or may adopt secondary structures, which may affect the transcription or translation of an operably linked transcribed DNA molecule. The leader sequence shown as SEQ ID NO: 3, or any of the leader elements included in SEQ ID NO: 3, can be used according to the invention to create chimeric regulatory elements that affect the transcription or translation of an operably linked transcribed DNA molecule.

[0040] As used herein, the term "intron" refers to a DNA molecule that can be isolated from or identified within a gene, and can be generally defined as the region that undergoes splicing during messenger RNA (mRNA) processing prior to translation. Alternatively, an intron may be a synthetically produced or processed DNA element. An intron may contain enhancer elements that affect the transcription of functionally related genes. An intron can be used as a regulatory element to modulate the expression of an operably linked transcribed DNA molecule. The construct may contain an intron, and this intron may or may not be heterologous with respect to the transcribed DNA molecule. Examples of introns in the art include the rice actin intron and the HSP70 maize intron.

[0041] In plants, the inclusion of certain introns in genetic constructs results in increased mRNA and protein accumulation compared to constructs without an intron. This effect has been termed "intron-mediated enhancement" (IME) of gene expression. Introns known to stimulate expression in plants have been identified in maize genes ( e.g. tubA1, Adh1, Sh1 and Ubi1), in rice genes ( e.g. tpi), and in dicotyledonous plant genes such as petunia genes ( e.g. rbcS), potato ( eg st-ls1), and Arabidopsis thaliana ( eg ubq3 and pat1). Deletions or mutations in intron splicing sites have been shown to reduce gene expression, suggesting that splicing may be required for IME. However, in dicotyledonous plants, IME has been demonstrated with point mutations in the splicing sites of the pat1 gene from A. thaliana . It has been shown that multiple use of the same intron in one plant has disadvantages. In such cases, it is necessary to have a collection of basic control elements in order to design suitable recombinant DNA elements.

[0042] An intron that can be used to implement the present invention is shown as SEQ ID NO:4. Intron-derived compositions set forth in SEQ ID NO:4 may contain internal deletions or duplications of cis regulatory elements. In addition, 5' and 3' sequence changes containing intron/exon splicing boundaries can be used to enhance expression or expression specificity when operably linked to a promoter+leader or chimeric promoter+leader and a coding sequence. When modifying intron/exon boundary sequences, it may be useful to avoid using an AT nucleotide sequence or an A nucleotide sequence immediately before the 5' end of the splice site (GT) and a G nucleotide or TG nucleotide sequence immediately after the 3' end of the splicing site (AG) to eliminate the possibility the appearance of unwanted start codons during the processing of messenger RNA into the final transcript. In this manner, DNA sequences near the 5' or 3' ends of splicing boundary sites can be modified. The intron and intron variants altered as described herein and corresponding methods known in the art can be tested empirically, as described in the Working Examples, to determine the effect of the intron on the expression of an operably linked DNA molecule. Alterations to the 5' and 3' regions that contain the intron/exon splicing interface can also be made to reduce the potential introduction of spurious start and stop codons generated during transcript processing and messenger RNA splicing. Introns can be tested empirically, as described in the Working Examples, to determine the effect of an intron on transgene expression.

[0043] In one embodiment, fragments of the intron sequence described herein are provided. Fragments of an intron may have the activity of a full length intron sequence and may be used alone or in combination with other introns or regulatory elements or expression element fragments. In certain embodiments, intron fragments are provided that contain at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175 at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at at least about 750, at least about 800, at least about 900, or at least about 1000 or more contiguous nucleotides of a DNA molecule having an intron activity as described herein. In certain embodiments, provided herein are fragments of any of SEQ ID NO: 4 having full-length sequence activity. Methods for obtaining such fragments from the original intron molecule are well known in the art.

[0044] As used herein, the terms "3' transcription termination molecule", "3' untranslated region" or "3'UTR" refer to a DNA molecule that is used during transcription to the untranslated region of the 3' portion of an mRNA molecule. The 3' untranslated region of an mRNA molecule can be created by specific cleavage and 3' polyadenylation, also known as a poly(A) tail. The 3'-UTR may be operably linked to and downstream of the transcribed DNA molecule and may have a polyadenylation signal and other regulatory signals capable of influencing transcription, mRNA processing, or gene expression. Poly(A) tails are believed to be involved in mRNA stability and translation initiation. Examples of art-known 3' transcription termination molecules are the nopaline synthase 3' region, the wheat hsp17 3' region, the pea ribulose 1,5-biphosphate carboxylase/oxygenase small subunit 3' region, the pea E6 3' region, and the 3' - NTO coixin.

[0045] 3'-UTRs generally find useful use for the recombinant expression of certain DNA molecules. A weak 3'-UTR has the potential to create a read-through that can affect the expression of a DNA molecule located in adjacent expression cassettes. Appropriate control of transcription termination can prevent read-through in DNA sequences ( e.g. , other expression cassettes) located in the forward direction, and can also promote efficient RNA polymerase cycle restart, thereby enhancing gene expression. Efficient transcription termination (release of RNA polymerase II from DNA) is a precondition for transcription re-initiation and thus directly affects the overall transcript level. After transcription is terminated, the mature mRNA leaves the site of synthesis and is transported into the cytoplasm. Eukaryotic mRNAs accumulate in vivo in the form of poly(A), making it difficult to determine transcription termination sites by standard methods. In addition, the prediction of functional and effective 3'-UTR using bioinformatics methods is difficult due to the lack of conserved DNA sequences that would easily predict effective 3'-UTR.

[0046] From a practical point of view, it is generally useful that the 3'-UTR used in the expression cassette has the following characteristics. First, the 3'-UTR must be able to quickly and efficiently terminate transgene transcription and prevent the transcript from being read through into any adjacent DNA sequence that may contain a different expression cassette, as is the case with multiple expression cassettes located in the same transport DNA (T- DNA), or in neighboring chromosomal DNA into which T-DNA is embedded. Secondly, the 3'-UTR should not cause a decrease in transcriptional activity due to the promoter, leader, enhancers and introns that are used to stimulate the expression of the DNA molecule. Finally, in the field of plant biotechnology, 3'-UTRs are often used to prime amplification reactions of reverse transcribed RNA isolated from a transformed plant, which is used to: (1) evaluate the transcriptional activity or expression of an expression cassette inserted into the plant's chromosome; (2) estimates of the number of copies of insertions in the DNA of the plant; and (3) assessing the zygosity of post-multiplication seeds. The 3'-UTR is also used in amplification reactions of DNA isolated from a transformed plant to characterize the retention of the inserted cassette. An example of a 3'-UTR useful in the practice of the present invention is shown as SEQ ID NO: 5.

[0047] In one embodiment, fragments of the 3'-UTR disclosed herein are provided. 3'-UTR fragments may have the activity of the full-length 3'-UTR sequence and may be used alone or in combination with other regulatory elements or expression element fragments. In certain embodiments, 3' UTR fragments are provided containing at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700 , at least about 750, at least about 800, at least about 900, or at least about 1000 or more contiguous nucleotides of a DNA molecule having 3' UTR activity as described herein. In certain embodiments, provided herein are fragments of any of SEQ ID NO: 5 having full-length sequence activity. Methods for obtaining such fragments from the parent 3' UTR molecule are well known in the art.

[0048] As used herein, the term "enhancer" or "enhancer element" refers to a cis -active regulatory element of an operably linked transcribed DNA molecule, also known as a cis -element, that provides one aspect of the overall expression pattern, but is usually insufficient alone for stimulation of transcription. Unlike promoters, enhancer elements typically do not contain a transcription start site (TSS), TATA box, or equivalent DNA sequence. A promoter or promoter fragment naturally may contain one or more enhancer elements that affect the transcription of an operably linked DNA sequence. An enhancer element can also be coupled to a promoter to produce a chimeric cis -element promoter that provides one aspect of overall modulation of gene expression. An example of an enhancer element used to implement the invention, derived from the putative Bouteloua gracilis ubiquitin gene promoter, is shown as SEQ ID NO: 7.

