EP4087935A1 - Method for producing bioluminescent plants - Google Patents
Method for producing bioluminescent plantsInfo
- Publication number
- EP4087935A1 EP4087935A1 EP21700110.6A EP21700110A EP4087935A1 EP 4087935 A1 EP4087935 A1 EP 4087935A1 EP 21700110 A EP21700110 A EP 21700110A EP 4087935 A1 EP4087935 A1 EP 4087935A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- seq
- plant
- chloroplasts
- chloroplast
- genes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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Classifications
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- 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
- C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/375—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Basidiomycetes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- 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
- C12N15/8214—Plastid transformation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
Definitions
- the field of the invention is that of plant biology.
- Bioluminescence is the production and emission of light by a living organism through a chemical reaction in which chemical energy is converted into light energy.
- the basic substrate that produces luminescence is luciferin, which emits light by oxidizing by the action of the enzyme luciferase.
- luciferin which emits light by oxidizing by the action of the enzyme luciferase.
- the expression of genes related to bioluminescence is controlled by the lux operon.
- Other organisms are also capable of bioluminescence, such as certain fungi or multicellular beings, especially many marine species.
- luciferases There are a large number of naturally occurring luciferases that are found in species from very different backgrounds. It is found in different families of insects such as the firefly (Photinus pyralis) or in a family of beetles, elaterides (click beetle) or even worms (Phrixothrix hirtus). Luciferases are also found in various marine species (Renilla reniformis, Gaussia princeps, Cypridina hilgendorfii ”) and in certain bacteria (Photobacterium phosphoreum or Vibrio harveyi).
- luciferin varies from species to species and may be called coelenterazine.
- the identification of the metabolic pathway of synthesis of coelenterazine or luciferin is important to allow the development of bioluminescent systems to which it is not necessary to add luciferin to obtain the bioluminescent effect.
- Mitiouchkina et al (BioRxiv, 2019, doi.org/10.1101/809376) described that the expression of the HispS, H3H, and Luz genes of Neonothopanus nambi in tobacco cells (integration into the nuclear genome) made it possible to generate plants luminescent. The authors did not use the NpgA gene.
- Bioluminescent plants can be used in decoration and events. Preferably, such plants should be capable of producing light autonomously, or after providing an inexpensive substrate. The amount of light that needs to be produced should be sufficient for the observed effect to be aesthetic. It is also preferred that these plants are sterile in order to avoid any contamination in the environment.
- the Applicant has developed a plant in which at least one chloroplast of a cell contains the genes encoding the proteins hispidine-3-hydroxylase (H3H, GenBank: BBH43497.1) and luciferase (Luz, GenBank: BBH43509.1) under the control of promoters active in the chloroplast.
- the chloroplast contains an expression cassette for the H3H and Luz proteins in the chloroplasts.
- chloroplasts of the cell contain the expression cassette.
- several cells of the plant contain at least one chloroplast containing the cassette expression.
- all the cells of the plant contain at least one chloroplast containing the expression cassette.
- at least 50% of the chloroplasts of the plant contain the expression cassette, that is, if a part of the plant is taken and the presence of the expression cassette in the chloroplasts of this plant part, at least 50% contain this cassette. This can be easily verified by any method known in the art such as quantitative PCR, the preferred method.
- chloroplasts are present in the cytoplasm of the cell and are not transmissible by pollen, which prevents contamination to other plants of the same species
- Chloroplasts can be transformed biolistically. Meristematic tissues (cells) are bombarded. With DNA coated gold microbeads. After divisions, the number of transformed plastids will increase faster than untransformed plastids (especially when using a selection medium), and untransformed plastids will be "lost" on dilution.
- PEGs polyethylene glycols
- the invention thus relates to a plant in which at least one chloroplast of a cell contains the genes encoding the proteins hispidine-3-hydroxylase (H3H) and luciferase (Luz) under the control of promoters active in the chloroplast.
- H3H hispidine-3-hydroxylase
- Luz luciferase
- the invention also relates to a plant cell in which at least one chloroplast of a cell contains the genes encoding the proteins hispidine-3-hydroxylase (H3H) and luciferase (Luz) under the control of promoters active in the chloroplast. It is possible to regenerate a whole plant from this cell.
- H3H hispidine-3-hydroxylase
- Luz luciferase
- the cell thus described can produce light by adding hispidin to the culture medium.
- the hispidin present in the culture medium reaches the cell via the sap.
- hispidin is then converted to luciferin by the enzyme H3H, and then luciferin is oxidized to oxyluciferin by luciferase Luz, producing light.
- This embodiment makes it possible to obtain luminescence only when the substrate (hispidin) is supplied to the plant.
- the chloroplasts of the plant cells contain, in addition to the genes encoding the enzymes H3H and Luz, a gene encoding the hispidine synthase enzyme HispS, under the control of a promoter active in the chloroplasts.
- luminescence will be initiated by the caffeic acid naturally present in the plant cell (cytoplasm) and in the chloroplast. This is then transformed into hispidin, which then gives luciferin.
- luminescence can be initiated by adding caffeic acid to the culture medium.
- the cell can thus produce luminescence autonomously, that is to say that it contains all the genes allowing the synthesis of luciferin, as well as its recycling.
- the chloroplast contains the genes encoding the proteins caffeyl-pyruvate hydrolase (CPH, GenBank: BBH43519.1), hispidine synthase (HispS, GenBank: BBH43485.1), under the control of promoters active in chloroplasts.
- CPH caffeyl-pyruvate hydrolase
- HispS hispidine synthase
- sequence of luciferase is SEQ ID NO: 20 (with or without the poly_histidine tag, His-tag).
- sequence of the H3H enzyme is SEQ ID NO: 21 (with or without the poly_histidine tag, His-tag).
- sequence of the CPH enzyme is SEQ ID NO: 22 (with or without the poly_histidine tag, His-tag).
- sequence of the HispS enzyme is SEQ ID NO: 23 (with or without the poly_histidine tag, His-tag).
- the chloroplast also contains a gene encoding a phosphopantetheinyl transferase (NpgA, NCBI Reference Sequence: XP_663744.1), also under the control of a promoter active in the plastids.
- the gene encoding NpgA can be added in each embodiment as described above.
