WO2000012734A1 - Etiquetage de transposon et transport de genes dans des cereales a petit grain - Google Patents

Etiquetage de transposon et transport de genes dans des cereales a petit grain Download PDF

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WO2000012734A1
WO2000012734A1 PCT/US1999/019648 US9919648W WO0012734A1 WO 2000012734 A1 WO2000012734 A1 WO 2000012734A1 US 9919648 W US9919648 W US 9919648W WO 0012734 A1 WO0012734 A1 WO 0012734A1
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plant
plants
transposase
gene
nucleic acid
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Peggy Lemaux
David Mcelroy
Thomas Koprek
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The Regents Of The University Of California
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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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector

Definitions

  • This invention relates to the use of the maize Ac/Ds transposon system in small grain cereals, including, for example, barley (Hordium vulgare), wheat, and oats.
  • T-DNA insertion has been successfully used as a mutagen in dicot plant species.
  • T- DNA insertion is irreversible, excluding the use of T-DNA tagging systems to generate phenotypic revertants or new mutant alleles following excision and re-insertion.
  • most cereals are not yet routinely transformable with Agrobacterium.
  • DNA elements, able to insert at random in chromosomal DNA, e.g., transposons, are effective insertional mutagens.
  • Transposable elements or transposons, first discovered in maize, are mobile genetic factors that move around the genome.
  • the preference of certain TEs to move to linked sites makes it possible to map the initial introduced elements and use these mapped elements to generate secondary transpositions into nearby genes of interest.
  • the insertion site and related gene can then be readily recovered using standard cloning or PCR-based procedures.
  • TEs can also be used for gene transfer to introduce a heterologous nucleic acid sequence into cereal plants.
  • the advantages of such a gene delivery system are that: 1) the transgene of interest is physically moved away from the selection gene; 2) the generation of large numbers of plants with single copies of the transgene inserted in different chromosomal locations requires only two primary transformation events; and 3) the potential exists for the transgene to move to a genomic location that supports stable expression.
  • TEs occur in families of related sequences, defined by their ability to interact genetically. Within any one family, individual elements occur in two forms: one a structurally conserved element capable of promoting its own excision, termed the "autonomous" element, the other a structurally heterogeneous group of elements unable to promote their own excision, the so-called “non-autonomous” elements. Non- autonomous elements from one family can be trans-activated only by the autonomous member of the same family.
  • TEs examples include Activator- Dissociation (AciDs), Enhancerlnhibitor/Suppressor-mutator (En/Spin) and Mutator (Mu/dMu) from maize and Transposon Antirrhinum majus (Tarn) from Antirrhinum majus.
  • AciDs Activator- Dissociation
  • En/Spin Enhancerlnhibitor/Suppressor-mutator
  • Mutator Mu/dMu
  • Tarn Transposon Antirrhinum majus
  • the autonomous member of a TE family encodes a trans-acting factor (transposase) that is required for transposition.
  • TEs can either excise somatically, giving rise to sectors of various phenotypes in the plant body, or germinally, in either cell lineages that undergo meiosis or in the gametes themselves. Somatic excision of a TE from a gene whose phenotype can be readily visualized can result in a variegated pattern of excision-mediated gene expression, with clonal sectors of revertant cells on a mutant background. The size and shape of clonal sectors of revertant cells are determined by the developmental timing of TE excision and by the pattern of cell division within the host tissue (for a review, see Federoff, 1989).
  • Progeny from a plant in which the TE has undergone germinal excision- mediated reversion will have a stable phenotype, ranging from null to full function depending on the "footprint" left behind by the transposon. These "footprints” result from excision-mediated deletions and/or non-template base additions. TEs themselves can undergo deletions, internal rearrangements and/or methylation-mediated inactivation converting an autonomous element into a non-autonomous element and/or altering the trans-activation pattern of non-autonomous elements.
  • transposons to tag genes in plants was first applied to facilitate gene cloning in maize and Antirrhinum where mutated alleles were already available and their endogenous TEs were well-characterized at both the genetic and molecular levels. For most higher plant species however, active transposons are either not available or not sufficiently characterized to be used to generate mutants or as gene delivery vehicles. Therefore, maize transposons have been used.
  • Maize Ac was the first to be introduced successfully into a heterologous host, tobacco (Baker et al, 1986) Subsequently, the Ac- Ds system was used in other dicotyledenous species, including Arabidopsis thalliana and carrot (Nan Sluys et al, 1987), potato (Knapp et al, 1988), tomato (Yoder et al, 1988), petunia (Gerats et al, 1989; Haring et al, 1989), soybean (Zhou et al, 1990), flax (Ellis et al, 1992; Lawrence et al, 1994), and lettuce (Yang et al, 1993).
  • Arabidopsis thalliana and carrot Arabidopsis thalliana and carrot (Nan Sluys et al, 1987), potato (Knapp et al, 1988), tomato (Yoder et al, 1988), petunia (Gerats et al, 1989; Haring
  • the Ac-Ds transposable element system has been used in dicots to tag genes.
