Patent Application
20110162114
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 plastids and which comprises a selection gene, for example isopentenyl transferase (IPT), and a transgene. After selection for transformed plastids, expression of a recombinase is induced in the plant cell, which leads to the excision of the selection gene from the plastid and the expression of the transgene in the plastid. The invention also provides cells and plants comprising the Transformation Cassette.
The use of genetically modified (GM) food crops in agriculture is rapidly increasing with an approximate £14 billion world market in 2005. There is, however, a growing concern amongst scientists, politicians, regulatory agencies and the general public regarding the risk of the spread of transgenes by pollen from GM crops to other plant species generating so-called “superweeds”.
All commercial GM crops used in today's agriculture have engineered nuclear genomes containing transgenes conferring desirable traits such as resistance to disease, insects, harsh environmental conditions and increased vitamin content, flavour, and storage time. However, there are several serious problems with nuclear transgenes, including gene silencing and low levels of transgene expression. One way of resolving the problems associated with nuclear transgenes is to insert transgenes into the plastid genome of plants.
Plastids are organelles unique to plants. Each plant cell contains approximately 100 plastids each of which contains approximately 100 genomes. This in effect means that when a gene is inserted into the plastid genome each plant cell contains approximately 10,000 copies of that gene compared to plant nuclear transformation where each plant cell would have at best 2-3 copies of the gene.
The expression of transgenes in plastids has numerous advantages over nuclear gene expression: (i) transgenes are not spread by pollen; (ii) proteins are expressed to high levels (up to 47% of total cellular protein); (iii) “toxic” effects of proteins are reduced due to plastid containment; (iv) the simultaneous expression of several genes allows for biochemical pathway engineering; and (v) gene silencing is eliminated. Plastid genetic engineering has clear fundamental and applied applications in that potentially any protein can be produced to high levels with little environmental risk opening up the possibility of producing edible vaccines, biopharmaceuticals and products of agronomical value.
Currently, the only reliable selection systems that are used to select for plastids, which have been transformed with transgenes, are based on the use of antibiotics such as spectinomycin. A disadvantage of such selection systems is that they will also select for spontaneous ribosomal mutants which are resistant to spectinomycin. This is a large problem, given the time-scales involved.
The invention is based on a selection and regeneration system for plastid transformation based on the over-expression of a gene such as the isopentenyl transferase (IPT) gene (cytokinin biosynthesis) in plastids. This system allows for the direct selection of cells containing transformed plastids on media lacking cytokinin due to cytokinin production within plastids. The system therefore provides an antibiotics-free selection and regeneration system which will overcome problems with spontaneous spectinomycin mutants and concerns regarding the use of antibiotic resistance genes in GMOs.
Whilst IPT has previously been used as a selectable marker in plant nuclear transformation (e.g. EP 1 069 855 A), its use in plastid transformation has not previously been suggested. The person skilled in the art will be aware of the fact that plastids are semi-autonomous organelles within plant cells with their own genomes and metabolism. In particular, it has not previously been shown that IPT precursors are made in plastids or that any IPT produced is capable of diffusing out of the plastid into the cell cytoplasm in order to initiate cytokinin signalling to stimulate shoot regeneration. Hence the criteria for choosing plastid-selection genes are distinct from those for choosing genome-selection genes.
Due to the fact that over-expression of plant-hormone biosynthetic polypeptides in adult plants leads to impaired development, 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.
In one embodiment, 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 plastid which is present in the plant cell,
wherein the Transformation Cassette comprises:
In some embodiments, the method additionally comprises the step:
(ii) selecting for transformed plant cells on media which lacks one or more of the plant-hormone biosynthetic polypeptides or antibiotic.
In other embodiments, the method additionally comprises the step:
(iii) expressing a recombinase in the plant cell, wherein the recombinase is one which recognises the first and second site-specific recombination elements.
The method of the invention is suitable for all plants that can be transformed and regenerated. The plant may be a monocot or dicot.
Examples of 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. Preferably, 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. Preferably, the cells are present in leaves that are removed from intact plants. It is preferable to use actively-growing leaves.
The term “plastid” is intended to cover all organelles which are found in the cytoplasm of eukaryotic plants, which contain DNA, which are bounded by a double membrane, and develop from a common type, i.e. a proplastid. Plastids may contain pigments and/or storage materials.
Examples of plastids include chloroplasts, leucoplasts, amyloplasts, etioplasts, chromoplasts, elaioplasts and gerontoplasts. Preferably, the plastid is a green plastid, most preferably a chloroplast.
As used herein, 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. Generally, 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 plastid which is present in the plant cell.
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 plastid or plastids, resulting in the insertion of the Transformation Cassette into the genome of the selected plastid or plastids.