- sequence of the NpgA is SEQ ID NO: 24 (with or without the poly_histidine tag, His-tag).
- the plant is such that all of its cells contain at least one chloroplast (and preferably at least 50% of its chloroplasts) transformed by the genes encoding the proteins H3H, Luz, CPH, HispS and NpgA.
- the invention also relates to a plant cell in which at least 50% of the chloroplasts are transformed by the genes encoding the proteins H3H, Luz, CPH, HispS and NpgA.
- the invention also relates to a vector containing
- nucleic acid sequence encoding the H3H protein in particular SEQ ID NO: 2
- a promoter functional in chloroplasts two nucleic acid sequences present in a chloroplast, preferably trnl (SEQ ID NO: 6) and trnA (SEQ ID NO: 7), flanking said nucleic acid sequences (b) and (c), these sequences (b ) and (c) therefore being located between these two sequences (d).
- the vector also contains
- nucleic acid sequence encoding a Cph protein (in particular SEQ ID NO: 3), located between sequences (d) with sequences (b) and (c).
- the vector also contains
- the vector also contains
- nucleic acid sequence encoding an NpgA protein (in particular SEQ ID NO: 5), located between the sequences (d) with the sequences (b) and (c).
- the vector contains the origin of replication (a), as well as sequences (b), (c), (e), (f) and (g) flanked by sequences (d).
- the invention also relates to a host cell containing a vector as described above.
- This cell is preferably a bacterial cell, preferably Escherichia coli transformed with the vector.
- nucleic acid sequence of the genes has been optimized for expression in chloroplasts (adaptation of the codon usages of chloroplasts).
- sequences SEQ ID NO: 1 to SEQ ID NO: 5 are thus sequences optimized for expression in chloroplasts. We can use these sequences, or sequences having at least 90% identity, preferably at least 95% identity, preferably at least 98% identity, more preferably at least 99% identity with these sequences and which encode the proteins SEQ ID NO: 20 to SEQ ID NO: 24 respectively.
- the genes are optimized to obtain a GC level of between 35% and 40%.
- the codons preferentially expressed in the chloroplasts are also chosen, in particular by taking into account the data of Liu et al.
- the system used is a system derived from fungi (fungus).
- fungi fungus
- Neonothopanus nambi it is preferred when all of the enzymes used (HispS, H3H, Luz, and CPH) originate from the same organism, preferably Neonothopanus nambi.
- npgA (4'-phosphopantetheinyl transferase) gene comes from Aspergillus nidulans.
- the different genes are expressed under the control of a single promoter, in the form of an operon, as is known in the art, for genes involved in the same metabolic pathway ( cf Saxena et al., 2014 J Biosci. 2014 Mar; 39 (1): 33-41; Kumar et al, 2012 Metab Eng. 2012 Jan; 14 (1): 19-28).
- a promoter chosen from the Patp1, Prrn, PrbcL or PpsbA promoters (in particular those described by the sequences SEQ ID NO: 8 to SEQ ID NO: 12).
- each transgene introduced into the chloroplast of the plant is under the control of its own promoter (a promoter of its own, it being understood that the same promoter can be used for two genes, but whereas it is preferred that at least two transgenes are under the control of two different promoters).
- the transgenes are therefore not expressed within an operon.
- This embodiment is preferred. Indeed, the fact of attributing, to each gene, an optimal regulatory system (promoters and terminators) which is specific to it, makes it possible to increase the levels of expression, compared to the use of a system based on an operon. This mode of operation thus goes against the modes generally observed in the art, summarized in Boehm and Bock (Plant Physiol.
- Boehm and Bock also recall the latest advances to improve the efficiency of synthetic operons; by adding intercistronic expression elements (IEE) (Zhou et al., Plant J. 2007 Dec; 52 (5): 961-972), or by stabilizing messenger RNAs with proteins which bind to them ( PRRs), (Legen et al., Plant J. 2018 Apr; 94 (1): 8-21). More recently still, they recall that Fuentes et al. (op. cit.) have shown that the complexity and the number of accessible pathways have been extended thanks to a trick that combines chloroplast transformation with nuclear transformation (“combinatorial supertransformation of transplastomic receptacle Unes (COSTREL)”).
- the art proposes rather to use operons and to optimize them (manufacture of synthetic operons), when it is desired to express several genes in chloroplasts, in particular when the genes considered must cooperate with each other in a given metabolic pathway.
- Patpl Patpl, Prrn, PrbcL or PpsbA promoters.
- the H3h gene is under the control of the PpsbA promoter, in particular the part specified in SEQ ID NO: 9.
- the Luz gene is under the control of the PpsbA promoter, in particular the part specified in SEQ ID NO: 9.
- the Cph gene is under the control of the PrbcL promoter, in particular the part specified SEQ ID NO: 10.
- the HispS gene is under the control of the Prrn promoter, in particular the part specified in SEQ ID NO: 11.
- the npgA gene is under the control of the Patp1 promoter, in particular the part specified in SEQ ID NO: 12.
- the sequences are optimized, and in particular the promoters by choosing particular 5′UTR sequences, in order to optimize the expression of the genes in the chloroplasts (De Costa et al. Genes Genet Syst. 2001 Dec. ; 76 (6): 363-71); Drechsel and Bock Nucleic Acids Res. 2011 Mar; 39 (4): 1427-38); Shinozaki and Sugiura Gene. 1982 Nov 20 (1) 91-102 and Nucleic Acids Res. 1982 Aug 25; 10 (16): 4923-34; Kuroda and Maliga, Plant Physiol. 2001 Jan; 125 (1): 430-6).
- sequences SEQ ID NO: 9 to SEQ ID NO: 12 represent such optimized promoters with added 5′UTRs to improve expression.
- the H3h gene is under the control of the optimized promoter described by SEQ ID NO: 9.
- the Luz gene is under the control of the optimized PpsbA promoter described by SEQ ID NO: 9.
- the Cph gene is under the control of the optimized PrbcL promoter described by SEQ ID NO: 10.
- the HispS gene is under the control of the optimized Prrn promoter described by SEQ ID NO: 11.
- the npgA gene is under the control of the optimized Patpl promoter described by SEQ ID NO: 12.
