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WO2007019616A2 - Modification d'infection endophyte - Google Patents

Modification d'infection endophyte Download PDF

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Publication number
WO2007019616A2
WO2007019616A2 PCT/AU2006/001161 AU2006001161W WO2007019616A2 WO 2007019616 A2 WO2007019616 A2 WO 2007019616A2 AU 2006001161 W AU2006001161 W AU 2006001161W WO 2007019616 A2 WO2007019616 A2 WO 2007019616A2
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WIPO (PCT)
Prior art keywords
nucleic acid
plant
seq
functionally active
construct
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/AU2006/001161
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WO2007019616A3 (fr
Inventor
Nicholas John Roberts
Chunhong Chen
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Agriculture Victoria Services Pty Ltd
New Zealand Institute for Bioeconomy Science Ltd
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Agriculture Victoria Services Pty Ltd
AgResearch Ltd
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Priority claimed from AU2005904429A external-priority patent/AU2005904429A0/en
Application filed by Agriculture Victoria Services Pty Ltd, AgResearch Ltd filed Critical Agriculture Victoria Services Pty Ltd
Priority to NZ565733A priority Critical patent/NZ565733A/en
Priority to AU2006281976A priority patent/AU2006281976B2/en
Publication of WO2007019616A2 publication Critical patent/WO2007019616A2/fr
Publication of WO2007019616A3 publication Critical patent/WO2007019616A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H3/00—Processes for modifying phenotypes, e.g. symbiosis with bacteria
    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to nucleic acid fragments encoding amino acid sequences for apyrase enzymes in plants.
  • the present invention also relates to the use of such nucleic acid fragments in the modification of plant/endophyte symbioses in plants, particularly in temperate grasses.
  • the Epichloe and Neotyphodium species form agronomically important symbiotic relationships with the major subfamily of temperate grasses, the Po ⁇ ideae (Schardl et a/., 2004).
  • These symbioses have a reasonably high degree of host-strain specificity and can be mutualistic, antagonistic or both; in some cases the fungal endophyte is seed transmissible.
  • the mechanisms by which this symbiosis is established and maintained are very poorly understood and this is reflected by our comparatively limited ability to manipulate them.
  • the identification of specific symbiosis genes in either the host or endophyte would generate exploitable properties including the potential to create new symbioses.
  • the grass/endophyte symbiosis is thought to have begun approximately 40 million years ago; hence this symbiosis is younger than either the plant/mycorrhizal or legume/rhizobium symbioses. It is possible that the grass/endophyte symbiosis may utilise some plant genes common in the plant/mycorrhizal and legume/rhizobium symbioses, such as the gene encoding Lectin Nucleotide Phosphohydrolases (LNPs).
  • LNPs Lectin Nucleotide Phosphohydrolases
  • LNPs and their cDNAs were originally isolated from a variety of legume species. These were characterised as peripherally bound membrane proteins belonging to the apyrase category of enzymes (Etzier et al., 1999; Roberts et al., 1999). Apyrases appear to be present in all eukaryotic organisms; and are involved in a wide variety of functions, it was recently demonstrated that LNP from the legume Lotus japonicus is essential for both the rhizobial- and mycorrhizal-legume symbioses (Etzier and Murphy, 2002; Etzier and Roberts, 2001 ; 2005).
  • LNPs appear to constitute a specialized category of apyrases unique to the legumes (Roberts et al., 1999; Cohn et al., 2001). However, since the ability to undergo symbiosis with mycorrhizal fungi is not confined to leguminous plants, Etzier and Roberts (2001 ; 2005) suggested that non leguminous Myc + plants possess a related apyrase that performs a similar function to LNP in this symbiosis.
  • the ability to manipulate host/strain specificity may provide a mechanism to alter, for example, abiotic stress tolerance, alkaloid production, and pathogenic resistance.
  • alkaloids with biological activity conferring resistance to grazing and insect predation.
  • the alkaloids belong to several distinct classes; the lolines and peramines are involved in preventing invertebrate predation; the indolediterpene and ergot alkaloids posses both anti-insect and anti-vertebrate activities. While the latter is particularly problematic in the search environment it can be useful in other grassed areas such as in turfed areas of golf courses and airports where the anti-vertebrate properties act as a deterrent to unwanted grazing birds.
  • the type and amount of alkaloid produced is determined by the strain of epichloe (Schardl et al., 2004). The ability to create and or manipulate the plant/epichloe symbioses may provide a mechanism to produce the desired alkaloid profile.
  • epichloe confer resistance to attack by pathogenic nematodes and pathogenic fungi.
  • the mechanisms by which resistance is developed is not fully elucidated.
  • the type and efficacy of resistance to pathogenic attack is determined by the strain of epichloe (Schardl et a/., 2004).
  • the ability to create and or manipulate the plant/epichloe symbioses may provide a mechanism that produced the desired resistance to pathogenic attack.
  • the present invention provides a substantially purified, recombinant, synthetic or isolated nucleic acid encoding an amino acid sequence of an apyrase enzyme or complementary or antisense to a nucleic acid sequence encoding an amino acid sequence of an apyrase enzyme; and functionally active fragments and variants of the nucleic acid.
  • the apyrase may have the enzyme nomenclature 3.6.1.5 (International Union of Biochemistry and Molecular Biology).