The nucleotide sequences of the homologous recombination elements are selected such that the Transformation Cassette is specifically targeted to one or more selected plastids. In particular, 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. In other words, the nucleotide sequences of the homologous recombination elements are preferably plastid-specific, i.e. corresponding sequences are not present in the nuclear genome and preferably not present in the mitochondrial 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 mitochondrial genome. The skilled person will readily be able to detect whether a specific sequence is or is not present in the nuclear genome by standard means, for example, by Southern Blotting of the nuclear genome with a labelled sequence probe or by sequence analysis.
Apart from the above, any sequences can be used from the plastid 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. Preferably, the insertion sites are not in coding regions of plastid genes.
The orientation of the sequences of the first and second homologous recombination elements should be the same as the orientation in the plastid genome to allow for efficient homologous recombination.
In order to target the Transformation Cassette to the plastid genome, the 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 plastid.
In the context of the present invention, the term “substantially identical” means that the nucleotide sequences of the first and second homologous recombination sequences are independently more than 95%, preferably more than 98% or more than 99% and particularly preferably 100% identical to sequences which are present in the plastid to be transformed. Percentage sequence identities may be determined using the Clustal method of alignment with default parameters, e.g. KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
Similarly, the 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. In this context, the term “substantially identical” means that the nucleotide sequences of the first and second homologous recombination sequences are independently less than 50%, more preferably less than 70% or less than 90% identical to sequences which are present in the nuclear genome of the plant to be transformed. Percentage sequence identities may be determined using the Clustal method of alignment with default parameters, e.g. KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
Preferably, the lengths of 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 plastid 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 (i.e. genetic elements (a)-(d)) is preferably less than 4000 nucleotides.
Preferably, the first homologous recombination sequence is nucleotides 104091-105380 of the Nicotiana tabacum (accession no. Z00044) chloroplast genome DNA; and/or preferably, the second homologous recombination sequence is nucleotides 105381-106370 of the Nicotiana tabacum (accession no. Z00044) chloroplast genome DNA.
In yet other embodiments, the first homologous recombination sequence is preferably nucleotides 102925-101857 of the Nicotiana tabacum (accession no. Z00044) choloroplast genome DNA; and/or the second homologous recombination sequence is nucleotides 100933-100130 of the Nicotiana tabacum (accession no. Z00044) choloroplast genome DNA.
The Transformation Cassette promoter (a) must be one that is operable in the selected plant plastid. 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. Preferably, the promoter is plant specific.
Examples of suitable promoters include PpsbA, RbcL and Prrn promoters.
Preferably, the promoter is a Prrn promoter (e.g. Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
Other preferred promoters include the Prrn-rbcl-ds promoter construct as shown in
In some embodiments, the promoter is an inducible promoter. This allows inducible, controlled expression of the selection gene(s). For example, 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. Alternatively, 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. elements (b2) (if used), (b3), (b4), any other desired elements. The sequences of the 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′).
When the recombinase is Cre, 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, Niu Q W, Møller S G, Chua N H (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 plastid. 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. Preferably, the promoter is plant specific. Examples of suitable promoters include PsbA, RbcL and Prrn promoters. Preferably, the promoter is a Prrn promoter (e.g. Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
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.
Preferably, 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. Alternatively, the plant-hormone biosynthetic polypeptide nucleotide sequences may each have their own promoters and terminator elements. A further option is that 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. a poly-glycine linker). In other embodiments, some of the 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.
In some embodiments, the or a plant-hormone biosynthetic polypeptide is IPT (isopentenyl transferase) which is an enzyme involved in cytokinin biosynthesis. 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 chloroplasts.
Preferably, the IPT nucleotide sequence is from Agrobacterium tumefaciens or from a plant (e.g. from the plant which is being transformed).
In other embodiments, 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.
Preferably, the iaaH and/or iaaM nucleotide sequences are from Agrobacterium tumefaciens.
In other embodiments of the invention where auxin biosynthetic enzymes are involved, the transformed plants may be selected on media lacking auxins. Examples of naturally-occurring auxins include 4-chloro indoleacetic acid, phenyl acetic acid (PAA) and indole-3-butyric acid (IBA).
In some preferred embodiments of the invention, the plant hormone biosynthetic enzymes are iaaH and iaaM, preferably from Agrobacterium 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.
Examples of suitable terminators include those of rrn, psbA, rbcL and T7.
The preferred terminator is a TrbcL terminator (e.g. Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
In some embodiments of the invention, the promoter and terminator used in the Excision Cassette do not both originate from the same plastid gene.
In the context of the present invention, the term “transgene” (i.e. element (c)) is used to refer to a nucleic acid molecule which is being introduced into the genome of the plastid. 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.
