WO2024099765A2 - Séquences nucléotidiques régulant la transcription et procédés d'utilisation - Google Patents

Séquences nucléotidiques régulant la transcription et procédés d'utilisation Download PDF

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WO2024099765A2
WO2024099765A2 PCT/EP2023/079618 EP2023079618W WO2024099765A2 WO 2024099765 A2 WO2024099765 A2 WO 2024099765A2 EP 2023079618 W EP2023079618 W EP 2023079618W WO 2024099765 A2 WO2024099765 A2 WO 2024099765A2
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plant
nucleic acid
expression
seed
promoter
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PCT/EP2023/079618
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WO2024099765A3 (fr
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Shirong Zhang
Peter Denolf
Marie-Laure SAUER
Soundarya SRIRANGAN
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BASF Agricultural Solutions Seed US LLC
Basf Se
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Publication of WO2024099765A3 publication Critical patent/WO2024099765A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells

Definitions

  • Modification of plants to alter and/or improve phenotypic characteristics requires the overexpression or down-regulation of endogenous genes or the expression of heterologous genes in plant tissues.
  • Such genetic modification relies on the availability of a means to drive and to control gene expression as required.
  • genetic modification relies on the availability and use of suitable promoters which are effective in plants and which regulate gene expression so as to give the desired effect(s) in the plant.
  • tissue-specific or tissue-preferential expression profiles can be advantageous, at least in part because beneficial effects of expression in one tissue may have disadvantages in others.
  • Constitutive promoters can be useful for regulating expression of genes, e.g., expressing or downregulating genes, in different tissues throughout the plant to achieve a desired function or effect.
  • an isolated nucleic acid having constitutive promoter activity selected from the group consisting of: a) a nucleic acid comprising a nucleotide sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 12 or a functional fragment thereof; and b) a nucleic acid comprising a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 1 to SEQ ID NO: 12, or a functional fragment thereof.
  • a recombinant nucleotide for regulating expression of a polynucleotide of interest comprising the isolated nucleic acid having constitutive promoter activity selected from the group consisting of: a) a nucleic acid comprising a nucleotide sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 12 or a functional fragment thereof; and b) a nucleic acid comprising a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 1 to SEQ ID NO: 12, or a functional fragment thereof.
  • the recombinant nucleotide can optionally include one or more of the following features.
  • the nucleic acid having constitutive promoter activity can be operably linked to a heterologous nucleic acid sequence.
  • the recombinant nucleotide can further comprise a transcription termination and polyadenylation sequence, optionally wherein the transcription termination and polyadenylation region is functional in plants.
  • the heterologous nucleic acid sequence can encode an expression product of interest.
  • the expression product of interest can be an RNA molecule capable of modulating the expression of a gene or is a protein.
  • the heterologous nucleic acid sequence can be an enhancer DNA.
  • the polynucleotide of interest can encode an herbicide selectable marker, an insecticidal protein, antibiotic resistance, herbicide resistance, insect resistance protein, disease resistance, herbicide tolerance.
  • an expression cassette for regulating constitutive expression of a polynucleotide of interest comprising the isolated nucleic acid having constitutive promoter activity selected from the group consisting of: a) a nucleic acid comprising a nucleotide sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 12 or a functional fragment thereof; and b) a nucleic acid comprising a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 1 to SEQ ID NO: 12, or a functional fragment thereof.
  • an expression cassette for regulating constitutive expression of a polynucleotide of interest comprising the recombinant nucleotide for regulating expression of a polynucleotide of interest, comprising the isolated nucleic acid having constitutive promoter activity selected from the group consisting of: a) a nucleic acid comprising a nucleotide sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 12 or a functional fragment thereof; and b) a nucleic acid comprising a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 1 to SEQ ID NO: 12, or a functional fragment thereof.
  • a vector comprising any one of the expression cassettes described above.
  • the vector can be an expression vector.
  • a host cell comprising the isolated nucleic acid having constitutive promoter activity selected from the group consisting of: a) a nucleic acid comprising a nucleotide sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 12 or a functional fragment thereof; and b) a nucleic acid comprising a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 1 to SEQ ID NO: 12, or a functional fragment thereof.
  • a host cell comprising the recombinant nucleotide for regulating expression of a polynucleotide of interest, comprising the isolated nucleic acid having constitutive promoter activity selected from the group consisting of: a) a nucleic acid comprising a nucleotide sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 12 or a functional fragment thereof; and b) a nucleic acid comprising a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 1 to SEQ ID NO: 12, or a functional fragment thereof.
  • a host cell comprising any one of the expression cassettes or vectors described above.
  • the host cell can be an E. coli cell, an Agrobacterium cell, an algal cell, a yeast cell, or a plant cell.
  • a planttissue, plant organ, plant part, plant, or seed comprising the isolated nucleic acid having constitutive promoter activity selected from the group consisting of: a) a nucleic acid comprising a nucleotide sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 12 or a functional fragment thereof; and b) a nucleic acid comprising a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 1 to SEQ ID NO: 12, or a functional fragment thereof.
  • a planttissue, plant organ, plant part, plant, or seed comprising the recombinant nucleotide for regulating expression of a polynucleotide of interest, comprising the isolated nucleic acid having constitutive promoter activity selected from the group consisting of: a) a nucleic acid comprising a nucleotide sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 12 or a functional fragment thereof; and b) a nucleic acid comprising a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 1 to SEQ ID NO: 12, or a functional fragment thereof.
  • a planttissue, plant organ, plant part, plant, or seed comprising any one of the expression cassettes or vectors described above.
  • the plant tissue, plant organ, plant part, plant, or seed can be a dicotyledonous plant tissue, plant organ, plant part, plant, or seed.
  • the plant tissue, plant organ, plant part, plant, or seed can be a soybean plant tissue, plant organ, plant part, plant, or seed or oilseed rape plant tissue, plant organ, plant part, plant, or seed.
  • a method for producing a plant tissue, plant organ, plant part, plant, or seed, the method comprising: a) introducing or providing the nucleic acid having constitutive promoter activity of claim 1 , the recombinant nucleic acid of any one of claims 2 to claim 8, the expression cassette of claim 9, or the vector of any one of claims 10 to 11 to a plant cell to create a modified plant cell; and b) regenerating the modified plant cell to form a plant tissue, plant organ, plant part, plant, or seed.
  • the method can optionally further comprise selecting said plant cell to form a plant tissue, plant organ, plant or seed for the presence of the isolated nucleic acids described above, the recombinant nucleotides described above, the expression cassettes described above, or the vectors described above.
  • a method for expressing a polynucleotide of interest in a host cell comprising: (a) introducing any of the recombinant nucleotides described above, any of the expression cassettes described above, or any of the vectors described above into the host cell, and (b) expressing at least one polynucleotide of interest in said host cell.
  • the host cell can be a plant cell.
  • a method for effecting constitutive expression of a nucleic acid comprising introducing any of the isolate nucleic acids having constitutive promoter activity described above, or any of the recombinant nucleotides described above, into the genome of a plant.
  • nucleic acid having constitutive promoter activity selected from the group consisting of: a) a nucleic acid comprising a nucleotide sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 12 or a functional fragment thereof; and b) a nucleic acid comprising a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 1 to SEQ ID NO: 12, or a functional fragment thereof, to regulate expression of an operably linked nucleic acid sequence in a plant.
  • nucleic acid having constitutive promoter activity selected from the group consisting of: a) a nucleic acid comprising a nucleotide sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 12 or a functional fragment thereof; and b) a nucleic acid comprising a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 1 to SEQ ID NO: 12, or a functional fragment thereof, to identify other nucleic acids having constitutive promoter activity.
  • the plant can optionally be a dicotyledonous plant. In some embodiments or the methods or uses described above, the plant can optionally be a soybean plant or an oilseed rape plant.
  • a method for producing food, feed, or an industrial product comprising: a) obtaining the plant tissue, plant organ, plant part, plant, or seed comprising the recombinant nucleotide for regulating expression of a polynucleotide of interest, comprising the isolated nucleic acid having constitutive promoter activity selected from the group consisting of a nucleic acid comprising a nucleotide sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 12 or a functional fragment thereof; and a nucleic acid comprising a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 1 to SEQ ID NO: 12, or a functional fragment thereof; or obtaining the plant tissue, plant organ, plant part, plant, or seed comprising any one of the expression cassettes or vectors described above; and b) preparing the food, feed or industrial product from the plant tissue, plant organ, plant part, plant, or seed.
  • the method comprising: a) obtaining the plant tissue, plant organ
  • FIG. 1 is a graph showing expression data (RNA-seq) for the native soybean gene Glyma02g43790 from which Promoter 7 (SEQ ID NO: 1) is derived, as described in Examples 1 and 9.
  • FIG. 2 is a graph showing expression data (RNA-seq) for the native soybean gene Glyma04g00500 from which Promoter 8 (SEQ ID NO: 2) is derived, as described in Examples 2 and 9.
  • FIG. 3 is a graph showing expression data (RNA-seq) for the native soybean gene Glyma06g09420 from which Promoter 9 (SEQ ID NO: 3) is derived, as described in Examples 3 and 9.
  • FIG. 4 is a graph showing expression data (RNA-seq) for the native soybean gene Glyma08g23830 from which Promoter 11 (SEQ ID NO: 5) is derived, as described in Examples 4 and 9.
  • FIG. 5 is a graph showing expression data (RNA-seq) for the native soybean gene Glymal 2g03470 from which Promoter 12 (SEQ ID NO: 6) is derived, as described in Examples 5 and 9.
  • FIG. 6 is a graph showing expression data (RNA-seq) for the native soybean gene Glymal 3g 18830 from which Promoter 13 (SEQ ID NO: 7) is derived, as described in Examples 6 and 9.
  • FIG. 7 is a graph showing expression data (RNA-seq) for the native soybean gene Glymal 9g37000 from which Promoter 15 (SEQ ID NO: 8) is derived, as described in Examples 7 and 9.
  • FIG. 8 is a graph showing expression data (RNA-seq) for the native Brassica napus gene from which Promoters 16 and 17 (SEQ ID NO: 9 and 10) are derived, as described in Example 10.
  • FIG. 9 is a graph showing expression data (RNA-seq) for the native Brassica napus gene from which Promoter 18 (SEQ ID NO: 11) is derived, as described in Example 10.
  • FIG. 10 is a graph showing expression data (RNA-seq) for the native Brassica napus gene from which Promoter 19 (SEQ ID NO: 12) is derived, as described in Example 10.
  • the present invention is based on the discovery that SEQ ID NO: 1 through SEQ ID NO: 12 have constitutive promoter activity in plants.
  • nucleic acids having constitutive promoter activity selected from the group consisting of a) a nucleic acid includes a nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 12 or a functional fragment thereof, and b) a nucleic acid includes a nucleotide sequence having at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 96%, 97%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or more sequence identity to any one of SEQ ID NO: 1 to SEQ ID NO: 12, or a functional fragment thereof.
  • nucleic acids having constitutive promoter activity described herein can, in some embodiments, also be comprised in a larger DNA molecule.
  • DNA refers to a physical structure comprising an orderly arrangement of nucleotides, e.g., deoxyribonucleotides or ribonucleotides.