[0049] Many promoter enhancer elements are believed to bind DNA binding proteins and/or influence DNA topology by giving rise to local conformations that selectively allow or restrict access of the RNA polymerase to the DNA template, or promote selective opening of the double helix at the initiation site. transcription. The function of an enhancer element may be to bind transcription factors that regulate transcription. Some enhancer elements bind more than one transcription factor, and transcription factors can interact with different affinities with more than one enhancer domain. Enhancer elements can be identified by a variety of techniques, including deletion analysis, i.e. removal of one or more nucleotides from the 5' end or from the promoter side, DNA-binding protein analysis using DNase I footprinting, methylation interference, mobility change analysis at electrophoresis, in vivo genomic footprinting by ligation-mediated polymerase chain reaction (PCR) and other standard assays, or by DNA sequence similarity analysis using known cis -element motifs as the target sequence or enhancer elements as the target motif using standard methods DNA sequence comparisons such as BLAST. The fine structure of the enhancer domain can be further explored by mutagenesis (or substitution) of one or more nucleotides, or other standard techniques known in the art. Enhancer elements can be obtained by chemical synthesis or isolation from regulatory elements that contain such elements, and they can be synthesized with additional flanking nucleotides that contain useful restriction enzyme sites to facilitate subsequent work. The design, construction and use of enhancer elements to modulate the expression of operably linked transcribed DNA molecules are included within the scope of the present invention.

[0050] As used herein, the term "chimeric" refers to a single DNA molecule obtained by hybridization of a first DNA molecule with a second DNA molecule, wherein neither the first nor the second DNA molecule typically occurs in such a configuration, i. not merged with each other. Thus, a chimeric DNA molecule is a new DNA molecule that does not normally occur naturally. As used herein, the term "chimeric promoter" refers to a promoter resulting from such manipulation of DNA molecules. A chimeric promoter may combine two or more DNA fragments; for example, a promoter may be hybridized to an enhancer element. The design, construction and use of chimeric promoters to modulate the expression of operably linked transcribed DNA molecules are within the scope of the present invention.

[0051] Chimeric regulatory elements can be engineered to contain various constituent elements that can be operably linked using various methods known in the art, such as restriction enzyme digestion and ligation, ligation-free cloning, modular assembly of PCR products during amplification or direct chemical synthesis of the regulatory element, as well as other methods known in the art. The resulting different chimeric regulatory elements may have the same constituent elements or variants thereof, but differ in DNA sequence or DNA sequences that contain the binding sequence(s) or DNA sequences that allow the constituent parts to be operably linked. According to the invention, the DNA sequences provided as SEQ ID NOs: 1-5 or 7 may provide reference sequences for regulatory elements, where constituent elements that contain the reference sequence may be joined by methods known in the art and may contain substitutions, deletions and/or insertions of one or more nucleotides or mutations that naturally occur during the bacterial-plant transformation of the cell.

[0052] As used herein, the term "variant" refers to a second DNA molecule, such as a regulatory element, that is similar in composition to, but not identical to, the first DNA molecule, while the second DNA molecule still retains overall functionality, i. the same or similar expression profile, for example, as a result of greater or lesser equivalence of transcriptional activity, the first DNA molecule. The variant may be a shortened or truncated version of said first DNA molecule, or an altered version of the sequence of the first DNA molecule, eg with different restriction enzyme sites, or internal deletions, substitutions or insertions. A "variant" may also include a regulatory element having a nucleotide sequence containing a substitution, deletion, or insertion of one or more nucleotides of the reference sequence, wherein the derived regulatory element has greater or lesser or equivalent transcriptional activity than the corresponding parental regulatory molecule. "Variants" of a regulatory element also encompasses variants resulting from mutations that naturally occur during bacterial-plant transformation of a cell. According to the present invention, polynucleotide sequences provided as SEQ ID NOs: 1-5 or 7 can be used to generate variants that are similar, but not identical in composition, to the DNA sequence of the parent regulatory element, while maintaining overall functionality, i.e. the same or similar expression pattern as the original regulatory element. The preparation of such embodiments according to the invention is well known to those skilled in the art in light of the description of the present invention and is within the scope of the invention.

[0053] The effect of modifications, duplications, or deletions described herein on desirable aspects of expression of a particular transgene can be empirically investigated in plant stable and transient expression assays, such as those described in the Working Examples of this document, for confirmation of the results, which may vary depending on the changes made and the purpose of the change in the original DNA molecule.

Constructs

[0054]As used herein, the term "construct" means any recombinant DNA molecule, such as a plasmid, cosmid, virus, phage, or linear or circular DNA or RNA molecule, obtained from any source, capable of genome integration or autonomous replication, which contains a DNA molecule , wherein at least one DNA molecule is linked to another DNA molecule in a functional manner,those.functionally related. In this document, the term "vector" means any construct that can be used to transform,those. insertion of heterogeneous DNA or RNA into the host cell. The construct typically contains at least one or more expression cassettes. As used herein, an "expression cassette" refers to a DNA molecule containing at least one transcribed DNA molecule operably linked to one or more regulatory elements, typically at least a promoter and a 3'-UTR.

[0055] As used herein, the term "operably linked" refers to a first DNA molecule attached to a second DNA molecule, wherein the first and second DNA molecules are arranged such that the first DNA molecule affects the function of the second DNA molecule. The two DNA molecules may or may not be part of a single continuous DNA molecule, and may or may not be adjacent. For example, a promoter is operably linked to a transcribed DNA molecule if the promoter modulates the transcription of the transcribed DNA molecule of interest in a cell. A leader, for example, is operably linked to a DNA sequence if it has the ability to influence the transcription or translation of the DNA sequence.

[0056] The constructs of the invention can be provided, in one embodiment, as double bordered tumor-inducing (Ti) plasmid constructs that have a right border (PG or AGRtu.RB) and a left border (LG or AGRtu.LB) regions A Ti plasmid isolated from Agrobacterium tumefaciens containing T-DNA which, along with transport molecules from A. tumefaciens cells, allows the integration of T-DNA into the plant cell genome (see, for example, US Pat. No. 6,603,061). The constructs may also contain plasmid DNA backbone segments that provide replication function and antibiotic selection in bacterial cells, an Escherichia coli origin of replication such as ori322, a wide host range origin of replication such as oriV or oriRi, and a selection marker coding region, such as Spec/Strp which encodes a Tn7 selective aminoglycoside adenyl transferase ( aadA ) marker gene conferring resistance to spectinomycin or streptomycin or gentamicin (Gm, Gent). When transforming plants, the host bacterial strain is often A. tumefaciens ABI, C58, or LBA4404; however, other strains known to those skilled in the art may function according to the invention.

[0057] Methods for assembling and introducing constructs into a cell such that a transcribed DNA molecule is transcribed into a functional mRNA molecule that is translated and expressed as a protein are known in the art. Standard compositions and methods for preparing and using constructs and host cells in the practice of the invention are well known to those skilled in the art. Exemplary vectors used to express nucleic acids in higher plants are well known in the art and include vectors derived from the Agrobacterium tumefaciens Ti plasmid and the pCaMVCN transport control vector.

[0058] The construct may contain various regulatory elements, including all proposed herein. Any such regulatory elements may be provided in combination with other regulatory elements. Such combinations can be designed or modified to obtain the desired regulatory properties. In one embodiment, the constructs of the invention comprise at least one regulatory element operably linked to a transcribed DNA molecule operably linked to a 3'-UTR.

[0059] The constructs of the invention may contain any promoter or leader as provided herein or known in the art. For example, a promoter of the invention may be operably linked to a heterologous 5' untranslated leader, such as a leader derived from a heat shock protein gene. Alternatively, the leader of the invention may be operably linked to a heterologous promoter, such as the cauliflower mosaic virus 35S transcript promoter.

[0060] Expression cassettes may also contain a transit peptide coding sequence that encodes a peptide that can be used to target an operably linked protein to a subcellular unit, such as a chloroplast, leukoplast, or other plastid organelle; mitochondria; peroxisome; vacuole or extracellular space. Many chloroplast-localized proteins are expressed as precursors from nuclear genes, and the chloroplast transit protein (CTP) helps them bind to the chloroplast. Examples of such isolated chloroplast proteins include, but are not limited to, those proteins associated with the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase, ferredoxin, ferredoxin oxidoreductase, light-absorbing complex protein I and protein II, thioredoxin F, and enolpyruvylshikimate phosphate synthase (EPSPS) . Chloroplast transit proteins are described, for example, in US Pat. No. 7,193,133. It has been demonstrated that the chloroplast can be targeted by non-chloroplast proteins through the expression of a heterologous CTP operably linked to a transgene encoding non-chloroplast proteins.

transcribed DNA molecules

[0061] As used herein, the term "transcribed DNA molecule" refers to any DNA molecule that can be transcribed into an RNA molecule, including, but not limited to, those molecules that contain protein-coding sequences and those molecules on which molecules are synthesized. RNA containing sequences suitable for gene suppression. The type of DNA molecule may include, but is not limited to, a DNA molecule derived from the same plant, a DNA molecule derived from a different plant, a DNA molecule derived from another organism, or a synthetic DNA molecule such as a DNA molecule containing the antisense code of a gene. , or a DNA molecule encoding an artificial, synthetic, or otherwise modified version of a transgene. Examples of transcribed DNA molecules for insertion into the construct of the present invention include, for example , DNA molecules or genes of a species different from the species into which the DNA molecule is inserted, or genes that are present in or derived from the same species but which inserted into recipient cells using genetic engineering methods, rather than classical selection methods.