Examples of 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).
The invention also relates to the production or overexpression of proteins/enzymes in plastids that make the plants more resistant to biotic and abiotic stress, such as salts and metals. Examples of this include the generation of transplastomic 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 plastids.
One or more transgenes may be inserted in the Transformation Cassette. Preferably, the transgene sequences are contiguous.
The transgene sequence may additionally encode a protein purification tag fused to the polypeptide of interest. Examples of protein purification tags include the N-terminal influenza haemagglutinin-HA-epitope (HA) and a sequence of six histidine amino acids (HIS6) and the Strep tag. Each of the transgene products may have a different affinity tag.
Once the Excision Cassette has been excised, the first promoter is capable of driving the expression of the transgene, leading to the accumulation of the product of the transgene in the plastid. The product of the transgene may be purified or isolated from the plant cell by any, suitable means.
In some embodiments of the invention, the transgene is constitutively expressed independently of whether or not the Excision Cassette has been removed. In such embodiments, 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 (genetic element (d)) 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.
Examples of suitable terminators include TrbcL or TspbA polyA addition sequences. The preferred terminator is the psbA polyA addition sequence.
In the Transformation Cassette, the elements are preferably operably linked in the order (a), (b), (c), (d).
In the Excision Cassette, 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.
In some embodiments of the invention, 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.
Thus in some embodiments of the invention, the Transformation Cassette comprises:
(a) a first promoter which is operable in said plant cell,
(b) an Excision Cassette comprising:
(c) one or more transgenes,
(d) a first terminator element,
operably linked in the order specified above in a 5′-3′ direction.
In this embodiment of the invention, the first promoter is capable of driving expression of the nucleotide sequence encoding the plant-hormone biosynthetic polypeptides (for example, as shown in
In other embodiments of the invention, the Excision Cassette will be in the reverse orientation compared to the first promoter, transgene(s) and first terminator element. In this embodiment, 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.
In such embodiments, the Excision Cassette will comprise a second promoter, capable of driving the expression of the nucleotide sequences encoding the plant-hormone biosynthetic polypeptides.
In this embodiment of the invention, the Transformation Cassette comprises:
(a) a first promoter which is operable in said plant cell,
(b) an Excision Cassette,
(c) one or more transgenes,
(d) a first terminator element,
wherein (a), (b), (c) and (d) are operably linked in the order specified above in a 5′-3′ direction,
wherein the Excision Cassette comprises:
In this embodiment, the second promoter drives expression of the nucleotide sequences encoding the plant-hormone biosynthetic polypeptides (for example, as shown in
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/or 3′-additional amino acids to increase protein stability.
In some embodiments of the invention, 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).
In yet other embodiments, the Transformation Cassette excludes a second selectable marker gene and/or excludes a nucleotide sequence which confers resistance to an antibiotic.
In some embodiments, the Transformation Cassette additionally comprises the LacI gene, preferably under control of an appropriate promoter (e.g. T7), in order to allow for inducible expression.
After successful delivery of the Transformation Cassette to the plastid(s), transformed cells are selected on media lacking the plant-hormone biosynthetic polypeptide. In general, plants are regenerated by adding cytokinin and auxin. Because the transformed plastids 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.
In some embodiments of the invention, 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.
In some embodiments of the invention, the method additionally comprises the step:
(iii) expressing 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. In the present invention, the expression of the recombinase leads to the excision of the Excision Cassette from the plastid genome.
In the context of the present invention, 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.
Examples of site-specific recombination elements/site-specific recombinases include Cre-lox, the FLP-FRP system from Saccharomyces cerevisae (O'Gorman S, Fox D T, Wahl G M. (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. (1991) The Gin recombinase of phage Mu can catalyse site-specific recombination in plant protoplasts. Mol 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, 6373-6378.).
The nucleotide sequence which codes for the recombinase may comprise 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. Preferably, the recombinase sequence used is a cDNA sequence encoding a Cre polypeptide.
Preferably, all or substantially all of the Excision Cassettes are excised from the plastid 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 plastid genome. The recombinase may be expressed in the cell by any suitable means.
In some embodiments of the invention, 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 plastid-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. Such 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.
In other embodiments of the invention, the plant cell is one which already comprises an expressible construct which is integrated into the genome of the desired plastid(s), wherein the expressible construct comprises a nucleotide sequence encoding a recombinase. The expressible construct might, for example, have been introduced into the plastid genome by homologous recombination. In such cases, the recombinase must be under the control of an inducible promoter. Such an inducible promoter may have been introduced with the construct or the construct may have been integrated adjacent to an endogenous inducible promoter.
Thus in some embodiments of the invention, 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.