- Terminators positioned at 3 'of the coding nucleic sequences are also used.
- the terminators represented by the sequences SEQ ID NO: 13 to SEQ ID NO: 18 can be used.
- a construct (5′-3 ′) SEQ ID NO: 8-SEQ ID NO: 19-SEQ ID NO: 17 is used to express the aadA selection gene.
- Integration of the Expression Cassette into the Chloroplasts As seen above, in a particular embodiment, all of the genes present in the chloroplasts form an expression cassette, that is to say that these genes are present one after the other on a DNA fragment. Thus, the chloroplasts are transformed with this expression cassette, in order to obtain the expression of the genes encoded in this expression cassette.
- genes are integrated into the genome of chloroplasts.
- homologous recombination is used to introduce the genes to a selected location in the genome of the chloroplast.
- insertion sites are possible in the genome of the chloroplast. It is preferentially chosen to integrate the expression cassette into a non-coding region of the genome of the chloroplast. It is thus possible to use inter-gene sequences comprised between two sequences encoding the chloroplast transfer RNAs.
- the trnl (SEQ ID NO: 6) and trnA (SEQ ID NO: 7) sites are chosen, encoding the transfer RNAs of isoleucine and alanine. It is thus possible to use the sequences SEQ ID NO: 6 or SEQ ID NO: 7, or sequences containing these sequences. It is also possible to use sequences included in SEQ ID NO: 6 or SEQ ID NO: 7.
- sequences SEQ ID NO: 6 and SEQ ID NO: 7 are obtained from tobacco (Nicotiana benthamiana). They are therefore particularly suitable for integration by homologous recombination in tobacco chloroplasts. However, they can be used for other plants, due to the high homology between these sequences and the trnl and trnA sequences of chloroplasts from other plants. Thus, one can use sequences having at least 99% identity, more preferably at least 99.45% identity, more preferably at least 99.5% identity, more preferably at least 99.7% identity. identity with SEQ ID NO: 6 or SEQ ID NO: 7.
- Max matches in a query range 0 Scoring Parameters Match / M ismatch Scores: 1, -2 Gap Costs: Linear Filters and Masking Filter: Low complexity regions filter: on
- compositional adjustments Conditional compositional score matrix adjustment
- the plant according to the invention is preferably an ornamental plant. It is preferably chosen from the group consisting of Hedera helix, Petunia axillaris subsp. axillaris, Nicotiana benthamiana, Ficus benjamina, ficus elastica, Ficus microcarpa, Chlorophytum comosum, Monstera deliciosa, Sansevieria socotrana, Pelargonium x hortorum, Spathiphyllum wallisii, Dracaena draco, Dracaena angustoifolia, Wormittonia almataarnonia, Dracaena angustoifolia, Yuccaellonium venaittonia, Dracaena angustoifolia, Synoellolia, Dracaena, Dracaena angustoifolia, Spathiphyllum wallisii, Dracaena draco, Dracaena angusteaifolia
- the plant is Nicotiana benthamiana.
- the plant is petunia (Petunia axillaris subsp. Axillaris).
- the plant is ivy (Hedera helix).
- the invention also relates to a method for producing a plant as described above, comprising a step of inserting the transgenes as described above into the genome of chloroplasts of plant cells.
- the method also preferably comprises the step of regenerating a plant by culturing callus.
- the method comprises inserting the transgenes encoding the enzymes H3H and Luz into the genome of chloroplasts of plant cells.
- the method comprises inserting the transgenes encoding the enzymes Cph, H3H and Luz into the genome of chloroplasts of plant cells.
- the method comprises inserting the transgenes encoding the enzymes Cph, HispS, H3H and Luz into the genome of chloroplasts of plant cells.
- the method comprises inserting transgenes encoding the enzymes Cph, HispS, H3H and Luz into the genome of chloroplasts of plant cells, as well as a transgene encoding NpgA.
- integration of the transgenes is accomplished by bombarding plant leaves with a plasmid using a particle gun. To do this, we prepare the expression cassette (DNA fragment carrying the transgenes that we want to integrate into the genome of the chloroplast), and we cover metal microbeads (preferably gold, but can also be tungsten. ) which are then projected onto plant cells.
- the expression cassette DNA fragment carrying the transgenes that we want to integrate into the genome of the chloroplast
- metal microbeads preferably gold, but can also be tungsten.
- the plant cells are contacted with polyethylene glycol (PEG), which destabilizes the plasma membranes and allows the entry of DNA fragments carrying the transgenes to integrate into the plastid genome.
- PEG polyethylene glycol
- transgenes into the chloroplast genome is accomplished by homologous recombination.
- the transgenes are flanked by sequences homologous to sequences of the chromosome of the chloroplast.
- the integration of transgenes is therefore carried out by the machinery of the organism by homologous recombination at the site
- the transformed cells are cultured under callus conditions, which are cultured, and from which a plant is regenerated by methods known in the art.
- the culture of the calli is carried out on a selective medium.
- a selective medium is a medium containing a selective element (often an antibiotic or a herbicide) on which only plants can grow. cells containing a gene for resistance to the selective element, while cells without this gene cannot grow or grow sluggishly.
- antibiotics neomycin / kanamycin and nptll (aminoglycoside 3'-phosphotransferase), betain and badh (betain aldehyde dehydrogenase), hygromycin B and hph (hygromycin B phosphotransferase), spectinomycin / alycosadosAlycin 3 (aminoglycoside 3'-phosphotransferase) and '- adenyltransferase), chloramphenicol and cat (chloramphenicol acetyltransferase), amikacin and aphA6 (3'-aminoglycoside phosphotransferase), blasticidin S and bsr (blasticidin S deaminase), sulfonamides and sull (dihydropteorate acetaminate Camycamyferase and
- aadA aminoglycoside 3'-adenyltransferase gene which confers resistance to spectinomycin, a coding sequence of which is represented by SEQ ID NO: 19.
- the resistance gene is introduced into the expression cassette containing the transgenes of interest, under the control of a promoter active in chloroplasts. It is also possible to implement a system in which the resistance gene can be excised after transformation, in particular by following the teaching of Scutt et al (Biochimie 84 (2002) 1119-1126) or Lantham et al (Nature Biotechnology, 2000, (18), 1172-76).