  • the nucleic acid may be obtained from ryegrass (Lolium) or fescue (Festuca) species. These species may be of any suitable type, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Preferably the species is a ryegrass, more preferably perennial ryegrass (L. perenne).
  • the nucleic acid may be obtained from clover (Trifolium) species.
  • clover Trifolium
  • the species is white clover (T. repens).
  • Nucleic acids according to the invention may be full-length genes or part thereof, and are also referred to as “nucleic acid fragments" and “nucleotide sequences" in this specification.
  • the nucleic acid may also be part of a DNA sequence including regulatory sequences such as a promoter and a terminator.
  • the nucleic acid may be of any suitable type and includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA) or interfering RNA (RNAi) that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases, and combinations thereof.
  • DNA such as cDNA or genomic DNA
  • RNA such as mRNA
  • RNAi interfering RNA
  • isolated means that the material is removed from its original environment (eg. the natural environment if it is naturally occurring).
  • a naturally occurring nucleic acid or polypeptide present in a living plant is not isolated, but the same nucleic acid or polypeptide separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such an isolated nucleic acid could be part of a vector and/or such nucleic acid could be part of a composition, and still be isolated in that such a vector or composition is not part of its natural environment.
  • substantially purified refers to a material which is removed from its original environment and which is substantially free of any compound normally associated with the material in its natural state.
  • the material may be homogeneous by one or more purity or homogeneity characteristics used by those of skill in the art.
  • the term is not meant to exclude artificial or synthetic mixtures of the material with other compounds.
  • the term is also not meant to exclude the presence of minor impurities which do not interfere with the biological activity of the material and which may be present, for example, due to incomplete purification.
  • the substantially purified material is at least approximately 80% pure, more preferably at least approximately 90% pure, most preferably at least approximately 95% pure.
  • the fragment or variant in respect of a nucleotide sequence is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of phosphohydrolase activity in a plant.
  • Such variants include naturally occurring allelic variants and non- naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant.
  • the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned sequence, more preferably at least approximately 90% identity, most preferably at least approximately 95% identity.
  • Such functionally active variants and fragments include, for example, those having nucleic acid changes which result in conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence.
  • the fragment has a size of at least 30 nucleotides, more preferably at least 45 nucleotides, most preferably at least 60 nucleotides.
  • the fragment or variant has one or more of the biological properties of the enzyme apyrase. Additions, deletions, substitutions and derivatizations of one or more of the amino acids are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant.
  • the functionally active fragment or variant has at least approximately 60% identity to the relevant part of the above mentioned sequence, more preferably at least approximately 80% identity, most preferably at least approximately 90% identity.
  • Such functionally active variants and fragments include, for example, those having conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence.
  • the fragment has a size of at least 10 amino acids, more preferably at least 15 amino acids, most preferably at least 20 amino acids.
  • operably linked is meant that a regulatory element is capable of causing expression of a nucleic acid in a plant cell with which it is operably linked and a terminator is capable of terminating expression of a nucleic acid in a plant cell.
  • the regulatory element is upstream of the nucleic acid and the terminator is downstream of said nucleic acid.
  • an effective amount is meant an amount sufficient to result in an identifiable phenotypic trait in said plant, or a plant, plant seed or other plant part derived there from. Such amounts can be readily determined by an appropriately skilled person, taking into account the type of plant, the route of administration and other relevant factors. Such a person will readily be able to determine a suitable amount and method of administration. See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, the entire disclosure of which is incorporated herein by reference. It will also be understood that the term “comprises” (or its grammatical variants) as used in this specification is equivalent to the term “includes” and should not be taken as excluding the presence of other elements or features.
  • the substantially purified, recombinant, synthetic or isolated nucleic acid encoding an apyrase protein or complementary or antisense to a sequence encoding an apyrase protein includes a nucleotide sequence selected from the group consisting of (a) the sequence shown in Figure 2 hereto (SEQ ID NO: 1); (b) the complement of the sequence recited in (a); (c) a sequence antisense to the sequences recited in (a) and (b); (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c); and (e) interfering RNA (RNAi) derived from the sequences recited in (a) and (b).
  • the substantially purified, recombinant, synthetic or isolated nucleic acid fragment encoding an apyrase protein or complementary or antisense to a sequence encoding an apyrase protein includes a nucleotide sequence selected from the group consisting of (a) the sequence shown in Figure 3 hereto (SEQ ID NO: 3); (b) the complement of the sequence recited in (a); (c) a sequence antisense to the sequences recited in (a) and (b); (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c); and (e) interfering RNA (RNAi) derived from the sequences recited in (a) and (b).
  • the substantially purified, recombinant, synthetic or isolated nucleic acid fragment encoding an apyrase protein or complementary or antisense to a sequence encoding an apyrase protein includes a nucleotide sequence selected from the group consisting of (a) the sequence shown in Figure 4 hereto (SEQ ID NO: 5); (b) the complement of the sequence recited in (a); (c) a sequence antisense to the sequences recited in (a) and (b); (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c); and (e) interfering RNA (RNAi) derived from the sequences recited in (a) and (b).
  • oligonucleotide probes based upon the nucleic acid sequences of the present invention may be designed and synthesized by methods known in the art. Moreover, the entire sequences may be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labelling, nick translation, or end- labelling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers may be designed and used to amplify a part or all of the sequences of the present invention. The resulting amplification products may be labelled directly during amplification reactions or labelled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.