  • the DNA sequence or nucleotide sequence can be contained within a larger nucleotide molecule, vector, or the like.
  • orderly arrangement of nucleic acids in these sequences can be depicted in the form of, e.g., a sequence listing, figure, table, electronic medium, or the like.
  • Isolated nucleic acid used interchangeably with “isolated DNA” as used herein refers to a nucleic acid not occurring in its natural genomic context, irrespective of its length and sequence.
  • Isolated DNA can, for example, refer to DNA which is physically separated from the genomic context, such as a fragment of genomic DNA.
  • Isolated DNA can also be an artificially produced DNA, such as a chemically synthesized DNA, or such as DNA produced via amplification reactions, such as polymerase chain reaction (PCR) well- known in the art.
  • Isolated DNA can further refer to DNA present in a context of DNA in which it does not occur naturally.
  • isolated DNA can refer to a piece of DNA present in a plasmid or in a transgenic host cell.
  • the isolated DNA can refer to a piece of DNA present in a chromosomal context other than the context in which it occurs naturally, such as, for example, at a position in the genome other than the natural position, in the genome of a species other than the species in which it occurs naturally, or in an artificial chromosome.
  • transcription regulating nucleotide sequence refers to a nucleotide sequences that influences the transcription, RNA processing or stability, or translation of the associated (or functionally linked) nucleotide sequence to be transcribed.
  • the transcription regulating nucleotide sequence can have various localizations with the respect to the nucleotide sequences to be transcribed.
  • the transcription regulating nucleotide sequence can be located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of the sequence to be transcribed (e.g., a coding sequence).
  • the transcription regulating nucleotide sequences can be selected from the group comprising enhancers, promoters, translation leader sequences, introns, 5’-untranslated sequences, 3’-untranslated sequences, and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences that are a combination of synthetic and natural sequences.
  • transcription regulating nucleotide sequence is not limited to promoters. However, preferably a transcription regulating nucleotide sequence of the invention described herein comprises at least one promoter sequence (e.g., a sequence localized upstream of the transcription start of a gene capable to induce transcription of the downstream sequences).
  • a transcription regulating nucleotide sequence described herein comprises the promoter sequence of the corresponding gene and - optionally and preferably - the native 5’- untranslated region of said gene.
  • the 3’-untranslated region and/or the polyadenylation region of said gene can also be employed.
  • promoter means a region of a DNA sequence that is essential for the initiation of transcription of DNA, resulting in the generation of an RNA molecule that is complementary to the transcribed DNA; this region may also be referred to as a "5' regulatory region.” Promoters are usually located upstream of the coding sequence to be transcribed and can have regions that act as binding sites for RNA polymerase II and other proteins such as transcription factors (trans-acting protein factors that regulate transcription) to initiate transcription of an operably linked gene. In some instances, promoters themselves can contain subelements (i.e. promoter motifs) such as cis-elements or enhancer domains that regulate the transcription of operably linked genes.
  • Promoters can be derived in their entirety from a native gene, or be composed of different elements, derived from different promoters found in nature, or even be comprised of synthetic DNA segments.
  • the promoters described herein can, in some embodiments, be altered to contain "enhancer DNA” to assist in elevating gene expression.
  • certain DNA elements can be used to enhance the transcription of DNA. These enhancers often are found 5' to the start of transcription in a promoter that functions in eukaryotic cells but can often be inserted upstream (5') or downstream (3') to the coding sequence. In some instances, these 5' enhancer DNA elements are introns.
  • a promoter or promoter region can, in some embodiments, include variations of promoters derived by inserting or deleting regulatory regions, subjecting the promoter to random or site-directed mutagenesis, etc.
  • the activity or strength of a promoter may be measured in terms of the amounts of RNA it produces, or the amount of protein accumulation in a cell or tissue, relative to a promoter whose transcriptional activity has been previously assessed or relative to a promoter driving the expression of a housekeeping gene.
  • a promoter as used herein can thus, in some embodiments, include sequences downstream of the transcription start, such as sequences coding the 5’ untranslated region (5’ UTR) of the RNA, introns located downstream of the transcription start, or even sequences encoding the protein.
  • Promoter activity for a functional promoter fragment, for an equivalent promoter or for a promoter comprising insertion, deletion, or substitution in its sequence can be determined by those skilled in the art, for example using analysis of RNA accumulation produced from the nucleic acid which is operably linked to the promoter as described herein, whereby the nucleic acid which is operably linked to the promoter can be the nucleic acid which is naturally linked to the promoter, i.e. the endogenous gene of which expression is driven by the promoter.
  • constitutive promoter refers to a promoter that is able to express the open reading frame (ORF) in all or nearly all of the plant tissues, such as, for example, apical meristem, flower buds, cotyledons, flowers, pods, roots, seeds, stems, leaves, during all or nearly all developmental stages of the plant.
  • ORF open reading frame
  • a constitutive promoter can, in some instances have activity in all tissues.
  • a constitutive promoter can have activity in most tissues, such as, e.g., flower buds, cotyledons, flowers, pods, seeds, stems, and leaves, but limited or no activity in another tissue, such as, e.g., roots.
  • Each of the transcription-activating elements do not exhibit an absolute tissue-specificity, but mediate transcriptional activation in most plant tissues at a level of at least 1 % reached in the plant tissue in which transcription is most active.
  • Constutive expression refers to expression using a constitutive promoter.
  • the constitutive expression capacity of the identified or generated fragments, equivalents or sequences comprising insertion, deletion, substitution of the promoters described herein can be conveniently tested by determining levels of the transcript of which expression is naturally driven by the respective promoter described herein, i.e. endogenous transcript levels, such as, for example, using the methods as described herein in the Example.
  • constitutive expression capacity of the identified or generated fragments of the promoters described herein can be conveniently tested by operably linking such DNA molecules to a nucleotide sequence encoding an easily scorable marker, e.g. a beta-glucuronidase gene, introducing such a chimeric gene into a plant and analyzing the expression pattern of the marker in different tissues or organs of the plant as compared with the expression pattern of the marker in other tissues or organs of the plant.
  • an easily scorable marker e.g. a beta-glucuronidase gene
  • a marker or a reporter gene
  • chloramphenicol acetyl transferase CAT
  • proteins with fluorescent properties such as green fluorescent protein (GFP) from Aequora victoria, or proteins with luminescent properties such as the Renilla luciferase or the bacterial lux operon.
  • GFP green fluorescent protein
  • a DNA segment representing the promoter region is removed from the 5’ region of the gene of interest and operably linked to the coding sequence of a marker (reporter) gene by recombinant DNA techniques well known to the art.
  • the reporter gene is operably linked downstream of the promoter, so that transcripts initiating at the promoter proceed through the reporter gene.
  • Reporter genes generally encode proteins, which are easily measured, including, but not limited to, chloramphenicol acetyl transferase (CAT), beta-glucuronidase (GUS), green fluorescent protein (GFP), beta-galactosidase (beta- GAL), and luciferase.
  • CAT chloramphenicol acetyl transferase
  • GUS beta-glucuronidase
  • GFP green fluorescent protein
  • beta-galactosidase beta-galactosidase
  • luciferase The expression cassette containing the reporter gene under the control of the promoter can be introduced into an appropriate cell type by transfection techniques well known to the art.
  • To assay for the reporter protein cell lysates can be prepared and appropriate assays, which are well known in the art, for the reporter protein can be performed.
  • CAT were the reporter gene of choice
  • the lysates from cells transfected with constructs containing CAT under the control of a promoter under study are mixed with isotopically labeled chloramphenicol and acetyl-coenzyme A (acetyl-CoA).
  • the CAT enzyme transfers the acetyl group from acetyl-CoA to the 2- or 3-position of chloramphenicol.
  • the reaction is monitored by thin-layer chromatography, which separates acetylated chloramphenicol from unreacted material.
  • the reaction products are then visualized by autoradiography.
  • the level of enzyme activity corresponds to the amount of enzyme that was made, which in turn reveals the level of expression and the constitutive functionality from the promoter or promoter fragment of interest. This level of expression can also be compared to other promoters to determine the relative strength of the promoter under study.
  • additional mutational and/or deletion analyses can be employed to determine the minimal region and/or sequences required to initiate transcription. Thus, sequences can be deleted at the 5’ end of the promoter region and/or at the 3’ end of the promoter region, and nucleotide substitutions introduced. These constructs are then again introduced in cells and their activity and/or functionality determined.
  • the activity or strength of a promoter can be measured in terms of the amount of mRNA or protein accumulation it specifically produces, relative to the total amount of mRNA or protein.
  • the promoter can express an operably linked nucleic acid sequence at a level greater than about 0.01 %, about 0.02%, or greater than about 0.05% of the total mRNA.
  • the activity or strength of a promoter can be expressed relative to a well-characterized promoter (for which transcriptional activity was previously assessed).
  • equivalent constitutive promoters can be isolated from other plants.
  • equivalent promoters can be isolated using the coding sequences of the genes driven by the promoters of any one of SEQ ID NO: 1 to SEQ ID NO: 12 or a functional fragment having at least 300 consecutive nucleotides thereof as a probe and identifying nucleotide sequences from these other plants which hybridize under the hybridization conditions described herein.
  • a promoter described herein can be used to screen a genomic library of a crop or plant of interest to isolate corresponding promoter sequences according to techniques well known in the art.
  • a promoter sequence described herein can be used as a probe for hybridization with a genomic library under medium to high stringency conditions.
  • equivalent promoters can be isolated using the coding sequences of the genes driven by the promoters of any one of SEQ ID NO: 1 to SEQ ID NO: 12 to screen a genomic library (e.g. by hybridization or in silico) of a crop of interest. When sufficient identity between the coding sequences is obtained (for example, higher than 80% identity) then promoter regions can be isolated upstream of the orthologous genes.
  • nucleic acids comprising constitutive promoter activity which comprise a nucleotide sequence having at least 40%, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity to the herein described promoters and promoter regions or functional fragments thereof and are also referred to as variants.
  • variants with respect to the transcription regulating nucleotide sequences of any one of SEQ ID NO: 1 to SEQ ID NO: 12 as described herein is intended to mean substantially similar sequences.
  • variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) as herein outlined before.
  • Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis of any one of SEQ ID NO: 1 to SEQ ID NO: 12.
  • nucleotide sequence variants of the invention will have at least 40%, 50%, 60%, to 70%, e.g., preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81% to 84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99% nucleotide sequence identity to the native (wild type or endogenous) nucleotide sequence or a functional fragment thereof.
  • Derivatives of the DNA molecules described herein may include, but are not limited to, deletions of sequence, single or multiple point mutations, alterations at a particular restriction enzyme site, addition of functional elements, or other means of molecular modification which may enhance, or otherwise alter promoter expression. Techniques for obtaining such derivatives are well-known in the art (see, for example, J. F. Sambrook, D. W. Russell, and N. Irwin (2000) Molecular Cloning: A Laboratory Manual, 3rd edition Volumes 1 , 2, and 3. Cold Spring Harbor Laboratory Press). For example, one of ordinary skill in the art may delimit the functional elements within the promoters disclosed herein and delete any non-essential elements.