- the invention also relates to a light system comprising a plant emitting bioluminescence as described above, that is to say in which at least one cell contains at least one chloroplast containing the genes mentioned above, and which emits bioluminescence by oxidation of luciferin by the enzyme Luz.
- the system contains a plant of which at least 50% of the chloroplasts contains the enzymatic system mentioned above.
- the invention also relates to a method for producing light, comprising the step of adding hispidin to the culture medium of a plant as described above, and of which at least one chloroplast contains at least the genes encoding the enzymes H3H and Luz (preferably integrated into its genome).
- the invention also relates to a method for producing light (by a plant), comprising the step of adding caffeic acid to the culture medium of a plant as described above, and of which at least a chloroplast contains at least the genes encoding the enzymes HispS, H3H and Luz (preferably integrated into its genome).
- the invention also relates to a method for producing light (by a plant), comprising the step of adding caffeic acid to the culture medium of a plant as described above, and of which at least a chloroplast contains the genes encoding the enzymes HispS, H3H, Luz, Cph (preferably integrated into its genome).
- the chloroplast also contains and the gene encoding NpgA, in the above methods.
- Example 1 Preparation of the plasmid containing the H3H and Luz genes and the sequences allowing the integration of these genes into the genome of the chloroplast as well as their expression
- the sequences of the H3H and Luz genes were adapted with codons for use by chloroplasts and then synthesized (SEQ ID NO: 2 and SEQ ID NO: 1).
- the promoters were chosen from among all the promoters present in the genome of the chloroplast and modified in their 5′UTR sequence so as to maximize the expression of the genes under their controls. These modified promoters were then synthesized.
- the terminators were chosen from among all the terminators present in the chloroplast genome and some have been optimized to be as short as possible while keeping their functions. They have also been synthesized.
- the trnl and trnA sequences (SEQ ID NO: 6 and SEQ ID NO: 7) were chosen to allow integration of the H3H and Luz genes into the genome of the chloroplast. They were amplified by PCR (Polymerase Chain Reaction) from chloroplast DNA of Nicotiana benthamiana. The spectinomycin / streptomycin resistance gene called aadA was amplified by PCR from a plasmid containing it (SEQ ID NO: 19). To develop the plasmid vector containing all of these sequences, the plasmid pUC19 was used.
- the promoters, genes and terminators were amplified by PCR and each part of the trio were ligated together: promoter, gene and terminator with the ln-fusion method from Takara. 50 ng or 100ng of DNA were incubated at 50 ° C. for 1 hour with the ligation enzymes from the ln-fusion kit. The ligation products were then amplified by PCR.
- the three genes (aadA, H3H, and Luz) thus fused with their respective promoters and terminators, and the trnI and trnA sequences were then cloned into the plasmid pUC19 linearized by PCR, following the NEBuilder protocol.
- the NEBuilder is based on the “Gibson assembly.
- the primers were designed so that the fragments show a 25bp sequence overlap with each other and with the insertion site sequence of the plasmid pUC19.
- the gold beads are prepared following the Biorad protocol supplied with the beads.
- the plasmid DNA is then precipitated on the gold beads (for 5 samples): 1. Vortex 50 ⁇ L of gold beads for 1 minute. 2. Add 10 ⁇ L of DNA plasmid (at 1 pg / pL) and vortex the mixture. 3. Add 50mI of 2.5M Cacl2 and vortex the mixture. 4. Add 20 ml of 0.1 M spermidine and vortex the mixture. The beads are then washed with 100% ethanol and then resuspended in 40 ml of 100% ethanol
- Preparation of the bombardment chamber 1. Wash the chamber and screens in 70% ethanol. 2. Place the gold beads coated with the plasmid DNA on the grid provided. 3. Place the intact sheet on Whatman No. 1 filter paper placed itself on medium without antibiotics. Place the sample and close the bombardment chamber. 4. Turn on the pump to reach the expected pressure and press the button to fire. 5. Stop the pump to relieve pressure and open the chamber. 6. Incubate the bombarded plate samples for 2 days in the dark. On the third day, cut the explants 3-5mm aside and place them on selection medium (MS supplemented with 3% sucrose and hormones: 1 mg / L BAP, and 0.1 mg / IAA, with 500 mg / L of spectinomycin 3. Transgenic stems appear after 3 to 5 weeks of transformation Cut the leaves of the transgenic stems that have appeared into small squares 2 mm apart and place them in a new selection medium, to achieve homoplasmy Regenerate plants according to known methods.
- Plant cells can be verified to produce light when grown on medium containing hispidin.
- Example 2 Preparation of the plasmid containing the genes H3H, Luz, CPH, HispS, NpgA and the sequences allowing the integration of these genes into the genome of the chloroplast as well as their expression
- the sequences of the Luz, H3H, CPH, HispS, NpgA genes were adapted with codons for use by chloroplasts and then synthesized (SEQ ID NO: 1 to SEQ ID NO: 5 respectively).
- the promoters chosen are SEQ ID NO: 9 to SEQ ID NO: 12 respectively and the terminators SEQ ID NO: 14 (Luz and HispS), SEQ ID NO: 15 (H3H), SEQ ID NO: 16 (CPH) and SEQ ID NO: 18 (NpgA).
- the trnl and trnA sequences (SEQ ID NO: 6 and SEQ ID NO: 7) were chosen to allow integration of the H3H and Luz genes into the genome of the chloroplast. They were amplified by PCR (Polymerase Chain Reaction) from chloroplast DNA of Nicotiana benthamiana. The resistance gene spectinomycin / streptomycin named aadA was amplified by PCR from a plasmid containing it (SEQ ID NO: 19).
- An expression cassette was prepared as described above and integrated into a plasmid.
- chloroplasts The transformation of chloroplasts was carried out biolistically on tobacco leaves, as described above.
- Samples were collected, cultured (several times to achieve homoplasmia) on medium containing spectinomycin.
- Example 3 Obtaining bioluminescent chloroplasts (from plant cells containing bioluminescent chloroplasts
- the chloroplasts of Nicotiana benthamiana leaves originating from shoots were visualized 15 weeks after transformation by bombardment.