Preferably, the inducible promoter operably linked to a nucleotide sequence encoding a recombinase is present in the nuclear genome of the plant cell.
In other preferred embodiments, the inducible promoter operably linked to a nucleotide sequence encoding a recombinase is present in a plastid genome of the plant cell.
In yet other embodiments of the invention, 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).
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 plastids or is targeted specifically or substantially specifically to plastids.
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. Preferably, the promoter is plant-specific or plastid-specific. Most preferably, the promoter is an inducible promoter such as XVE (Zuo J, Niu Q W, Chua N H. (2000) Technical advance: An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J. 24, 265-273.).
In the context of the present invention, the term “plant-specific” means plant-specific or substantially plant-specific. Similarly, the term “plastid-specific” means specific or substantially specific to plastids.
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.
Preferably, the promoter is an inducible promoter, for example an XVE promoter (Zuo J, Niu Q W, Chua N H. (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.
In one embodiment of the invention, the Recombinase Vector comprises a promoter, operably linked to a nucleotide sequence encoding a plastid-targeting transit peptide and a recombinase.
Upon expression, a polypeptide product will be produced comprising a plastid-targeting transit peptide operably linked to a recombinase polypeptide. In this embodiment, the promoter may or may not be plastid-specific.
In the context of the present invention, the term “plastid-targeting transit peptide” means a peptide sequence which is capable of targeting the recombinase polypeptide to a plastid in a specific or substantially specific manner. Upon expression, the recombinase polypeptide will be produced and specifically imported into plastids by means of the plastid-targeting peptide.
Examples of plastid-targeting transit peptides include plastid-targeting transit peptides from plastid-targeted proteins.
Preferably, the plastid-targeting transit peptide is one which is capable of targeting the recombinase polypeptide to a chloroplast.
Most preferably, the plastid-targeting transit peptide is a plastid-targeting transit peptide from a stromal plastid targeted protein.
Specific examples of plastid-targeting transit peptides include: Transit peptide from AtABC 1 (Simon Geir Møller, Tim Kunkel and Nam-Hai Chua. (2001) “A plastidic ABC protein involved in intercompartmental communication of light signaling”, Genes and Dev. 15, 90-103.); from AtMinE 1 (Jodi Maple, Nam-Hai Chua and Simon Geir Møller (2002) “The topological specificity factor AtMinE 1 is required for correct plastid division site placement in Arabidopsis”, Plant J. 31, 269-277); and from GIANT CHLOROPLAST 1 (Jodi Maple, Makoto T. Fujiwara, Nobutaka Kitahata, Tracey Lawson, Neil Baker, Shigeo Yoshida and Simon Geir Møller (2004) “GIANT CHLOROPLAST 1 is essential for correct plastid division in Arabidopsis”. Current Biology. 14, 776-781)).
Most preferably, the Recombinase Vector comprises an XVE promoter, operably linked to a nucleotide sequence encoding a plastid-targeting transit peptide and CRE recombinase.
The Recombinase Vector may also comprise other elements, for example, the nptII gene (kanamycin resistance) to allow for selection of transformed cells.
Thus in some embodiments of the invention, step (iii) comprises:
(iii) transforming the plant cell with a Recombinase Vector comprising a promoter, a nucleotide sequence encoding a plastid-targeting transit peptide and a recombinase, wherein the recombinase is one which recognises the first and second site-specific recombination elements.
Particularly preferably, step (iii) of the invention comprises:
(iii) transforming the plant cell with a Recombinase Vector comprising a promoter, a nucleotide sequence encoding a plastid-targeting transit peptide and a recombinase, wherein the promoter is capable of driving the expression of the nucleotide sequence encoding the plastid-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 plastid. More preferably, the promoter is an inducible promoter.
In other embodiments of the invention, 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, microparticle 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.
For transformation of the plant cell containing the selected plastid, any such suitable method may be used.
In some embodiments of the invention, 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 should 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).
For targeting the genetic construct to plastids, biolistic transformation is preferred. This involves shooting nucleic acid vector-coated gold particles (micro-projectiles) into plastids of plant tissues, followed by selection of the transformed plastids and plant regeneration. Preferably, 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 plastid genome).
Once the nucleotide sequences encoding the plant-hormone biosynthetic polypeptides have been removed from the plastid 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.
In general, plants are regenerated in the presence of cytokinin (shoot formation) and auxin (root formation). In the case of regenerating plants containing the IPT gene (producing cytokinin), appropriate cells/tissues (e.g. leaf segments) will be placed on media containing auxin only (for root regeneration) whilst 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 plastids.