  • Functional elements may be modified or combined to increase the utility or expression of the sequences of the invention for any particular application.
  • Those of skill in the art are familiar with the standard resource materials that describe specific conditions and procedures for the construction, manipulation, and isolation of macromolecules (e.g., DNA molecules, plasmids, etc.), as well as the generation of recombinant organisms and the screening and isolation of DNA molecules.
  • a nucleic acid comprising a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 1 to SEQ ID NO: 12 can thus be a nucleic acid comprising a nucleotide sequence having at least at least 80%, or at least 85% , or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to any one of SEQ ID NO: 1 to SEQ ID NO: 12.
  • Sequence identity usually is provided as “% sequence identity” or “% identity”.
  • % sequence identity or “% identity”.
  • a pairwise sequence alignment is generated between those two sequences, wherein the two sequences are aligned over their complete length (i.e., a pairwise global alignment).
  • EMBOSS European Molecular Biology Open Software Suite
  • Seq B GATCTGA length : 7 bases
  • sequence B is sequence B.
  • the symbol in the alignment indicates gaps.
  • the number of gaps introduced by alignment within the Seq B is 1 .
  • the number of gaps introduced by alignment at borders of Seq B is 2, and at borders of Seq A is 1.
  • the alignment length showing the aligned sequences over their complete length is 10.
  • the alignment length showing the shorter sequence over its complete length is 8 (one gap is present which is factored in the alignment length of the shorter sequence). Accordingly, the alignment length showing Seq A over its complete length would be 9 (meaning Seq A is the sequence of the invention). Accordingly, the alignment length showing Seq B over its complete length would be 8 (meaning Seq B is the sequence of the invention).
  • an identity value is determined from the alignment produced.
  • sequence identity in relation to comparison of two nucleic acid sequences according to this embodiment is calculated by dividing the number of identical residues by the length of the alignment region which is showing the respective sequence of this invention over its complete length. This value is multiplied with 100 to give “%-identity”.
  • an isolated nucleic acid described herein, having constitutive promoter activity can include a functional fragment of a nucleotide sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 12, or a functional fragment of a nucleic acid comprising a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 1 to SEQ ID NO: 12.
  • a “functional fragment” of a nucleic acid comprising constitutive promoter denotes a nucleic acid comprising a stretch of the nucleic acid sequences of any one of SEQ ID NO: 1 to SEQ ID NO: 12, or of the nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to any one of SEQ ID NO: 1 to SEQ ID NO: 12 which still exerts the desired function, i.e. which has constitutive promoter activity.
  • a functional fragment can be a nucleic acid sequence that is shorter in length than the transcription regulating nucleotide sequence yet retains the activity of the transcription regulating nucleotide sequence.
  • the functional fragment of the constitutive promoter contains the conserved promoter motifs, such as, for example, conserved promoter motifs as described in DoOP (doop.abc.hu, databases of Orthologous Promoters, Barta E. et al (2005) Nucleic Acids Research Vol. 33, D86-D90).
  • conserved promoter motifs such as, for example, conserved promoter motifs as described in DoOP (doop.abc.hu, databases of Orthologous Promoters, Barta E. et al (2005) Nucleic Acids Research Vol. 33, D86-D90).
  • a functional fragment can be a fragment of at least about 500 bp, at least about 540 bp, at least about 544 bp, at least about 550 bp, at least about 600 bp, at least about 650 bp, at least about 700 bp, at least about 750 bp, at least about 800 bp, at least about 850 bp, at least about 900 bp, or at least about 1000 bp in length, or a fragment of at least about 500 bp, least 540 bp, at least 544, at least 550 bp, at least 600 bp, at least 650 bp, at least 700 bp, at least 750 bp, at least 800 bp, at least 850 bp, at least 900 bp, or at least 1000 bp from the translation start site, which retains the activity of the transcription regulating nucleotide sequence, e.g., having constitutive promoter activity.
  • a functional fragment can include a nucleic acid comprising the nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 12 which further comprises insertion, deletion, substitution of at least 1 nucleotide up to 20 nucleotides, at least 1 nucleotide up to 15 nucleotides, at least 1 nucleotide up to 10 nucleotides, at least 1 nucleotide up to 5 nucleotides, at least 1 nucleotide up to 4 nucleotides, at least 1 nucleotide up to 3 nucleotides, or even at least 1 nucleotide up to 2 nucleotides and is a fragment of at least about 500 bp, at least about 540 bp, at least about 544 bp, at least about 550 bp, at least about 600 bp, at least about 700 bp, at least about 800 bp, at least about 900 bp, or at least 1000
  • a recombinant nucleotide comprising the nucleic acid having constitutive promoter activity described herein.
  • the nucleic acid having constitutive promoter activity is operably linked to a heterologous nucleic acid sequence.
  • the recombinant nucleotide can further comprise a transcription termination and polyadenylation sequence.
  • the transcription termination and polyadenylation sequence can be a transcription termination and polyadenylation region functional in plants or plant cells.
  • the heterologous nucleic acid sequence can encode an expression product of interest, which can, for example, be an RNA molecule capable of modulating the expression of a gene or a protein.
  • the heterologous nucleic acid sequence can also, in some embodiments, encode an enhancer DNA.
  • recombinant nucleotide refers to any nucleotide that contains: a) DNA sequences, including regulatory and coding sequences that are not found together in nature, or b) sequences encoding parts of proteins not naturally adjoined, or c) parts of promoters that are not naturally adjoined. Accordingly, a recombinant nucleotide can comprise regulatory sequences and coding sequences that are derived from different sources, or comprise regulatory sequences, and coding sequences derived from the same source, but arranged in a manner different from that found in nature.
  • operably linked refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences.
  • a promoter region can be positioned relative to a nucleic acid sequence such that transcription of a nucleic acid sequence is directed by the promoter region.
  • a promoter region is “operably linked” to the nucleic acid sequence. “Functionally linked” is an equivalent term.
  • heterologous refers to the relationship between two or more nucleic acid or protein sequences that are derived from different sources.
  • a promoter is heterologous with respect to an operably linked DNA region, such as a coding sequence if such a combination is not normally found in nature.
  • a particular sequence can be “heterologous” with respect to a cell or organism into which it is inserted (i.e. does not naturally occur in that particular cell or organism or at that position in that particular cell or organism).
  • nucleic acid molecule or DNA refers to a nucleic acid molecule which is operably linked to, or is manipulated to become operably linked to, a second nucleic acid molecule to which it is not operably linked in nature, or to which it is operably linked at a different location in nature.
  • a promoter of the invention in its natural environment is functionally linked to its native nucleic acid sequence encoding an expression product, whereas in some embodiments described herein, it can be linked to another nucleic acid sequence encoding an expression product which might be derived from the same organism or from a different organism.
  • nucleic acid sequence encoding an expression product functionally linked to a promoter of the invention is heterologous to the promoter as its sequence has been manipulated by, for example, mutation such as insertions, deletions and the like so that the natural sequence of the nucleic acid molecule under control of the promoter is modified and therefore has become heterologous to a promoter of the invention.
  • a “transcription termination and polyadenylation region” as used herein is a sequence that drives the cleavage of the nascent RNA, whereafter a poly(A) tail is added at the resulting RNA 3’ end, functional in plant cells.
  • Transcription termination and polyadenylation signals functional in plant cells include, but are not limited to, 3’nos, 3’35S, 3’his and 3’g7.
  • the term "expression product” refers to a product of transcription, the expression product can be the transcribed RNA. It is understood that the RNA which is produced is a biologically active RNA.
  • the expression product can also be a peptide, a polypeptide, ora protein, when the biologically active RNA is an mRNA and the protein is produced by translation of the mRNA.
  • the heterologous nucleic acid, operably linked to the promoters of the invention can, in some embodiments, also code for an RNA molecule capable of modulating the expression of a gene.
  • the RNA molecule capable of modulating the expression of a gene can be an RNA which reduces expression of a gene.
  • the RNA can reduce the expression of a gene for example through the mechanism of RNA-mediated gene silencing.
  • the RNA molecule capable of modulating the expression of a gene can be a silencing RNA down-regulating expression of a target gene.
  • silencing RNA or “silencing RNA molecule” refers to any RNA molecule, which upon introduction into a plant cell, reduces the expression of a target gene.
  • silencing RNA can e.g. be so-called “antisense RNA”, whereby the RNA molecule comprises a sequence of at least 20 consecutive nucleotides having 95% sequence identity to the complement of the sequence of the target nucleic acid, preferably the coding sequence of the target gene.
  • antisense RNA can also be directed to regulatory sequences of target genes, including the promoter sequences and transcription termination and polyadenylation signals.
  • Silencing RNA further includes so-called “sense RNA” whereby the RNA molecule comprises a sequence of at least 20 consecutive nucleotides having 95% sequence identity to the sequence of the target nucleic acid.
  • Other silencing RNA can be “unpolyadenylated RNA” comprising at least 20 consecutive nucleotides having 95% sequence identity to the complement of the sequence of the target nucleic acid, such as described in WO01/12824 or US6423885 (both documents herein incorporated by reference).
  • RNA molecule as described in W003/076619 (herein incorporated by reference) comprising at least 20 consecutive nucleotides having 95% sequence identity to the sequence of the target nucleic acid or the complement thereof, and further comprising a largely-double stranded region as described in W003/076619 (including largely double stranded regions comprising a nuclear localization signal from a viroid of the Potato spindle tuber viroid-type or comprising CUG trinucleotide repeats).
  • Silencing RNA can also be double stranded RNA comprising a sense and antisense strand as herein defined, wherein the sense and antisense strand are capable of base-pairing with each other to form a double stranded RNA region (in some embodiments, the the at least 20 consecutive nucleotides of the sense and antisense RNA are complementary to each other).
  • the sense and antisense region can also be present within one RNA molecule such that a hairpin RNA (hpRNA) can be formed when the sense and antisense region form a double stranded RNA region.
  • hpRNA hairpin RNA
  • the hpRNA can be classified as long hpRNA, having long, sense and antisense regions which can be largely complementary, but need not be entirely complementary (generally larger than about 200 bp, ranging between 200 and 1000 bp). hpRNA can also be rather small ranging in size from about 30 to about 42 bp, but not much longer than 94 bp (see W004/073390, herein incorporated by reference). Silencing RNA can also be artificial micro-RNA molecules as described e.g. in W02005/052170, W02005/047505 or US 2005/0144667, or ta-siRNAs as described in W02006/074400 (all documents incorporated herein by reference). The RNA molecule capable of modulating the expression of a gene can also be an RNA ribozyme.
  • the RNA molecule capable of modulating the expression of a gene can modulate, e.g., down-regulate, the expression of other genes (i.e. target genes) comprised within the seeds or even of genes present within a pathogen or pest that feeds upon the seeds of the transgenic plant such as a virus, fungus, insect, bacteria.
  • protein interchangeably used with the term “polypeptide,” as used herein describes a group of molecules consisting of more than 30 amino acids, whereas the term “peptide” describes molecules consisting of up to 30 amino acids. Proteins and peptides can further form dimers, trimers and higher oligomers, i.e. consisting of more than one (poly)peptide molecule. Protein or peptide molecules forming such dimers, trimers, etc., can be identical or non-identical. The corresponding higher order structures are, consequently, termed homo- or heterodimers, homo- or heterotrimers etc.