- Imaging was performed with a Nikon Eclipse Ti microscope with a 100x 1.49 NA immersion objective.
- the 405 nm laser (cw, Oxxius) was used to visualize the chloroplasts (10 W / cm 2 ).
- the emission of the samples was spectrally filtered using a dichroic mirror (Di01-R488-25x36, Semrock) and then imaged on a Hamamatsu EM-CCD camera (ImagEM).
- ImagEM Hamamatsu EM-CCD camera
- the 405nm laser was blocked with a mechanical shutter.
- An additional lens was used to obtain a final magnification of 150X corresponding to a pixel size of 106.67 nm.
- the acquisition time was 1s.
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Abstract
The invention relates to a method for producing bioluminescent plants by introducing luciferase genes and luciferin biosynthesis genes into plant chloroplasts.
Description
MÉTHODE DE FABRICATION DE PLANTES BIOLUMINESCENTES METHOD OF MANUFACTURING BIOLUMINESCENT PLANTS
Le domaine de l’invention est celui de la biologie végétale. The field of the invention is that of plant biology.
La bioluminescence est la production et l’émission de lumière par un organisme vivant via une réaction chimique au cours de laquelle l’énergie chimique est convertie en énergie lumineuse. Bioluminescence is the production and emission of light by a living organism through a chemical reaction in which chemical energy is converted into light energy.
Le substrat de base à l’origine de la luminescence est la luciférine, qui émet de la lumière en s’oxydant par l’action de l’enzyme luciférase. Chez les bactéries, l’expression des gènes liés à la bioluminescence est contrôlée par l’opéron lux. D’autres organismes sont également capables de bioluminescence, tels que certains champignons ou des êtres multicellulaires, notamment un grand nombre d’espèces marines. The basic substrate that produces luminescence is luciferin, which emits light by oxidizing by the action of the enzyme luciferase. In bacteria, the expression of genes related to bioluminescence is controlled by the lux operon. Other organisms are also capable of bioluminescence, such as certain fungi or multicellular beings, especially many marine species.
Il existe un grand nombre de luciférases naturelles que l’on retrouve chez des espèces provenant de milieux très différents. On en trouve chez différentes familles d’insectes comme la luciole ( Photinus pyralis) ou chez une famille de scarabées, les élatérides (click beetle) ou encore de vers ( Phrixothrix hirtus). Les luciférases se retrouvent aussi dans différentes espèces marines ( Renilla reniformis, Gaussia princeps, Cypridina hilgendorfii...) et dans certaines bactéries ( Photobacterium phosphoreum ou Vibrio harveyi). On retrouve encore ce genre de mécanisme dans certains champignons (ex : Panellus stipticus) ou encore dans du phytoplancton (Haddock et al., Ann Rev Mar Soi. 2010;2:443-93; Wilson and Hastings, Annu Rev Cell Dev Biol. 1998;14:197-230). There are a large number of naturally occurring luciferases that are found in species from very different backgrounds. It is found in different families of insects such as the firefly (Photinus pyralis) or in a family of beetles, elaterides (click beetle) or even worms (Phrixothrix hirtus). Luciferases are also found in various marine species (Renilla reniformis, Gaussia princeps, Cypridina hilgendorfii ...) and in certain bacteria (Photobacterium phosphoreum or Vibrio harveyi). We still find this kind of mechanism in certain fungi (eg: Panellus stipticus) or in phytoplankton (Haddock et al., Ann Rev Mar Soi. 2010; 2: 443-93; Wilson and Hastings, Annu Rev Cell Dev Biol. 1998; 14: 197-230).
La structure chimique de la luciférine varie d’une espèce à l’autre et peut être appelée coelentérazine. L’identification de la voie métabolique de synthèse de la coelentérazine ou de la luciférine est importante pour permettre de mettre en place des systèmes bioluminescents auxquels il n’est pas nécessaire d’apporter la luciférine pour obtenir l’effet bioluminescent. The chemical structure of luciferin varies from species to species and may be called coelenterazine. The identification of the metabolic pathway of synthesis of coelenterazine or luciferin is important to allow the development of bioluminescent systems to which it is not necessary to add luciferin to obtain the bioluminescent effect.
Kotlobay et al (Proc Natl Acad Soi U S A. 2018 Dec 11;115(50) :12728- 12732) ont décrit des mécanismes de formation et recyclage de la luciférine chez les champignons et ont identifié trois enzymes, en plus de la luciférase (Luz). L’hispidine synthase (HispS) transforme l’acide caféique en hispidine, elle-même substrat de l’enzyme hispidine-3-hydroxylase (H3H) qui transforme l’hispidine en luciférine (3-hydroxyhispidine). Enfin, la caffeylpyruvate hydrolase (CPH)
transforme l’oxyluciférine (acide caféil-pyruvique), obtenue après oxydation de la luciférine par la luciférase, en acide caféique. Les auteurs n’ont toutefois pas décrit que ce système puisse être utilisé dans des végétaux, en général et des chloroplastes en particulier. Kotlobay et al (Proc Natl Acad Soi US A. 2018 Dec 11; 115 (50): 12728-12732) described mechanisms of formation and recycling of luciferin in fungi and identified three enzymes, in addition to luciferase ( Luz). Hispidine synthase (HispS) converts caffeic acid into hispidine, itself a substrate for the enzyme hispidine-3-hydroxylase (H3H) which converts hispidine into luciferin (3-hydroxyhispidine). Finally, caffeylpyruvate hydrolase (CPH) converts oxyluciferin (caffeil-pyruvic acid), obtained after oxidation of luciferin by luciferase, into caffeic acid. However, the authors have not described that this system can be used in plants, in general and chloroplasts in particular.
Mitiouchkina et al (BioRxiv, 2019, doi.org/10.1101/809376) ont décrit que l’expression des gènes HispS, H3H, and Luz de Neonothopanus nambi dans des cellules de tabac (intégration dans le génome nucléaire) permettait de générer des plantes luminescentes. Les auteurs n’ont pas utilisé le gène NpgA. Mitiouchkina et al (BioRxiv, 2019, doi.org/10.1101/809376) described that the expression of the HispS, H3H, and Luz genes of Neonothopanus nambi in tobacco cells (integration into the nuclear genome) made it possible to generate plants luminescent. The authors did not use the NpgA gene.