  • Examples include the ⁇ viral resistance gene from tobacco (Whitham et al, 1994, U.S. Patent No. 5,571,706), the petunia Ph6 coloration gene (Chuck et al, 1993), the tomato Cf-9 fungal resistance gene (Jones et al, 1994), the flax L ⁇ 5 gene for rust resistance (Lawrence et al, 1993) and developmental (Bancroft et al, 1993) and male- sterility (Aarts et al, 1993) genes from Arabidopsis.
  • the Ac-Ds system has been proposed as a means of obtaining transgenic plants that are free of potentially problematic selectable marker genes that are typically used in transformation vectors (see U.S. Patent Nos. 5,225,341 and 5,482,852).
  • This strategy incorporates the transgene of interest into a Ds element, and introduces the construct either into plants that already contain an Ac- transposase gene, or co-transforms this construct with an Ac-transposase gene into the plant species of interest.
  • a plant containing the Ds element including the transgene may be crossed with a plant containing the Ac-transposase gene .
  • the present inventors have shown for the first time that the maize Ac/Ds transposable element system is active in stably transformed barley, and have demonstrated that Ds elements can excise from one position in the genome and integrate at another. In some instances, reintegration of the Ds element occurs at sites in the genome that are unlinked to the original (excision) site; in other cases the sites are linked.
  • the Ac/Ds system may be used to tag genes in barley and other cereals such as wheat and oats, and may be used for delivering transgenes to new genomic locations.
  • the latter capability can be used to obtain integration of a transgene contained within a Ds element (a "Ds-transgene”) at a position unlinked to the site at which the transformation vector originally integrated.
  • Ds-transgene a transgene contained within a Ds element
  • plants may be obtained that contain only the Ds-transgene and not the other nucleic acid sequences contained in the original transformation vector.
  • This approach will be particularly useful for obtaining transgenic plants that express a beneficial transgene but do not contain the selectable resistance or screenable markers used in the transformation vector.
  • the invention thus provides small grain cereal plants, such as barley, wheat, and oat, containing an Ac and/or Ds element, as well as methods of producing transgenic plants that have integrated Ds elements capable of excising and reinserting into the genome at a new location.
  • Such plants include, for example, barley plants having stable insertion mutations.
  • the invention provides a small grain cereal plant, preferably barley, wheat, or oat, comprising at least one Ds element integrated into its genome, wherein the Ds element is integrated into the genome at a position to which the element transposed (rather than the site at which the transformation vector integrated).
  • the Ds element is integrated at a genomic position that is unlinked to the position from which the element transposed.
  • the Ds element may include a heterologous gene sequence, such as the bar herbicide resistance gene or other gene of interest.
  • the invention also provides a method of mobilizing a Ds element that is integrated in the genome of a barley, or other small grain cereal plant, by supplying an Ac transposase enzyme to the Ds element.
  • the Ac transposase may be encoded by a construct that comprises the open reading frame of the transposase operably linked to a promoter sequence that is active in the cereal, or the intact Ac element may be used.
  • the invention provides a method of creating an insertional mutation in a small grain cereal plant, the method comprising introducing into a cereal plant, for example barley or wheat, at least one copy of a nucleic acid encoding maize Ac transposase and at least one Ds element or another gene, enclosed in the Ds ends. While this two element system is likely to be advantageous because of the ability to stabilize a Ds insertion by outcrossing to remove the transposase gene, transposon tagging in small grain cereals may also be effected using a single Ac element approach.
  • the single Ac element or a vector containing transposase and a functional Ds element in the same plasmid minimally encodes a functional transposase and includes the terminal repeat sequences necessary for transposition.
  • the invention also provides a method of creating an insertional mutation in a cereal plant, e.g., barley, the method comprising introducing into a cereal plant at least one Ac element.
  • aspects of the invention include alternative methods of obtaining an insertion mutation in a cereal plant, preferably barley, wheat or oat, using the Ac-Ds system.
  • One such embodiment involves crossing a first generation cereal plant carrying at least one nucleic acid -molecule encoding an Ac transposase with a first generation cereal plant carrying at least one Ds element; and then selecting at least one second generation progeny plant carrying at least one nucleic acid molecule encoding an Ac transposase and at least one Ds element.
  • This method may further comprise breeding the selected first generation progeny plant by selfing or outcrossing; and then obtaining at least one second generation progeny plant that carries at least one Ds element, wherein the Ds element has transposed within the genome of the cereal plant.
  • the selected second generation progeny plant contains no Ac transposase sequences. Crossing procedures such as these may also be carried out using later generation plants.
  • the invention also encompasses a method for identifying and isolating a gene in a cereal plant such as barley, wheat or oat, the method comprising providing a cereal plant having an insertional mutation resulting from the insertion of an Ac or a Ds element, and isolating the cereal gene into which the Ac or Ds element is inserted. Also encompassed by the invention is a cereal gene isolated according to this method.
  • Fig. 1 shows schematic representations of vectors used in certain embodiments of the invention.