[0062] "Transgene" refers to a transcribed DNA molecule that is heterologous to a host cell, at least relative to its position in the genome of said host cell, or a transcribed DNA molecule artificially inserted into the genome of a host cell in the current or any previous generation of the cell .

[0063] A regulatory element, such as a promoter of the invention, may be operably linked to a transcribed DNA molecule that is heterologous to the regulatory element. As used herein, the term "heterologous" refers to a combination of two or more DNA molecules where such a combination is not normally found in natural conditions. For example, the two DNA molecules may be from different species, or the two DNA molecules may be from different genes, such as different genes from the same species or the same genes from different species. A regulatory element is thus heterologous to an operably linked transcribed DNA molecule if such a combination is not normally found in nature, ie, the transcribed DNA molecule is not normally operably linked to the regulatory element.

[0064] The DNA molecule to be transcribed can generally be any DNA molecule for which expression of the transcript is desired. Such expression of a transcript can result in translation of the resulting mRNA molecule and thus in protein expression. Alternatively, for example, a transcribed DNA molecule can be designed to ultimately result in a reduction in the expression of a particular gene or protein. In one embodiment, this can be achieved by using a transcribed DNA molecule that is oriented in the antisense direction. One of ordinary skill in the art is familiar with the use of such antisense technology. Any gene can be negatively regulated in this way, and in one embodiment, a transcribed DNA molecule can be designed to suppress a particular gene by expressing a dsRNA, small RNA, or microRNA molecule.

[0065] Thus, one embodiment of the invention is a recombinant DNA molecule containing a regulatory element according to the invention, such as the regulatory elements proposed as SEQ ID NO: 1-5 or 7, operably linked to a heterologous transcribed DNA molecule, in order to to modulate the transcription of the DNA molecule at the desired level or pattern when the construct is integrated into the genome of the transgenic plant cell. In one embodiment, the transcribed DNA molecule contains a protein-coding region of a gene, and in another embodiment, the transcribed DNA molecule contains an antisense region of a gene.

Genes of interest from an agronomic point of view

[0066] The DNA molecule to be transcribed may be a gene of interest from an agronomic point of view. As used herein, a "gene of agronomic interest" refers to a transcribed DNA molecule that, when expressed in a particular plant tissue, cell, or cell type, provides a desired characteristic. A gene product of agronomic interest may act within a plant to affect the morphology, physiology, growth, development, yield, grain composition, nutritional characteristics, disease or pest resistance, environmental or chemical resistance of the plant. , or may act as a pesticide when feeding on a pest that eats the plant. In one embodiment, the regulatory element of the invention is incorporated into the construct such that the regulatory element is operably linked to a transcribed DNA molecule that is a gene of interest from an agronomic point of view. In a transgenic plant containing such a construct, expression of a gene of agronomic interest can confer an agronomically beneficial property. An agronomically useful trait may include, for example, but not limited to, herbicide resistance, insect pest control, modified yield, disease resistance, pathogen resistance, modified plant growth and development, modified starch content, modified oil content. , modified fatty acid content, modified protein content, modified fruit ripening, improved animal and human nutritional value, biopolymer production, environmental stress tolerance, pharmaceutical peptides, improved processing performance, improved taste, hybrid seed technology, improved fiber production, and desired product biofuels.

[0067] Known in the art examples of genes of interest from an agronomic point of view include herbicide resistance genes ( e.g. , US Patent Nos. , increased yields ( e.g. , US Patent Nos. USRE38446, 6716474, 6663906, 6476295, 6441277, 6423828, 6399330, 6372211, 6235971, 6222098, and 5716837), pest control ( e.g. 6713063, 6686452, 6657046, 6645497, 6642030, 6639054, 6620988, 6593293, 6555655, 6538109, 6537756, 6521442, 6501009, 6468523, 6326351, 6313378, 6284949, 6281016, 6248536, 6242241, 6221649, 6177615, 6156573, 6153814, 6110464, 6093695, 6063756, 6063597, 6023013, 5959091, 5942664, 5942658 5880275, 5763245, и 5763241), устойчивости к грибковым заболеваниям ( например , Патенты США №№. 6653280, 6573361, 6506962, 6316407, 6215048, 5516671, 5773696, 6121436, 6316407 , and 6506962), virus resistance ( e.g. Pat US credit no. 6617496, 6608241, 6015940 , 6013864 , 5850023, and 5304730), nematode resistance ( e.g. US Pat. 6,723,897 and 6,518,488 ), starch products ( e.g. , US Pat. например , Патенты США №№. 6495739, 5608149, 6483008, и 6476295), содержания модифицированных жирных кислот ( например , Патенты США №№. 6828475, 6822141, 6770465, 6706950, 6660849, 6596538, 6589767, 6537750, 6489461, и 6459018) , high protein production ( e.g. US Patent No. 6380466), fruit ripening (U.S. Patent No. 5512466), increased nutritional value for animals and humans ( e.g. US Patent Nos. 6723837, 6653530, 6541259, 5985605, and 6171640), biopolymers ( e.g. , US Patent Nos. USRE37543, 6228623, and 59 58745 and 6946588), resistance to environmental stresses ( e.g. US Pat. No. 6,072,103), pharmaceutical peptides, and secreted peptides ( e.g. , US Pat. Nos. 6,812,379 ; 6,774,283 ; US Patent No. 5,543,576), improved taste ( eg , US Patent No. 6,011,199), nitrogen fixation ( eg , US Patent No. 5,229,114), hybrid seed production ( eg , US Patent No. 5,689,041), fiber production ( eg , US Patent No. 6,576,818, 6271443, 5981834, and 5869720) and biofuel products ( e.g. US Pat. No. 5,998,700).

[0068] Alternatively, a gene of interest from an agronomic point of view can influence the aforementioned plant characteristics or phenotypes by encoding an RNA molecule that causes targeted modulation of endogenous gene expression, such as by antisense ( see, for example , US Pat. 5107065); inhibitory RNA ("RNAi", including modulation of gene expression by mechanisms mediated by microRNAs, miRNAs, trans-acting miRNAs, and phased small RNAs, for example , as described in US Published Applications 2006/0200878 and 2008/0066206 and US Patent Application 11/974 469); or via cosuppression-mediated cosuppression-mediated mechanisms. The RNA can also be a catalytic RNA molecule ( e.g. ribozyme or riboswitch; see e.g. US 2006/0200878) designed to cleave the desired endogenous mRNA product. Methods for constructing and introducing constructs into a cell such that a transcribed DNA molecule is transcribed into a molecule that is capable of inducing gene suppression are known in the art.

selective markers

[0069] A transgene selectable marker can also be used with the regulatory elements of the invention. As used herein, the term "transgene selectable marker" refers to any transcribed DNA molecule whose expression or absence in a transgenic plant, tissue, or cell can be detected by screening or otherwise assessed. Selectable gene markers and related selection and screening methods used in the practice of the invention are known in the art and include, but are not limited to, transcribed DNA molecules encoding β-glucuronidase (GUS), green fluorescent protein (GFP), proteins , which confer antibiotic resistance, and proteins that confer herbicide resistance. An example of a transgene selectable marker is provided as SEQ ID NO: 6.

Cellular transformation

[0070] The present invention also relates to a method for obtaining transformed cells and plants that contain at least one or more regulatory element(s) operably linked(x) to the transcribed DNA molecule.

[0071] The term "transformation" refers to the introduction of a DNA molecule into a recipient host. As used herein, the term "host" refers to bacteria, fungi, or plants, including any cells, tissues, organs, or progeny of bacteria, fungi, or plants. Plant cells and plant tissues of particular interest include protoplasts, calli, roots, tubers, seeds, stems, leaves, seedlings, embryos, and pollen.

[0072] As used herein, the term "transformed" refers to a cell, tissue, organ, or organism into which a foreign DNA molecule, such as a construct, has been introduced. The introduced DNA molecule can be integrated into the genomic DNA of the recipient cell, tissue, organ, or organism such that the introduced DNA molecule is inherited by subsequent offspring. A "transgenic" or "transformed" cell or organism may also include the offspring of that cell or organism and the offspring resulting from breeding programs using such a transgenic organism as the parent in the crossing and exhibiting an altered phenotype resulting from the presence of a foreign DNA molecule. The introduced DNA molecule can also be temporarily integrated into the recipient cell so that the introduced DNA molecule is not inherited by subsequent offspring. The term "transgenic" refers to a bacterium, fungus or plant containing at least one or more heterologous(s) DNA molecule(s).