L’enseignement de W02020005120A1 est similaire à celui des deux documents ci-dessus. The teaching of W02020005120A1 is similar to that of the two documents above.
Krichevsky et al (PLoS ONE 5(11): e15461) décrivent des plantes auto luminescentes en utilisant l’opéron lux de Photobacterium leiognathi (luxCDABEG) pour transformer les chloroplastes. Les différents transgènes utilisés (gène de résistance aux antibiotiques spectinomycine et streptomycine (aadA) et opéron lux (luxCDABEG)) sont sous le contrôle du même promoteur de chloroplaste. Ainsi, 7 gènes ont été introduits sous forme d’opéron sous le contrôle d’un seul promoteur de chloroplaste. W02009017821 présente le même enseignement que le document précédent. Krichevsky et al (PLoS ONE 5 (11): e15461) describe self-luminescent plants using the lux operon of Photobacterium leiognathi (luxCDABEG) to transform chloroplasts. The different transgenes used (spectinomycin and streptomycin antibiotic resistance gene (aadA) and lux operon (luxCDABEG)) are under the control of the same chloroplast promoter. Thus, 7 genes were introduced as an operon under the control of a single chloroplast promoter. W02009017821 presents the same teaching as the previous document.
Les plantes bioluminescentes peuvent être utilisées dans le domaine de la décoration et de l’événementiel. Préférentiellement, de telles plantes doivent être capables de produire de la lumière de façon autonome, ou après fourniture d’un substrat peu onéreux. La quantité de lumière qui doit être produite devrait être suffisante afin que l’effet observé soit esthétique. Il est aussi préféré que ces plantes soient stériles afin d’éviter toute contamination dans l’environnement. Bioluminescent plants can be used in decoration and events. Preferably, such plants should be capable of producing light autonomously, or after providing an inexpensive substrate. The amount of light that needs to be produced should be sufficient for the observed effect to be aesthetic. It is also preferred that these plants are sterile in order to avoid any contamination in the environment.
Afin de résoudre les questions ci-dessus, la Demanderesse a développé une plante dont au moins un chloroplaste d’une cellule contient les gènes codant pour les protéines hispidine-3-hydroxylase (H3H, GenBank : BBH43497.1) et luciférase (Luz, GenBank: BBH43509.1) sous le contrôle de promoteurs actifs dans le chloroplaste. Ainsi, le chloroplaste contient une cassette d’expression des protéines H3H et Luz dans les chloroplastes. In order to resolve the above questions, the Applicant has developed a plant in which at least one chloroplast of a cell contains the genes encoding the proteins hispidine-3-hydroxylase (H3H, GenBank: BBH43497.1) and luciferase (Luz, GenBank: BBH43509.1) under the control of promoters active in the chloroplast. Thus, the chloroplast contains an expression cassette for the H3H and Luz proteins in the chloroplasts.
Dans un mode de réalisation particulier, plusieurs chloroplastes de la cellule contiennent la cassette d’expression. Dans un autre mode de réalisation, plusieurs cellules de la plante contiennent au moins un chloroplaste contenant la cassette
d’expression. Dans un mode de réalisation particulier, toutes les cellules de la plante contiennent au moins un chloroplaste contenant la cassette d’expression. Dans ce mode de réalisation, au moins 50% des chloroplastes de la plante contiennent la cassette d’expression, c’est-à-dire que, si l’on prend une partie de la plante et que l’on investigue la présence de la cassette d’expression dans les chloroplastes de cette partie de plante, au moins 50% contiennent cette cassette. Ceci peut être vérifié aisément par toute méthode connue dans l’art telle que la PCR quantitative, méthode préférée. In a particular embodiment, several chloroplasts of the cell contain the expression cassette. In another embodiment, several cells of the plant contain at least one chloroplast containing the cassette expression. In a particular embodiment, all the cells of the plant contain at least one chloroplast containing the expression cassette. In this embodiment, at least 50% of the chloroplasts of the plant contain the expression cassette, that is, if a part of the plant is taken and the presence of the expression cassette in the chloroplasts of this plant part, at least 50% contain this cassette. This can be easily verified by any method known in the art such as quantitative PCR, the preferred method.
Il est intéressant de transformer les chloroplastes (ou plastes) des plantes pour plusieurs raisons : It is interesting to transform the chloroplasts (or plastids) of plants for several reasons:
- ceux-ci sont présents en grande quantité dans chaque cellule, ce qui permet de multiplier la quantité de points de réactions enzymatiques produisant la bioluminescence - these are present in large quantities in each cell, which makes it possible to multiply the quantity of enzymatic reaction points producing bioluminescence
- les chloroplastes sont présents dans le cytoplasme de la cellule et ne sont pas transmissibles par le pollen, ce qui évite la contamination à d'autres plantes de même espèce - chloroplasts are present in the cytoplasm of the cell and are not transmissible by pollen, which prevents contamination to other plants of the same species
- il y a plusieurs copies du génome dans chaque chloroplaste, ce qui permet également de multiplier la quantité de points de réactions enzymatiques produisant la bioluminescence. - there are several copies of the genome in each chloroplast, which also makes it possible to multiply the quantity of enzymatic reaction points producing bioluminescence.
Transformation des chloroplastes Chloroplast transformation
Bien que des méthodes de transformation des plastes soient connues dans l’art, la transformation des chloroplastes est, généralement, bien plus compliquée, longue et avec un taux de succès beaucoup plus faible que les transformations nucléaires. Ceci est d’autant plus vrai lorsque l’on veut intégrer des gènes de grande taille ou de multiples gènes. Although methods of transformation of plastids are known in the art, transformation of chloroplasts is generally much more complicated, time consuming, and with a much lower success rate than nuclear transformations. This is all the more true when we want to integrate large genes or multiple genes.