  • short segments of the nucleic acid fragments of the present invention may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA.
  • the polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the nucleic acid fragments of the present invention, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding plant genes.
  • the second primer sequence may be based upon sequences derived from the cloning vector. For example, those skilled in the art can follow the RACE protocol (Frohman et a/.
  • the ryegrass (Lolium) or fescue (Festuca) species may be of any suitable type, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. In one embodiment the species is perennial ryegrass (L. perenne).
  • the clover (Trifolium) species may be of any suitable type.
  • the species is white clover (T. repens).
  • a substantially purified, recombinant, synthetic or isolated apyrase polypeptide including an amino acid sequence selected from the amino acid sequence shown in Figure 2 hereto (SEQ ID NO: 2), and functionally active fragments and variants thereof.
  • a substantially purified recombinant, synthetic or isolated apyrase polypeptide including an amino acid sequence selected from the amino acid sequence shown in Figure 3 hereto (SEQ ID NO: 4), and functionally active fragments and variants thereof.
  • a substantially purified recombinant, synthetic or isolated apyrase polypeptide including an amino acid sequence selected from the amino acid sequence shown in Figure 4 hereto (SEQ ID NO: 6), and functionally active fragments and variants thereof.
  • polypeptide recombinantly produced from a nucleic acid according to the present invention.
  • Techniques for recombinantly producing polypeptides are known to those skilled in the art.
  • nucleotide sequences of the present invention facilitates immunological screening of cDNA expression libraries.
  • Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunise animals to produce polyclonal or monoclonal antibodies with specificity for peptides and/or proteins comprising the amino acid sequences. These antibodies can be then used to screen cDNA expression libraries to isolate full-length cDNA clones of interest.
  • a genotype is the genetic constitution of an individual or group. Variations in genotype are essential in commercial breeding programs, in determining parentage, in diagnostics and fingerprinting, and the like. Genotypes can be readily described in terms of genetic markers.
  • a genetic marker identifies a specific region or locus in the genome. The more genetic markers, the finer defined is the genotype.
  • a genetic marker becomes particularly useful when it is allelic between organisms because it then may serve to unambiguously identify an individual.
  • a genetic marker becomes particularly useful when it is based on nucleic acid sequence information that can unambiguously establish a genotype of an individual and when the function encoded by such nucleic acid is known and is associated with a specific trait.
  • nucleic acids and/or nucleotide sequence information including single nucleotide polymorphisms (SNPs), variations in single nucleotides between allelic forms of such nucleotide sequence, can be used as perfect markers or candidate genes for the given trait.
  • SNPs single nucleotide polymorphisms
  • nucleic acids of the present invention including SNP's, and/or nucleotide sequence information thereof, as molecular genetic markers.
  • nucleic acid of the present invention including a single nucleotide polymorphism (SNP).
  • SNP single nucleotide polymorphism
  • Nucleic acids and fragments thereof from a nucleic acid library may desirably be sequenced.
  • the nucleic acid library may be of any suitable type and is preferably a cDNA library.
  • the nucleic acid fragments may be isolated from recombinant plasmids or may be amplified, for example using polymerase chain reaction. The sequencing may be performed by techniques known to those skilled in the art.
  • nucleic acid according to the present invention and/or nucleotide sequence information thereof, as a molecular genetic marker.
  • nucleic acids according to the present invention and/or nucleotide sequence information thereof may be used as a molecular genetic marker for quantitative trait loci (QTL) tagging, QTL mapping, DNA fingerprinting and in marker assisted selection, particularly in ryegrasses and fescues.
  • QTL quantitative trait loci
  • nucleic acids according to the present invention and/or nucleotide sequence information thereof may be used as molecular genetic markers in forage and turf grass improvement, e.g.
  • sequence information revealing SNPs in allelic variants of the nucleic acids of the present invention and/or nucleotide sequence information thereof may be used as molecular genetic markers for QTL tagging and mapping and in marker assisted selection, particularly in ryegrasses and fescues.
  • a construct including a nucleic acid according to the present invention may be a vector.
  • the vector may include at least one regulatory element, such as a promoter, a nucleic acid according to the present invention and a terminator; said regulatory element, nucleic acid and terminator being operably linked.
  • the vector may be of any suitable type and may be viral or non-viral.
  • the vector may be an expression vector.
  • Such vectors include chromosomal, non-chromosomal and synthetic nucleic acid sequences, eg. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium tumefaciens, derivatives of the Ri plasmid from Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA.
  • any other vector may be used as long as it is replicable, or integrative or viable in the plant cell.
  • the regulatory element and terminator may be of any suitable type and may be endogenous to the target plant cell or may be exogenous, provided that they are functional in the target plant cell.
  • the vector may include more than one nucleic acid.
  • the nucleic acids within the same vector may have identical or differing sequences.
  • the vector has at least two nucleic acids encoding functionally similar enzymes.
  • one of the regulatory elements is a promoter.
  • a variety of promoters which may be employed in the vectors of the present invention are well known to those skilled in the art. Factors influencing the choice of promoter include the desired tissue specificity of the vector, and whether constitutive or inducible expression is desired and the nature of the plant cell to be transformed (eg. monocotyledon or dicotyledon).
  • Particularly suitable constitutive promoters include the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter, the maize Ubiquitin promoter, and the rice Actin promoter.