[0073] There are many ways of introducing DNA molecules into plant cells, well-known specialists. Typically, this process includes the steps of selecting a suitable host cell, transforming the host cell with a vector, and obtaining a transformed host cell. Methods and materials for transforming plant cells by introducing a plant construct into the plant genome in the practice of the present invention may include any of the well-known and demonstrated methods. Suitable methods include, but are not limited to, bacterial infection ( e.g. , Agrobacterium ), BAC binary vectors, direct DNA delivery ( e.g. , PEG-mediated transformation, drying/inhibition-mediated DNA uptake, electroporation, silicon carbide fiber excitation, and plated acceleration). DNA particles), gene editing ( e.g. CRISPR-Cas systems), among others.

[0074] The host cells can be any cell or organism, such as a plant cell, an algae cell, an algae, a fungal cell, a fungus, a bacterial cell, or an insect cell. In specific embodiments, host cells and transformed cells may include cells derived from cultured plants.

[0075] The transgenic plant can then be regenerated from the transgenic plant cell of the present invention. Using conventional breeding or self-pollination methods, it is possible to obtain seeds from such a transgenic plant. Such seeds and the resulting progeny plant grown from such seeds will contain the recombinant DNA molecule of the present invention and will therefore be transgenic.

[0076] The transgenic plants of the present invention may self-pollinate, providing seeds for homozygous transgenic plants of the present invention (homozygous for the recombinant DNA molecule), or may be crossed with non-transgenic plants or different transgenic plants, providing seeds for heterozygous transgenic plants, according to of the present invention (heterozygous for a recombinant DNA molecule). Both homozygous and heterozygous transgenic plants are referred to herein as "progeny plants". Progeny plants are transgenic plants derived from the original transgenic plant and containing a recombinant DNA molecule according to the invention. Seeds produced using a transgenic plant according to the invention can be harvested and used to grow generations of transgenic plants, i.e. plant progeny according to the invention, including the construct according to the present invention and the expression of a gene of interest from the point of view of agronomy. Descriptions of propagation methods that are commonly used for different crops can be found in one of several reference books: see, for example , Allard, Principles of Plant Breeding , John Wiley & Sons, NY, U. of CA, Davis, CA, 50-98 (1960); Simmonds, Principles of Crop Improvement , Longman, Inc., NY, 369-399 (1979); Sneep and Hendriksen, Plant breeding Perspectives , Wageningen (ed), Center for Agricultural Publishing and Documentation (1979); Fehr, Soybeans: Improvement, Production and Uses , 2nd edition, monograph, 16:249 (1987); Fehr, Principles of Variety Development , Theory and Technique, (Vol. 1) and Crop Species Soybean (Vol. 2), Iowa State Univ., Macmillan Pub. Co., NY, 360-376 (1987).

[0077] Transformed plants can be examined for the presence of the gene or genes of interest and the level and expression profile conferred by the regulatory elements of the present invention. Those skilled in the art are aware of the numerous methods available for examining transformed plants. For example, plant analysis methods include, but are not limited to, Southern blots or Northern blots, PCR-based assays, biochemical assays, phenotypic screening methods, field assessments, and immunodiagnostic assays. Expression of the transcribed DNA molecule can be measured using TaqMan® (Applied Biosystems, Foster City, CA), reagents and methods described by the manufacturer, and by timing PCR cycles using the TaqMan® TestingMatrix. Alternatively, Invader® (Third Wave Technologies, Madison, WI) can be used to assess transgene expression, reagents and methods are described by the manufacturer.

[0078] The present invention also provides plant parts of the invention. Plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovule, and pollen. Parts of plants according to the invention may be viable, non-viable, regenerative and non-regenerate. The invention also includes and provides transformed plant cells containing the DNA molecule of the invention. Transformed or transgenic plant cells according to the invention include regenerative and non-regenerate plant cells.

[0079] According to the invention, a commercial product is also provided, which is obtained from a transgenic plant or part thereof containing(s) a recombinant DNA molecule according to the present invention. Commercial products according to the invention contain a detectable amount of DNA containing a DNA sequence that is selected from the group consisting of SEQ ID NO: 1-5 or 7. In this document, "commercial product" refers to any composition or product, which(th) consists of material obtained from a transgenic plant, seed, plant cell or plant part containing(s) a recombinant DNA molecule according to the present invention. Marketable products include, but are not limited to, processed seeds, grains, plant parts, and meal. A commercial product according to the invention will contain a detectable amount of DNA corresponding to the recombinant DNA molecule according to the invention. Detection of one or more of these DNAs in a sample can be used to determine the content of a commercial product or its source. Any standard method for detecting DNA molecules can be used, including the detection methods described herein.

[0080] The invention can be better understood by referring to the following examples, which are given by way of illustration and are not intended to limit the scope of the present invention unless otherwise indicated. Those skilled in the art will appreciate that the techniques described in the following examples are techniques that the inventors have found to work well in carrying out the invention. However, those skilled in the art, in light of the present disclosure, will appreciate that many changes can be made to the specific embodiments described herein and still obtain similar or similar results without departing from the spirit and scope of the invention, so all the information set forth or shown here should be interpreted as illustrative and not as limiting.

EXAMPLES

Example 1

Identification and cloning of regulatory elements

[0081] New transcriptional regulatory elements and a group of expression regulatory elements (EXPs) have been identified and cloned from the genomic DNA of the monocot plants Bouteloua gracilis .

[0082] Using the identified sequences, bioinformatic analysis was performed to identify regulatory elements in the amplified DNA. For example, a bioinformatics analysis was performed to identify the transcription start site (TSS) and any bidirectional, intron or upstream coding sequence present within the sequence. Using the results of this analysis, regulatory elements were identified in the DNA sequence and primers were designed to amplify the regulatory elements. The respective DNA molecules for the regulatory elements were amplified using standard polymerase chain reaction conditions with primers containing unique restriction enzyme sites and genomic DNA isolated from Bouteloua gracilis . The resulting DNA fragments were ligated into base plant expression vectors using standard restriction enzyme digestion of a compatible restriction site and DNA ligation.

[0083] Analysis of the TSS regulatory element and intron/exon splicing boundaries can be performed using transformed plant tissue. Briefly, plants are transformed with plant expression vectors containing cloned DNA fragments operably linked to a heterologous transcribed DNA molecule. The 5'-RACE System for Rapid Amplification of cDNAEnds, Invitrogen, Carlsbad, CA 92008 is then used to confirm the TSS regulatory element and the intron/exon splicing boundary by DNA sequence analysis. from the resulting mRNA transcripts.

[0084] A DNA sequence encoding a transcriptional expression regulatory element group or an EXP sequence derived from the putative Bouteloia gracilis ubiquitin gene that contains a promoter element operably linked to an intron element is shown in Table 1, along with its respective promoter, leader, and intron. The 3'-UTR corresponding to the putative Bouteloua gracilis ubiquitin gene is also shown in Table 1.

Table 1. Transcriptional group of expression regulatory elements, promoter, leader, intron, and 3'-UTR isolated from Bouteloua gracilis

Description SEQID NO: Description and/or regulatory elements of EXP linked in 5' → 3' direction (SEQ ID NO:) EXP-BOUgr.Ubq:2 one EXP: P-BOUgr.Ubq:2 (SEQ ID NO: 2); L-BOUgr.Ubq:1 (SEQ ID NO: 3); I-BOUgr.Ubq:1 (SEQ ID NO: 4) P-BOUgr.Ubq:2 2 promoter L-BOUgr.Ubq:1 3 Leader I-BOUgr.Ubq:1 four Intron T-BOUgr.Ubq:1 5 3'-UTR

Example 2

Analysis of Regulatory Elements Stimulating GUS Expression in Stably Transformed Corn Plants

[0085] Maize plants were transformed with a vector, namely a plant expression vector, containing a regulatory element of interest that stimulates the expression of the β-glucuronidase (GUS) transgene. In the obtained plants, the GUS protein expression was analyzed to evaluate the influence of the selected regulatory elements on the expression.