On peut transformer les chloroplastes par biolistique. On bombarde les tissus (cellules) méristématiques. Avec des microbilles d’or enrobées d’ADN. Après divisions, le nombre de plastes transformés augmentera plus rapidement que les plastes non transformés (notamment lorsque l’on utilise un milieu de sélection), est les plastes non transformés vont se « perdre » par dilution. Chloroplasts can be transformed biolistically. Meristematic tissues (cells) are bombarded. With DNA coated gold microbeads. After divisions, the number of transformed plastids will increase faster than untransformed plastids (especially when using a selection medium), and untransformed plastids will be "lost" on dilution.
Alternativement, on peut utiliser la méthode des PEGs (polyéthylène glycols). La déstabilisation des membranes plasmiques en présence de PEG permet l’entrée des transgènes dans les chloroplastes.
Transgènes Alternatively, one can use the method of PEGs (polyethylene glycols). The destabilization of the plasma membranes in the presence of PEG allows the entry of the transgenes into the chloroplasts. Transgenes
L’invention se rapporte ainsi à une plante dont au moins un chloroplaste d’une cellule contient les gènes codant pour les protéines hispidine-3-hydroxylase (H3H) et luciférase (Luz) sous le contrôle de promoteurs actifs dans le chloroplaste. The invention thus relates to a plant in which at least one chloroplast of a cell contains the genes encoding the proteins hispidine-3-hydroxylase (H3H) and luciferase (Luz) under the control of promoters active in the chloroplast.
L’invention se rapporte également à une cellule de plante dont au moins un chloroplaste d’une cellule contient les gènes codant pour les protéines hispidine-3- hydroxylase (H3H) et luciférase (Luz) sous le contrôle de promoteurs actifs dans le chloroplaste. Il est possible de régénérer une plante complète à partir de cette cellule. The invention also relates to a plant cell in which at least one chloroplast of a cell contains the genes encoding the proteins hispidine-3-hydroxylase (H3H) and luciferase (Luz) under the control of promoters active in the chloroplast. It is possible to regenerate a whole plant from this cell.
La cellule ainsi décrite peut produire de la lumière par ajout d’hispidine dans le milieu de culture. Lorsque cette cellule est présente dans une plante, l’hispidine présente dans le milieu de culture atteint la cellule via la sève. The cell thus described can produce light by adding hispidin to the culture medium. When this cell is present in a plant, the hispidin present in the culture medium reaches the cell via the sap.
Dans le chloroplaste, l’hispidine est alors transformée en luciférine par l’enzyme H3H, puis la luciférine est oxydée en oxyluciférine par la luciférase Luz, en produisant de la lumière. Ce mode de réalisation permet d’obtenir la luminescence uniquement lorsque le substrat (hispidine) est apporté à la plante. In the chloroplast, hispidin is then converted to luciferin by the enzyme H3H, and then luciferin is oxidized to oxyluciferin by luciferase Luz, producing light. This embodiment makes it possible to obtain luminescence only when the substrate (hispidin) is supplied to the plant.
Dans un autre mode de réalisation, les chloroplastes des cellules de plantes contiennent, outre les gènes codant pour les enzymes H3H et Luz, un gène codant pour l’enzyme hispidine synthase HispS, sous le contrôle d’un promoteur actif dans les chloroplastes. Dans ce mode de réalisation, la luminescence sera initiée grâce à l’acide caféique naturellement présent dans la cellule végétale (cytoplasme) et dans le chloroplaste. Celui-ci est alors transformé en hispidine, qui donne ensuite la luciférine. Alternativement, on peut initier la luminescence en ajoutant de l’acide caféique dans le milieu de culture. In another embodiment, the chloroplasts of the plant cells contain, in addition to the genes encoding the enzymes H3H and Luz, a gene encoding the hispidine synthase enzyme HispS, under the control of a promoter active in the chloroplasts. In this embodiment, luminescence will be initiated by the caffeic acid naturally present in the plant cell (cytoplasm) and in the chloroplast. This is then transformed into hispidin, which then gives luciferin. Alternatively, luminescence can be initiated by adding caffeic acid to the culture medium.
Dans ce mode de réalisation, la cellule peut ainsi produire la luminescence de façon autonome, c’est-à-dire qu’elle contienne l’ensemble des gènes permettant la synthèse de luciférine, ainsi que son recyclage. In this embodiment, the cell can thus produce luminescence autonomously, that is to say that it contains all the genes allowing the synthesis of luciferin, as well as its recycling.
Dans un autre mode de réalisation, on préfère toutefois qu’elle contienne l’ensemble des gènes permettant la synthèse de luciférine, ainsi que les gènes permettant le recyclage de l’acide caféil-pyruvique, produit de réaction de la luciférase sur la luciférine, afin d’éviter son accumulation et un risque de déficit en acide caféique. In another embodiment, however, it is preferred that it contains all of the genes allowing the synthesis of luciferin, as well as the genes allowing the recycling of caffeil-pyruvic acid, reaction product of luciferase on luciferin, in order to avoid its accumulation and a risk of caffeic acid deficiency.
Dans ce mode de réalisation, le chloroplaste contient les gènes codant pour les protéines caféyl-pyruvate hydrolase (CPH, GenBank: BBH43519.1), hispidine
synthase (HispS, GenBank: BBH43485.1), sous le contrôle de promoteurs actifs dans les chloroplastes. In this embodiment, the chloroplast contains the genes encoding the proteins caffeyl-pyruvate hydrolase (CPH, GenBank: BBH43519.1), hispidine synthase (HispS, GenBank: BBH43485.1), under the control of promoters active in chloroplasts.
Dans un mode de réalisation particulier, la séquence de la luciférase est SEQ ID NO: 20 (avec ou sans l’étiquette poly_histidine, His-tag). In a particular embodiment, the sequence of luciferase is SEQ ID NO: 20 (with or without the poly_histidine tag, His-tag).
Dans un mode de réalisation particulier, la séquence de l’enzyme H3H est SEQ ID NO: 21 (avec ou sans l’étiquette poly_histidine, His-tag). In a particular embodiment, the sequence of the H3H enzyme is SEQ ID NO: 21 (with or without the poly_histidine tag, His-tag).
Dans un mode de réalisation particulier, la séquence de l’enzyme CPH est SEQ ID NO: 22 (avec ou sans l’étiquette poly_histidine, His-tag). In a particular embodiment, the sequence of the CPH enzyme is SEQ ID NO: 22 (with or without the poly_histidine tag, His-tag).