  • protein and “peptide” also refer to naturally modified proteins or peptides wherein the modification is affected e.g. by glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art.
  • the nucleic acid sequence heterologous to the promoters as described herein can generally be any nucleic acid sequence effecting increased, altered (e.g. in a different organ) or reduced level of transcription of a gene for which such expression modulation is desired.
  • the nucleic acid sequence can for example encode a protein of interest.
  • the recombinant nucleotide can regulate expression of a polynucleotide of interest, e.g., a polynucleotide that encodes an herbicide selectable marker, an insecticidal protein, antibiotic resistance, herbicide resistance, insect resistance protein, disease resistance, herbicide tolerance, or the like.
  • a polynucleotide of interest e.g., a polynucleotide that encodes an herbicide selectable marker, an insecticidal protein, antibiotic resistance, herbicide resistance, insect resistance protein, disease resistance, herbicide tolerance, or the like.
  • polynucleotide of interest refers to a nucleic acid which can be expressed under the control of the transcription regulating nucleotide sequence referred to herein.
  • the polynucleotide of interest encodes a polypeptide, the presence of which is desired in a plant cell, a plant, or a plant part as referred to herein.
  • a polypeptide may be an enzyme which is required for the synthesis of seed storage compounds or may be a seed storage protein. It is to be understood that if the polynucleotide of interest encodes a polypeptide, transcription of the nucleic acid in RNA and translation of the transcribed RNA into the polypeptide may be required.
  • a polynucleotide of interest also preferably, includes biologically active RNA molecules and, more preferably, antisense RNAs, ribozymes, micro RNAs or siRNAs.
  • biologically active RNA molecules include biologically active RNA molecules and, more preferably, antisense RNAs, ribozymes, micro RNAs or siRNAs.
  • an undesired enzymatic activity in a seed can be reduced due to the seed specific expression of an antisense RNAs, ribozymes, micro RNAs or siRNAs.
  • the underlying biological principles of action of the aforementioned biologically active RNA molecules are well known in the art.
  • the person skilled in the art is well aware of how to obtain nucleic acids which encode such biologically active RNA molecules. It is to be understood that the biologically active RNA molecules may be directly obtained by transcription of the nucleic acid of interest, i.e. without translation into a polypeptide.
  • the polynucleotide of interest is obtained from an insect resistance gene; a disease resistance gene such as, for example, a bacterial disease resistance gene, a fungal disease resistance gene, a viral disease resistance gene, or a nematode disease resistance gene; a herbicide resistance gene; a gene affecting grain composition or quality; a nutrient utilization gene; a mycotoxin reduction gene; a male sterility gene; a selectable marker gene; a screenable marker gene; a negative selectable marker; a positive selectable marker; a gene affecting plant agronomic characteristics, i.e., yield, standability, and the like; or an environment or stress resistance gene, i.e., one or more genes that confer herbicide resistance or tolerance, insect resistance or tolerance, disease resistance or tolerance (viral, bacterial, fungal, oomycete, or nematode), stress tolerance or resistance (as exemplified by resistance or tolerance to drought, heat, chilling, freezing,
  • a disease resistance gene such
  • resistant is meant a plant, which exhibits substantially no phenotypic changes as a consequence of agent administration, infection with a pathogen, or exposure to stress.
  • tolerant is meant a plant, which, although it may exhibit some phenotypic changes as a consequence of infection, does not have a substantially decreased reproductive capacity or substantially altered metabolism.
  • the polynucleotide of interest is a selectable marker gene.
  • selectable marker gene refers to a gene that--in the presence of the corresponding selection compound (e.g., herbicide) in the growing medium-confers a growth advantage to a plant or plant cell transformed with a plant expression cassette for the selectable marker as compared to a plant or plant cell not been transformed with the plant expression cassette and which, thus, does not comprise the selectable marker gene.
  • the selectable marker gene and/or plant expression cassette for the marker gene is heterologous to the plant to be transformed, and thus is not naturally present in the plant to be transformed.
  • the selectable marker gene is a negative selection marker gene.
  • Negative selection marker genes confer a resistance and/or increased tolerance to a selection compound (e.g., herbicide).
  • exemplary selectable marker genes include, but are not limited to, Phosphinothricin acetyltransferases (PAT; also named Bialaphoeresistance; bar; De Block et al. (1987) Plant Physiol 91 :694- 701; EP 0 333 033; U.S. Pat. No. 4,975,374) 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; U.S. Pat. No.
  • Phosphinothricin acetyltransferases Phosphinothricin acetyltransferases
  • EPSPS 5-enolpyruvylshikimate-3-phosphate synthase
  • GlyphosateTM N-(phosphonomethyl)glycine
  • Glyphosate.TM. degrading enzymes for example mutated ALS variants with, for example, the S4 and/or Hra mutation BromoxynilTM degrading nitrilases (bxn) Kanamycin- or.
  • G418-resistance genes NPTII; NPTI
  • NPTII G418-resistance genes coding e.g., for neomycin phosphotransferases (Fraley et al. (1983) Proc Natl Acad Sci USA 80:4803), which expresses an enzyme conferring resistance to the antibiotic kanamycin and the related antibiotics neomycin, paromomycin, gentamicin, and G418, Dicamba degrading enzymes (O-demethylase, oxygenase, ferredoxin) (Behrens et al. 2007 Science 316:1185-1188; U.S. Pat. No.
  • marker genes that confer resistance against the toxic effects imposed by D-amino acids like e.g., D-alanine and D-serine (W003/060133).
  • D-amino acids like e.g., D-alanine and D-serine (W003/060133).
  • Especially preferred as marker genes in this contest are the daol gene (EC: 1.4. 3.3: GenBank Acc.-No.: U60066) from the yeast Rhodotorula gracilis (Rhodosporidium toruloides) and the E. coli gene dsdA (D- serine dehydratase (D-serine deaminase) [EC: 4.3. 1.18; GenBank Acc.-No.: J01603).
  • the selectable marker gene is a positive selection marker, which confers a growth advantage to a transformed plant in comparison with a non-transformed one.
  • positive selection markers include, but are not limited to, mannose-6-phosphate isomerase (in combination with mannose), UDPgalactose-4-epimerase (in combination with e.g., galactose), wherein mannose-6-phosphate isomerase in combination with mannose is especially preferred.
  • the selectable marker gene is the acetohydroxy acid synthase (AHAS) gene, or a mutated AHAS gene.
  • the acetohydroxy acid synthase enzyme also known as acetolactate synthase, or ALS
  • ALS acetolactate synthase
  • the mutated AHAS protein preferably, confers resistance to at least one imidazolinone herbicide.
  • Imidazolinone herbicides are well known in the art, and, preferably, include imazapyr, imazaquin, imazethapyr, imazapic, imazamox and imazamethabenz.
  • the imidazolinone herbicide is imazaquin. More preferably, the imidazolinone herbicide is imazethapyr. Most preferably, the imidazolinone herbicide is imazapyr.
  • Exemplary mutated AHAS genes are disclosed in W02004/005516 or W02008/124495 which herewith is incorporated by reference with respect to its entire disclosure content. Further preferred mutated AHAS genes are disclosed in W02006/015376 or W02007/054555 or US20100287641. The mutated AHAS enzyme confers resistance to imidazolinone herbicides.
  • Further selection marker genes are marker genes that confer resistance or increased tolerance to the toxic effects imposed by D-amino acids.
  • Such preferred marker genes preferably, encode for proteins which are capable of metabolizing D-amino acids.
  • Preferred D-amino acids are D-alanine and D-serine.
  • Particularly preferred marker genes encode for D-serine ammonialyases, D-amino acid oxidases and D- alanine transaminases.
  • Preferred examples for such marker genes encoding for proteins which are capable of metabolizing D-amino acids are those which are as disclosed in International Patent Publication Nos. WO 03/060133, WO 05/090584, WO 07/107,516 and WO 08/077,570 which are incorporated herein by reference in their entirety.
  • the polynucleotide of interest in a herbicide resistant gene encoding a herbicide resistant protein include, but are not limited to, the genes encoding phosphinothricin acetyltransferase (bar and pat), glyphosate tolerant EPSP synthase genes, the glyphosate degradative enzyme gene gox encoding glyphosate oxidoreductase, deh (encoding a dehalogenase enzyme that inactivates dalapon), herbicide resistant (e.g., sulfonylurea and imidazolinone) acetolactate synthase, and bxn genes (encoding a nitrilase enzyme that degrades bromoxynil).
  • phosphinothricin acetyltransferase bar and pat
  • glyphosate tolerant EPSP synthase genes the glyphosate degradative enzyme gene gox encoding glyphosate oxid
  • the bar and pat genes code for an enzyme, phosphinothricin acetyltransferase (PAT), which inactivates the herbicide phosphinothricin and prevents this compound from inhibiting glutamine synthetase enzymes.
  • PAT phosphinothricin acetyltransferase
  • the enzyme 5- enolpyruvylshikimate 3-phosphate synthase (EPSP Synthase) is normally inhibited by the herbicide N- (phosphonomethyl)g lycine (glyphosate).
  • genes are known that encode glyphosate-resistant EPSP Synthase enzymes.
  • the deh gene encodes the enzyme dalapon dehalogenase and confers resistance to the herbicide dalapon.
  • the bxn gene codes for a specific nitrilase enzyme that converts bromoxynil to a non- herbicidal degradation product.
  • the polynucleotide of interest is an insect resistant gene or a variant thereof encoding an insect resistant protein.
  • Such variants can include synthetically derived sequences including but not limited to sequences that are a fusion of two or more polynucleotides of interest (e.g., two or more insect resistant genes).
  • Exemplary insect resistant genes include, but are not limited to, genes that encode insecticidal proteins such as the Cry and Cyt proteins as well as genes that encode insecticidal proteins such as the “Vip” proteins.
  • genes include Cry1, such as members of the Cry1A, Cry1 B, Cry1C, Cry 1 D, Cry 1 E, Cry 1 F, and Cr11 families; Cry2, such as members of the Cry2A family; Cry 9, such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families; and members of the Vip3 family, etc.
  • Cry2A family such as members of the Cry2A family
  • Cry 9, such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families
  • members of the Vip3 family etc.
  • Exemplary insect resistant genes include, but are not limited to, Bacillus thuringiensis crystal toxin genes or Bt genes (Watrud 1985).
  • Bt genes may provide resistance to lepidopteran or coleopteran pests such as European Corn Borer (ECB) and corn rootworm (CRW).
  • Preferred Bt toxin genes for use in such embodiments include the CrylA(b) and CrylA(c) genes.
  • Endotoxin genes from other species of B. thuringiensis, which affect insect growth or development, may also be employed in this regard.
  • Protease inhibitors may also provide insect resistance (Johnson 1989) and will thus have utility in plant transformation.
  • the use of a protease inhibitor II gene, pin 11, from tomato or potato is envisioned to be particularly useful.
  • Other genes, which encode inhibitors of the insects' digestive system, or those that encode enzymes or cofactors that facilitate the production of inhibitors may also be useful.