[0086] Maize plants were transformed with the GUS plant expression construct. The regulatory elements were cloned into a basic plant expression vector using standard techniques known in the art. The resulting plant expression vector contained the left border region from Agrobacterium tumefaciens (left border B-AGRtu), the first transgene selective cassette used to select transformed plant cells that confers resistance to the herbicide glyphosate; a second transgenic cassette for evaluating the activity of the regulatory element, which contained EXP-BOUgr.Ubq:2 (SEQ ID NO: 1) operably linked 5' to a synthetic coding sequence designed for expression in a plant cell encoding β-glucuronidase (GUS, GOI -Ec.uidA+St.LS1.nno:1, SEQ ID NO: 6) and containing a processable intron derived from the light-induced tissue-specific potato gene ST-LS1 (Genbank Accession: X04753) operably linked 5' to a 3' termination site, T-BOUgr.Ubq:1 (SEQ ID NO: 5); and right border region of Agrobacterium tumefaciens (right border of B-AGRtu).

[0087] Maize plant cells were transformed using the binary vector construct described above using Agrobacterium -mediated transformation, as is generally known in the art. From the obtained transformed cells, the formation of a whole maize plant was stimulated.

[0088] GUS histochemical analysis was used to qualitatively and quantitatively analyze the expression of transformed plants. Whole tissue sections were incubated with GUS staining solution X-Gluc (5-bromo-4-chloro-3-indolyl-b-glucuronide) (1 mg/mL) for an appropriate length of time, washed and visually checked for blue staining. GUS activity was qualitatively determined by direct visual inspection or microscopic examination using selected plant organs and tissues.

[0089] To quantify GUS expression, total protein was extracted from selected tissues of transformed corn plants. One microgram of total protein was used with the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide (MUG) in a total reaction volume of 50 microliters. The reaction product, 4-methylumbelliferone (4-MU), exhibits a maximum level of fluorescence at high pH when the hydroxyl group is ionized. The addition of a stock sodium carbonate solution simultaneously stops the assay and adjusts the pH to quantify the fluorescence product. Fluorescence was measured with emission at 365 nm, emission at 445 nm using a Fluoromax-3 with a Micromax Reader, with the slit width set at 2 nm emission and at 3 nm emission. Values are given in units of nmol GUS/hr/mg total protein.

[0090] The following tissues were selected for GUS expression in the R ₀ generation: leaf and root stage V4; stage V7 leaves and roots; VT stage of flower/anthers, leaves and roots; R1 cob stage and filiform stigmas; stage R3 of seed embryos and endosperm 21 days after pollination (DAP). Table 2 below shows the average quantitative GUS expression for all selected tissues stimulated with a regulatory element transgenic cassette containing EXP-BOUgr.Ubq:2 (SEQ ID NO: 1) and T-BOUgr.Ubq:1 (SEQ ID NO: 5 ).

Table 2 Average Quantitative GUS Expression in Stably Transformed Maize Stimulated with Regulatory Elements Derived from Bouteloua gracilis

Stage Organ Average standard error V4 Sheet 2552.55 895.34 Root 6476.58 1383.15 V7 Sheet 3622.05 1091.84 Root 3953.61 1121.73 VT flower, anthers 9207.81 1587.9 Sheet 8795.48 1795.25 Root 4123.47 1207.1 R1 spadix, filiform stigma 5994.73 1548.68 R3 Seed, 21DAP, Em 10338.8 1592.33 Seed, 21DAP, En 11249.6 746.08

[0091] As can be seen from Table 2, the highest level of expression associated with regulatory elements was observed in seed embryos and endosperm at stages R3, 21 DAP. High expression was also observed in the VT stage of flowers, anthers, and leaves. Expression in leaves appears to have increased in leaves from stage V4 to stage V7. Expression in roots appeared to decrease from the V4 stage to the V7 stage and then remained the same from the V7 stage to the VT stage.

Example 3

Enhancer elements derived from regulatory elements

[0092] Enhancers derived from a promoter element are shown as SEQ ID NO: 2. An enhancer element may contain one or more cis regulatory elements that, when operably linked 5' or 3' to the promoter element, or operably linked 5' or 3 ' with additional enhancer elements that are operably linked to a promoter can enhance or modulate the expression levels of a transcribed DNA molecule, or allow the expression of a transcribed DNA molecule in a specific cell type or plant organ, or at a specific time point in development or circadian rhythm. Enhancers are created by removing the TATA box or functionally similar elements from the promoters and any sequence in the forward reading direction, which allows initiation of transcription from the promoter represented by SEQ ID NO: 2 or fragments thereof.

[0093] The TATA box in plant promoters is not as highly conserved as in some other eukaryotic organisms. Therefore, to identify a fragment as an enhancer, the transcription start site (TSS) of the gene must first be identified, with the 5'-UTR transcribed first. For example, the transcriptional regulatory element, EXP-BOUgr.Ubq:2 (SEQ ID NO: 1), contains a promoter element, P-BOUgr.Ubq:2 (SEQ ID NO: 2), operably linked to a 5'-UTR or leader element , L-BOUgr.Ubq:1 (SEQ ID NO: 3), which is operably linked to the intron element, I-BOUgr.Ubq:1 (SEQ ID NO: 4). Within 1095 base pairs of the promoter element, P-BOUgr.Ubq:2 (SEQ ID NO: 2), a putative core TATA-like element is found within nucleotides 1046 to 1053. An enhancer fragment derived from P-BOUgr.Ubq:2 , may contain nucleotides 1 to 1045 of SEQ ID NO: 2, giving the sequence shown as SEQ ID NO: 7 (E-BOUgr.Ubq). Enhancers derived from the promoter, P-BOUgr.Ubq:2 (SEQ ID NO: 2), may further contain smaller fragments of E-BOUgr.Ubq (SEQ ID NO: 7). The potency of enhancer elements derived from the promoter, P-BOUgr.Ubq:2, is empirically determined by constructing a chimeric transcriptional regulatory element containing an enhancer derived from the promoter, P-BOUgr.Ubq:2, which is operably linked to the promoter and leader sequence and used to stimulate the expression of a transcribed DNA molecule, such as GUS, in a stable or transient plant assay.

[0094] It may be necessary to further improve the enhancer element, empirically verified. In addition, the position of the enhancer element relative to other elements in the chimeric transcriptional regulatory element is also determined empirically, since the order of each element in the chimeric transcriptional regulatory element may have a different effect depending on the relative position of each element. Some promoter elements will contain many TATA boxes or TATA box-like elements and possibly many transcription start sites. In such circumstances, it may be necessary to first identify the location of the first TSS and then start designing enhancers using the first TSS to prevent possible transcription initiation at the putative enhancer element.

[0095] Enhancer elements derived from the promoter element shown in SEQ ID NO: 2 are cloned using methods known in the art such that they are 5' or 3' operably linked to the promoter element, or 5' or 3' with additional enhancer elements that are operably linked to the promoter. Alternatively, enhancer elements can be cloned using methods known in the art to provide a larger enhancer element that contains two or more copies of the enhancer and cloned using methods known in the art so as to be operably linked. 5' or 3' with additional enhancer elements that are operably linked to the promoter, resulting in a chimeric transcriptional regulatory element. Enhancer elements can also be cloned using methods known in the art so that they are 5' operably linked to a promoter element derived from an organism of a different genus, or 5' or 3' operably linked to additional enhancer elements derived from organisms of a different genus. , which are operably linked to a promoter derived either from an organism of the same genus or from an organism of another genus, resulting in a chimeric transcriptional regulatory element. A plant transformation GUS expression vector can be constructed using methods known in the art similar to the construct described in Example 2, wherein the resulting plant expression vectors contain the left border region from Agrobacterium tumefaciens (B-AGRtu.1 left border), the first transgenic selective cassette used to select transformed plant cells that confers resistance to the herbicide glyphosate; and a second transgenic cassette for assessing the activity of a regulatory element, containing an enhancer element operably linked 5' or 3' to a promoter element or operably linked 5' or 3' to additional enhancer elements, which in turn are operably linked to a promoter that is operably linked 5' with a leader element operably linked 5' to an intron element operably linked to a β-glucuronidase coding sequence (GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO: 6) containing a processable intron derived from a light-induced the potato tissue-specific gene ST-LS1 (Genbank Accession: X04753) operably linked 3' to a termination site from a gene for a protein similar to the lipid transfer protein Oryza sativa (T-Os.LTP:1, SEQ ID NO: 8); and a right border area from A. tumefaciens (B-AGRtu. right border). The resulting plasmids are used to transform corn plants or other plant genera using the methods described above. Alternatively, protoplast cells derived from the soybean plant or other plant genera are transformed using methods known in the art to perform expression transient assays.

[0096] GUS expression mediated by a regulatory element containing at least one enhancer is evaluated in plant expression stability or transient assays to determine the effect of the enhancer element on the expression of the transcribed DNA molecule. Modifications to one or more enhancer elements or duplication of one or more enhancer elements can be made based on empirical experimentation and the resulting regulation of gene expression that is observed with each regulatory element composition. Changing the relative position of one or more enhancers in the resulting regulatory or chimeric regulatory elements can affect the transcriptional activity or specificity of the regulatory or chimeric regulatory element, and this is determined empirically to identify the best enhancers for the desired expression profile of the transgene in a corn plant or plant of another genus.