  • the maize Ac element has been extensively characterized (see, for example Federoff, 1989, Gierl and Sadler, 1992, Coupland et al, 1988).
  • the 4.6 kb autonomous Ac element encodes an active transposase (contained on a 3.5 kb transcript), whereas the non-autonomous Ds elements carry internal deletions and can only transpose when the Ac transposase is provided in trans.
  • Transposition of the Ac element requires the presence of the 11 bp inverted terminal repeats that are present at each end of the element.
  • the Ac transposase may be provided in trans in a plant by introducing a fully functional Ac element, by introducing a crippled Ac element (lacking one or both of the terminal repeat regions), or by introducing the transposase open reading frame operably linked to a promoter sequence.
  • Suitable promoters include the cauliflower mosaic virus 35S promoter, and the native Ac transposase promoter (see, for example, Shimamoto et al, 1993; McElroy et al, 1997).
  • Ac element refers to an intact autonomous Ac transposon
  • a nucleic acid molecule encoding an Ac transposase refers to any nucleic acid molecule that encodes a functional transposase enzyme.
  • a functional transposase enzyme is one that can trans-activate a Ds element in barley or other cereals; this may be conveniently determined using the transient assay described by McElroy et al, 1997.
  • Ds elements do not encode a functional Ac transposase, but they do include the inverted terminal repeat sequences and associated sequences that are required for transactivation by an Ac transposase (see Federoff, 1989, Gierl and Sadler, 1992, Coupland et al, 1988). Ds elements may be engineered to carry heterologous nucleic acid sequences between their terminal repeats, as described in U.S. Patent Nos. 5,225,341 and 5,482,852. As used herein, the term "Ds element” refers to any form of Ds non-autonomous element that can transpose in small grain cereals such as barley, wheat, or oat, including forms of Ds carrying heterologous sequences between the terminal repeats. The ability of a Ds element to transpose under the influence of a trans-acting Ac transposase may be conveniently determined using the transient assay described by McElroy et al, 1997.
  • a polynucleotide sequence is "heterologous to" an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form (e.g. does not naturally occur linked to the second sequence).
  • a transposon comprising a heterologous sequence refers to a transposon comprising a sequence that is not naturally linked to the transposon, but is inserted into the transposon as a result of genetic engineering techniques.
  • the inserted sequence is typically a recombinant expression cassette.
  • Probes and primers may readily be prepared based on the nucleic acids provided by this invention.
  • a probe comprises an isolated nucleic acid attached to a detectable label or reporter molecule.
  • Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, e.g., in Sambrook et al, 1989 and Ausubel et al, 1987.
  • Primers are short nucleic acids, preferably DNA oligonucleotides 15 nucleotides or more in length.
  • Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme.
  • Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.
  • PCR polymerase chain reaction
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, ⁇ 1991, Whitehead
  • a primer comprising 20 consecutive nucleotides of the maize Ac sequence will anneal to a related target sequence with a higher specificity than a corresponding primer of only 15 nucleotides.
  • probes and primers may be selected that comprise 20, 25, 30, 35, 40, 50 or more consecutive nucleotides of a selected sequence.
  • Oligonucleotide A linear polynucleotide sequence of up to about 100 nucleotide bases in length.
  • a nucleic acid molecule as introduced into a host cell, thereby- producing a transformed host cell may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector may also include one or more selectable marker genes and other genetic elements known in the art.
  • Transformed A transformed cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, Agrobacterium infection, and particle gun acceleration. Methods for transformation of barley include those described by Wan et al, 1994, Lemaux et al, 1996, and Tingay et al, 1997.
  • Isolated An "isolated" biological component (such as a nucleic acid or protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles.
  • Nucleic acids and proteins that have been "isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified protein preparation is one in which the subject protein is more enriched than the protein is in its natural environment within a cell.
  • a preparation of a protein is purified such that the subject protein represents at least 50% of the total protein content of the preparation. For particular applications, higher purity may be desired, such that preparations in which the subject protein represents at least 75% or at least 90% of the total protein content may be employed.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary or desirable, join two protein-coding regions, in the same- reading frame.
  • Recombinant A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • cDNA complementary DNA: A piece of DNA lacking internal, non- coding segments (introns) and regulatory sequences that determine transcription. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.
  • Transgenic plant As used herein, this term refers to a plant that contains recombinant genetic material not normally found in plants of this type and which has been introduced into the plant in question (or into progenitors of the plant) by human manipulation. Thus, a plant that is grown from a plant cell into which recombinant DNA is introduced by transformation is a transgenic plant, as are all offspring of that plant that contain the introduced transgene (whether produced sexually or asexually).
  • the Ac-Ds system may be employed in barley, wheat, or oat to introduce heterologous gene sequences that do not include selectable marker genes used in transformation vectors, using the approached described by Yoder et al in U.S. Patent Nos. 5,225,341 and 5,482,852.