  • CaMV 35S Cauliflower Mosaic Virus 35S
  • terminators which may be employed in the vectors of the present invention are also well known to those skilled in the art. It may be from the same gene as the promoter sequence or a different gene. Particularly suitable terminators are polyadenylation signals, such as the CaMV 35S polyA and other terminators from the nopaline synthase (nos) and the octopine synthase (ocs) genes.
  • the vector in addition to the regulatory element, the nucleic acid of the present invention and the terminator, may include further elements necessary for expression of the nucleic acid, in different combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize Ubiquitin Ubi intron), antibiotic resistance genes and other selectable marker genes (such as the neomycin phosphotransferase (npt2) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or pat) gene), and reporter genes (such as green fluorescence protein (GFP), beta- glucuronidase (GUS) gene (gusA)).
  • the vector may also contain a ribosome binding site for translation initiation.
  • the vector may also include appropriate sequences for amplifying expression.
  • the presence of the vector in transformed cells may be determined by other techniques well known in the art, such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, histochemical GUS assays, northern and Western blot hybridisation analyses.
  • the vectors of the present invention may be incorporated into a variety of plants, including monocotyledons (such as grasses from the genera Lolium, Festuca, Paspalum, Pennisetum, Panicum and other forage and turfgrasses, corn, rice, sugarcane, oat, wheat and barley) dicotyledons (such as arabidopsis, tobacco, soybean, canola, cotton, potato, chickpea, medics, white clover, red clover, subterranean clover, alfalfa, eucalyptus, poplar, hybrid aspen, and gymnosperms (pine tree)).
  • monocotyledons such as grasses from the genera Lolium, Festuca, Paspalum, Pennisetum, Panicum and other forage and turfgrasses
  • dicotyledons such as arabidopsis, tobacco, soybean, canola, cotton, potato,
  • the vectors are used to transform monocotyledons, preferably grass species such as ryegrasses (Lolium species) and fescues (Festuca species), even more preferably a ryegrass, most preferably perennial ryegrass, including forage- and turf-type cultivars.
  • grass species such as ryegrasses (Lolium species) and fescues (Festuca species)
  • fescues Festuca species
  • a ryegrass most preferably perennial ryegrass, including forage- and turf-type cultivars.
  • Techniques for incorporating the vectors of the present invention into plant cells are well known to those skilled in the art. Such techniques include Agrobacterium mediated introduction, electroporation to tissues, cells and protoplasts, protoplast fusion, injection into reproductive organs, injection into immature embryos and high velocity projectile introduction to cells, tissues, calli, immature and mature embryos. The choice of technique will depend largely on the type of plant to be transformed.
  • Cells incorporating the vectors of the present invention may be selected, as described above, and then cultured in an appropriate medium to regenerate transformed plants, using techniques well known in the art.
  • the culture conditions such as temperature, pH and the like, will be apparent to the person skilled in the art.
  • the resulting plants may be reproduced, either sexually or asexually, using methods well known in the art, to produce successive generations of transformed plants.
  • a plant cell, plant, plant seed or other plant part including, e.g. transformed with, a vector of the present invention.
  • the plant cell, plant, plant seed or other plant part may be from any suitable species, including monocotyledons, dicotyledons and gymnosperms.
  • the plant cell, plant, plant seed or other plant part is from a monocotyledon, preferably a grass species, more preferably a ryegrass (Lolium species) or fescue (Festuca species), even more preferably a ryegrass, most preferably perennial ryegrass, including both forage- and turf-type cultivars.
  • the present invention also provides a plant, plant seed or other plant part derived from a plant cell of the present invention.
  • the present invention also provides a plant, plant seed or other plant part derived from a plant of the present invention.
  • a method of modifying apyrase activity in a plant including introducing into said plant an effective amount of a nucleic acid, construct and/or vector according to the present invention.
  • the plant is a temperate grass, such as a ryegrass or fescue.
  • a method of modifying plant/endophyte symbioses in a plant including introducing into said plant an effective amount of a nucleic acid, construct and/or vector according to the present invention.
  • the plant is a temperate grass, such as a ryegrass or fescue.
  • the endophyte is an Epichloe or Neotypodium species.
  • the apyrase content of a plant for example L. perenne may be modified by over expressing the transcribed region of an apyrase sequence according to the present invention or by silencing the homologous gene sequence in the plant. Varying the apyrase content of a plant enables the manipulation of plant/epichloe symbioses.
  • a method of modifying ryegrass/epichloe symbiosis including introducing into a plant an effective amount of a nucleic acid, construct and/or vector according to the present invention.
  • a method of modifying fescue/epichloe symbiosis including introducing into a plant an effective amount of a nucleic acid, construct and/or vector according to the present invention.
  • the temperate grass (Po ⁇ ideae)/ Epichloe and Neotyphodium host/endophyte symbiosis may be reduced or eliminated by the suppression of apyrase expression.
  • the host/strain specificity may be manipulated by the expression of the complete open reading frame of an apyrase (using either a constitutive promoter or the native promoter) from one temperate grass species in a separate temperate grass species. This may allow the transgenic grass to form symbiotic relationships with strains of Epichloe and Neotyphodium species that were previously not possible.
  • Plants may be transformed with constructs containing either over expression cassettes or silencing constructs. Apyrase expression may then be analysed, for example by Northern blot analysis, RT-PCR or by using antibodies to apyrase.
