WO2009150441A1 - Transformation mitochondriale - Google Patents

Transformation mitochondriale Download PDF

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Publication number
WO2009150441A1
WO2009150441A1 PCT/GB2009/001510 GB2009001510W WO2009150441A1 WO 2009150441 A1 WO2009150441 A1 WO 2009150441A1 GB 2009001510 W GB2009001510 W GB 2009001510W WO 2009150441 A1 WO2009150441 A1 WO 2009150441A1
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Prior art keywords
plant
recombinase
promoter
site
plant cell
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PCT/GB2009/001510
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English (en)
Inventor
Simon Geir Moller
Xiang Ming Xu
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University Of Stavanger
Webber, Philip, Michael
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Application filed by University Of Stavanger, Webber, Philip, Michael filed Critical University Of Stavanger
Publication of WO2009150441A1 publication Critical patent/WO2009150441A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • 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

Definitions

  • the invention relates to a method for producing a transformed plant cell. More particularly, the method involves the transformation of a plant cell with a Transformation Cassette which is targeted to plant mitochondria and which comprises a selection gene, for example isopentenyl transferase (IPT), and a transgene. After selection for transformed mitochondria, expression of a recombinase may be induced in the plant cell, which leads to the excision of the selection gene from the mitochondria and the expression of the transgene in the mitochondria.
  • the invention also provides cells and plants comprising the Transformation Cassette.
  • GM genetically modified
  • Mitochondrial transformation has clear advantages over nuclear transformation in that high levels of proteins can most probably be produced and that the transformed mitochondria are maternally inherited (i.e. from the mother) providing genetic control. Mitochondrial transformation will also be useful for assaying mitochondrial functions in terms of agricultural traits.
  • the invention is based on a selection and regeneration system for mitochondrial transformation based on the over-expression of a gene such as the isopentenyl transferase (IPT) gene (cytokinin biosynthesis) in mitochondria.
  • IPT isopentenyl transferase
  • This system allows, for example, for the direct selection of cells containing transformed mitochondria on media lacking cytokinin due to cytokinin production within mitochondria.
  • the system therefore provides an antibiotics-free selection and regeneration system which will overcome concerns regarding the use of antibiotic resistance genes in GMOs.
  • IPT has previously been used as a selectable marker in plant nuclear transformation (e.g. EP 1 069 855 A), its use in mitochondrial transformation has not previously been suggested.
  • mitochondria are semi-autonomous organelles within plant cells with their own genomes and metabolism.
  • IPT precursors are made in mitochondria or that any IPT produced is capable of diffusing out of the mitochondria into the cell cytoplasm in order to initiate cytokinin signalling to stimulate shoot regeneration.
  • the criteria for choosing mitochondrial - selection genes are distinct from those for choosing genome-selection genes.
  • the plant- hormone biosynthetic gene is preferably removed after selection and initial regeneration has occurred.
  • the transgene in question (expressing the polypeptide of interest) might only be activated after the removal of the plant- hormone biosynthetic gene, so that any adverse effects due to transgene expression during selection and regeneration are also eliminated.
  • the invention provides a method for producing a transformed plant cell, the method comprising the step: (i) transforming the plant cell with a genetic construct, wherein the genetic construct comprises first and second homologous recombination elements flanking a Transformation Cassette, wherein the first and second homologous recombination elements are capable of directing the integration of the Transformation Cassette into the genome of at least one mitochondria which is present in the plant cell, wherein the Transformation Cassette comprises: (a) a first promoter which is operable in said plant cell,
  • Excision Cassette comprises: (bl) a first site-specific recombination element
  • nucleotide sequences encoding one or more plant- hormone biosynthetic polypeptides and optionally a nucleotide sequence encoding a polypeptide which confers resistance to an antibiotic
  • the method additionally comprises the step:
  • the method additionally comprises the step:
  • the method of the invention is suitable for all plants that can be transformed and regenerated.
  • the plant may be a monocot or dicot.
  • suitable plants are cereals (rice, wheat, barley, oats, sorghum, corn), legumes (alfalfa, lentils, peanut, pea, soybean), oil crops (palm, sunflower, coconut, canola, olive), cash crops (cotton, sugar cane, cassava), vegetable crops (potato, tomato, carrot, sweet potato, sugar-beet, squash, cucumber, lettuce, broccoli, cauliflower, snap bean, cabbage, celery, onion, garlic), fruits/trees and nuts (banana, grape cantaloupe, muskmelon, watermelon, strawberry, orange, apple, mango, avocado, peach, grapefruit, pineapple, maple, almond), beverages (coffee, tea, cocoa), and timber trees (oak, black walnut, sycamore).
  • Other suitable plants include mosses and duckweed.
  • the plant is tobacco or lettuce.
  • the plant cells which are being transformed may be used in any convenient form, for example, as individual cells, groups of cells, in dissociated form or undissociated form, or as part of a plant tissue or plant part.
  • the cells are present in leaves that are removed from intact plants. It is preferable to use actively-growing leaves.
  • the term "genetic construct” refers to a nucleic acid molecule comprising the specified elements and Cassettes.
  • the genetic construct may, for example, be in the form of a vector or a plasmid. It may also contain other elements which enable its handling and reproduction, such as an origin of replication, selection elements, and multiple cloning sites.
  • the genetic construct will be a double-stranded nucleic acid molecule, preferably a dsDNA molecule.
  • the first and second homologous recombination elements are ones that are capable of directing the integration of the Transformation Cassette into the genome of at least one mitochondria which is present in the plant cell.
  • the first and second homologous recombination elements Upon transformation of the genetic construct into the plant cell, the first and second homologous recombination elements recombine with corresponding sequences in the genome of the selected mitochondria, resulting in the insertion of the Transformation Cassette into the genome of the selected mitochondria.