  • Cystatin and amylase inhibitors such as those from wheat and barley, may exemplify this group.
  • genes encoding lectins may confer additional or alternative insecticide properties.
  • Lectins (originally termed phytohemagglutinins) are multivalent carbohydrate-binding proteins, which have the ability to agglutinate red blood cells from a range of species. Lectins have been identified recently as insecticidal agents with activity against weevils, ECB and rootworm (Murdock 1990; Czapla & Lang, 1990). Lectin genes contemplated to be useful include, for example, barley and wheat germ agglutinin (WGA) and rice lectins (Gatehouse 1984), with WGA being preferred.
  • WGA barley and wheat germ agglutinin
  • rice lectins Gatehouse 1984
  • Genes controlling the production of large or small polypeptides active against insects when introduced into the insect pests form another aspect of the invention.
  • insect pests such as, e.g., lytic peptides, peptide hormones and toxins and venoms
  • the expression of juvenile hormone esterase, directed towards specific insect pests may also result in insecticidal activity, or perhaps cause cessation of metamorphosis (Hammock 1990).
  • the promoters, recombinant nucleotides, or heterologous nucleic acid sequences described above can be provided in a recombinant vector.
  • a recombinant vector typically comprises, in a 5' to 3' orientation: a promoter to direct the transcription of a nucleic acid sequence and a nucleic acid sequence.
  • the recombinant vector can, in some embodiments, further comprise a 3' transcriptional terminator, a 3' polyadenylation signal, other untranslated nucleic acid sequences, transit and targeting nucleic acid sequences, selectable markers, enhancers, and operators, or combinations thereof, as desired.
  • 5' UTR refers to the untranslated region of DNA upstream, or 5' of the coding region of a gene and "3' UTR” refers to the untranslated region of DNA downstream, or 3' of the coding region of a gene.
  • Means for preparing recombinant vectors are well known in the art. Methods for making recombinant vectors particularly suited to plant transformation are described, for example, in US4971908, US4940835, US4769061 and US4757011 .
  • Typical vectors useful for expression of nucleic acids in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens.
  • one or more additional promoters can also be provided in the recombinant vector.
  • These promoters can be operably linked, for example, without limitation, to any of the nucleic acid sequences described above.
  • the promoters can be operably linked to other nucleic acid sequences, such as those encoding transit peptides, selectable marker proteins, or antisense sequences.
  • These additional promoters can be selected on the basis of the cell type into which the vector will be inserted. Additionally, promoters which function in bacteria, yeast, and plants are all well taught in the art.
  • the additional promoters can also be selected on the basis of their regulatory features. Example of such features include enhancement of transcriptional activity, inducibility, tissue specificity, and developmental stage-specificity.
  • the recombinant vector can also contain one or more additional nucleic acid sequences.
  • additional nucleic acid sequences can, in some embodiments, be any sequences suitable for use in a recombinant vector.
  • Such nucleic acid sequences include, without limitation, any of the nucleic acid sequences, and modified forms thereof, described above.
  • the additional structural nucleic acid sequences can also be operably linked to any of the promoters described herein.
  • the one or more structural nucleic acid sequences can each be operably linked to separate promoters. Alternatively, the structural nucleic acid sequences can be operably linked to a single promoter (i.e. a single operon).
  • Vectors can include phage, plasmid, viral or retroviral vectors as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes.
  • Vectors can also include targeting constructs which allow for random or site- directed integration of the targeting construct into genomic DNA.
  • target constructs preferably, comprise DNA of sufficient length for either homologous or heterologous recombination as described in detail below.
  • the vector encompassing the nucleic acid having constitutive promoter activity or the polynucleotides of interest described herein may comprise selectable markers for propagation and/or selection in a host.
  • the vector can be incorporated into a host cell by various techniques well known in the art.
  • the vector may reside in the cytoplasm or may be incorporated into the genome. In the latter case, it is to be understood that the vector can further comprise nucleic acid sequences which allow for homologous recombination or heterologous insertion.
  • Vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques well known to those skilled in the art, such as, e.g., calcium phosphate, rubidium chloride or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, carbon-based clusters, chemically mediated transfer, electroporation or particle bombardment (e.g., “gene-gun”).
  • plasmid vector can be introduced by heat shock or electroporation techniques. Should the vector be a virus, it can be packaged in vitro using an appropriate packaging cell line prior to application to host cells. Retroviral vectors can be replication competent or replication defective. In the latter case, viral propagation generally occurs only in complementing host/cells.
  • vectors described herein can be suitable as cloning vectors, i.e. replicable in microbial systems.
  • Such vectors ensure efficient cloning in bacteria and, preferably, yeasts or fungi and make possible the stable transformation of plants, and can include various binary and co-integrated vector systems which are suitable for the T DNA-mediated transformation.
  • Such vector systems are, as a rule, characterized in that they contain at least the vir genes, which are required for the Agrobacterium-mediated transformation, and the sequences which delimit the T-DNA (T-DNA border).
  • These vector systems can also comprise further cis-regulatory regions such as promoters and terminators and/or selection markers with which suitable transformed host cells or organisms can be identified.
  • the expression cassette of the invention can be introduced into host cells or organisms such as plants or animals and, thus, be used in the transformation of plants, such as those which are published, and cited, in: Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), chapter 6/7, pp. 71-119 (1993); F.F. White, Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, vol. 1 , Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press, 1993, 15-38; B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press (1993), 128- 143; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205 225.
  • the vector of the present invention is an expression vector.
  • the expression cassette comprises a transcription regulating nucleotide sequence as specified above allowing forexpression in eukaryotic cells or isolated fractions thereof.
  • An expression vector can, in addition to the expression cassette of the invention, also comprise further regulatory elements including transcriptional as well as translational enhancers.
  • the expression vector is also a gene transfer or targeting vector.
  • Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, can be used for delivery of the expression cassettes or vector of the invention into targeted cell population.
  • Suitable expression vector backbones can, in some embodiments, be derived from expression vectors known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNAI, pcDNA3 (Invitrogene) or pSPORTI (GIBCO BRL). Further examples of typical fusion expression vectors are pGEX (Pharmacia Biotech Inc; Smith, D.B., and Johnson, K.S.
  • the target gene expression of the pTrc vector is based on the transcription from a hybrid trp-lac fusion promoter by host RNA polymerase.
  • the target gene expression from the pET 11d vector is based on the transcription of a T7-gn 10-lac fusion promoter, which is mediated by a coexpressed viral RNA polymerase (T7 gn 1 ).
  • This viral polymerase is provided by the host strains BL21 (DE3) or HMS174 (DE3) from a resident X-prophage which harbors a T7 gn1 gene under the transcriptional control of the laclIV 5 promoter.
  • Examples of vectors for expression in the yeast S. cerevisiae comprise pYepSecI (Baldari et al. (1987) Embo J.
  • Vectors and processes for the construction of vectors which are suitable for use in other fungi, such as the filamentous fungi, comprise those which are described in detail in: van den Hondel, C.A.M.J.J., & Punt, P.J. (1991) “Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of fungi, J.F. Peberdy et al., Ed., pp.
  • yeast vectors are, for example, pAG-1 , YEp6, YEp13 or pEMBLYe23.
  • the vector (or vectors) described herein comprising the expression cassette can be propagated and amplified in a suitable organism, i.e. expression host. In some embodiments, one copy of the vector is propagated and amplified in a suitable organism. In some embodiments, two or more (e.g., 3, 4, 5, 6 7, 8 or more) copies of the vector are propagated and amplified in a suitable organism.
  • expression cassette refers to a linear or circular nucleic acid molecule. It encompasses DNA as well as RNA sequences which are capable of directing expression of a particular nucleotide sequence in an appropriate host cell. In general, it comprises a promoter operably linked to a polynucleotide of interest, which is - optionally - operably linked to termination signals and/or other regulatory elements.
  • the expression cassette of the present invention is characterized in that it comprises a transcription regulating nucleotide sequence (e.g., nucleic acid having constitutive promoter activity) as described herein.
  • An expression cassette can also comprise sequences required for proper translation of the nucleotide sequence.
  • the coding region can, in some embodiments, code for a protein of interest. In some embodiments, the coding region can code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction.
  • the expression cassette comprising the polynucleotide sequence of interest can be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression cassette can also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • An expression cassette can be assembled entirely extracellularly (e.g., by recombinant cloning techniques). However, an expression cassette can also be assembled using in part endogenous components.
  • an expression cassette can be obtained by placing (or inserting) a promoter sequence upstream of an endogenous sequence, which thereby becomes functionally linked and controlled by said promoter sequences.
  • a nucleic acid sequence to be expressed can be placed (or inserted) downstream of an endogenous promoter sequence thereby forming an expression cassette.
  • such expression cassettes will comprise a transcriptional initiation region linked to a nucleotide sequence of interest.
  • Such an expression cassette is preferably provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions.
  • the expression cassette can additionally contain selectable marker genes.
  • the cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants.
  • the termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source.
  • Convenient termination regions are available from the Ti-plasmid of A tumefaciens, such as the octopine synthase and nopaline synthase termination regions and others described below (see also, Guerineau 1991; Proudfoot 1991; Sanfacon 1991; Mogen 1990; Munroe 1990; Ballas 1989; Joshi 1987).
  • the expression cassette can also comprise a multiple cloning site.
  • the multiple cloning site is, preferably, arranged in a manner as to allow for operative linkage of a polynucleotide to be introduced in the multiple cloning site with the nucleic acid having constitutive promoter activity.
  • the expression cassette described herein can, in some embodiments, comprise components required for homologous recombination, i.e. flanking genomic sequences from a target locus.
  • an expression cassette which essentially consists of the nucleic acid having constitutive promoter activity, as described herein.
  • a host cell such as an E. coli cell, an Agrobacterium cell, a yeast cell, an algae, or a plant cell, comprising the isolated nucleic acids having constitutive promoter activity described herein, or the recombinant nucleotides described herein. Also provided are methods for expressing a polynucleotide of interest in a host cell comprising introducing a recombinant nucleotide or an expression cassette or vector described herein into the host cell and expressing the polynucleotide of interest in the host cell.
  • expression refers to the transcription and/or translation of an endogenous gene, ORF or portion thereof, or a transgene in plants.
  • expression may refer to the transcription of the antisense DNA only.
  • expression refers to the transcription and stable accumulation of sense (mRNA) or functional RNA. Expression may also refer to the production of protein.
  • the "expression pattern" of a promoter is the pattern of expression levels, which shows where in the plant and in what developmental stage transcription is initiated by said promoter. Expression patterns of a set of promoters are said to be complementary when the expression pattern of one promoter shows little overlap with the expression pattern of the other promoter.
  • the level of expression of a promoter can be determined by measuring the 'steady state' concentration of a standard transcribed reporter mRNA. This measurement is indirect since the concentration of the reporter mRNA is dependent not only on its synthesis rate, but also on the rate with which the mRNA is degraded. Therefore, the steady state level is the product of synthesis rates and degradation rates.
  • the rate of degradation can however be considered to proceed at a fixed rate when the transcribed sequences are identical, and thus this value can serve as a measure of synthesis rates.