  • apyrase null plants e.g. ryegrass
  • symbioses may be demonstrated by performing inoculations with suitable strains of endophyte, mycorrhizae and rhizobium and comparing the level of symbiotic formation with wild type plants.
  • plants expressing an apyrase from a different grass species may be able to form a symbiotic relationship with strains of Epichloe and Neotyphodium species that were previously not possible. This may be demonstrated by performing inoculations with suitable strains of endophyte and comparing the level of symbiotic formation with wild type plants.
  • RNAi constructs designed using the nucleic acids or nucleic acid fragments of the present invention.
  • the RNAi construct contains the apyrase sequence-derived nucleic acid fragments shown in Figure 28 (SEQ ID NO: 16), or functionally active fragments or variants thereof.
  • RNAi construct containing the apyrase sequence-derived nucleic acid fragments shown in Figure 30 SEQ ID NO: 17 or functionally active fragments or variants thereof.
  • RNAi construct containing the apyrase sequence-derived nucleic acid fragments shown in Figure 32 SEQ ID NO: 18
  • functionally active fragments or variants thereof SEQ ID NO: 18
  • RNAi construct containing the apyrase sequence-derived nucleic acid fragments shown in Figure 34 SEQ ID NO: 19
  • functionally active fragments or variants thereof SEQ ID NO: 19
  • RNAi construct containing the apyrase sequence-derived nucleic acid fragments shown in Figure 36 SEQ ID NO: 20
  • functionally active fragments or variants thereof SEQ ID NO: 20
  • Figure 1 Dendrogram analysis of aligned apyrase sequences.
  • FIG. 1 Nucleic acid sequence of 6RG cDNA (open reading frame indicated by overhead bar) (SEQ ID NO: 1 ), and (B) translated sequence of 6RG open reading frame (SEQ ID NO: 2).
  • FIG. 3 Nucleic acid sequence of 4WC cDNA (open reading frame indicated by overhead bar) (SEQ ID NO: 3) and (B) translated sequence of 4WC open reading frame (SEQ ID NO: 4).
  • Figure 4 (A) Nucleic acid sequence of 7WC cDNA (open reading frame indicated by overhead bar) (SEQ ID NO: 5) and (B) translated sequence of 7WC open reading frame (SEQ ID NO: 6).
  • Figure 5. Vector map of C-terminal 6RG::His in pET28.
  • Figure 7 Vector map of mature 4WC:: His in pET28.
  • Figure 8 Sequence feature map of mature 4WC::His in pET28 (SEQ ID NOS: 9 [nucleotide sequence] and 10 [amino acid sequence]).
  • Figure 9 Vector map of mature 7WC::His in pET28.
  • FIG. 11 Coomassie stained SDS-PAGE analysis of recombinant C-terminal 6RG in pET28 expressed in E. coli. Arrows indicate recombinant 6RG
  • Lanes 0, 5 protein marker Lanes 1 , 2, 3, 4, 6: pET28-7WC Lane 1 induction 0 hr Lane 2 induction 3 hr
  • Lane 3 soluble proteins Lane 4 inclusion bodies Lane 6: Nickel column eluate (major fraction)
  • FIG. 12 Coomassie stained SDS-PAGE analysis of recombinant mature 4WC in pET28 expressed in E. coli. Arrows indicate recombinant 4 WC.
  • Lane 0 protein marker Lane 1 to lane 5: pET28 without insert Lane 6 to lane 11 : pET28 - 4WC Lane 1 and lane 6: induction 0 hr Lane 2 and lane 7: induction 1 hr Lane 3 and lane 8: induction 3 hr Lane 4 and lane 9: soluble proteins Lane 5 and lane 10: inclusion bodies Lane 11 : whole gel elution(major fraction)
  • FIG. 13 Coomassie stained SDS-PAGE analysis of recombinant mature 7WC in pET28 expressed in E. coli. Arrows indicate recombinant 7WC.
  • Lane 1 to lane 5 pET28 without insert
  • Lane 6 to lane 11 pET28 - 7WC
  • Lane 4 and lane 10 soluble proteins
  • Lane 5 and lane 9 inclusion bodies
  • Lane 11 whole gel elution(major fraction)
  • FIG. 14 lmmunoblot analysis to determine antisera titre of anti-6RG.
  • Figure 15. lmmunoblot analysis to determine anti-6RG sensitivity to 6RG. Titre: 1 :1000 exposure time 2 min.
  • Figure 16. lmmunoblot analysis to determine antisera titre of anti-4WC. Purified antigen (protein 4WC) was run a 10% SDS PAGE as a preparative gel then was transferred on the Sequi-Blot PVDF membrane that was cut to many stripes. Every strip was tested a different titre. Blotting was detected by chemiluminescence's method (100 ng antigen, 30 sec exposure).
  • Figure 17 lmmunoblot analysis to determine anti-4WC sensitivity to 4WC.
  • a range of purified antigen (100ng, 50ng 25ng, 12.5ng and 6.25ng) was run a 10% SDS PAGE and then transferred on the membranes.
  • Figure 18 lmmunoblot analysis to determine anti 4WC specificity. Purified proteins 6rg and 7wc (250ng) were run on SDS PAGE and transferred on membrane. High titres of antibody 4wc (1 :100 and 1 :200) were used to against the membrane. The blot was detected by chemiluminescence's method (30 sec exposure).