  • Means for introducing heterologous recombinant expression cassettes into small grain cereals are known. These methods include bombardment-mediated techniques (see, e.g. Lemaux et al, 1996), electroporation (see, e.g., Salmenkallio- Marttila et al, 1995), ox Agrobacterium infection (see, e.g., Tingay et al, 1997).
  • a DNA construct comprising a transposon containing an expression cassette designed for initiating transcription or translation of a polynucleotide of interest is introduced into cereal plants.
  • Such polynucleotides can, for example include ⁇ -amylase (see, e.g., Kihara et al, 1997), lipoxygenase (see, e.g., Voeroes et al., 1998), and ⁇ -glucanase (see, e.g., Jensen et al., 1998) for barley; and wheat-starch-branching enzyme (see, e.g., Rahman et al, 1999), ADP-glucose pyrophosphorylase (see, e.g., Lalonde et al., 1997), and waxy (see, e.g., Miura et al, 1994) for wheat.
  • ⁇ -amylase see, e.g., Kihara et al, 1997)
  • lipoxygenase see, e.g., Voeroes et al., 1998)
  • ⁇ -glucanase see
  • the construct will also typically contain an ancillary selectable marker gene by which transformed plant cells can be identified in culture.
  • the marker gene will encode antibiotic or herbicide resistance. These markers include resistance to G418, hygromycin, bleomycin, kanamycin, methotrexate, chlorsulfuron, lincomycin, clindamycin, spectinomycin, phosphinotricin, glyphosate and gentamicin.
  • G418, hygromycin, bleomycin, kanamycin, methotrexate, chlorsulfuron, lincomycin, clindamycin, spectinomycin, phosphinotricin, glyphosate and gentamicin After transforming the plant cells, those cells that have the vector and express the selectable transgene will be identified by their ability to grow on a medium containing the particular antibiotic or herbicide.
  • the recombinant expression cassette will typically contain in addition to the desired sequence, a plant promoter region, an intron
  • a transcription initiation site (if the sequence to be transcribed lacks one), and a transcription termination sequence.
  • Unique restriction enzyme sites at the 5' and 3' ends of the cassette are typically included to allow for easy insertion into a pre-existing vector.
  • the particular promoter used in the expression cassette is not a critical aspect of the invention. Any of a number of promoters which direct transcription in plant cells is suitable. For example, for overexpression, a plant promoter fragment may be employed which will direct expression of the gene in all tissues of a regenerated plant. Such promoters are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation.
  • the plant promoter may direct expression of the heterologous nucleic acid in a specific tissue or may be otherwise under more precise environmental or developmental control.
  • environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, elevated temperature, or the presence of light.
  • inducible or tissue- specific promoters.
  • tissue-specific promoter may drive expression of operably linked sequences in tissues other than the target tissue.
  • a tissue-specific promoter is one that drives expression preferentially in the target tissue, but may also lead to some expression in other tissues as well.
  • intron regions are often included between the promoter and the transgene in order to enhance expression. Examples of such introns are the maize ubiquitin 1 intron and the rice actin intron.
  • a polyadenylation region at the 3'-end of the coding region should be included.
  • the polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.
  • the expression cassettes of the invention need not encode functional polypeptides and can be used, for example, to control, transcription, RNA accumulation, translation, and the like of endogenous genes.
  • a number of methods can be used to inhibit gene expression in plants. For instance, antisense technology can be conveniently used. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the antisense strand of RNA will be transcribed.
  • RNA translation see, Bourque, 1995; Pantopoulos In Progress in Nucleic Acid Research and Molecular Biology, Vol. 48. Cohn, W. E. and K. Moldave (Ed.). Academic Press, Inc.: San Diego, California, USA; London, England, UK. p.
  • Oligonucleotide-based triple-helix formation can also be used to disrupt gene expression.
  • Triplex DNA can inhibit DNA transcription and replication, generate site-specific mutations, cleave DNA, and induce homologous recombination (see, e.g., Havre and Glazer, 1993; Scanlon et al, 1995; Giovannangeli et al, 1996; Chan and Glazer, 1997).
  • Triple helix DNAs can be used to target the same sequences identified for antisense regulation.
  • RNA molecules or ribozymes can also be used to inhibit expression of endogenous genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. Thus, ribozymes can be used to target the same sequences identified for antisense regulation.
  • RNA-specific ribozymes The design and use of target RNA-specific ribozymes is described in Zhao and Pick, 1993; Eastham and Ahlering, 1996; Sokol and Murray, 1996; Sun et al, 1997; and Haseloff et al, 1988). Another method of suppression is sense co-suppression. Introduction of nucleic acid configured in the sense orientation has been recently shown to be an effective means by which to block the transcription of target genes. For an example of the use of this method to modulate expression of endogenous genes (see, Assaad et al, 1993; Flavell, 1994; Stam et al, 1997; Napoli et al, 1990; and U.S. Patents Nos. 5,034,323, 5,231,020, and 5,283,184).