Example 4

Analysis of intron enhancement of GUS activity using plant-derived protoplasts.

[0097] The intron is selected based on experimentation and comparison with an intron-deprived control expression vector to empirically select the intron and element configuration within the DNA transport vector (T-DNA) for optimal expression of the transgene. For example, when expressing a herbicide resistance gene, such as CP4, which provides glyphosphate tolerance, it is desirable to obtain transgene expression in reproductive tissues as well as vegetative tissues to prevent loss of yield when herbicides are applied. The intron is here selected for its ability, when operably linked to a constitutive promoter, to enhance the expression of a transgene conferring herbicide resistance, particularly in the reproductive cells and tissues of the transgenic plant, thereby providing both vegetative and reproductive tolerability of herbicide treatment in the transgenic plant. In most ubiquitin genes, the 5'-UTR contains a leader that has an intron sequence inside. Therefore, regulatory elements derived from such genes are examined using the complete 5'UTR containing the promoter, leader, and intron. To achieve different expression profiles or to modulate the expression level of a transgene, an intron from such a regulatory element can be removed or replaced with a heterologous intron.

[0098] The I-BOUgr.Ubq:1 intron, represented herein as SEQ ID NO: 4, is identified using genomic DNA contigs versus clusters of expressed cDNA tagged or contig sequences to identify exon and intron sequences in the genomic DNA. In addition, the 5'-UTR or leader is also used to define the intron/exon splicing boundary of one or more introns under conditions where the gene sequence encodes a leader sequence that is interrupted by one or more introns. The introns are cloned using methods known in the art into a plant transformation vector such that they are 3' operably linked to a regulatory element and a leader fragment and 5' operably linked to either a second leader fragment or coding sequences.

[0099] As discussed above, it may be preferable to avoid using an AT nucleotide sequence or an A nucleotide sequence immediately before the 5' end of the splice site (GT), and a G nucleotide or TG nucleotide sequence, respectively, immediately after the 3' end of the splicing site (AG) to eliminate the possibility of unwanted start codons during the processing of messenger RNA into the final transcript. Thus, it is possible to modify DNA sequences near the 5' or 3' ends of the splice boundary sites.

[00100] The enhancing effect of introns is assessed by their ability to enhance expression in a stably transformed plant assay. For a transient intron enhancement assay, a plant base vector is constructed using methods known in the art, the intron is cloned into a plant base vector similar to that described in Example 2, which has an expression cassette containing a constitutive promoter such as the cauliflower mosaic virus enhancer promoter, and a P+L-CaMV.35S-enh leader (SEQ ID NO: 9) operably linked 5' to the intron element of interest ( e.g. , SEQ ID NO: 4) operably linked to a GUS coding sequence that has a processable intron (GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO: 6) operably linked to the 3'-UTR of nopaline synthase from A. tumefaciens (T-AGRtu.nos-1:1:13, SEQ ID NO : ten); and a second expression cassette for selecting transformed plant cells. Maize plant cells are transformed using the binary vector construct described above using Agrobacterium mediated transformation as is generally known in the art. From the obtained transformed cells, the formation of a whole maize plant was stimulated. Single copy or low copy number transformants are selected for comparison with single copy or low copy number plants transformed with a plant transformation vector identical to the test vector but without the test intron to determine if the intron provides an intron-mediated enhancement effect.

[00101] The I-BOUgr.Ubq:1 intron, represented herein as SEQ ID NO: 4, can be altered in a variety of ways, such as removing fragments in the intron sequence that can reduce expression or duplication of fragments with an intron that can enhance expression. In addition, DNA sequences within the intron, which may affect the specificity of expression in certain cell types or tissues and organs, can be duplicated or altered or deleted to affect the expression and expression patterns of the transgene. In addition, the intron provided herein can be modified to remove any potential start codons (ATGs) that could give rise to unscheduled transcripts when expressed from misplanned introns, such as different, longer, or truncated proteins. Once the intron has been empirically explored or altered based on experiments, the intron is used to enhance transgene expression in stably transformed plants, which can be both monocot and dicot genera, as long as the intron provides transgene amplification. An intron can also be used to enhance expression in other organisms, such as algae, fungi, or animal cells, as long as the intron provides increased or decreased expression or specificity for the expression of the transgene to which it is operatively linked.

[00102] Based on the illustration and description of the principles of the present invention, those skilled in the art will appreciate that the present invention may be modified in structure and detail without departing from its principles. We claim all modifications, within the spirit and scope of the claims. All publications and published patent documents cited herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and separately indicated as being incorporated by reference.

--->

SEQUENCE LIST

<110> Monsanto Technology, LLC

<120> PLANT REGULATORY ELEMENTS AND THEIR APPLICATIONS

<130> MONS:419WO

<150> US 62/340,656

<151> 2016-05-24

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 2066

<212> DNA

<213> Bouteloua gracilis

<220>

<221> other_attribute

<222> (1)..(2066)

<223> The DNA sequence of a group of expression regulatory elements containing a promoter, leader, and intron operably linked and derived from the putative ubiquitin gene buteloa finesse.

<400> 1

tacgagcaaa cgcacaaccg ggatacagat ctacggtaca gaggttactt ccaacgggaa 60

ggttcccgtc cctctagcgc aagcaagacg acgcagagat ggtgaccaaa aacgattctt 120

tgctttacct ggtcatgaca ccaccggata cggataagaa ggaaaaccgg agcatctcta 180

gcaatagaga aatcgacgtt gcctcctaga ggaagaaagc gacggcctat gcttttttct 240

tttgtcgatg attccactga attgtttctc tatttttttc gttgtgtaat ggtcctggta 300

caaacgttca tccaatcatg caacttgcaa gaataaaaat aaaaaatata atctcagacg 360

tccaatcatg gcttcctaaa aaaataaaaa ggacgagaaa ctactcagaa aaaataaata 420

aatacacaac tgtaggaacc aaagcaaagt tgacaaggaa ccaaagcaag gcaggtccac 480

atgtgctgcg agcaggtgcg cctcctcctc cgtttcttcg ccttgctaat cacgtcgcta 540

aatacagagg ccgacttaag aatgccgaca tggcaatttt gctggatatt tttgcttcct 600

tgtagtatca aacagaagaa tattaacgtg ggagtataca gtaattttct tttaccattt 660

ttctttaatc gcgtttttgt tcaaccaaac acaactttag acgtactagt actccactat 720

tttttatttt attttctcct acagtacttt tgaaaaaaag gaatcttgtc atgcatgatt 780

gagatcacgg taacggcgac tcacgccgcc gcacgggtaa cggccaccaa ccaaaagtag 840

caaacggcgt caacctcctc gacatctccg cgtcgctttt ctttttctcc gcagagaatg 900

agtggcgggg agggacccca cgacgcaacc ccacgacgca accgcttatc gcagccggggt 960

ccacaccgca ccgctgccgt agcgtcatgg gactcggccc gcggcccgcc ctccgacccg 1020

gacccgcttt ccttcccctc ccgtgtataa ataccgcccc ccctgctcgc ctcctcccca 1080

atccatccga tccccaatcc agtcccgcga gcgaaatcgc cgacgaccga tcgaagcgaa 1140

gcagccttcc cccatcctct caaggtatgc gaattctcga tcctcctctc ctatgttcgt 1200

1260

tcctcagatc tatagtcggt ttgcacaggt agaggggctaa tctctagtcg atctgcgggt 1320

agagttgttg aatcatgtgc tgccttcagt tcatgtggtt tagatccgtg ttgcttgttg 1380

cggtcatgtg ttcacacaac acgtacggtt cctgccgtgc gcggattagc cgcgctgagg 1440

ctctgacgtt gtcggtgtgg gcgtgatcgc gcgggctgcc taggcttgtt attgttgaat 1500

taggcttggt cgattgggtc tgtttctatt cacggatggt cttcgatggt tcatgtgcat 1560

gctgttggtc gctggttagg tggtgatacc gttcgatttt tgtttaatct gtctagaatt 1620

ccttagatct gaaattctgt gatgtctaca tccagctgct tgttgttgaa tattttagct 1680

atgttaatct gccatggtct tcgtagtttc catgcataaa gccatgcaaa tacgttgtat 1740

atttactgtt gctgcaaccc atctcagatt ctagtttgct gtgtactatt aggccttgta 1800

aatgcttgtt aaaatggtta taaattgtgt tcatggttct gtgcctgtta agtcgtcatg 1860

tttattttct ggctgatgct atctgtgggt agaatttgca taggcttcaa tctactgact 1920

ctatgggtag gatctgtgta gcgttgcatc aaacatggca gtgtcttaat gctttcacat 1980

aaaatgtgga actgtttata ttattattat gtactgcatg ttttggtggc ctaactttgc 2040

ctctgctgct aaatttgcag gtgcag 2066

<210> 2

<211> 1095

<212> DNA

<213> Bouteloua gracilis

<220>

<221> other_attribute

<222> (1)..(1095)

<223> Promoter sequence derived from the putative ubiquitin gene buteloa finesse.