  • Figure 19 lmmunoblot analysis to determine antisera titre of anti-7WC.
  • Purified antigen protein 7wc
  • Figure 20 lmmunoblot analysis to determine anti-7WC sensitivity to 7WC.
  • a range of purified antigen (100ng, 50ng 25ng, 12.5ng and 6.25ng) was run a 10% SDS PAGE and then transferred on the membranes.
  • Figure 22 Region of 6RG genomic sequence used to probe ryegrass Northern blot. Probe covers part of the second last exon, as well as the last exon and 3'UTR (SEQ ID NO: 13 [nucleotide sequence], SEQ ID NO: 14 [amino acid sequence, second last exon], SEQ ID NO: 15 [amino acid sequence, last exon]).
  • FIG. 23 Northern blot analysis showing spatial and temporal expression of 6RG in ryegrass.
  • Total RNAs were extracted by Trizol method. 10 ⁇ g of Total RNA was loaded on a 1.2% of formaldehyde denature agarose gel and transferred to the membrane by 20 ⁇ SSC. The membrane was hybridisated by the probe 6RG (610bp fragment with two exons and one intron of 3'-end) in Church Buffer at 65 0 C overnight following by high stringency washing.
  • Figure 24 lmmunoblot analysis showing expression of 6RG in ryegrass (Nui cultivar). Total proteins were extracted from seedlings germinated in either light or dark conditions. Proteins were loaded on a 10% SDS PAGE and then transferred to the membrane. Antibody 6RG (1 :1000) was against the membrane and the blot was detected by chemiluminescence's method. Arrows indicated the presence of immunoreactive bands of the correct predicted size.
  • Figure 25 lmmunoblot analysis showing spatial and temporal expression of 4WC in white clover.
  • Two different concentrations of total proteins were loaded on a 10% SDS PAGE and then transferred to the membrane.
  • Antibody 4WC (1 :1000) was against the membrane and the blot was detected by chemiluminescence's method (2 min exposure).
  • Figure 26 lmmunoblot analysis showing spatial and temporal expression of 7WC in white clover.
  • Two different concentrations of total proteins were loaded on a 10% SDS PAGE and then transferred to the membrane.
  • Antibody 7WC (1 :1000) was against the membrane and the blot was detected by chemiluminescence's method (2 min exposure).
  • FIG. 27 Vector map of pCH04.
  • Figure 28 Sequence feature map of pCH04 (SEQ ID NO: 16).
  • Figure 29 Vector map of pCH05.
  • Figure 30 Sequence feature map of pCH05 (SEQ ID NO: 17).
  • Figure 31 Sequence feature of map pCH05.
  • Figure 32 Sequence feature map of pCH17b (SEQ ID NO: 18). Plant binary vector containing the RNAi construct of 7WC coding region spanning coding region between the last apyrase domain and the first hydrophobic domain, designed to specifically silence the expression of 7WC transcripts.
  • Figure 33 Vector map of pCH18b. Plant binary vector containing the RNAi construct of 7WC coding region spanning apyrase domains 1-2, designed to specifically silence the expression of 7WC transcripts.
  • Figure 34 Sequence feature map of pCH18b (SEQ ID NO: 19). Plant binary vector containing the RNAi construct of 7WC coding region spanning apyrase domains 1-2, designed to specifically silence the expression of 7WC transcripts
  • Figure 35 Vector map of pCH19b. Plant binary vector containing the RNAi construct of 4WC coding region between the last apyrase domain and the first hydrophobic domain, designed to specifically silence the expression of 4WC transcripts.
  • Figure 36 Sequence feature map of pCH19b (SEQ ID NO: 20). Plant binary vector containing the RNAi construct of 4WC coding region between the last apyrase domain and the first hydrophobic domain, designed to specifically silence the expression of 4WC transcripts.
  • Figure 37 PCR - gel analysis of transgenic ryegrass.
  • A) genomic samples amplified using the hygromycin primers, expected band size 375bp (arrow).
  • B) genomic samples amplified using the ocs terminator primers, expected band size 439bp (arrow).
  • Figure 38 Southern blot analysis of RNAi white clover plants using the BAR ORF as the probe.
  • Clones were chosen from ryegrass and white clover EST libraries by BLAST (Altschul et al., 1990) searching the EST databases using translated exon sequences from public domain apyrase sequences.
  • EST sequences were translated and aligned to published apyrase peptide sequences and to translated sequences from published apyrase cDNAs. Sequences were aligned using the PiIeUp programme on the Wisconsin Package Version 10.3, Accelrys Inc., San Diego, CA. This analysis generates a single alignment output and a non statistically analysed dendogram tree based on the alignment ( Figure 1).
  • Three clones were selected; including: 4WC, 7WC, 6RG
  • the white clover clones (4WC and 7WC) were chosen since they align closely with the apyrases Db-LNP and Lj-LNP (predicted secretory apyrases) that are involved in the establishment of symbiotic relations and possibly in cell-cell interaction.
  • Two white clover clones were selected since one belonged to a clade containing sequences from only indeterminate nodule forming legume species and the other belonged to a clade that contains only legume sequences but from both determinate and indeterminate nodule forming species.