  • the nucleotide sequences of the homologous recombination elements are selected such that the Transformation Cassette is specifically targeted to one or more selected mitochondria.
  • the nucleotide sequences of the homologous recombination elements are selected such that no or essentially no Transformation Cassettes become integrated into the nuclear genome of the plant or into the mitochondrial genome of the plant.
  • the nucleotide sequences of the homologous recombination elements are preferably mitochondria-specific, i.e. corresponding sequences are not present in the nuclear genome and preferably not present in the plastid genome of the plant in question. This may be done by avoiding sequences which are present in the nuclear genome of the plant and optionally in the plastid genome also.
  • the skilled person will readily be able to detect whether a specific sequence is or is not present in the nuclear/plastid genome by standard means, for example, by Southern Blotting of the nuclear/plastid genome with a labelled sequence probe or by sequence analysis.
  • any sequences can be used from the mitochondrial genome as long as the selected insertion site is not lethal to the cell, i.e. it does not result in the death of the cell.
  • the insertion sites are not in coding regions of mitochondrial genes.
  • the orientation of the sequences of the first and second homologous recombination elements should be the same as the orientation in the mitochondrial genome to allow for efficient homologous recombination.
  • nucleotide sequences of the first and second homologous recombination elements must be identical or substantially identical to sequences in the genome of the selected plant mitochondria.
  • nucleotide sequences of the first and second homologous recombination elements should preferably not be identical or substantially identical to sequences in the nuclear genome of the selected plant.
  • first and second homologous recombination sequences will independently be 50-2500, 50-2000, 50-1500 or 50-1000 nucleotides each, more preferably about 150, about 1000 or about 1200 nucleotides in length.
  • the distance between the first and second homologous recombination sequences in the mitochondrial genome may be 0-4000 nucleotides or more. Preferably, the distance is about 1-100, 100-500, 500-1000 or 1000-3000 nucleotides.
  • the total length of the genetic elements which are present between the first and second homologous recombination is preferably less than 4000 nucleotides.
  • the first homologous recombination sequence is nucleotides
  • the Transformation Cassette promoter (a) must be one that is operable in the selected plant mitochondria.
  • the promoter is one which is capable of initiating transcription of the transgene once the Excision Cassette has been excised; and of initiating the transcription of the nucleotide sequence encoding a plant-hormone biosynthetic polypeptide, in cases where the Excision Cassette does not contain its own promoter.
  • the promoter might, for example, be one derived from a plant or bacterial gene.
  • the promoter is plant specific.
  • the promoter is an inducible promoter. This allows inducible, controlled expression of the selection gene(s).
  • the inducible promoter may be inducible by IPTG, e.g. the PrrnL promoter.
  • Other inducible promoters include those inducible by light, dark, ethanol, drought, metals, pathogens, growth regulators, heat, cold, galactose and other sugars.
  • the promoter is a high-expression level promoter.
  • the Excision Cassette comprises a first site-specific recombination element; optionally, a second promoter; a nucleotide sequence encoding a plant-hormone biosynthetic polypeptide; a terminator sequence; and a second site-specific recombination element.
  • the first and second site-specific recombination elements are sequences of nucleotides which are capable of being recognised and/or bound by the site-specific recombinase which is produced by a recombinase.
  • the site-specific recombination elements must flank the genetic elements in the Excision Cassette, e.g.
  • first and second recombination elements will be identical or substantially identical to each other; and will be in the same orientation relative to each other (e.g. both 5'-3' or both 3'-5').
  • the site-specific recombination sequences are preferably lox sequences.
  • Site-specific lox recombination sites are 34 bp sequences; these act as binding sites for the Cre recombinase polypeptide.
  • Wild- type lox sequences are preferred (Zuo J 3 Niu QW, M ⁇ ller SG, Chua NH (2001) Chemical-regulated, site-specific DNA excision in transgenic plants. Nat. Biotechnol. 19, 157-161.)
  • the Excision Cassette promoter when present, must be one that is operable in the selected plant mitochondria.
  • the promoter is one which is capable of initiating transcription of the plant-hormone biosynthetic gene.
  • the promoter might be one derived from a plant or bacterial gene.
  • the promoter is plant specific. Examples of suitable promoters include PsbA, RbcL and Prrn promoters.
  • the plant-hormone biosynthetic polypeptides act as selection markers, allowing the selection of plant cells which have been transformed with the Transformation Cassette.
  • the plant-hormone biosynthetic polypeptides may be any polypeptides which are involved in the synthesis of a plant cytokinin or auxin or other plant growth regulator, or which regulate the production or metabolism of a plant cytokinin or auxin or other plant growth regulator.
  • nucleotide sequences encoding 1, 2, 3, or 4 plant- hormone biosynthetic polypeptides there are nucleotide sequences encoding 1, 2, 3, or 4 plant- hormone biosynthetic polypeptides.
  • the nucleotide sequences encoding the plant-hormone biosynthetic polypeptides may be present in an operon, with a single optional promoter and terminator element.
  • the plant- hormone biosynthetic polypeptide nucleotide sequences may each have their own promoters and terminator elements.
  • two or more of the nucleotide sequences encoding the plant-hormone biosynthetic polypeptides are present as fusion proteins, optionally with a short linker sequence joining the proteins (e.g. encoding a 1-10 amino acid linker sequence, e.g.
  • nucleotide sequences encoding the plant-hormone biosynthetic polypeptides may be present in an operon and/or as fusion proteins, and others have their own promoters and/or terminators.
  • the or a plant-hormone biosynthetic polypeptide is IPT (isopentenyl transferase) which is an enzyme involved in cytokinin biosynthesis.
  • IPT isopentenyl transferase
  • the IPT nucleotide sequence may be from any suitable source. Due to codon usage, bacterial IPT genes are preferred, because nuclear genes may not be expressed to maximum levels in mitochondria.