  • transformed plant cells or plants containing the desired gene After transformation, transformed plant cells or plants containing the desired gene must be identified. A selectable marker, such as those discussed, supra, is typically used. Transformed plant cells can be selected by growing the cells on growth medium containing the appropriate antibiotic. Means for regenerating transgenic cereal plants, e.g., barley, are described, for example, in Jahne et al, 1994).
  • the two element Ac-Ds system in which the trans-activating transposase is provided not by an autonomous Ac element, but rather by a nucleic acid molecule encoding the Ac-transposase in a separate vector, is likely to be a more useful system for gene isolation.
  • Various forms of Ac-transposase nucleic acids may be employed, including Ac elements lacking terminal repeats, and the Ac-transposase open reading frame operably linked to a promoter sequence.
  • the F] progeny may be analyzed for molecular evidence of transposition of the Ds elements (by Southern blotting or PCR), and these lines may either be screened for mutations, or may be selfed to produce an F generation that is screened for mutations, the tagged gene being more easily identified in the F 2 populations.
  • the F 2 generation will include plants that are homozygous for Ds insertions, allowing recessive mutations to be detected.
  • the type of mutation screen employed will vary depending on the type of mutation that is being sought. For example, if one is looking for a cereal plant such as a barley plant that produces seeds earlier in the growing season, then visual inspection of the crop during the growing season could be used as a simple screen. On the other hand, the seeds might be sown in medium having an acidic pH to select for plants that have enhanced ability to grow in acidic soils.
  • the F 2 population plants will be identified in which only the Ds element with its associated sequences is present.
  • a plant having a particular desired phenotype is selfed and the progeny analyzed for co-segregation of a particular Ds insertion with the desired phenotype.
  • techniques such as IPCR (Earp et al, 1990) may be employed in conjunction with primers that read out from the Ds element to clone plant, e.g., barley, sequences that flank the Ds element. These barley sequences may then be used to confirm segregation of the inserted Ds element with the observed phenotype.
  • the barley gene containing the cloned flanking regions may then be cloned using conventional techniques such as PCR or hybridization with a barley genomic library.
  • This may be achieved by outcrossing the plant containing the Ds-tagged gene and screening progeny plants by Southern hybridization or PCR to obtain progeny plants that carry the Ds-tagged gene but not the Ac-transposase sequences.
  • This screening may be facilitated by use of an Ac-transposase construct that is linked to a negative selectable marker, such as codA (Stougaard, 1993), as illustrated in Fig. lb and Fig. lc.
  • plants carrying the Ac-transposase may be killed by application of the appropriate agent for the negative selectable marker gene (in the case of CodA, 5-fluorocytosine), leaving a population of plants that is then screened for the presence of the Ds-tagged gene.
  • the appropriate agent for the negative selectable marker gene in the case of CodA, 5-fluorocytosine
  • the gene or its corresponding cDNA may be isolated and introduced into a barley, wheat, or oat plant, or other plant species, in order to modify that particular plant characteristic.
  • the basic approach is to clone the cDNA or gene into a transformation vector, such that it is operably linked to control sequences (e.g., a promoter) that direct expression of the cDNA or gene in appropriate plant cells.
  • the transformation vector is then introduced into plant cells by one of a number of techniques (e.g., electroporation, microparticle bombardment, or Agrobacterium infection) and progeny plants containing the introduced cDNA or gene are selected.
  • that part of the transformation vector containing the transgene of interest will stably integrate into the genome of the plant cell.
  • That part of the transformation vector which integrates into the plant cell and which contains the introduced cDNA or gene and associated sequences for controlling expression (the introduced "transgene”) may be referred to as the recombinant expression cassette.
  • Selection of progeny plants containing the introduced transgene may be made based upon the detection of an altered phenotype.
  • Such a phenotype may result directly from the cDNA or gene cloned into the transformation vector or may be manifested as enhanced resistance to a chemical agent (such as an antibiotic or herbicide) as a result of the inclusion of a dominant selectable marker gene incorporated into the transformation vector.
  • a chemical agent such as an antibiotic or herbicide
  • genes isolated from, e.g., barley by the described methods may be re-introduced in various forms into barley, the genes may also be usefully introduced into a wide range of other plant species including monocotyledonous and dicotyledenous plants, such as maize, wheat, oat, millet, rice, soybean, cotton, beans in general, rape/canola, alfalfa, flax, sunflower, safflower, brassica, tobacco, peanut, clover, cowpea, grapes; vegetables such as lettuce, tomato, cucurbits, cassava, potato, carrot, radish, pea, lentils, cabbage, cauliflower, broccoli, Brussels sprouts, peppers; tree fruits such as citrus, apples, pears, peaches, apricots, walnuts; and flowers such as carnations and roses.
  • monocotyledonous and dicotyledenous plants such as maize, wheat, oat, millet, rice, soybean, cotton, beans in general, rape/
  • plant transformation vectors include one or more cloned plant genes (or cDNAs) under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker.