  • Db-LNP and Lj-LNP are from determinate nodule forming legumes and have been shown to have roles in establishing legume/rhizobium and legume/mycorhizzal symbioses (Etzler and Murphy 2002; Etzler and Roberts 2001; 2005). Furthermore, Kalsi and Etzler (2000) have shown that Db-LNP is a peripherally membrane bound protein.
  • the white clover clones serve as controls from which to compare the analysis of the ryegrass apyrase clone 6RG. 6RG was chose as it contains secretory elements and therefore has the potential to be on the cell surface and involved in cell-cell interaction.
  • the 6RG clone contains the full length open reading frame ( Figure 2).
  • the translated sequence of 6RG clusters with a clade that contains other sequences from monocotyledons only, all of which are predicted to be secretory proteins (Figure 1).
  • the 4WC clone contains the full length open reading frame ( Figure 3).
  • the 7WC clone contains the full length open reading frame ( Figure 4).
  • a 711 base pair section of the C-terminal of 6RG was cloned into the pET28 expression vector ( Figures 5 & 6) to express a 237 amino acid 6RG peptide with a C-terminal 6xHis tag.
  • a culture of pCH25 in BL21 E. coli cells was initiated and incubated at 37°C.
  • the culture had grown to an OD 6 oo of 0.6-0.7, expression of C-terminal 6RG was induced by the addition of IPTG (isopropyl ⁇ -D-thiogalactopyranoside), and the culture was incubated for a further 3h at 37°C.
  • IPTG isopropyl ⁇ -D-thiogalactopyranoside
  • the culture was harvested and inclusion bodies isolated. Samples taken during the induction period and a sample from the inclusion body prep were analysed on an SDS-PAGE gel ( Figure 11 ).
  • Proteins from the inclusion body prep were solubilised in buffered 6M urea and passed down a Nickel Affinity Column. Selected fractions were dialysed against PBS and concentrated to 0.35 ⁇ g/ ⁇ L by lyophilisation. These fractions were further purified by whole gel elution from an SDS-PAGE gel ( Figure 11 ).
  • Sufficient quantities (3-400/yg) of the truncated C-terminal 6RG protein was purified using a 3 step process utilizing inclusion body preparation, affinity purification (Ni3 + column for His tag) and gel elution. 300 ⁇ g of purified protein was injected into a rabbit over a period of 3 injections (4 weeks) to generate anti-apyrase polyclonal antibodies. Prior to immunisation a 'pre-bleed 1 blood sample was removed from the rabbit for comparison of specificity between pre- and post-immunisation antibody titres.
  • 300 ⁇ g of each purified protein was injected into separate rabbits over a period of 3 injections (4 weeks) to generate anti-apyrase polyclonal antibodies. Prior to immunisation a 'pre-bleed' blood sample was removed from the rabbit for comparison of specificity between pre- and post-immunisation antibody titres.
  • Rabbit polyclonal antibodies generated against E. coli expressed, C-terminal 6RG were analysed by immunoblot to determine titre and specificity.
  • the maximum titre that was still able to detect 100ng (-0.0001 OD 2 so) of purified recombinant 6RG was between 1 :12,800 and 1 :25,000 ( Figure 14).
  • the maximum sensitivity by immunoblot was 12.5ng using a 1 :1000 dilution ( Figure 15).
  • the pre-immune antibody serum has a negligible cross reactivity to recombinant 6RG.
  • Rabbit polyclonal antibodies generated against E. coli expressed, mature 4WC were analysed by immunoblot (chemiluminesence detection of Horse Radish Peroxidase activity) to determine titre and specificity.
  • the maximum titre that was still able to detect 100ng (-0.0001 OD 28 o) of purified recombinant 4WC was between 1 :3,200 and 1 :6,400 ( Figure 16).
  • the maximum sensitivity by immunoblot was 12.5ng using a 1 :500 dilution ( Figure 17).
  • Antiserum generated against 4WC is also able to detect recombinant 7WC but not 6RG ( Figure 18).
  • Tissue included: shoots from seedlings germinated and grown for 1 week in 16hr day light conditions; roots from the same seedlings; whole seedlings grown for 1 week in the dark; first fully expanded leaves (3rd node below the apex) from plants grown in the glass house.
  • Anti 4WC detected 2 bands in the root and 3 or more bands in the whole seedlings ( Figure 25). In the roots these bands corresponded to approximately 46 and 55kDa, in the whole seedling the bands corresponded to approximately 46, 55 and 6OkDa. No immunogenic protein was detected in the shoots or leaves. Antiserum against 7WC detected 1 band in the root and 1 band in the whole seedlings ( Figure 26). In both the roots and the whole seedlings the band corresponded to approximately 55kDa. Like anti 4WC, anti 7WC did not detect any immunogenic protein in the shoots or leaves.
  • the N-terminal half of the apyrase coding sequence encodes for 4 apyrase domains; this region is highly conserved (Handa and Guidotti, 1996; Roberts et al., 1999; Etzler and Roberts, 2002). In comparison, the C-terminal half of the nucleotide sequence, coding for the conserved cysteines, is relatively divergent. In order to generate plants with effective apyrase silencing we created a number of constructs using different regions of the cDNA clones; these are summarised in Table 1.
  • Lolium perenne was transformed using an adapted protocol from Altpeter et al, 2000 and Klein et al, 1992, using co-transformation of plasmids.