A 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 plastid which is present in the plant cell,
wherein the Transformation Cassette comprises:
(a) a first promoter which is operable in said plant cell,
(b) an Excision Cassette,
(c) one or more transgenes,
(d) a first terminator element,
wherein the Excision Cassette comprises:
A 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 plastid which is present in the plant cell,
wherein the Transformation Cassette comprises:
(b) an Excision Cassette,
(c) one or more transgenes,
(d) a first terminator element,
wherein the Excision 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 plastid which is present in the plant cell,
wherein the Transformation Cassette comprises:
(a) a first promoter which is operable in said plant cell,
(b) an Excision Cassette,
(c) one or more transgenes,
(d) a first terminator element,
wherein the Excision Cassette comprises:
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 plastid which is present in the plant cell,
wherein the Transformation Cassette comprises:
(a) a first promoter which is operable in said plant cell,
(b) an Excision Cassette,
(c) one or more transgenes,
(d) a first terminator element,
wherein the Excision Cassette comprises:
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 Transformation Cassette and a Recombinase Vector of the invention.
The invention further provides a plant plastid comprising a Transformation Cassette of the invention, a plant plastid comprising a Recombinase Vector of the invention, and a plant plastid comprising a Transformation Cassette and a Recombinase Vector of the invention.
Additionally, the invention provides a plant cell obtainable or obtained using a method of the invention.
pPT1001-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.
Bright field (A) and fluorescence (B) images of E. coli DH5α cells containing the pPT1001-YFP vector. Bright field (C) and fluorescence (D) images of E. coli DH5α cells containing both the pPT1001-YFP and pER10-TP.CRE vectors (
HOM1: Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacterium tumefaciens; TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); YFP: Yellow fluorescence protein; TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
(A) pPT1001a-YFP, pPT1001-YFP containing an N-terminal influenza hemagglutinin-HA-epitope tag (HA3).
(B) pPT1001b-YFP, pPT1001-YFP containing six N-terminal histidine amino acids (HIS6).
(C) pPTI001c-YFP, pPT1001-YFP containing a C-terminal influenza hemagglutinin-HA-epitope tag (HA3).
(D) pPT1001d-YFP, pPT1001-YFP containing six C-terminal histidine amino acids (HIS6).
To allow for the use of EcoRV as a cloning site downstream from YFP in (C) pPT1001c-YFP and (D) pPTI001d-YFP the IPT DNA sequence was modified changing adenine to guanidine at nucleotide position 519 (+1 taken as adenine in the start codon) thereby removing an endogenous EcoRV site.
HOM1: Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacterium tumefaciens; TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); YFP: Yellow fluorescence protein; TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
The construction of the vector series pPT1002 (see also
(A) pPT1002a-YFP, pPT1002-YFP containing an N-terminal influenza hemagglutinin-HA-epitope tag (HA).
(B) pPT1002b-YFP, pPT1002-YFP containing six N-terminal histidine amino acids (HIS6).
(C) pPT1002c-YFP, pPT1002-YFP containing a C-terminal influenza hemagglutinin-HA-epitope tag (HA).
(D) pPT1002d-YFP, pPT1002-YFP containing six C-terminal histidine amino acids (HIS6).
To allow for the use of EcoRV as a cloning site downstream from YFP in (C) pPT1002c-YFP and (D) pPT1002d-YFP, the IPT DNA sequence was modified changing adenine to guanidine at nucleotide position 519 (+1 taken as adenine in the start codon) thereby removing an endogenous EcoRV site.
XVE acts as the inducible promoter that drives TP-CRE (Transit peptide fused to CRE) expression. Selection of this transgene is by Kanamycin resistance conferred by the nptII gene.
A) First round of selection after plastid transformation showing the presence of new shoots in the absence of cytokinin.
B) Second round of selection from the primary transformant shown in A.
C) Regenerated transplastomic tobacco plant used for CRE infiltration and GFP activation (
Lanes shown: WT, wild-type; #1, regenerant 1; #2 regenerant 2; M, marker.
Regenerants transplastomic tobacco plant harbouring the pPT1001/YFP transformation cassette analysed by fluorescence microscopy after infiltration and induction of pER10/TP.CRE. Extended focus images of reconstituted YFP fluorophore (YFP) and chlorophyll autofluorescence (Chlorophyll) were captured by epifluorescence microscopy using Volocity II software. Scale bar=5 μm.