  • the IPT nucleotide sequence is from Agrobacteriwn tumefaciens or a plant (e.g. from the plant which is being transformed).
  • the or a plant-hormone biosynthetic polypeptide is iaaH (indoleacetamide hydrolase) and/or iaaM (tryptophan mono-oxygenase), which are enzymes involved in auxin biosynthesis.
  • the nucleotide sequences may be from any source. Due to codon usage, bacterial iaaH and/or iaaM genes are preferred.
  • the iaaH and/or iaaM nucleotide sequences are from Agrobacteriwn tumefaciens or a plant (e.g. from the plant which is being transformed).
  • the or a plant-hormone biosynthetic polypeptide is iaaH (indoleacetamide hydrolase) and/or iaaM (tryptophan
  • the transformed plants may be selected on media lacking auxins.
  • auxins include 4-chloro indoleacetic acid, phenyl acetic acid (PAA) and indole-3-butyric acid (IBA).
  • the plant hormone biosynthetic enzymes are iaaH and iaaM, preferably from Agrobacteriwn tumefaciens.
  • the Excision Cassette terminator prevents the premature expression of the transgene(s) prior to the excision of the Excision Cassette. Any terminator can be used for this provided that it is recognised in the plant cell being transformed.
  • the terminator may be a plant terminator or a bacterial terminator, inter alia.
  • Suitable terminators include those of rrn, psbA, rbcL, T3, T7 and ATPase.
  • the preferred terminator is a T3 or T7 terminator.
  • the promoter and terminator used in the Excision Cassette do not both originate from the same mitochondrial gene.
  • transgene i.e. element (c)
  • the transgene may, for example, be a genomic DNA, cDNA or synthetic nucleic acid molecule coding for a peptide or polypeptide; a nucleic acid molecule encoding a mRNA, tRNA or ribozyme; or any other nucleic acid molecule.
  • transgenes include those coding for antibodies, antibiotics, herbicides, vaccine antigens, enzymes, enzyme inhibitors and design peptides.
  • Single or multiple antigens may be produced from viridae, bacteria, fungi or other pathogens.
  • the antigens may be expressed as single units or as multiple units of several antigens, e.g. for broad-spectrum vaccine development.
  • Enzymes may be produced for use in cosmetics (e.g. superoxide dismutase, peroxidase, etc.). Enzymes may also be produced for use in detergent compositions.
  • the invention particularly relates to the production of proteins/enzymes with specific activities, for example, immunostimulants to boost immune responses, such as interferons; and growth factors, e.g. transforming growth factor-beta (TGF-beta), bone morphogenic protein (BMP), neurotrophins (NGF, BDNF, NT3), fibroblast growth factor (FGF), proteolytic enzymes (papain, bromelain), and food supplement enzymes (protease, lipase, amylase, cellulase).
  • TGF-beta transforming growth factor-beta
  • BMP bone morphogenic protein
  • NGF neurotrophins
  • BDNF BDNF
  • NT3 fibroblast growth factor
  • FGF proteolytic enzymes
  • proteolytic enzymes papain, bromelain
  • food supplement enzymes protease, lipase, amylase, cellulase.
  • the invention also relates to the production or over-expression of proteins/
  • trans-mitochondrial plants that chelate iron (Fe) for mopping up excess metal in agriculturally important areas for future planting.
  • the invention further relates to the use of transgenes encoding polypeptides which modify fatty acid biosynthesis in mitochondria.
  • transgenes may be inserted in the Transformation Cassette.
  • the transgene sequences are contiguous.
  • the transgene sequence may additionally encode a protein purification tag fused to the polypeptide of interest.
  • protein purification tags include the N-terminal influenza haemagglutinin-HA-epitope (HA) and a sequence of six histidine amino acids (HIS 6).
  • the transgene is constitutively expressed independently of whether or not the Excision Cassette has been removed.
  • the transgene may be expressed from its own promoter. Such embodiments are useful if the plant does not show any detrimental effects of the transgene expression during regeneration.
  • the Transformation Cassette terminator terminates the expression of the transgene (s). Any terminator can be used for this provided that it is recognised in the plant cell being transformed.
  • the terminator may be a plant terminator or a bacterial terminator, inter alia.
  • Suitable terminators include TrbcL or TspbA polyA addition sequences and the ATPase terminator.
  • the preferred terminator is the psbA polyA addition sequence.
  • the elements are preferably operably linked in the order (a), (b), (c), (d).
  • the first and second site-specific recombination elements must flank the optional promoter (when present), the nucleotide sequence encoding a plant-hormone biosynthetic polypeptide and the terminator element.
  • the nucleotide sequence encoding a plant-hormone biosynthetic polypeptide and the terminator element will be downstream (i.e. 3') to the promoter of the Transformation Cassette and hence the latter promoter will be capable of driving expression of the plant-hormone biosynthetic polypeptide.
  • the Transformation Cassette the transformation
  • Cassette comprises:
  • a first site-specific recombination element (b3) one or more nucleotide sequences encoding one or more plant-hormone biosynthetic polypeptides, (b4) a second terminator element, (b5) a second site-specific recombination element, wherein the first and second site-specific recombination elements are capable of being recognised by a recombinase; (c) one or more transgenes, (d) a first terminator element, operably linked in the order specified above in a 5'-3' direction.
  • the first promoter is capable of driving expression of the nucleotide sequence encoding the plant-hormone biosynthetic polypeptides. After the removal of the Excision Cassette, the first promoter drives expression of the transgene(s).
  • the Excision Cassette will be in the reverse orientation compared to the first promoter, transgene(s) and first terminator element.
  • the expressed parts of the Excision Cassette will be present in the nucleotide strand which is complementary to that which codes for the first promoter, transgene(s) and first terminator element, and in the reverse direction.