  • techniques available to those skilled in the art are hybridization S1-RNAse analysis, northern blots and competitive RT- PCR. This list of techniques in no way represents all available techniques, but rather describes commonly used procedures used to analyze transcription activity and expression levels of mRNA.
  • the analysis of transcription start points in practically all promoters has revealed that there is usually no single base at which transcription starts, but rather a more or less clustered set of initiation sites, each of which accounts for some start points of the mRNA.
  • GUS betaglucuronidase
  • CAT chloramphenicol acetyl transferase
  • GFP green fluorescent protein
  • Detection systems can readily be created or are available which are based on, e.g., immunochemical, enzymatic, fluorescent detection and quantification. Protein levels can be determined in plant tissue extracts or in intact tissue using in situ analysis of protein expression.
  • individual transformed lines with one chimeric promoter reporter construct may vary in their levels of expression of the reporter gene. Also frequently observed is the phenomenon that such transformants do not express any detectable product (RNA or protein). The variability in expression is commonly ascribed to 'position effects', although the molecular mechanisms underlying this inactivity are usually not clear.
  • the expression of the polynucleotide of interest can be determined by various well-known techniques, e.g., by Northern Blot or in situ hybridization techniques as described in WO 02/102970.
  • the nucleic acid molecules described herein can be "optimized" for enhanced expression in plants of interest (see, for example, WO 91/16432; Perlak 1991 ; Murray 1989). In this manner, the open reading frames in genes or gene fragments can be synthesized utilizing plant-preferred codons (see, for example, Campbell & Gowri, 1990 for a discussion of host-preferred codon usage).
  • the nucleotide sequences can be optimized forexpression in any plant. It is recognized that all or any part of the gene sequence can be optimized or synthetic. That is, synthetic or partially optimized sequences can also be used.
  • Variant nucleotide sequences and proteins also encompass sequences and protein derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different coding sequences can be manipulated to create a new polypeptide possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo.
  • the host cell is a plant cell, plant, a plant seed, a non-human animal or a multicellular microorganism.
  • plant refers to a photosynthetic, eukaryotic multicellular organism. Plants encompass green algae (Chlorophyta), red algae (Rhodophyta), Glaucophyta, mosses and liverworts (bryophytes), seedless vascular plants (horsetails, club mosses, ferns) and seed plants (angiosperms and gymnosperms).
  • plant or “plants” according to the invention.
  • plant parts cells, tissues or organs, seed pods, seeds, severed parts such as roots, leaves, flowers, pollen, etc.
  • progeny of the plants which retain the distinguishing characteristics of the parents, such as seed obtained by selfing or crossing, e.g. hybrid seed (obtained by crossing two inbred parental lines), hybrid plants and plant parts derived there from are encompassed herein, unless otherwise indicated.
  • plant also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen, microspores and propagules, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.
  • a “Propagule” is any kind of organ, tissue, or cell of a plant capable of developing into a complete plant.
  • a propagule can be based on vegetative reproduction (also known as vegetative propagation, vegetative multiplication, or vegetative cloning) or sexual reproduction.
  • a propagule can therefore be seeds or parts of the non-reproductive organs, like stem or leave.
  • suitable propagules can also be sections of the stem, i.e., stem cuttings.
  • the plant cells described herein as well as plant cells generated according to the methods described herein can be non-propagating cells.
  • the obtained plants described herein can be used in a conventional breeding scheme to produce more plants with the same characteristics or to introduce the same characteristic in other varieties of the same or related plant species, or in hybrid plants.
  • the obtained plants can further be used for creating propagating material.
  • Plants according to the invention can further be used to produce gametes, seeds (including crushed seeds and seed cakes), seed oil, embryos, either zygotic or somatic, progeny or hybrids of plants obtained by methods described herein. Seeds obtained from the plants according to the invention are also encompassed by the methods described herein.
  • Creating propagating material relates to any means know in the art to produce further plants, plant parts or seeds and includes inter alia vegetative reproduction methods (e.g. air or ground layering, division, (bud) grafting, micropropagation, stolons or runners, storage organs such as bulbs, corms, tubers and rhizomes, striking or cutting, twin-scaling), sexual reproduction (crossing with another plant) and asexual reproduction (e.g. apomixis, somatic hybridization).
  • vegetative reproduction methods e.g. air or ground layering, division, (bud) grafting, micropropagation, stolons or runners, storage organs such as bulbs, corms, tubers and rhizomes, striking or cutting, twin-scaling
  • sexual reproduction crossing with another plant
  • asexual reproduction e.g. apomixis, somatic hybridization
  • the nature of the plant, or transgenic plant, cells, plants, and plant parts are not limited; for example, the plant cell can be monocotyledonous or dicotyledonous.
  • “Monocotyledonous plants”, also known as “monocot plants” or “monocots” are well known in the art and are plants of which the seed typically has one cotyledon.
  • Examples of monocotyledons plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as festuca, lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn).
  • “Dicotyledonous plants”, also known as “dicot plants” or “dicots” are well known in the art and are plants of which the seed typically has two cotyledons. Examples of families of dicotyledonous plants are Brassicaceae, Solanaceae, Fabaceae, Malvaceae.
  • Brassicaceae or “Brassicaceae plant” as used herein refers to plants belonging to the family of Brassicaceae plants, also called Cruciferae or mustard family.
  • Exemplary Brassicaceae are, but are not limited to, Brassica species, such as Brassica napus, Brassica oleracea, Brassica rapa, Brassica carinata, Brassica nigra, and Brassica juncea; Raphanus species, such as Raphanus caudatus, Raphanus raphanistrum, and Raphanus sativus; Matthiola species; Cheiranthus species; Camelina species, such as Camelina sativa; Crambe species, such as Crambe abyssinica and Crambe hispanica; Eruca species, such as Eruca vesicaria; Sinapis species such as Sinapis alba; Diplotaxis species; Lepidium species; Nasturtium species; Orychophragmus species; Armoracia species, Eutrem
  • Said Brassicaceae plant can be a Brassica plant.
  • Crop plants of the Brassica species are, for example, Brassica napus, Brassica juncea, Brassica carinata, Brassica rapa (syn. B. campestris), Brassica oleracea or Brassica nigra.
  • Fabaceae refers to the plant commonly known as the legume, pea, or bean family plants.
  • Examples of Fabaceae are, but are not limited to, Glycine max (soybean), Phaseolus (beans), Pisum sativum (pea), Cicer arietinum (chickpeas), Medicago sativa (alfalfa), Arachis hypogaea (peanut), Lathyrus odoratus (sweet pea), Ceratonia siliqua (carob), and Glycyrrhiza glabra (liquorice).
  • Malvaceae refers to plants belonging to the family of Malvaceae plants, also called mallows family.
  • Malvaceae are, but are not limited to, Gossypium species, such as Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum and Gossypium herbaceum or progeny from crosses of such species with other species or crosses between such species.
  • Cotton or “cotton plant” as used herein can be any variety useful for growing cotton.
  • the most commonly used cotton varieties are Gossypium barbadense, G. hirsutum, G. arboreum and G. herbaceum. Further varieties include G. africanum and G. raimondii. Also included are progeny from crosses of any of the above species with other species or crosses between such species.
  • cotton genotypes which can be used for transformation: Coker 312, Coker310, Coker 5Acala SJ-5, GSC25110, Siokra 1-3, T25, GSA75, Acala SJ2, Acala SJ4, Acala SJ5, Acala SJ-C1 , Acala B1644, Acala B1654-26, Acala B1654-43, Acala B3991, Acala GC356, Acala GC510, Acala GAM1, Acala C1, Acala Royale, Acala Maxxa, Acala Prema, Acala B638, Acala B1810, Acala B2724, Acala B4894, Acala B5002, Acala 1517-88, Acala 1517-91 , Acala 1517-95, non Acala "picker” Siokra, "stripper” variety FC2017, Coker 315, STONEVILLE 50
  • MON/DP 10R020 MON/DP 10R030, MON/DP 10R051, MON/DP 10R052, MON/DP 11 R112, MON/DP
  • MON/DP 12R224 MON/DP 12R242, MON/DP 12R244, MON/DP 12R249, MON/DP 12R251, 12R254,
  • the plant cell or plant comprising the isolated nucleic acid described herein can be a plant cell or a plant comprising the promoter described herein, which is heterologous with respect to the plant cell or which is at another position in the genome than the natural position.
  • Such plant cells or plants may be mutant, intragenic, cisgenic or transgenic plants in which the isolated nucleic acid is introduced via mutagenesis, transformation or gene editing.
  • the plant cell or plant comprising the recombinant nucleotide according to the invention can be a plant cell or a plant comprising a recombinant nucleotide of which either a promoter described herein, or the heterologous nucleic acid sequence operably linked to the promoter, are heterologous with respect to the plant cell.
  • Such plant cells or plants may be transgenic, intragenic or cisgenic plants in which the recombinant nucleotide is introduced via transformation or gene editing.
  • the plant cell of plant may comprise a promoter described herein derived from the same species operably linked to a nucleic acid which is also derived from the same species, i.e.
  • neither the promoter nor the operably linked nucleic acid is heterologous with respect to the plant cell, but the promoter is operably linked to a nucleic acid to which it is not linked in nature.
  • a recombinant nucleotide can be introduced in the plant or plant cell via transformation or gene editing, such that both the promoter and the operably linked nucleotide are at a position in the genome in which they do not occur naturally.
  • a promoter described herein can be integrated in a targeted manner in the genome of the plant or plant cell upstream of an endogenous nucleic acid encoding an expression product of interest, i.e. to modulate the expression pattern of an endogenous gene.
  • the promoter that is integrated in a targeted manner upstream of an endogenous nucleic acid can be integrated in cells of a plant species from which it is originally derived, or in cells of a heterologous plant species.
  • a heterologous nucleic acid can be integrated in a targeted manner in the genome of the plant or plant cell downstream of the promoter according to the invention, such that said heterologous nucleic acid is expressed constitutively.
  • This heterologous nucleic acid is a nucleic acid which is heterologous with respect to the promoter, i.e. the combination of the promoter with said heterologous nucleic acid is not normally found in nature.
  • This heterologous nucleic acid can be a nucleic acid which is heterologous to said plant species in which it is inserted, but it can also naturally occur in said plant species at a different location in the plant genome.
  • a promoter described herein or said heterologous nucleic acid can be integrated in a targeted manner in the plant genome via targeted sequence insertion, using, for example, the methods as described in W02005/049842.
  • the plants and plant cells described herein can also comprise other nucleic acid sequences which may have been introduced into the host cell along with the promoter and structural nucleic acid sequence, e. g. also in connection with the vector described herein. These other sequences may include 3' transcriptional terminators, 3' polyadenylation signals, other untranslated nucleic acid sequences, transit or targeting sequences, selectable markers, enhancers, and operators.
  • Preferred nucleic acid sequences of the present invention including recombinant vectors, structural nucleic acid sequences, promoters, and other regulatory elements, are described above.
  • Plants described herein can comprise one or more recombinant nucleotides as described herein and may in addition, contain a recombinant gene comprising a nucleic acid comprising promoter activity which is preferential or specific to other plant tissues, such as apical meristem, flower buds, cotyledons, flowers, pods, roots, and leaves or other seed developmental stages, operably linked to a nucleic acid sequence encoding an expression product of interest.