HOM1: Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); PpsbA: Plastidic psbA promoter; aadA: spectinomycin adenyltransferase gene; T7: T7 transcription terminator sequence: Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacterium tumefaciens; TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
HOM3: Homologous recombination sequence (nt 102925-101857, accession Z00044 Nicotiana tabacum chloroplast genome DNA); PpsbA: Plastidic psbA promoter; aadA: spectinomycin adenyltransferase gene; T7: T7 transcription terminator sequence: Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacterium tumefaciens; TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM4: Homologous recombination sequence (nt 100933-100130, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
HOM3: Homologous recombination sequence (nt 102925-101857, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacterium tumefaciens; aadA: spectinomycin adenyltransferase gene; TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM4: Homologous recombination sequence (nt 100933-100130, accession Z00044 Nicotiana tabacum chloroplast genome DNA). The IPT.aadA cassette is constructed as an operon. Both of the IPT and aadA genes are preceded by a shine delgardo sequence and a six base pair spacer.
HOM3: Homologous recombination sequence (nt 102925-101857, accession. Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacterium tumefaciens; aadA: spectinomycin adenyltransferase gene; TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM4: Homologous recombination sequence (nt 100933-100130, accession Z00044 Nicotiana tabacum chloroplast genome DNA). The IPT.aadA cassette is expressed as a fusion protein. The IPT a gene is preceded by a shine delgardo sequence and a six base pair spacer and the two open reading frames are separated by an eight glycine linker sequence.
Each pPTI vector contains a constitutively expressed LacI gene. pPT1003 is shown as an example. Modified Prrn promotors (PrrnL) will be inserted upstream of the IPT.aadA cassette, allowing inducible, controlled expression of the IPT.aadA cassette. (A) pind1PTI, pPTI containing the LacI open reading frame under the control of the Prrn promoter sequence and modified PrrnL promoter upstream of the IPT.aadA cassette. (B) pind2PTI, pPTI containing the LacI open reading frame under the control of the T7 promoter sequence and modified PrrnL promoter upstream of the IPT.aadA cassette. HOM1: Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacterium tumefaciens; aadA: spectinomycin adenyltransferase gene; TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA). The IPT aadA cassette is constructed as an operon. Both of the IPT and aadA genes are preceded by a shine delgardo sequence and a six base pair spacer.
Each pPTI vector will be modified to contain a modified Prrn-trbcL (Prrn promoter construct, composed of the Prrn promter and rbcL 5′ translation control region, to ultimately produce higher levels of expression of the foreign proteins. pPT1003 is shown as an example. HOM1: Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn-t: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA) and rbcL 5′ translation control region; IPT: isopentenyltranferase gene from Agrobacterium tumefaciens; aadA: spectinomycin adenyltransferase gene; TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA). The IPT.aadA cassette is constructed as an operon. Both of the IPT and aadA genes are preceded by a shine delgardo sequence and a six base pair spacer.
HOM1: Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacterium tumefaciens; aadA: spectinomycin adenyltransferase gene; TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA). The IPT.aadA cassette is constructed as an operon. Both of the IPT and aadA genes are preceded by a shine delgardo sequence and a six base pair spacer.
pPT1004
HOM1: Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacterium tumefaciens; aadA: spectinomycin adenyltransferase gene; TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA). The IPT.aadA cassette is expressed as a fusion protein. The IPT a gene is preceded by a shine delgardo sequence and a six base pair spacer and the two open reading frames are separated by an eight glycine linker sequence.
HOM1: Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); iaaH: indoleacetamide hydrolase from Agrobacterium tumefaciens; iaaM: tryptophan monooxygenase from Agrobacterium tumefaciens; TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA). The iaaH.iaaM cassette is constructed as an operon. Both of the iaaH and iaaM genes are preceded by a shine delgardo sequence and a six base pair spacer.
HOM1: Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); iaaH: indoleacetamide hydrolase from Agrobacterium tumefaciens; iaaM: tryptophan monooxygenase from Agrobacterium tumefaciens; TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA). The iaaH.iaaM cassette is expressed as a fusion protein. The iaaH a gene is preceded by a shine delgardo sequence and a six base pair spacer and the two open reading frames are separated by an eight glycine linker sequence.
HOM1: Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); iaaH: indoleacetamide hydrolase from Agrobacterium tumefaciens; iaaM: tryptophan monooxygenase from Agrobacterium tumefaciens; Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA). The iaaH.iaaM cassette is constructed as an operon. Both of the iaaH and iaaM genes are preceded by a shine delgardo sequence and a six base pair spacer.
HOM1: Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); iaaH: indoleacetamide hydrolase from Agrobacterium tumefaciens; iaaM: tryptophan monooxygenase from Agrobacterium tumefaciens; Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA). The iaaH.iaaM cassette is expressed as a fusion protein. The iaaH a gene is preceded by a shine delgardo sequence and a six base pair spacer and the two open reading frames are separated by an eight glycine linker sequence.