  • Such plant transformation vectors typically also contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally-or developmentally-regulated, or cell- or tissue-specific expression), an intron, a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
  • a promoter regulatory region e.g., a regulatory region controlling inducible or constitutive, environmentally-or developmentally-regulated, or cell- or tissue-specific expression
  • constitutive plant promoters examples include: the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues (see, e.g., Odel et al, 1985, Dekeyser et al, 1990, Terada and Shimamoto, 1990; Benfey and Chua, 1990); the nopalme synthase promoter (An et al, 1988); the octopine synthase promoter (Fromm et al, 1989), the maize ubiquitin 1 promoter and intron (Christensen and Quail, 1996) or the rice actin 1 promoter and intron (McElroy et al, 1991).
  • CaMV cauliflower mosaic virus
  • tissue specific (root, leaf, flower, and seed for example) promoters (Carpenter et al, 1992, Denis et al, 1993, Opperman et al, 1993, Stockhause et al, 1997; Roshal et al, 1987; Schernthaner et al, 1988; Bustos et al, 1989; and Cho et al, 1999) can be fused to the coding sequence to obtained particular expression in respective organs.
  • Plant transformation vectors may also include RNA processing signals, for example, introns (such as the maize ubiquitin- 1 intron and the rice actin- 1 intron), which may be positioned upstream or downstream of the ORF sequence in the transgene.
  • the expression vectors may also include additional regulatory sequences from the 3 '-untranslated region of plant genes, e.g., a 3' terminator region to increase mRNA stability of the mRNA, such as the PI-II terminator region of potato or the octopine or nopaline synthase (NOS) 3' terminator regions.
  • plant transformation vectors may also include dominant selectable marker genes to allow for the ready selection of transformants.
  • genes include those encoding antibiotic resistance genes (e.g., resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin) and herbicide resistance genes (e.g., phosphinothricin acetyltransferase).
  • antibiotic resistance genes e.g., resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin
  • herbicide resistance genes e.g., phosphinothricin acetyltransferase.
  • the ORF of the tagged sequence may be operably linked to a constitutive high-level promoter such as the CaMV 35S, maize ubiquitin, or rice actin promoter.
  • reducing the expression of the native homolog of the tagged gene in the transgenic plant may be obtained by introducing into plants antisense constructs based on the tagged gene sequence.
  • Use of antisense, ribozymes, and sense suppression constructs are described above.
  • Constructs expressing an untranslatable form of the tagged gene's mRNA may also be used to suppress the expression of the native homolog. Methods for producing such constructs are described in U.S. Patent No. 5,583,021 to Dougherty et al.
  • such constructs are made by introducing a premature stop codon into the ORF of the tagged gene.
  • Transformation and regeneration of both monocotyledonous and dicotyledonous plant cells is now routine, and the appropriate transformation technique will be determined by the practitioner.
  • the choice of method will vary with the type of plant to be transformed; those skilled in the art will recognize the suitability of particular methods for given plant types.
  • Suitable methods may include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and Agrobacterium tumefaciens (AT) mediated transformation.
  • Typical procedures for transforming and regenerating plants are described in the patent documents listed at the beginning of this section. e. Selection of Transformed Plants
  • transformed plants are usually selected using a dominant selectable marker incorporated into the transformation vector.
  • a dominant selectable marker will confer antibiotic or herbicide resistance on the seedlings of transformed plants, and selection - of transformants can be accomplished, by exposing the seedlings to appropriate concentrations of the antibiotic or herbicide.
  • the selectable marker will be operably linked to the transgene of interest.
  • Examples 1-6 demonstrate that the Ac transposase gene is stably expressed over several generations and that the expression level is sufficient to transactivate Ds elements in transient assays as well as in vivo.
  • Example 1 Generation of barley plants with a stably integrated Ac/Ds maize TE system. Construction of vectors
  • plasmids pUC-codA-Act-AcAc and pBS-codA-Act-UbiAc were generated by cloning a 3.6 kbp Smal-Bgl ⁇ l fragment, which was derived from pTps (Wirtz et al, 1997) and contained the Ac transposase coding region and nos terminator sequence, into either a pUC28- or pBS-derived plasmid.
  • Each of these plasmids contains the codA coding region, which was cloned as a 1.3 kbp Hz ' «dIII/-EcoRI fragment from pN ⁇ 3 (kind gift of Dr. J. Stougaard), under the transcriptional control of the actin promoter Actl and first intron from rice (McElroy et al, 1990); and the nos terminator, which was derived from plasmid pNG72 (McElroy et al, 1995).
  • hygroscopicus phosphinothricin acetyl transferase gene (bar) and the nos terminator as a 0.9 kbp Clal-Notl restriction fragment derived from pBARGUS (Fromm et al, 1990).
  • the bar gene is under control of the Ubil promoter and first intron from maize, which was derived as a 2.0 kbp Pstl fragment from plasmid pAHC27 (Christensen and Quail, 1996).
  • the Ubil-bar-nos cassette is flanked by 254 bp Ds 5' sequence and 320 bp Ds 3' sequence derived from pDs7 (Wirtz et al, 1997) as a 0.59 kbp SaR-BamHl restriction fragment.