  • the recombinant nucleotide according to the invention and the recombinant gene comprising a nucleic acid comprising another promoter activity may be present at one locus and may be derived from the same transforming DNA molecule.
  • the plant cells or transgenic plant transgenic plant tissue, plant organ, plant or seed is a monocotyledonous plant or a plant cell, plant tissue, plant organ, plant seed from a monocotyledonous plant.
  • the transgenic plant transgenic plant tissue, plant organ, plant or seed is a dicotyledonous plant or a plant cell, plant tissue, plant organ, plant seed from a dicotyledonous plant.
  • transgenic plant cells useful herein include, but are not limited to, cells (or entire plants or plant parts) derived from the genera: Ananas, Musa, Vitis, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Carica, Persea, Prunus, Syragrus, Theobroma, Coffea, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Mangifera, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucur
  • the plant cells or transgenic plant cells include cells (or entire plants or plant parts) from the family of poaceae, such as the genera Hordeum, Secale, Avena, Sorghum, Andropogon, Holcus, Panicum, Oryza, Zea, Triticum, for example the genera and species Hordeum vulgare, Hordeum jubatum, Hordeum murinum, Hordeum secalinum, Hordeum distichon, Hordeum aegiceras, Hordeum hexastichon, Hordeum hexastichum, Hordeum irregulare, Hordeum sativum, Hordeum secalinum, Secale cereale, Avena sativa, Avena fatua, Avena byzantina, Avena fatua var.
  • poaceae such as the genera Hordeum, Secale, Avena, Sorghum, Andropogon, Holcus, Panicum, Oryza, Zea, Triticum, for example
  • plants to be used as transgenic plants are oil fruit crops which comprise large amounts of lipid compounds, such as peanut, oilseed rape, canola, sunflower, safflower, poppy, mustard, hemp, castor-oil plant, olive, sesame, Calendula, Punica, evening primrose, mullein, thistle, wild roses, hazelnut, almond, macadamia, avocado, bay, pumpkin/squash, linseed, soybean, pistachios, borage, trees (oil palm, coconut, walnut) or crops such as maize, wheat, rye, oats, triticale, rice, barley, cotton, cassava, pepper, Tagetes, Solanaceae plants such as potato, tobacco, eggplant and tomato, Vicia species, pea, alfalfa or bushy plants (coffee, cacao, tea), Salix species, and perennial grasses and fodder crops.
  • lipid compounds such as peanut, oilseed rape, canola, sunflower,
  • Preferred plants according to the invention are oil crop plants such as peanut, oilseed rape, canola, sunflower, safflower, poppy, mustard, hemp, castor-oil plant, olive, Calendula, Punica, evening primrose, pumpkin/squash, linseed, soybean, borage, trees (oil palm, coconut).
  • oil crop plants such as peanut, oilseed rape, canola, sunflower, safflower, poppy, mustard, hemp, castor-oil plant, olive, Calendula, Punica, evening primrose, pumpkin/squash, linseed, soybean, borage, trees (oil palm, coconut).
  • the plants according to the invention can additionally contain an endogenous or a transgene, which confers herbicide resistance, such as the bar or pat gene, which confer resistance to glufosinate ammonium (Liberty®, Basta® or Ignite®) [EP 0 242 236 and EP 0 242 246 incorporated by reference]; or any modified EPSPS gene, such as the 2mEPSPS gene from maize [EP0 508 909 and EP 0 507 698 incorporated by reference], or glyphosate acetyltransferase, or glyphosate oxidoreductase, which confer resistance to glyphosate (RoundupReady®), or bromoxynitril nitrilase to confer bromoxynitril tolerance, or any modified AHAS gene, which confers tolerance to sulfonylureas, imidazolinones, sulfonylaminocarbonyltriazolinones, tri
  • the plants according to the invention may additionally contain an endogenous or a transgene which confers increased oil content or improved oil composition, such as a 12:0 ACP thioesteraseincrease to obtain high laureate, which confers pollination control, such as such as barnase under control of an anther-specific promoter to obtain male sterility, or barstar under control of an anther-specific promoter to confer restoration of male sterility, or such as the Ogura cytoplasmic male sterility and nuclear restorer of fertility.
  • an endogenous or a transgene which confers increased oil content or improved oil composition such as a 12:0 ACP thioesteraseincrease to obtain high laureate, which confers pollination control, such as such as barnase under control of an anther-specific promoter to obtain male sterility, or barstar under control of an anther-specific promoter to confer restoration of male sterility, or such as the Ogura cytoplasmic male sterility and nuclear restorer of
  • the plants or seeds of the plants described herein can be further treated with a chemical compound, such as a chemical compound selected from the following lists: Herbicides: Clethodim, Clopyralid, Diclofop, Ethametsulfuron, Fluazifop, Glufosinate, Glyphosate, Metazachlor, Quinmerac, Quizalofop, Tepraloxydim, Trifluralin.
  • a chemical compound selected from the following lists: Herbicides: Clethodim, Clopyralid, Diclofop, Ethametsulfuron, Fluazifop, Glufosinate, Glyphosate, Metazachlor, Quinmerac, Quizalofop, Tepraloxydim, Trifluralin.
  • Fungicides / PGRs Azoxystrobin, N-[9-(dichloromethylene)-1, 2,3,4- tetrahydro-1,4-methanonaphthalen-5-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide (Benzovindiflupyr, Benzodiflupyr), Bixafen, Boscalid, Carbendazim, Carboxin, Chlormequat-chloride, Coniothryrium minitans, Cyproconazole, Cyprodinil, Difenoconazole, Dimethomorph, Dimoxystrobin, Epoxiconazole, Famoxadone, Fluazinam, Fludioxonil, Fluopicolide, Fluopyram, Fluoxastrobin, Fluquinconazole, Flusilazole, Fluthianil, Flutriafol, Fluxapyroxad, Iprodione, Isopyra
  • Insecticides Acetamiprid, Aldicarb, Azadirachtin, Carbofuran, Chlorantraniliprole (Rynaxypyr), Clothianidin, Cyantraniliprole (Cyazypyr), (beta-)Cyfluthrin, gamma-Cyhalothrin, lambda- Cyhalothrin, Cypermethrin, Deltamethrin, Dimethoate, Dinetofuran, Ethiprole, Flonicamid, Flubendiamide, Fluensulfone, Fluopyram, Flupyradifurone, tau-Fluvalinate, Imicyafos, Imidacloprid, Metaflu mizone, Methiocarb, Pymetrozine, Pyrifluquinazon, Spinetoram, Spinosad, Spirotetramate, Sulfoxaflor, Thiacloprid, Thiamethoxam, 1-(3-chloro
  • Methods are provided for producing a plant or transgenic plant tissue, plant organ, plant or seed comprising the steps of (a) introducing or providing an isolated nucleic acid, a recombinant nucleotide, or an expression cassette or vector described herein to a plant cell to create modified cells; and (b) regenerating plants from said modified cell to form, e.g., a plant tissue, plant organ, plant or seed.
  • “Introducing” as used herein relates to the placing of genetic information in a plant cell or plant by artificial means. This can be affected by any method known in the art for introducing RNA or DNA into plant cells, protoplasts, calli, roots, tubers, seeds, stems, leaves, seedlings, embryos, pollen and microspores, other plant tissues, or whole plants. "Introducing” also includes stably integrating into the plant's genome. Introducing the isolated nucleic acid or recombinant nucleotide can be performed by mutagenesis, by transformation or by gene editing.
  • the term “providing” can refer to introduction of a construct to a plant cell by transformation, optionally followed by regeneration of a plant from the transformed plant cell.
  • the term can also refer to introduction of the construct by crossing of a plant comprising the construct with another plant and selecting progeny plants which have inherited the construct.
  • Yet another alternative meaning of providing refers to introduction of the construct by techniques such as protoplast fusion, optionally followed by regeneration of a plant from the fused protoplasts.
  • the construct can be provided to a plant cell by methods well-known in the art.
  • mutagenesis can be used.
  • “Mutagenesis”, as used herein, refers to the process in which plant cells (e.g., a plurality of seeds or other parts, such as pollen, etc.) are subjected to a technique which induces mutations in the DNA of the cells, such as contact with a mutagenic agent, such as a chemical substance (such as ethylmethylsulfonate (EMS), ethylnitrosourea (ENU), etc.) or ionizing radiation (neutrons (such as in fast neutron mutagenesis, etc.), alpha rays, gamma rays (such as that supplied by a Cobalt 60 source), X-rays, UV-radiation, etc.), T-DNA insertion mutagenesis (Azpiroz-Leehan et al.
  • a mutagenic agent such as a chemical substance (such as ethylmethylsulfonate (EMS), ethyl
  • plants are regenerated from the treated cells using known techniques. For instance, the resulting seeds can be planted in accordance with conventional growing procedures and following self-pollination seed is formed on the plants. Additional seed that is formed as a result of such self-pollination in the present or a subsequent generation can be harvested and screened for the presence of the mutation in a nucleic acid sequence of the promoter.
  • DeleteageneTM Delete-a-gene; Li et al., 2001, Plant J 27: 235-242
  • PCR polymerase chain reaction
  • the construct can be provided to a cell by transformation.
  • transformation herein refers to the introduction (or transfer) of nucleic acid into a recipient host such as a plant or any plant parts or tissues including plant cells, protoplasts, calli, roots, tubers, seeds, stems, leaves, seedlings, embryos and pollen. Plants resulting from transformation are referred to as cisgenic, intragenic or transgenic plants, depending on the origin of the nucleic acid compared to the transformed plant species.
  • Transformed, intragenic, cisgenic, transgenic and recombinant refer to a host organism such as a plant into which an isolated nucleic acid or a heterologous nucleic acid molecule (e.g. a recombinant nucleotide or vector) has been introduced.
  • the nucleic acid can be stably integrated into the genome of the plant.
  • Transformation methods are well known in the art and include Agrobacterium-vned'iated transformation.
  • Agrobacterium-mediated transformation of cotton has been described e.g. in US patent 5,004,863, in US patent 6,483,013 and W02000/71733.
  • Plants may also be transformed by particle bombardment: Particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. This method also allows transformation of plant plastids.
  • Viral transformation may be used for transient or stable expression of a gene, depending on the nature of the virus genome. The desired genetic material is packaged into a suitable plant virus and the modified virus is allowed to infect the plant.
  • the progeny of the infected plants is virus free and also free of the inserted gene.
  • Suitable methods for viral transformation are described or further detailed e. g. in WO 90/12107, WO 03/052108 or WO 2005/098004. Further suitable methods well-known in the art are microinjection, electroporation of intact cells, polyethyleneglycol-mediated protoplast transformation, electroporation of protoplasts, liposome- mediated transformation, silicon-whiskers mediated transformation etc.
  • Said gene may be stably integrated into the genome of said plant cell, resulting in a transformed plant cell.
  • the transformed plant cells obtained in this way may then be regenerated into mature fertile transformed plants.
  • the construct can be provided to a cell by gene editing techniques.