  • the Excision Cassette will comprise a second promoter, capable of driving the expression of the nucleotide sequences encoding the plant-hormone biosynthetic polypeptides.
  • the Transformation Cassette comprises:
  • the second promoter drives expression of the nucleotide sequences encoding the plant-hormone biosynthetic polypeptides.
  • the first promoter drives expression of the transgene(s).
  • the Transformation Cassette is not restricted to the parts (a)-(d) specified herein. It may, for example, additionally comprise a 5'-UTR to increase the expression level of the transgene(s) and 3'-additional amino acids to increase protein stability.
  • the Transformation Cassette additionally comprises a second selectable marker gene, e.g. an antibiotic resistance gene, preferably a nucleotide sequence encoding spectinomycin adenyltransferase (e.g. the aadA gene). This polypeptide confers resistance to the antibiotic spectinomycin.
  • the nucleotide sequence enoding spectinomycin adenyltransferase may be placed downstream of one of the promoters in the Transformation Cassette. It may, for example, be placed downstream and operably linked to the first promoter; or downstream and operably linked to the nucleotide sequences encoding the plant-hormone biosynthetic polypeptides (e.g. IPT); or upstream and operably linked to the nucleotide sequences encoding the plant-hormone biosynthetic polypeptides (e.g. IPT).
  • the plant-hormone biosynthetic polypeptides e.g. IPT
  • upstream and operably linked to the nucleotide sequences encoding the plant-hormone biosynthetic polypeptides e.g. IPT
  • the Transformation Cassette excludes a second selectable marker gene and/or excludes a nucleotide sequence which confers resistance to an antibiotic.
  • the Transformation Cassette additionally comprises the Lad gene, preferably under control of an appropriate promoter (e.g. T7), in order to allow inducible expression.
  • the promoter(s) and/or terminator(s) used in the Transformation Cassette are not from mitochondrial genes. This avoids/minimises vector rearrangement.
  • transformed cells are selected on media lacking the plant- hormone biosynthetic polypeptide.
  • plants are regenerated by adding cytokinin and auxin. Because the transformed mitochondria will preferably produce IPT and therefore cytokinin, plants can be selected and regenerated in the presence of auxin only.
  • the cells for selection will preferably be leaf cells.
  • plants are selected in the presence of cytokinin inhibitors (e.g. cytokinin oxidase or chemical inhibitors) in order to minimise leakage of the cytokinin from the site of biosynthesis to other parts of the plant. This reduced mosaicism in the plant.
  • the method additionally comprises the step:
  • a recombinase in the plant cell, wherein the recombinase is one which recognises the first and second site-specific recombination elements.
  • the recombinase is a site-specific recombinase.
  • a site-specific recombinase is a polypeptide which is capable of binding to site-specific recombination elements and inducing a cross-over event in the nucleic acid molecule in the vicinity of the site-specific recombination elements.
  • the expression of the recombinase leads to the excision of the Excision Cassette from the mitochondrial genome.
  • the recombinase is one which is capable of binding to the first and second site-specific recombination elements which are present in the Excision Cassette, leading to the excision of the Excision Cassette in a standard manner.
  • site-specific recombination elements/site-specific recombinases include Cre-lox, the FLP-FRP system from Saccharomyces cerevisae (O'Gorman S, Fox DT, Wahl GM. (1991) Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science. 25, 1351-1555.), the GIN/gix system from bacteriophage Mu (Maeser S and Kahmann R.
  • the Gin recombinase of phage Mu can catalyse site- specific recombination in plant protoplasts. MoI Gen Genet. 230, 170-176.) or the R/RS system from Zygosaccharomyces rouxii (Onouchi H, Yokoi K, Machida C, Matsuzaki H, Oshima Y, Matsuoka K, Nakamura K, Machida Y. (1991) Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells. Nucleic Acids Res. 19, 6373-
  • the nucleotide sequence which codes for the recombinase may comprise an intron, preferably a plant-specific intron.
  • an intron preferably a plant-specific intron. The presence of such an intron will suppress the expression of the recombinase polypeptide in prokaryotes, for example bacteria.
  • the preferred recombination site is lox in combination with the recombinase Cre.
  • the recombinase sequence used is a cDNA sequence encoding a Cre polypeptide.
  • Excision Cassettes are excised from the mitochondrial genome by the recombinase.
  • the skilled person will understand, however, that some or all of the sequences of one or more recombination elements might remain in the mitochondrial genome.
  • the recombinase may be expressed in the cell by any suitable means.
  • the plant cell is one which already comprises an expressible construct which is integrated into the nuclear genome, wherein the expressible construct comprises a nucleotide sequence encoding a mitochondria-targeting transit peptide and a recombinase (operably linked, i.e. in frame).
  • the expressible construct might, for example, have been introduced into the nuclear genome by homologous recombination. In such cases, the recombinase must be under the control of an inducible promoter.
  • an inducible promoter may have been introduced with the construct or the construct may have been integrated adjacent to an endogenous inducible promoter.
  • Plants containing nuclear-located sequences encoding recombinases may be removed from a desired population by crossing, wherein the sequences may be lost due to segregation of this trait.
  • the plant cell is one which already comprises an expressible construct which is integrated into the genome of the desired mitochondria, wherein the expressible construct comprises a nucleotide sequence encoding a recombinase.
  • the expressible construct might, for example, have been introduced into the mitochondrial genome by homologous recombination. In such cases, the recombinase must be under the control of an inducible promoter.
  • an inducible promoter may have been introduced with the construct or the construct may have been integrated adjacent to an endogenous inducible promoter.