  • Plasmid pAHC20 (Christensen and Quail, 1996), containing the S. hygroscopicus phosphinothricin acetyl transferase gene (bar) under control of the Ubil promoter and first intron from maize was used for selection after co-transformation with plasmids pUC -codA- Act- Ac Ac or pBS-codA-Act-UbiAc.
  • Figure 1 shows diagrams of all of the plasmids used in this study.
  • T 0 plants were regenerated from stably transformed callus following the protocol of Cho et al, 1998. Phenotypically normal T 0 plants were transferred to soil and grown under greenhouse conditions (14 h light/ 10 h dark, 15- 18°C, natural and supplemental light levels at 700 - 1000 ⁇ mol/m 2 /s).
  • Genomic DNA was isolated from two leaves when the plants were in the three-leaf stage (Cone, K., 1989) and digested with either Pstl to analyze whether the Ubi-Ac or Ac-Ac cassettes (6.0 kbp and 4.4 kbp, respectively) were intact, or with EcoRV and Htttdi ⁇ to release the intact Ds-Ubi-bar (3.6 kbp) cassette from Ds-positive plants. To detect transposition events and to determine the number of Ds copies, the DNA was digested with HmdIII, which does not cut within the Ds-Ubi-bar cassette.
  • the probe for Ds-Ubi-bar was PCR-amplified using plasmid DNA pSP-Ds-Ubi-bar as a template for the primers described below and purified using the QIAquick PCR purification kit from Qiagen.
  • Probe DsB was derived from restriction endonuclease- digested and electrophoretically-separated plasmid DNA. The probe was isolated from gel slices with the QIAquick gel extraction kit from Qiagen. Labeling of the probes was performed using the Promega Prime-a-Gene labeling kit (Promega, Madison, Wisconsin).
  • PCR analysis of transformants DNA was isolated (Cone, K., 1989) from leaf tissue when plants were in the three-leaf stage and 0.1 ⁇ g of genomic DNA was subjected to PCR amplification in a MJ Research thermocycler (PTC- 100). Fifty ⁇ l PCR reactions contained lx PCR Buffer (Promega, Madison, Wisconsin), 200 ⁇ M of each dNTP, 1.5 mM MgCl 2 , 1 ⁇ M primer, 1% DMSO and 2.5 U Taq DNA Polymerase (Promega, Madison, Wisconsin).
  • the primer pairs used for the PCR analysis were: 1.) for the Ac transposase coding region, Ac5' (5'-AAC CTA TTT GAT GTT GAG GGA TGC-3') and AcV (5'-ACC AcQ AGC Ac ⁇ GAA CGC AGA CTC-3'), to produce an expected PCR product of 852 bp; and 2.) for DsA, bar5' (5'-TGC AcC ATC GTC AAC CAC TA -3') and Ds3' (5'-AAC GTC AGT AGG GC TAA TCT TTT-3') to produce a 650 bp PCR fragment.
  • primers EDS5 ' (5 '-CGT CAG GGC GCG TCA GCG GGT GTT-3 ') and EDS3' (5'-AAT cG CAA ⁇ cC GCC TCT CCC CGC-3') were used to amplify a 300 bp fragment.
  • PCR reactions were performed with an initial denaturation at 94°C for 2 min followed by 10 cycles of a touch down program with decreasing annealing temperatures from 65 to 60°C in increments of -0.5°C per cycle for 45 sec, an extension at 72°C for 60 sec, a denaturation at 94°C for 45 sec, and subsequent 25 amplification cycles for 45 sec at 60°C, 60 sec at 72°C and 45 sec at 94°C and a final extension at 72°C for 5 mm.
  • PCR products were analyzed by gel electrophoresis in 1.1% agarose gels.
  • the number of blue spots which is an indicator of Ac transposase expression, showed a wide variability among plants from independent transformation events, as well as among plants derived from the same transformation event. Differences in the lc-transposase expression level appeared to be the result of plant-to-plant variation rather than the promoter driving the Ac transposase gene. Ac activity was detectable in embryos at different developmental stages. The number of blue spots per embryo, however, was in general too low to observe significant differences in v4c-transposase expression at different stages of embryo development.
  • the transposition frequencies in F2 plants derived from independent FI parents varied from 0% to 47%. Upon taking into account the differences in copy number of the Ds element in different FI plants, the observed transposition frequencies ranged from 0% to 38% per introduced Ds element. Transposition frequencies of F2 plants derived from FI plants with the same parents were similar (see, for example, the F2 plants of Al-1, Al-5 and Al-8 in Table 1) indicating that differences in transposition frequencies in these plants were more likely due to the c-transposase expression level in the original parental c-transposase-containing plant than to plant-to-plant variation in transposase expression.
  • A3-1 Ac 100 0 2 0 0
  • F2 plant Al-5-67 has two new Ds insertion sites as compared to the FI parent
  • transpositions resulted in a unique new banding pattern among the F2 plants. These unique integration sites could be due to either germinal transpositions in FI or to somatic transpositions in F2 plants. Germinal transpositions in FI resulted in F2 plants that are heterozygous for the new integration site of Ds. Selfing of these, c-transposase negative, plants resulted in a segregation ratio in the F3 generation close to 3:1 of plants carrying the transposed element versus plants without transposed Ds.