HOM1: Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); PpsbA: Plastidic psbA promoter; aadA: spectinomycin adenyltransferase gene; T7: T7 transcription terminator sequence: Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); iaaH: indoleacetamide hydrolase from Agrobacterium tumefaciens; iaaM: tryptophan monooxygenase from Agrobacterium tumefaciens; TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA). The iaaH.iaaM cassette is constructed as an operon. Both of the iaaH and iaaM genes are preceded by a shine delgardo sequence and a six base pair spacer.
HOM1: Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); PpsbA: Plastidic psbA promoter; aadA: spectinomycin adenyltransferase gene; T7: T7 transcription terminator sequence: Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); iaaH: indoleacetamide hydrolase from Agrobacterium tumefaciens; iaaM: tryptophan monooxygenase from Agrobacterium tumefaciens; TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA). The iaaH.iaaM cassette is expressed as a fusion protein. The iaaH a gene is preceded by a shine delgardo sequence and a six base pair spacer and the two open reading frames are separated by an eight glycine linker sequence.
HOM4L: Homologous recombination sequence (nt 104065-102312, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn-rbcl-ds: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA), rbcl is the 5′-UTR of the plastid rbcL gene and the downstream sequence (ds) sequence encodes RbcL amino acids 1-14 (Kuroda and Maliga (2001). Sequences downstream of the translation initiation codon are important determinants of translation efficiency in chloroplasts. Plant Physiol. volume 125(1) Page 431,
TpsbA: psbA polyA addition sequence; HOM4R: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
HOM4L: Homologous recombination sequence (nt 104065-102312, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn-atpB-ds: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA), atpb is the 5′-UTR of the plastid atpb gene and the downstream sequence (ds) sequence encodes AtpB amino acids 1-14 (Kuroda and Maliga (2001). Sequences downstream of the translation initiation codon are important determinants of translation efficiency in chloroplasts. Plant Physiol. volume 125(1) Page 431,
HOM4L: Homologous recombination sequence (nt 104065-102312, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn-T7g10: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA), T7g10 is the 5′-UTR of the E. coli phage T7 gene 10 (Kuroda and Maliga (2001) Complementarity of the 16S rRNA penultimate stem with sequences downstream of the AUG destabilizes the plastid mRNAs. Nucleic Acids Res. volume 29(4) page 972,
The present invention is further defined in the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.
The plastid transformation vectors pPT1001 and pPT1002 were constructed as detailed in
For proof-of-principle purposes, the Yellow Fluorescent Protein (YFP) reporter gene was cloned into pPT1001 and pPT1002 to generate pPT1001-YFP (
Because the pPT1001 and pPT1002 vector series also functions in bacteria, we tested whether the CRE-mediated excision of the IPT gene was functional ultimately leading to YFP gene activation and YFP fluorescence in E. coli cells. A schematic diagram showing the principal of the system is shown in
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 plastid genome where it remains dormant until the IPT selectable marker gene is removed by CRE/lox mediated recombination.
The transgene encoding the “plant-toxic” protein is inserted into one of the pPT1001 or pPT1002 vectors (
Plastids play an integral role during plant development and it may therefore be of interest to express proteins (plant or non-plant) inside plastids that would have a positive effect on plant growth, development and/or confer modified characteristics to the plant as whole. Due to the high expression levels of transgenes in plastids coupled to the fact that the IPT as a selectable marker gene does not lead to the generation of spontaneous ribosomal mutants (as with the common spectinomycin selectable marker gene), the present invention represents an ideal system for this application.
The transgene is inserted into one of the pPT1001 or pPT1002 vectors (
There are often problems associated with expression of eukaryotic proteins in bacterial systems due to insolubility and/or lack of post-translational modifications. Similarly, the expression of eukaryotic proteins in mammalian cells is expensive and labour intensive. The present system can be used for the expression of eukaryotic proteins in plastids using IPT marker gene selection and transgene activation.
As described in Examples 2 and 3, any gene encoding a eukaryotic protein may be inserted into one or all of the pPT1001 series or the pPT1002 series of vectors followed by transformation, selection and regeneration as described previously. Following regeneration, the expressed protein may be purified using the affinity tags shown in
A non-exclusive list of possible eukaryotic protein families that will be expressed includes antibodies, enzymes, enzyme inhibitors and design peptides.
Due to the endosymbiotic origin of plastids, it is possible to express prokaryotic proteins in plastids. As described in Examples 2 and 3, any gene encoding a prokaryotic protein may be inserted into one or all of the pPT1001 series or the pPT1002 series of vectors followed by transformation, selection and regeneration as described previously. Following regeneration, the expressed protein may be purified using the affinity tags shown in
Due to the endosymbiotic origin of plastids, it is possible to express operon-like gene structures in plastids. This entails the coordinated and simultaneous expression of multiple proteins under the regulation of a single promoter. As described in Examples 2 and 3, multiple genes encoding protein of both prokaryotic and eukaryotic origin (or a mix thereof) may be inserted into one or all of the pPT1001 series or the pPT1002 series of vectors followed by transformation, selection and regeneration as described previously.
pPT1001/YFP vector was bombarded into tobacco leaf cells and regenerants selected on media containing only auxin and on media lacking all hormones. Regenerants were obtained (
To optimize the selection and regeneration system further a new series of plastid transformation vectors have been constructed. The following modifications and additions have been made:
1) Inclusion of the aadA gene in addition to the IPT gene for double selection purposes.