  • step (iii) comprises: (iii) inducing the expression of a recombinase in the plant cell from an inducible promoter operably linked to a nucleotide sequence encoding a recombinase which is present in the plant cell, wherein the recombinase recognises the first and second site-specific recombination elements.
  • the inducible promoter operably linked to a nucleotide sequence encoding a recombinase is present in the nuclear genome of the plant cell.
  • the inducible promoter operably linked to a nucleotide sequence encoding a recombinase is. present in a mitochondrial genome of the plant cell.
  • the plant cell is transformed with a Recombinase Vector which comprises a promoter operably linked to a nucleotide sequence encoding a recombinase, either before step (i), simultaneously with step (i) or after step (i); or before step (ii), simultaneously with step (ii) or after step (ii).
  • a Recombinase Vector which comprises a promoter operably linked to a nucleotide sequence encoding a recombinase, either before step (i), simultaneously with step (i) or after step (i); or before step (ii), simultaneously with step (ii) or after step (ii).
  • the Recombinase Vector is a nucleic acid vector that comprises a promoter element that is capable of driving the expression of a downstream recombinase.
  • the vector is preferably designed such that the recombinase is either expressed only or substantially only in mitochondria or is targeted specifically or substantially specifically to mitochondria.
  • the promoter in the Recombinase Vector must be one that is operable in the plant cell which is to be transformed.
  • the promoter might, for example, be one derived from a plant or bacterial gene.
  • the promoter is plant-specific or mitochondria-specific.
  • the promoter is an inducible promoter such as XVE (Zuo J, Niu QW, Chua NH. (2000) Technical advance: An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J. 24,
  • plant-specific means plant-specific or substantially plant-specific.
  • mitochondria-specific means specific or substantially specific to mitochondria.
  • the promoter may or may not be an inducible promoter. If the Recombinase Vector is introduced to the plant cell before or during selection (step (ii)), it is preferable that the promoter is inducible. Examples of inducible promoters which are capable of operating in plants include light inducible promoters, metal inducible promoters, heat-shock promoters and other environmentally-inducible promoters.
  • the promoter is an inducible promoter, for example an XVE promoter (Zuo J, Niu QW, Chua NH. (2000) Technical advance: An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J. 24, 265-273.) or a lac promoter.
  • XVE promoter Zao J, Niu QW, Chua NH. (2000) Technical advance: An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J. 24, 265-273.
  • a lac promoter a lac promoter.
  • the Recombinase Vector comprises a promoter, operably linked to a nucleotide sequence encoding a mitochondria— targeting transit peptide and a recombinase.
  • a polypeptide product Upon expression, a polypeptide product will be produced comprising a mitochondria-targeting transit peptide operably linked to a recombinase polypeptide.
  • the promoter may or may not be mitochondria-specific.
  • mitochondria-targeting transit peptide means a peptide sequence which is capable of targeting the recombinase polypeptide to a mitochondria in a specific or substantially specific manner. Upon expression, the recombinase polypeptide will be produced and specifically imported into mitochondria by means of the mitochondria-targeting peptide.
  • mitochondria-targeting transit peptides examples include mitochondria-targeting transit peptides from mitochondria-targeted proteins.
  • Specific mitochondrial targeting transit peptides include: 1) 28 amino acid mt targeting peptide of mtSSB
  • the Recombinase Vector comprises an XVE promoter, operably linked to a nucleotide sequence encoding a mitochondria- targeting transit peptide and CRE recombinase.
  • the Recombinase Vector may also comprise other elements, for example, the nptll gene (kanamycin resistance) to allow for selection of transformed cells.
  • the nptll gene kanamycin resistance
  • step (iii) comprises: (iii) transforming the plant cell with a Recombinase Vector comprising a promoter, a nucleotide sequence encoding a mitochondria -targeting transit peptide and a recombinase, wherein the recombinase is one which recognises the first and second site-specific recombination elements.
  • step (iii) of the invention comprises: (iii) transforming the plant cell with a Recombinase Vector comprising a promoter, a nucleotide sequence encoding a mitochondria-targeting transit peptide and a recombinase, wherein the promoter is capable of driving the expression of the nucleotide sequence encoding the mitochondria-targeting transit peptide and the recombinase in the plant cell, and wherein, upon expression in the plant cell, the recombinase polypeptide is targeted by the transit peptide to the mitochondria. More preferably, the promoter is an inducible promoter.
  • the genetic construct additionally comprises the Recombinase Vector as defined herein.
  • the person skilled in the art will be aware of numerous methods for transforming plant cells with nucleic acid vectors. These include direct DNA uptake into protoplasts, PEG-mediated uptake to protoplasts, microparticle bombardment, electroporation, heat shock, micro-injection of DNA, micro- particle bombardment of tissue explants or cells, vacuum-infiltration of plant tissues, and T-DNA mediated transformation of plant tissues by Agrobacterium, and plant (preferably tobacco) liquid cultures.
  • any such suitable method may be used.
  • the plant cells to be transformed are guard cells, i.e. stomatal guard cells. Such cells have been shown to be totipotent and therefore regeneration will be more efficient.
  • Guard cells may be used as epidermal strips or as isolated guard cell protoplasts. (Hall et al. 1996. 112 889-892, Plant Physiology; Hall et al. 1996, 14. 1133-1138, Nature Biotechnology).
  • biolistic transformation is preferred. This involves shooting nucleic acid vector-coated gold particles (micro-projectiles) into mitochondria of plant tissues, followed by selection of the transformed mitochondria and plant regeneration.
  • the plant tissue is a plant leaf, although callus, as for rice transformation, may also be used.
  • the method of the invention preferably also comprises the additional step of inducing the expression of the recombinase in the plant cell.
  • This step will take place after the Recombinase Vector/expressible construct and Transformation Cassette are both present in the plant cell. Preferably, this step will take place after selection of the plant cells on media lacking the plant-hormone cytokinin.