  • Somatic transpositions in the F2 generation may have occurred early or late in plant development. Late transpositions affected only small sectors in F2 plants and were likely not transmitted through the germline and were therefore either not detectable in the F3 generation, or resulted in an aberrant segregation ratio of the new Ds integration site in F3 plants. Early transpositions, on the other hand, were much more likely to be transmitted to the next generation and resulted in an expected 3:1 segregation of the new Ds site among the F3 plants.
  • DNA hybridization analysis of F3 plants revealed that about 25% of all new Ds insertions observed in F2 were due to transpositions to unlinked sites (and, therefore, that about 75% of reinserted elements were linked to the original integration site). Independent segregation of a new and old Ds integration sites in progeny of a selfed F2 plant (Al-5-67) was observed, thereby demonstrating that transposition was to an unlinked site. Plants, containing two Ds loci after the transposition, segregated in a ratio which was similar to the expected 9:3:3:1 segregation ratio.
  • transposase expression is often associated with transpositions in developmentally earlier stages (Long, D. et al, 1993; Jones et al, 1989; Keller et al, 1993; Balcells, L. and Coupland, G., 1994) leading to large somatic sectors, which often transmit the transposed element through the germline and result in many siblings carrying the same transposition pattern.
  • the Ac-transposase/Ds maize TE system is also functional in wheat.
  • the functionality of the expressed c-transposase were monitored using a transient assay for Ac-transposase activity (McElroy, et al, 1997).
  • T1/T2 immature embryos (1.5 to 3.0 mm in length) of plants confirmed molecularly as containing either AcAc-transposase or UbiAc-transposase were bombarded with a test construct carrying an Actl-gusA expression cassette interrupted by aDs insert ( Figure 1, plasmid pSPWDN-Actl(Dsbar)- GUS. ⁇ ), histochemically stained (Jefferson, 1987), and incubated for 24 hours at 37°C; embryos were scored two days later.
  • transposon system of the invention has the full functionality of a two element transposon system and is functional in barley and wheat.

Abstract

La présente invention concerne le fonctionnement du système transposon Ac-Ds dans des céréales à petit grain telles que l'orge, le blé et l'avoine. En outre, cette composition concerne des procédés et des compositions d'utilisation de ce système pour introduire des cassettes d'expression de recombinaison et d'étiquetage de transposon de gènes dans des céréales à petit grain.
PCT/US1999/019648 1998-08-28 1999-08-27 Etiquetage de transposon et transport de genes dans des cereales a petit grain WO2000012734A1 (fr)

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US7164056B2 (en) 2002-05-03 2007-01-16 Pioneer Hi-Bred International, Inc. Gene targeting using replicating DNA molecules
WO2012030714A3 (fr) * 2010-08-30 2012-04-26 Agrigenetics, Inc. Plateforme de marquage d'activation pour maïs et population marquée résultante et plantes
EP2568048A1 (fr) * 2007-06-29 2013-03-13 Pioneer Hi-Bred International, Inc. Procédés de modification du génome d'une cellule de plante monocotylédone

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WO1992001370A1 (fr) * 1990-07-19 1992-02-06 The Regents Of The University Of California Systeme de transformation de plantes sans danger au niveau biologique

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WO1992001370A1 (fr) * 1990-07-19 1992-02-06 The Regents Of The University Of California Systeme de transformation de plantes sans danger au niveau biologique

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002016625A2 (fr) 2000-08-25 2002-02-28 Basf Plant Science Gmbh Polynucleotides vegetaux codant de nouvelles proteases prenyle
US7164056B2 (en) 2002-05-03 2007-01-16 Pioneer Hi-Bred International, Inc. Gene targeting using replicating DNA molecules
US7608752B2 (en) 2002-05-03 2009-10-27 Pioneer Hi-Bred International, Inc. Gene targeting using replicating DNA molecules
EP2568048A1 (fr) * 2007-06-29 2013-03-13 Pioneer Hi-Bred International, Inc. Procédés de modification du génome d'une cellule de plante monocotylédone
US8912392B2 (en) 2007-06-29 2014-12-16 Pioneer Hi-Bred International, Inc. Methods for altering the genome of a monocot plant cell
WO2012030714A3 (fr) * 2010-08-30 2012-04-26 Agrigenetics, Inc. Plateforme de marquage d'activation pour maïs et population marquée résultante et plantes
US8912393B2 (en) 2010-08-30 2014-12-16 Dow Agrosciences, Llc. Activation tagging platform for maize, and resultant tagged populations and plants
US9896692B2 (en) 2010-08-30 2018-02-20 Dow Agrosciences Llc Sugarcane bacilliform viral enhancer-based activation tagging platform for maize, and resultant tagged populations and plants

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