  • One plasmid (pAcH1) contained the hygromycin phosphotransferase gene conferring resistance to the antibiotic hygromycin expressed from the rice actin promoter and the second plasmid contained the genetic construct of interest for transformation. Plasmids were mixed in a one to one ratio at 1 ⁇ g/ ⁇ Land simultaneously coated onto the microcarriers.
  • Genomic DNA from hygromycin resistant (transformed) ryegrass plantlets was analysed by PCR using hygromycin specific primers which give a 375bp product:
  • HPT-2 CGCATAACAGCGGTCATTGACTGGAGC (SEQ ID NO: 22)
  • Genomic DNA from hygromycin resistant ryegrass plantlets was also analysed by PCR using terminator specific primers to confirm the gene of interest (RNAi construct) had been co-integrated with the hygromycin construct, the terminator specific primers give a 439bp product:
  • White Clover Transformation White clover was transformed according to the following procedure (modifed from Hiei eta/., 1994; Voisey eta/., 1994 and Larkin eta/., 1996)
  • Plants transformed with constructs containing either over expression cassettes or silencing may be analysed for apyrase expression by Northern blot analysis, RT-PCR or by using the antibodies developed above.
  • Ryegrass apyrase null plants will be endophyte- and mycorrhizae-(EtzIer and Roberts 2001; 2005), while white clover apyrase null plants will be nodulation- and mycorrhizee- (Etzler and Murphy 1999; Etzler and Roberts, 2002). This may be demonstrated by performing inoculations with suitable strains of endophyte, mycorrhizae and rhizobium and comparing the level of symbiotic formation with .
  • ryegrass plants expressing an apyrase from a different grass species may be able to form a symbiotic relationship with strains of Epichloe and Neotyphodium species that were previously not possible. This may be demonstrated by performing inoculations with suitable strains of endophyte and comparing the level of symbiotic formation with wild type ryegrass.

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Abstract

L'invention concerne des acides nucléiques codant des enzymes apyrase dans des végétaux et l'utilisation de ceux-ci aux fins de modification des symbioses de végétaux/endophytes.
PCT/AU2006/001161 2005-08-16 2006-08-14 Modification d'infection endophyte Ceased WO2007019616A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008002157A1 (fr) * 2006-06-27 2008-01-03 Agresearch Limited Apyrases et utilisations correspondantes
CN109068642A (zh) * 2015-11-12 2018-12-21 得克萨斯州大学系统董事会 含有三磷酸腺苷双磷酸酶基因组合的改良植物和用于制备具有三磷酸腺苷双磷酸酶组合的改良植物的方法
WO2019014126A1 (fr) * 2017-07-10 2019-01-17 The Texas A&M University System Événement transgénique de coton tam66274
WO2019018895A1 (fr) * 2017-07-28 2019-01-31 Agriculture Victoria Services Pty Ltd Transfert horizontal de gènes fongiques dans des plantes

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6465716B2 (en) * 1997-08-06 2002-10-15 The Regents Of The University Of California Nod factor binding protein from legume roots
US6849777B1 (en) * 2000-09-06 2005-02-01 The Regents Of The University Of California LNP, a protein involved in the initiation of mycorrhizal infection in plants
WO2003000898A1 (fr) * 2001-06-22 2003-01-03 Syngenta Participations Ag Genes de plantes intervenant dans la defense contre des pathogenes

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008002157A1 (fr) * 2006-06-27 2008-01-03 Agresearch Limited Apyrases et utilisations correspondantes
CN109068642A (zh) * 2015-11-12 2018-12-21 得克萨斯州大学系统董事会 含有三磷酸腺苷双磷酸酶基因组合的改良植物和用于制备具有三磷酸腺苷双磷酸酶组合的改良植物的方法
EP3373730A4 (fr) * 2015-11-12 2019-04-24 Board of Regents, The University of Texas System Plantes modifiées contenant une combinaison de gènes apyrase et procédé de fabrication de plantes modifiées avec une combinaison de gènes apyrase
US11203745B2 (en) 2015-11-12 2021-12-21 Board Of Regents, The University Of Texas System Modified plants containing combination of apyrase genes and method for making modified plants with combination of apyrase genes
CN114457096A (zh) * 2015-11-12 2022-05-10 得克萨斯州大学系统董事会 含有三磷酸腺苷双磷酸酶基因组合的改良植物和用于制备其的方法
EP4115734A1 (fr) * 2015-11-12 2023-01-11 Board of Regents, The University of Texas System Plantes modifiées contenant une combinaison de gènes apyrase et procédé de fabrication de plantes modifiées avec une combinaison de gènes apyrase
AU2016353320B2 (en) * 2015-11-12 2023-01-19 Board Of Regents, The University Of Texas System Modified plants containing combination of apyrase genes and method for making modified plants with combination of apyrase genes
US12297464B2 (en) 2015-11-12 2025-05-13 Board Of Regents, The University Of Texas System Modified plants containing combination of apyrase genes and method for making modified plants with combination of apyrase genes
WO2019014126A1 (fr) * 2017-07-10 2019-01-17 The Texas A&M University System Événement transgénique de coton tam66274
US10604764B2 (en) 2017-07-10 2020-03-31 The Texas A&M University System Cotton transgenic event TAM66274
WO2019018895A1 (fr) * 2017-07-28 2019-01-31 Agriculture Victoria Services Pty Ltd Transfert horizontal de gènes fongiques dans des plantes

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