  • the expression of the recombinase may be induced by applying an inducing agent which results in the activation of the promoter which is present in the Recombinase Vector or endogenous promoter or expressible construct.
  • the recombinase polypeptide is expressed and it then binds to the first and second site-specific recombination elements in the Excision Cassette, leading to the excision of that Cassette. (As will be understood by the person skilled in the art, one of the site-specific recombination elements and some adjacent sequence may be left in the mitochondria genome).
  • the promoter which is present in the Transformation Cassette will then be able to direct expression of the downstream transgene(s), thus producing the polypeptides (s) of interest.
  • plants are regenerated in the presence of cytokinin (shoot formation) and auxin (root formation).
  • cytokinin shoot formation
  • auxin root formation
  • appropriate cells/tissues e.g. leaf segments
  • auxin only for root regeneration
  • shoot regeneration will occur due to the presence of the IPT gene.
  • the invention also provides a method for making a transgene product, comprising the method for producing a transformed plant cell, as described hereinbefore, and additionally comprising purifying the transgene product from the mitochondria.
  • a particularly preferred embodiment of the invention includes a method for producing a transformed plant cell, the method comprising the step:
  • Transformation Cassette comprises:
  • a further particularly preferred embodiment of the invention includes a method for producing a transformed plant cell, the method comprising the step:
  • Transformation Cassette comprises:
  • a yet further particularly preferred embodiment of the invention includes a method for producing a transformed plant cell, the method comprising the step: (i) transforming the plant cell with a genetic construct, wherein the genetic construct comprises first and second homologous recombination elements flanking a Transformation Cassette, wherein the first and second homologous recombination elements are capable of directing the integration of the Transformation Cassette into the genome of at least one mitochondria which is present in the plant cell, wherein the Transformation Cassette comprises:
  • a first site-specific recombination element (b2) an optional second promoter (b3) one or more nucleotide sequence encoding one or more plant- hormone biosynthetic polypeptides, wherein one or more of the polypeptides is IPT, (b4) a second terminator element, (b5) a second site-specific recombination element, wherein the first and second site-specific recombination elements are capable of being recognised by a recombinase and wherein the first and second site- specific recombination elements are lox elements and the recombinase is Cre.
  • Yet a further particularly preferred embodiment provides a method for producing a transformed plant cell, the method comprising the steps: (i) transforming the plant cell with a genetic construct, wherein the genetic construct comprises first and second homologous recombination elements flanking a Transformation Cassette, wherein the first and second homologous recombination elements are capable of directing the integration of the Transformation Cassette into the genome of at least one mitochondria which is present in the plant cell, wherein the Transformation Cassette comprises:
  • Excision Cassette comprises:
  • a second site-specific recombination element wherein the first and second site-specific recombination elements are capable of being recognised by a recombinase and wherein the first and second site- specific recombination elements are lox elements and the recombinase is Cre, (ii) selecting for transformed plant cells on media which is lacking IPT; and (iii) expressing a Cre recombinase in the plant cell at a level which results in excision of the Excision Cassette from the mitochondrial genome.
  • the invention also provides a Transformation Cassette as herein defined, and a Recombinase Vector as herein defined.
  • the invention further provides a plant cell comprising a
  • Transformation Cassette of the invention a plant cell comprising a Recombinase Vector of the invention, and a plant cell comprising a Transformation Cassette and a Recombinase Vector of the invention.
  • the invention further provides a transgenic plant comprising a Transformation Cassette of the invention, a transgenic plant comprising a Recombinase Vector of the invention, and a transgenic plant comprising a Transformation Cassette and a Recombinase Vector of the invention.
  • the invention further provides a plant seed comprising a Transformation Cassette of the invention, a plant seed comprising a Recombinase Vector of the invention, and a plant seed comprising a
  • the invention further provides a plant mitochondria comprising a Transformation Cassette of the invention, a plant mitochondria comprising a Recombinase Vector of the invention, and a plant mitochondria comprising a Transformation Cassette and a Recombinase Vector of the invention.
  • the invention provides a plant cell obtainable or obtained using a method of the invention.
  • FIG. 1 Schematic diagram showing the overall principle of the
  • IPT gene excision and transgene activation IPT gene excision and transgene activation.
  • pPTI001-YFP containing the IPT gene sandwiched between two lox sites which allows for CRE-mediated IPT excision after regeneration.
  • the removal of the IPT gene results in simultaneous transgene (YFP) activation.
  • HOMl Left homologous region, 72001-72501, Nicotiana tobacum mitochondrial genome DNA, GenBank BA000042; HOM2: right homologous region, 72502-73000; Nicotiana tobacum mitochondrial genome DNA, GenBank BA000042.
  • P ⁇ 7 T7 promoter; aadA: aminoglycoside adenine transferase CDS; IPT: Tn 5 T3 terminator; T ⁇ 7 ⁇ T7 terminator.
  • IPT isopentenyltranferase gene from Agrobacterium tumefaciens. aadA and IPT are expressed as separate transgenes.
  • HOMl Left homologous region, 72001-72501, Nicotiana tobacum mitochondrial genome DNA, GenBank BA000042; HOM2: right homologous region, 72502-73000; Nicotiana tobacum mitochondrial genome
  • T 7 T7 promoter
  • aadA aminoglycoside adenine transferase CDS
  • IPT T ⁇ 3 5 T3 terminator
  • T ⁇ 7 T7 terminator
  • IPT isopentenyltranferase gene from Agrobacterium tumefaciens. IPT and aadA are expressed as an operon or as a fusion protein.
  • FIG. 4 Mitochondrial Transformation Vector pMTAOOl HOMl: Left homologous region, 72001-72501, Nicotiana tobacum mitochondrial genome DNA, GenBank BA000042; HOM2: right homologous region, 72502-73000; Nicotiana tobacum mitochondrial genome DNA, GenBank BA000042.