  • Introducing the isolated nucleic acid of the invention by gene editing allows the modulation of the expression of endogenous genes by introducing said isolated nucleic acid upstream of an endogenous nucleic acid encoding an expression product of interest.
  • the recombinant nucleotide of the invention can also be introduced by gene editing by introducing a heterologous nucleic acid encoding an expression product of interest downstream of the endogenous sequence of promoter described herein.
  • the isolated nucleic acid described herein can be introduced by gene editing by introducing point mutations in the genome of the plant or plant cell.
  • Gene editing refers to the targeted modification of genomic DNA using sequencespecific enzymes (such as endonuclease, nickases, base conversion enzymes) and/or donor nucleic acids (e.g. dsDNA, oligo’s) to introduce desired changes in the DNA.
  • sequencespecific enzymes such as endonuclease, nickases, base conversion enzymes
  • donor nucleic acids e.g. dsDNA, oligo’s
  • Sequence-specific nucleases that can be programmed to recognize specific DNA sequences include meganucleases (MGNs), zinc-finger nucleases (ZFNs), TAL-effector nucleases (TALENs) and RNA-guided or DNA-guided nucleases such as Cas9, Cpf1 , CasX, CasY, C2c1 , C2c3, certain Argonaut-based systems (see e.g. Osakabe and Osakabe, Plant Cell Physiol. 2015 Mar; 56(3):389-400; Ma et al., Mol Plant.
  • MGNs meganucleases
  • ZFNs zinc-finger nucleases
  • TALENs TAL-effector nucleases
  • RNA-guided or DNA-guided nucleases such as Cas9, Cpf1 , CasX, CasY, C2c1 , C2c3, certain Argonaut-based systems (see e.g. O
  • Donor nucleic acids can be used as a template for repair of the DNA break induced by a sequence specific nuclease, but can also be used as such for gene targeting (without DNA break induction) to introduce a desired change into the genomic DNA. Sequence-specific nucleases may also be used without donor nucleic acid, thereby allowing insertion or deletion mutations via non homologous end joining repair mechanism.
  • Modified cells can be created by the methods described herein.
  • a “modified cell” refers to a cell comprising the isolated nucleic acid, the recombinant nucleotide, the expression cassette, or the vector described herein.
  • Such modified cell can be a mutant cell, a transgenic cell or a cisgenic or intragenic cell.
  • the plant or plant cell is transgenic when the isolated nucleic acid, the promoter of the recombinant nucleotide or the heterologous nucleic acid sequence operably linked to said promoter in the recombinant nucleotide it comprises originates from a non-crossable species, i.e. a not sexually compatible species.
  • the plant or plant cell is cisgenic or intragenic when the isolated nucleic acid or the promoter of the recombinant nucleotide and the heterologous nucleic acid sequence operably linked to said promoter in the recombinant nucleotide it comprises originates from a crossable species, i.e. a sexually compatible species, or the same species.
  • the plant or plant cell is a mutant plant or mutant plant cell if the isolated nucleic acid is inserted by insertion of point mutations either by gene editing or by mutagenesis.
  • crossable species means the species within the taxonomic family of the organism.
  • non crossable species means species that are outside of the taxonomic family of the organism.
  • Also provided is a method of effecting constitutive expression of a nucleic acid comprising introducing the isolated nucleic acid according to the invention, or the recombinant nucleotide according to the invention into the genome of a plant or providing the plant according to the invention. Also provided is a method for altering properties of a plant or to produce a commercially relevant product in a plant, said method comprising introducing the isolated nucleic acid according to the invention, or the recombinant gene according to the invention into the genome of a plant, or providing the plant according to the invention. In another embodiment, said plant is a seed crop plant.
  • Properties of the plant that can be altered can include, for example, plant yield (biomass, seed yield), plant architecture (shoot and/or root branching, length), biotic stress tolerance, abiotic stress tolerance, and the like.
  • Seed properties can, for example, be seed yield, seed storage compound production, seed compound accumulation, seed nutrient accumulation; seed micronutrient accumulation; seed storage compound quality, seed compound composition, seed quality, biotic stress tolerance such as disease tolerance, abiotic stress tolerance, herbicide tolerance, seed dormancy, seed imbibition, seed germination, seed vigor.
  • Seed storage compounds can, for example, be, seed oil, seed starch, or seed protein.
  • Seed properties can be modulated by modulating metabolic pathways, such as starch metabolism, sugar metabolism, inositol phosphate metabolism, glycolysis, amino acid biosynthesis, carbon metabolism, nucleotide metabolism, oxidative pentose phosphate cycle, fatty acid biosynthesis, protein synthesis, or phytate metabolism, and modulating secondary metabolism pathways.
  • metabolic pathways such as starch metabolism, sugar metabolism, inositol phosphate metabolism, glycolysis, amino acid biosynthesis, carbon metabolism, nucleotide metabolism, oxidative pentose phosphate cycle, fatty acid biosynthesis, protein synthesis, or phytate metabolism
  • Another example is the methyl recycling metabolic activity impacting chromatin remodeling, phospholipid biosynthesis and cell wall lignification.
  • Such metabolic pathways can be modulated by, for example, overexpressing or down-regulating a gene involved in one or more of the metabolic pathways using the constitutive promoter according to the invention.
  • Yield includes, e.g., yield of the plant or plant part which is harvested, such as seed, including seed oil content, seed protein content, seed weight, seed number. Increased yield can be increased yield per plant, and increased yield per surface unit of cultivated land, such as yield per hectare. Yield can be increased by modulating, for example, by increasing seed size or oil content or indirectly by increasing the tolerance to biotic and abiotic stress conditions and decreasing seed abortion.
  • Quality includes, e.g., quality of the seed or grain such as beneficial carbohydrate composition or level, beneficial amino acid composition or level, beneficial fatty acid composition or level, nutritional value, seed, and fiber content.
  • Abiotic stress tolerance can include, e.g., resistance to environmental stress factors such as drought, extreme (high or low) temperatures, soil salinity or nutrient availability.
  • Biotic stress tolerance can include, e.g., pest resistance, such as resistance to fungal, bacterial, bacterial or viral pathogens or insects.
  • the promoters (isolated nucleic acids having constitutive promoter activity) described herein can further be used to create hybrid promoters, i.e. promoters containing (parts of) one or more of the promoters(s) of the current invention and (parts of) other promoter which can be newly identified or known in the art.
  • hybrid promoters can have optimized tissue specificity or expression level.
  • Also provided herein are methods for producing food, feed, or an industrial product comprising (a) obtaining the plant or a part thereof, or seed according to the invention; and (b) preparing the food, feed or industrial product from the plantor part thereof or seed.
  • said food or feed is oil, meal, grain, starch, flour or protein
  • said industrial product is biofuel, fiber, industrial chemicals, a pharmaceutical or a nutraceutical.
  • PCR products were resolved using electrophoresis on a 0.8% (w/v) agarose gel. Fragments with the size of around 1002 bp were excised from the gel, purified and assembled into a proprietary plant gene expression vector, which was pre-digested with Xho ⁇ and Pme ⁇ enzymes, with the Gibson assembly kit according to manufacturer's instructions (SGI-DNA, Madison, Wis., USA). The resulting vector was sequenced and aligned with the native promoter sequence from Thorne and a 100% alignment was found. [00154] Promoter 7 was originally cloned from soybean (Glycine Max cultivar Thorne) genomic DNA with PCR amplification.
  • Promoter 8 was originally cloned from soybean (Glycine Max cultivar Thorne) genomic DNA with PCR amplification.
  • Promoter 9 was originally cloned from soybean (Glycine Max cultivar Thorne) genomic DNA with PCR amplification.
  • Glyma08g23830 was originally cloned from soybean (Glycine Max cultivar Thorne) genomic DNA with PCR amplification.
  • Promoter 12 was originally cloned from soybean (Glycine Max cultivar Thorne) genomic DNA with PCR amplification.
  • Promoter 13 was originally cloned from soybean (Glycine Max cultivar Thorne) genomic DNA with PCR amplification.
  • Promoter 15 was originally cloned from soybean (Glycine Max cultivar Thorne) genomic DNA with PCR amplification.
  • DNA constructs having a luciferase expression cassette driven by different promoters disclosed herein were prepared using standard vector construction methods and introduced into Agrobacteria.
  • the Agrobacteria containing the vectors were infiltrated into N. Benthamiana leaves.
  • Leaf samples were collected 2 days after the infiltration from the infiltrated area and assayed for the luciferase reporter activity.
  • the luciferase reporter was driven by the respective promoter being assessed.
  • Promoter’s relative strength was calculated by dividing the corresponding promoter’s luciferase activity in the infiltrated tobacco cells with that of Pubi 10At (control). Table 1 shows the expression performance (based on relative luciferase activity) for each promoter. Each bar represents the average luciferase activity of four (4) infiltrated leaf samples: two (2) leaves per plant and total of two (2) plants infiltrated for each vector. A range is reported if the value differs in multiple experiments.
  • RNA-seq Expression analysis for the native soybean genes from which the Promoters are derived was performed. RNA-seq data were generated at GENEWIZ (South Plainfield, NJ) with soybean tissues (cultivar Thorne). The mRNA expression profiles of Promoters 7, 8, 9, 11, 12, 13 and 15 are shown in Figures 1-7.
  • the y-axis indicates the RNAseq counts in “fragments per kilobase million” (FPMKs).
  • FPMKs fragment per kilobase million
  • Promoter 7, 8, 9, 11, 12, 13 and 15 are constitutively expressed in nearly all plant tissues, including stem, leaf, root and flower seed/pod.
  • Table 3 Samples used for expression analysis of promoters in Figures 1 - 7.
  • RNA-seq expression analysis for the corresponding native oilseed rape genes was performed. Analysis of public available RNA-seq data was performed using Genevestigator (Hruz et al., 2008). As shown in Figures 8-10, Promoters 16, 17, 18 and 19 induce constitutive expression in nearly all plant tissues, including stem, leaf, root and flower seed/pod. The expression levels are shown on the x-axis in transcripts per million (tpm, log 2 scale). The y-axis shows the sample number which corresponds to a specific tissue as indicated in Table 5.
  • Table 4 Expression analysis of promoters derived from Brassica napus
  • Table 5 Samples used for expression analysis of promoters in Figures 8 - 10.

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Abstract

L'invention concerne des séquences d'acide nucléique ayant une activité de promoteur constitutif, ainsi que des nucléotides recombinants, des cassettes d'expression, des vecteurs, des cellules, des plantes, des parties de plante et des graines comprenant les séquences d'acide nucléique ayant une activité de promoteur constitutif. L'invention concerne également des procédés d'utilisation des séquences d'acides nucléiques, des nucléotides recombinants, des cassettes d'expression et des vecteurs décrits ici. Les séquences d'acide nucléique ayant une activité de promoteur constitutif peuvent être utilisées pour modifier l'expression d'un polynucléotide d'intérêt dans différents tissus et organes de plante, et pour modifier un caractère de plante, tel qu'une tolérance au stress biotique ou abiotique, un rendement, une qualité de graine ou des propriétés de graine.
PCT/EP2023/079618 2022-11-10 2023-10-24 Séquences nucléotidiques régulant la transcription et procédés d'utilisation WO2024099765A2 (fr)

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