2) Inclusion of new homologous recombination sites (HOM3 and HOM4) that may increase more efficient homologous recombination and transgene insertion.
3) Inclusion of an IPTG inducible promoter.
4) Inclusion of a modified Prrn promoter for higher expression levels.
The details of the vectors are shown in
The plastid transformation vectors pPTA001-pPTA004 are constructed as detailed in
These vectors are bombarded into tobacco leaves as detailed in Appendix 3. For transformation experiments, young actively growing leaves from plants growing in Magenta Box are used.
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 plastid genome where it remains dormant until 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 pPTA001-pPTA004 vectors (
Plastids play an integral role during plant development and it may therefore be of interest to express proteins (plant or non-plant) inside plastids that would have a positive effect on plant growth, development and/or confer modified characteristics to the plant as whole. Due to the high expression levels of transgenes in plastids coupled to the fact that the iaaH and iaaM as selectable marker genes does not lead to the generation of spontaneous ribosomal mutants (as with the common spectinomycin selectable marker gene), the present invention represents an ideal system for this application.
The transgene encoding the plastidic protein is inserted into one of the pPTA001-pPTA004 vectors (
There are often problems associated with expression of eukaryotic proteins in bacterial systems due to insolubility and/or lack of post-translational modifications. Similarly, the expression of eukaryotic proteins in mammalian cells is expensive and labour intensive. The present system can be used for the expression of eukaryotic proteins in plastids using iaaH and iaaM marker gene selection and transgene activation.
As described in Examples 10 and 11, any gene encoding a eukaryotic protein may be inserted into one or all of pPTA001-pPTA004 series of 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.
Due to the endosymbiotic origin of plastids, it is possible to express prokaryotic proteins in plastids. As described in Examples 10 and 11, any gene encoding a prokaryotic protein may be inserted into one or all of ppTA001-pTA004 vectors followed by transformation, selection and regeneration as described previously. Following regeneration, the expressed protein may be purified and used for downstream applications.
Due to the endosymbiotic origin of plastids, it is possible to express operon-like gene structures in plastids. This entails the coordinated and simultaneous expression of multiple proteins under the regulation of a single promoter. As described in Examples 10 and 11, multiple genes encoding protein of both prokaryotic and eukaryotic origin (or a mix thereof) may be inserted into one or all of the pPTA001-pPTA004 vectors followed by transformation, selection and regeneration as described previously.
To optimize the selection and regeneration system further a new series of plastid transformation vectors are constructed. The following modifications and additions are being made:
1) Inclusion of the aadA gene in addition to the iaaH and iaaM genes for double selection purposes (
2) Inclusion of new homologous recombination sites with varying lengths and composition that may increase the efficiency of homologous recombination and transgene insertion.
3) Inclusion of a variety of inducible promoters to drive transgene expression.
4) Inclusion of a modified Prrn promoter for higher expression levels.
Other preferred constructs of the invention are given in
The standard procedures produce transformed plants in 3-5 months.
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.
Each 30 μl Gold-DNA mix gives four or five bombardments. The remaining mixture is usually too dense for good results.
2 days
1MFB: media for bombardement
2MTS: media for transgenic selection
3MFR: media for rooting
4Time: time that tobacco leaves stay in the media
5This time includes second selection time
1. 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.
2. Each 30 μl Gold-DNA mix gives four or five bombardments. The remaining mixture is usually too dense for good results.
2 days
1MFB: media for bombardment
2MTS: media for transgenic selection
3MFR: media for rooting
4Time: time that tobacco leaves stay in the media
5This time includes second selection time
1. 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.
2. Each 30 μl Gold-DNA mix gives four or five bombardments. The remaining mixture is usually too dense for good results.
2 days
1MFB: media for bombardment
2MTS: media for transgenic selection
3MFR: media for rooting
4Time: time that tobacco leaves stay in the media
5This time includes second selection time
Place a stopping screen and macrocarrier with gold-DNA (in holder) into the retaining assembly and screw down.
2 days
4Time: time that tobacco leaves stay in the media
5This time includes second selection time
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB2009/001511 | 6/12/2009 | WO | 00 | 3/18/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61061405 | Jun 2008 | US |