  • iaaH and iaaM are expressed as an operon or as a fusion protein.
  • HOMl Left homologous region, 72001-72501, Nicotiana tobacum mitochondrial genome DNA, GenBank BA000042; HOM2: right homologous region, 72502-73000; Nicotiana tobacum mitochondrial genome DNA 3 GenBank BA000042.
  • P ⁇ 7 T7 promoter; aadA: aminoglycoside adenine transferase CDS; iaaH: indoleacetamide hydrolase from Agrobacte ⁇ um tumefaciens; iaaM: tryptophan monooxygenase from Agrobacte ⁇ um tumefaciens; T ⁇ 3, T3 terminator; Tn: T7 terminator.
  • iaaH and iaaM are expressed as as an operon or as a fusion protein.
  • FIG. 6 Mitochondrial Transformation Vector pMTA003 HOMl: Left homologous region, 72001-72501, Nicotiana tobacum mitochondrial genome DNA, GenBankBA000042; HOM2: right homologous region, 72502-73000; Nicotiana tobacum mitochondrial genome DNA, GenBank BA000042.
  • PT 7 T7 promoter; aadA: aminoglycoside adenine transferase CDS; iaaH: indoleacetamide hydrolase from Agrobacte ⁇ um tumefaciens; iaaM: tryptophan monooxygenase from Agrobacte ⁇ um tumefaciens; Tn, T3 terminator; Tn: T7 terminator.
  • aadA, iaaH and iaaM are expressed as an operon or as a fusion protein.
  • the mitochondria transformation vectors pPMTIOO 1 , pMTI002 and pMTA001-pMTA003 are constructed as detailed in Figures 2-6 using a 500 bp homologous recombination sequence (72001-72501 nt) and a 498 bp homologous recombination sequence (72502-73000 nt) from the mitochondrial genome from Nicotiana tabacum on either side of the gene cassette. Shorter or longer homologous recombination sequences are also used to ensure appropriate homologous recombination events.
  • the expression of high levels of foreign protein in plants can lead to detrimental effects on plant development because of toxic effects.
  • the described system overcomes this by combining insertion of the transgene(s) into the mitochondrial genome where it remains dormant until the cytokinin or the auxin selectable marker genes are removed by CRE/lox mediated recombination.
  • the transgene encoding the "plant-toxic" protein is inserted into one of the pPMTIOOl, pMTI002 and pMTA001-pMTA003 vectors ( Figures 2- 6) and the construct transformed into mitochondria using the protocol shown in the Appendices followed by cytokinin-, auxin- and/or antibioltics-mediated selection and regeneration. Once regenerated, the cytokinin and/or auxin biosynthetic genes are removed by CRE-mediated recombination. Once expressed, the transmitomic plants are used conferring a desired trait or the recombinant protein is purified.
  • Mitochondria play an integral role during plant development and it may therefore be of interest to express proteins (plant or non-plant) inside mitochondria that would have a positive effect on plant growth, development and/or confer modified characteristics to the plant as whole.
  • the transgene encoding the mitochondrial protein is inserted into one of the pPMTIOOl, pMTI002 and pMTA001-pMTA003 vectors ( Figures 2- 6) and the constructs are transformed into mitochondria using the protocol shown in the Appendices followed by cytokinin-, auxin- and/or antibioltics- mediated selection and regeneration. Once regenerated, the cytokinin and/or auxin biosynthetic genes are removed by CRE-mediated recombination. Once expressed, the transmitomic plants are used conferring a desired trait or the recombinant protein is purified.
  • the present system can be used for the expression of eukaryotic proteins in mitochondria using IPT, iaaH and/or iaaM marker gene selection.
  • IPT IPT
  • iaaH iaaM marker gene selection
  • any gene encoding a eukaryotic protein may be inserted into one or all of the pPMTIOOl, pMTI002 and pMTA001-pMTA003 vectors followed by transformation, selection and regeneration as described previously. Following regeneration, the expressed protein may be purified and used for downstream applications.
  • a non-exclusive list of possible eukaryotic protein families that will be expressed includes antibodies, enzymes, enzyme inhibitors and design peptides.
  • any gene encoding a prokaryotic protein may be inserted into one or all of the pPMTIOOl, pMTI002 and pMTA001-pMTA003 vectors followed by transformation, selection and regeneration as described previously. Following regeneration, the expressed protein may be purified and used for downstream applications.
  • Level of macrocarrier launch assembly 1 (from top) • Level of Petri dish holder: 4 (from top)
  • Vacuum release rate Attenuate the release so it approximates the speed of vacuum inflow.
  • the key to successful bombardment is usually in the spread of particles on the macrocarrier.
  • the ethanol/gold/DNA mixture should quickly spread out over the centre of the macrocarrier.
  • the resulting spread should be a very fine dusting of particles, evenly spread and containing few chunks. Chunk causes increased cell death.
  • Tobacco plants are micropropagated using sterile technique in magenta boxes containing MS media. • Expanded leaves are excised and placed abaxial surface up on a
  • Leaves from these shoots are cut up (2mm square) and subcultured in the same selective media for approx. 4 weeks.
  • Rooted shoots are transferred to soil approx. 3-5 weeks after isolation. Plants are allowed to grow in standard tobacco conditions (16:8 photoperiod at 25°C).
  • Tobacco leaves do not have to lay completely flat for bombardment. 2. Tobacco leaves will lose their turgor after 2 days on filter paper. This is OK.
  • Transformation frequency for an average experiment is anywhere from 1.5 stably transformed plants to 0.3 stably transformed plants per bombardment.
  • MFB media for bombardement
  • MTS media for transgenic selection
  • This time includes second selection time
  • Vacuum release rate Attenuate the release so it approximates the speed of vaccum inflow.