<400> 2

tacgagcaaa cgcacaaccg ggatacagat ctacggtaca gaggttactt ccaacgggaa 60

ggttcccgtc cctctagcgc aagcaagacg acgcagagat ggtgaccaaa aacgattctt 120

tgctttacct ggtcatgaca ccaccggata cggataagaa ggaaaaccgg agcatctcta 180

gcaatagaga aatcgacgtt gcctcctaga ggaagaaagc gacggcctat gcttttttct 240

tttgtcgatg attccactga attgtttctc tatttttttc gttgtgtaat ggtcctggta 300

caaacgttca tccaatcatg caacttgcaa gaataaaaat aaaaaatata atctcagacg 360

tccaatcatg gcttcctaaa aaaataaaaa ggacgagaaa ctactcagaa aaaataaata 420

aatacacaac tgtaggaacc aaagcaaagt tgacaaggaa ccaaagcaag gcaggtccac 480

atgtgctgcg agcaggtgcg cctcctcctc cgtttcttcg ccttgctaat cacgtcgcta 540

aatacagagg ccgacttaag aatgccgaca tggcaatttt gctggatatt tttgcttcct 600

tgtagtatca aacagaagaa tattaacgtg ggagtataca gtaattttct tttaccattt 660

ttctttaatc gcgtttttgt tcaaccaaac acaactttag acgtactagt actccactat 720

tttttatttt attttctcct acagtacttt tgaaaaaaag gaatcttgtc atgcatgatt 780

gagatcacgg taacggcgac tcacgccgcc gcacgggtaa cggccaccaa ccaaaagtag 840

caaacggcgt caacctcctc gacatctccg cgtcgctttt ctttttctcc gcagagaatg 900

agtggcgggg agggacccca cgacgcaacc ccacgacgca accgcttatc gcagccggggt 960

ccacaccgca ccgctgccgt agcgtcatgg gactcggccc gcggcccgcc ctccgacccg 1020

gacccgcttt ccttcccctc ccgtgtataa ataccgcccc ccctgctcgc ctcctcccca 1080

atccatccga tcccc 1095

<210> 3

<211> 69

<212> DNA

<213> Bouteloua gracilis

<220>

<221> other_attribute

<222> (1)..(69)

<223> Leader sequence derived from the putative ubiquitin gene buteloa gracilis.

<400> 3

aatccagtcc cgcgagcgaa atcgccgacg accgatcgaa gcgaagcagc cttcccccat 60

cctctcaag 69

<210> 4

<211> 902

<212> DNA

<213> Bouteloua gracilis

<220>

<221> other_attribute

<222> (1)..(902)

<223> Intron sequence derived from the putative ubiquitin gene buteloa gracilis.

<400> 4

gtatgcgaat tctcgatcct cctctcctat gttcgttgtg gatctgctgg ttaggattga 60

ttaggttgtc gtaccatgcc tttttcctgt tcgtgttcct cagatctata gtcggtttgc 120

acaggtagag ggctaatctc tagtcgatct gcgggtagag ttgttgaatc atgtgctgcc 180

ttcagttcat gtggtttaga tccgtgttgc ttgttgcggt catgtgttca cacaacacgt 240

acggttcctg ccgtgcgcgg attagccgcg ctgaggctct gacgttgtcg gtgtgggcgt 300

360

tctattcacg gatggtcttc gatggttcat gtgcatgctg ttggtcgctg gttaggtggt 420

gataccgttc gatttttgtt taatctgtct agaattcctt agatctgaaa ttctgtgatg 480

tctacatcca gctgcttgtt gttgaatatt ttagctatgt taatctgcca tggtcttcgt 540

agtttccatg cataaagcca tgcaaatacg ttgtatattt actgttgctg caacccatct 600

660

ttgtgttcat ggttctgtgc ctgttaagtc gtcatgttta ttttctggct gatgctatct 720

gtgggtagaa tttgcatagg cttcaatcta ctgactctat gggtaggatc tgtgtagcgt 780

tgcatcaaac atggcagtgt cttaatgctt tcacataaaa tgtggaactg tttatattat 840

tattatgtac tgcatgtttt ggtggcctaa ctttgcctct gctgctaaat ttgcaggtgc 900

ag902

<210> 5

<211> 498

<212> DNA

<213> Bouteloua gracilis

<220>

<221> other_attribute

<222> (1)..(498)

<223> 3' untranslated region derived from the putative ubiquitin gene buteloa gracilis.

<400> 5

gtttggtcag gagctgctct tgtctctggt ttcacaagta tggtgtctct ggtgtatggc 60

gtgtccagtc ccgttgtggt tcgactgatg tctctgtcgt gttatgagtc ctatgtcgtc 120

tggttgtccg tgtgaaacat tgctgcatgt gcagctaatt cgtgtgaaac attgctgcat 180

gtgcagctgg ttggtttatg aataagtgaa acctgaactt tgtgtgaatt ctgcttcctc 240

tatgcgctga atgttttgat tgtgatcttt tatctactca cagttgctga gatgataagg 300

ctgaatcaat gtagagctaa gattctctcg taaatgttta tattaaatac cttgtgtaat 360

tgcgagtaca aatcgacagt aattcaagct gcggttcttt gttcatccag aaggccaaga 420

ttgcaaattc aggtttcttg gcagtgaaag attgaacgca tgcaaatttc ctttgttctt 480

ttgatcacaa aattgatc 498

<210> 6

<211> 2001

<212> DNA

<213> Artificial

<220>

<223> A synthetic coding sequence encoding a beta-glucuronidase designed for expression in a plant cell.

<400> 6

atggtgaggc ccgttgagac cccgactagg gagatcaaga agctggacgg cctctgggcc 60

ttctccctcg accgtgagaa ctgcggcatc gaccagcgct ggtgggagtc cgccctccag 120

gagtctaggg ccatcgccgt gcccggttcc ttcaacgacc agttcgccga cgccgacatc 180

cgcaactacg cgggcaacgt ctggtatcag cgcgaggtgt tcatcccgaa gggctgggcg 240

ggccagcgca tcgtgctccg cttcgacgcc gtgacccact acggcaaggt ctgggtgaac 300

360

gtagtaatat aatatttcaa atattttttt caaaataaaa gaatgtagta tatagcaatt 420

gcttttctgt agtttataag tgtgtatatt ttaatttata acttttctaa tatatgacca 480

aaatttgttg atgtgcaggt gatggagcac cagggcggtt acaccccgtt cgaggccgac 540

gtgacgccgt acgtgatcgc cgggaagtcc gtccgcatca ccgtctgcgt gaacaatgag 600

ctgaactggc agaccatccc gcctggcatg gtcatcaccg acgagaacgg caagaagaag 660

cagtcctact tccacgactt cttcaactac gctggcatcc accgctccgt gatgctctac 720

accactccca aacacctgggt ggacgacatc accgtggtca cccacgtggc ccaggactgc 780

aaccacgcct ccgtggactg gcaagtcgtt gccaacggcg acgtcagcgt cgagctgcgc 840

gacgccgacc agcaagtcgt tgccaccggc cagggcacca gcggcaccct ccaagtcgtc 900

aaccctcacc tctggcagcc tggcgagggc tacctctacg agctgtgcgt caccgccaag 960

agccagactg agtgcgacat ctaccctctc cgcgtcggca tcaggagcgt cgctgtcaag 1020

ggcgagcagt tcctcatcaa ccacaagcct ttctacttca ctggtttcgg ccgccacgag 1080

1140 gacgctgacc tgaggggcaa gggtttcgac

atggactgga tcggtgccaa cagctacagg accagtcact acccgtacgc tgaggagatg 1200

1260

aacctgtctc tgggcattgg tttcgaggct gggaacaagc cgaaggagct gtactctgag 1320

gaagctgtca acggcgagac tcagcaagct catctccagg cgattaagga gctgattgcc 1380

agggacaaga accatccgtc tgtcgtgatg tggtctattg cgaatgagcc ggacaccaga 1440

ccgcaagggg cgcgtgaata cttcgcgccg ctggcggagg cgactcgcaa actggaccca 1500

acccgtccaa tcacgtgcgt caatgtcatg ttctgcgacg cccatacgga tacgatctcg 1560

gacctgttcg atgttctttg tctcaatcgg tactatgggt ggtatgttca gagcggggat 1620

1680

cagccgatca ttatcacgga gtacggggtt gacacacttg cgggccttca cagtatgtac 1740

acagatatgt ggtcggagga ataccagtgt gcatggttgg atatgtacca tcgtgtcttc 1800

1860

caagggatac tgcgggtagg agggaacaag aagggaatct tcacacggga tcggaagccc 1920

aagtcagcag ccttcctgtt gcagaagcga tggacaggaa tgaacttcgg agaaaagcca 1980

cagcaaggcg gaaagcagtg a 2001

<210> 7

<211> 1045

<212> DNA

<213> Bouteloua gracilis

<220>

<221> other_attribute

<222> (1)..(1045)

<223> Enhancer derived from the putative ubiquitin gene promoter buteloa gracilis.