  • the key to successful bombardment is usually in the spread of particles on the macrocarrier.
  • the ethanol/gold/DNA mixture should quickly spread out over the centre of the macrocarrier.
  • the resulting spread should be a very fine dusting of particles, evenly spread and containing few chunks. And chunk causes cell death.
  • Tobacco plants are micropropagated using sterile technique in magenta boxes containing MS media
  • Green shoots can be collected from the bleached explants in 3-8 weeks.
  • Leaves from these shoots are cut up (2mm square) and subcultured in the same selective media for approx. 4 weeks. • Typically 4 shoots are collected per initial subcultured shoots. These are rooted in tubes containing MS media + lmg/1 IBA and 2 ⁇ M 17- ⁇ - estradiol. • Rooted shoots are transferred to soil appox. 3-5 weeks after isolation.
  • Plants are allowed to grow in standard tobacco conditions (16:8 photoperiod at 25°C).
  • Transformation frequency for an average experiment is anywhere from 1.5 stably transformed plants to 0.3 stably transformed plants per bombardment.
  • MTS media for transgenic selection
  • This time includes second selection time
  • Vacuum release rate Attenuate the release so it approximates the speed of vaccum inflow.
  • the key to successful bombardment is usually in the spread of particles on the macrocarrier.
  • the ethanol/gold/DNA mixture should quickly spread out over the centre of the macrocarrier.
  • the resulting spread should be a very fine dusting of particles, evenly spread and containing few chunks. And chunk causes cell death.
  • MS MS salts and vitamins (IX) 30 g/1 sucrose
  • TISSUE CULTURE • Tobacco plants are micropropagated using sterile technique in magenta boxes containing MS media
  • Expanded leaves are excised and placed abaxial surface up on a Whatman filter paper laying on top of RMOP media.
  • the leaves are allowed to desiccate slightly (1-2 hours) prior to bombardment on the filter paper.
  • Each bombardment can treat 1-3 leaves covering approx.
  • Green shoots can be collected from the bleached explants in 3-8 weeks.
  • Leaves from these shoots are cut up (2mm square) and subcultured in the same selective media for approx. 4 weeks. • Typically 4 shoots are collected per initial subcultured shoots. These are rooted in tubes containing MS media + lmg/1 IBA and 2 ⁇ M 17- ⁇ - estradiol. Rooted shoots are transferred to soil appox. 3-5 weeks after isolation. Plants are allowed to grow in standard tobacco conditions (16:8 photoperiod at 25 0 C).
  • Transformation frequency for an average experiment is anywhere from 1.5 stably transformed plants to 0.3 stably transformed plants per bombardment.
  • MTS media for transgenic selection
  • This time includes second selection time
  • Vacuum release rate Attenuate the release so it approximates the speed of vaccum inflow.
  • BOMBARDMENT Snap the macrocarriers into their holders.
  • Tobacco plants are micropropagated using sterile technique in magenta boxes containing MS media
  • Expanded leaves are excised and placed abaxial surface up on a Whatman filter paper laying on top of RMOP media.
  • the leaves are allowed to desiccate slightly (1-2 hours) prior to bombardment on the filter paper.
  • Each bombardment can treat 1-3 leaves covering approx.
  • Green shoots can be collected from the bleached explants in 3-8 weeks.
  • Leaves from these shoots are cut up (2mm square) and subcultured in the same selective media for approx. 4 weeks. Typically 4 shoots are collected per initial subcultured shoots. These are rooted in tubes containing MS media + lmg/1 IBA and 2 ⁇ M 17- ⁇ - estradiol.
  • Rooted shoots are transferred to soil appox. 3-5 weeks after isolation. Plants are allowed to grow in standard tobacco conditions (16:8 photoperiod at 25 0 C).
  • Tobacco leaves do not have to lay completely flat for bombardment. 2. Tobacco leaves will lose their turgor after 2 days on filter paper. This is OK.
  • Transformation frequency for an average experiment is anywhere from 1.5 stably transformed plants to 0.3 stably transformed plants per bombardment.
  • MTS media for transgenic selection
  • This time includes second selection time

Abstract

L’invention concerne un procédé de fabrication d’une cellule de plante transformée. Plus particulièrement, le procédé comprend la transformation d’une cellule de plante avec une cassette de transformation qui cible les mitochondries des plantes et qui comprend un gène de sélection, par exemple isopentényle transférase (IPT), et un transgène. Après la sélection des mitochondries transformées, l’expression d’une recombinase est induite dans la cellule de plante, ce qui conduit à l’excision du gène de sélection de la mitochondrie et à l’expression du transgène dans la mitochondrie. L’invention concerne également des cellules et des plantes qui comprennent la cassette de transformation.
PCT/GB2009/001510 2008-06-13 2009-06-12 Transformation mitochondriale WO2009150441A1 (fr)

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RU2660715C2 (ru) * 2016-06-09 2018-07-09 Федеральное государственное учреждение "Федеральный исследовательский центр "Фундаментальные основы биотехнологии" Российской академии наук" (ФИЦ Биотехнологии РАН) Способ репликации человеческого митохондриального генома в клетках дрожжей Yarrowia lipolytica

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Publication number Priority date Publication date Assignee Title
US9986736B2 (en) 2016-03-11 2018-06-05 Timothy ST. GERMAIN Herbicide compositions and methods for controlling growth of plants of the buckthorn family
RU2660715C2 (ru) * 2016-06-09 2018-07-09 Федеральное государственное учреждение "Федеральный исследовательский центр "Фундаментальные основы биотехнологии" Российской академии наук" (ФИЦ Биотехнологии РАН) Способ репликации человеческого митохондриального генома в клетках дрожжей Yarrowia lipolytica

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