<400> 7

tacgagcaaa cgcacaaccg ggatacagat ctacggtaca gaggttactt ccaacgggaa 60

ggttcccgtc cctctagcgc aagcaagacg acgcagagat ggtgaccaaa aacgattctt 120

tgctttacct ggtcatgaca ccaccggata cggataagaa ggaaaaccgg agcatctcta 180

gcaatagaga aatcgacgtt gcctcctaga ggaagaaagc gacggcctat gcttttttct 240

tttgtcgatg attccactga attgtttctc tatttttttc gttgtgtaat ggtcctggta 300

caaacgttca tccaatcatg caacttgcaa gaataaaaat aaaaaatata atctcagacg 360

tccaatcatg gcttcctaaa aaaataaaaa ggacgagaaa ctactcagaa aaaataaata 420

aatacacaac tgtaggaacc aaagcaaagt tgacaaggaa ccaaagcaag gcaggtccac 480

atgtgctgcg agcaggtgcg cctcctcctc cgtttcttcg ccttgctaat cacgtcgcta 540

aatacagagg ccgacttaag aatgccgaca tggcaatttt gctggatatt tttgcttcct 600

tgtagtatca aacagaagaa tattaacgtg ggagtataca gtaattttct tttaccattt 660

ttctttaatc gcgtttttgt tcaaccaaac acaactttag acgtactagt actccactat 720

tttttatttt attttctcct acagtacttt tgaaaaaaag gaatcttgtc atgcatgatt 780

gagatcacgg taacggcgac tcacgccgcc gcacgggtaa cggccaccaa ccaaaagtag 840

caaacggcgt caacctcctc gacatctccg cgtcgctttt ctttttctcc gcagagaatg 900

agtggcgggg agggacccca cgacgcaacc ccacgacgca accgcttatc gcagccggggt 960

ccacaccgca ccgctgccgt agcgtcatgg gactcggccc gcggcccgcc ctccgacccg 1020

gacccgcttt ccttcccctc ccgtg 1045

<210> 8

<211> 300

<212> DNA

<213> Oryza sativa

<220>

<221> other_attribute

<222> (1)..(300)

<223> 3' non-translated region derived from the rice lipid transfer protein-like protein gene.

<400> 8

attaatcgat cctccgatcc cttaattacc ataccattac accatgcatc aatatccata 60

tatatataaa ccctttcgca cgtacttata ctatgttttg tcatacatat atatgtgtcg 120

aacgatcgat ctatcactga tatgatatga ttgatccatc agcctgatct ctgtatcttg 180

ttatttgtat accgtcaaat aaaagtttct tccacttgtg ttaataatta gctactctca 240

tctcatgaac cctatatata actagtttaa tttgctgtca attgaacatg atgatcgatg 300

<210> 9

<211> 631

<212> DNA

<213> Artificial

<220>

<223> Enhancer promoter and naive leader sequence derived from cauliflower mosaic virus.

<400> 9

ggtccgatgt gagacttttc aacaaagggt aatatccgga aacctcctcg gattccattg 60

cccagctatc tgtcacttta ttgtgaagat agtggaaaag gaaggtggct cctacaaatg 120

ccatcattgc gataaaggaa aggccatcgt tgaagatgcc tctgccgaca gtggtcccaa 180

agatggaccc ccacccga ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc 240

aaagcaagtg gattgatgtg atggtccgat gtgagacttt tcaacaaagg gtaatatccg 300

gaaacctcct cggattccat tgcccagcta tctgtcactt tattgtgaag atagtggaaa 360

aggaaggtgg ctcctacaaa tgccatcatt gcgataaagg aaaggccatc gttgaagatg 420

cctctgccga cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag 480

aagacgttcc aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa 540

gggatgacgc acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat 600

ttcatttgga gaggacacgc tgaacacgct g 631

<210> 10

<211> 253

<212> DNA

<213> Agrobacterium tumefaciens

<220>

<221> other_attribute

<222> (1)..(253)

<223> 3' untranslated region derived from nopaline synase gene

Agrobacterium tumefaciens.

<400> 10

gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 60

atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 120

atgacgttat ttatgagatg ggtttttatg

gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 240

atgttactag atc 253

<---

Claims (22)

1. A recombinant DNA molecule for expressing a heterologous transcribed polynucleotide molecule that contains a DNA sequence selected from the group consisting of: a) a sequence having at least 95% sequence identity with the full length of any of SEQ ID NO: 1, 2, 4, 5 or 7, where the sequence has promoter activity; b) a sequence containing any of SEQ ID NO: 1, 2, 4, 5 or 7; and c) a fragment containing at least 150 contiguous nucleotides of any of SEQ ID NO: 1, 2, 4, 5 or 7, where the fragment has promoter activity; wherein said sequence is operably linked to a heterologous transcribed DNA molecule. 2. A recombinant DNA molecule according to claim 1, characterized in that said sequence has at least 97% sequence identity with a DNA sequence of any of SEQ ID NOs: 1, 2, 4, 5, or 7. 3. A recombinant DNA molecule according to claim 1, characterized in that said sequence has at least 99% sequence identity with a DNA sequence of any of SEQ ID NOs: 1, 2, 4, 5, or 7. 4. A recombinant DNA molecule according to claim 1, characterized in that said sequence contains a DNA sequence of any of SEQ ID NOs: 1, 2, 4, 5, or 7. 5. The recombinant DNA molecule according to claim 1, characterized in that the heterologous transcribed DNA molecule contains a gene of interest from the point of view of agronomy. 6. A recombinant DNA molecule according to claim 5, characterized in that the gene of interest from the point of view of agronomy determines the resistance of plants to herbicides. 7. A recombinant DNA molecule according to claim 5, characterized in that the gene of interest from the point of view of agronomy determines the resistance of plants to pests. 8. Transgenic plant cell for the expression of a heterologous transcribed polynucleotide molecule, which contains a recombinant DNA molecule according to any one of paragraphs. 1-7. 9. A transgenic plant cell according to claim 8, characterized in that said transgenic plant cell is a monocotyledonous plant cell. 10. A transgenic plant cell according to claim 8, characterized in that said transgenic plant cell is a dicotyledonous plant cell. 11. A transgenic plant containing a recombinant DNA molecule according to claim 1, characterized in that said transgenic plant has an increased expression of a heterologous transcribed polynucleotide molecule compared to a plant that does not contain a recombinant DNA molecule according to claim 1. 12. Part of a transgenic plant containing a recombinant DNA molecule according to claim 1, characterized in that said part of the transgenic plant has an increased expression of a heterologous transcribed polynucleotide molecule compared to a plant part that does not contain a recombinant DNA molecule according to claim 1. 13. Transgenic seed for obtaining a plant with increased expression of a heterologous transcribed polynucleotide molecule, where the seed contains a recombinant DNA molecule according to claim 1. 14. A method for expressing a transcribed DNA molecule according to claim 1, comprising expressing a transcribed DNA molecule according to claim 1 in a transgenic plant according to claim 11 and cultivating the plant. 15. A method for producing a transgenic plant cell, which includes introducing a recombinant DNA molecule according to claim 1 into a plant cell. 16. The method of claim 15, characterized in that the introduction of said recombinant DNA molecule into said plant cell comprises transformation. 17. The method of claim 16, further comprising regenerating the transgenic plant from said plant cell. 18. The method of claim 15, characterized in that the introduction of said recombinant DNA molecule into said plant cell comprises crossing the transgenic plant of claim 11 with another plant to produce plant progeny containing said plant cell.