Dans un mode de réalisation particulier, la séquence de l’enzyme HispS est SEQ ID NO: 23 (avec ou sans l’étiquette poly_histidine, His-tag). In a particular embodiment, the sequence of the HispS enzyme is SEQ ID NO: 23 (with or without the poly_histidine tag, His-tag).
Dans un mode de réalisation, le chloroplaste contient également un gène codant pour une phosphopantetheinyl transferase (NpgA, NCBI Reference Sequence: XP_663744.1), également sous le contrôle d’un promoteur actif dans les plastes. Le gène codant pour NpgA peut être ajouté dans chaque mode de réalisation tel que décrit ci-dessus. In one embodiment, the chloroplast also contains a gene encoding a phosphopantetheinyl transferase (NpgA, NCBI Reference Sequence: XP_663744.1), also under the control of a promoter active in the plastids. The gene encoding NpgA can be added in each embodiment as described above.
Dans un mode de réalisation particulier, la séquence de la NpgA est SEQ ID NO: 24 (avec ou sans l’étiquette poly_histidine, His-tag). In a particular embodiment, the sequence of the NpgA is SEQ ID NO: 24 (with or without the poly_histidine tag, His-tag).
Dans un mode de réalisation particulier, la plante est telle que l’ensemble de ses cellules contient au moins un chloroplaste (et de préférence au moins 50% de ses chloroplastes) transformé par les gènes codant pour les protéines H3H, Luz, CPH, HispS et NpgA. In a particular embodiment, the plant is such that all of its cells contain at least one chloroplast (and preferably at least 50% of its chloroplasts) transformed by the genes encoding the proteins H3H, Luz, CPH, HispS and NpgA.
L’invention se rapporte également à une cellule de plante dont au moins 50% des chloroplastes sont transformés par les gènes codant pour les protéines H3H, Luz, CPH, HispS et NpgA. The invention also relates to a plant cell in which at least 50% of the chloroplasts are transformed by the genes encoding the proteins H3H, Luz, CPH, HispS and NpgA.
Vecteurs Vectors
L’invention se rapporte également à un vecteur contenant The invention also relates to a vector containing
(a) une origine de réplication dans une bactérie ou une levure, préférentiellement une origine de réplication dans Escherichia coli , (a) an origin of replication in a bacterium or a yeast, preferably an origin of replication in Escherichia coli,
(b) une séquence d’acide nucléique codant pour la protéine Luz (en particulier SEQ ID NO: 1) sous le contrôle d’un promoteur fonctionnel dans les chloroplastes, (b) a nucleic acid sequence encoding the Luz protein (in particular SEQ ID NO: 1) under the control of a promoter functional in chloroplasts,
(c) une séquence d’acide nucléique codant pour la protéine H3H (en particulier SEQ ID NO: 2) sous le contrôle d’un promoteur fonctionnel dans les chloroplastes,
(d) deux séquences nucléiques présentes dans un chloroplaste, de préférence trnl (SEQ ID NO: 6) et trnA (SEQ ID NO: 7), flanquant lesdites séquences d’acide nucléique (b) et (c), ces séquences (b) et (c) étant donc localisées entre ces deux séquences (d).(c) a nucleic acid sequence encoding the H3H protein (in particular SEQ ID NO: 2) under the control of a promoter functional in chloroplasts, (d) two nucleic acid sequences present in a chloroplast, preferably trnl (SEQ ID NO: 6) and trnA (SEQ ID NO: 7), flanking said nucleic acid sequences (b) and (c), these sequences (b ) and (c) therefore being located between these two sequences (d).
Dans un mode de réalisation particulier, le vecteur contient également In a particular embodiment, the vector also contains
(e) une séquence d’acide nucléique codant pour une protéine Cph (en particulier SEQ ID NO: 3), localisée entre les séquences (d) avec les séquences (b) et (c). (e) a nucleic acid sequence encoding a Cph protein (in particular SEQ ID NO: 3), located between sequences (d) with sequences (b) and (c).
Dans un mode de réalisation particulier, le vecteur contient également In a particular embodiment, the vector also contains
(f) une séquence d’acide nucléique codant pour une protéine HispS (en particulier SEQ ID NO: 4), localisée entre les séquences (d) avec les séquences (b) et (c). (f) a nucleic acid sequence encoding a HispS protein (in particular SEQ ID NO: 4), located between sequences (d) with sequences (b) and (c).
Dans un mode de réalisation particulier, le vecteur contient également In a particular embodiment, the vector also contains
(g) une séquence d’acide nucléique codant pour une protéine NpgA (en particulier SEQ ID NO: 5), localisée entre les séquences (d) avec les séquences (b) et (c). (g) a nucleic acid sequence encoding an NpgA protein (in particular SEQ ID NO: 5), located between the sequences (d) with the sequences (b) and (c).
Dans un mode de réalisation préféré, le vecteur contient l’origine de réplication (a), ainsi que les séquences (b), (c), (e), (f) et (g) flanquées par les séquences (d). In a preferred embodiment, the vector contains the origin of replication (a), as well as sequences (b), (c), (e), (f) and (g) flanked by sequences (d).
Cellules hôtes Host cells
L’invention se rapporte également à une cellule hôte contenant un vecteur tel que décrit ci-dessus. Cette cellule est préférentiellement une cellule bactérienne, de façon préférée Escherichia coli transformée par le vecteur. The invention also relates to a host cell containing a vector as described above. This cell is preferably a bacterial cell, preferably Escherichia coli transformed with the vector.
Optimisation des séquences codantes Optimization of coding sequences
Il est préférable que la séquence nucléique des gènes ait été optimisée pour expression dans les chloroplastes (adaptation des usages de codon des chloroplastes). It is preferable that the nucleic acid sequence of the genes has been optimized for expression in chloroplasts (adaptation of the codon usages of chloroplasts).
On peut se baser sur les informations données dans Nakamura et al (Plant J. 2007 Jan;49(1):128-34. 2006), Liu et al (J Genet. 2005 Apr;84(1):55-62) ou Zhang et al (Journal of Intégrative Plant Biology 2007, 49 (2): 246-254). En particulier on utilise les bases de données du Kazusa DNA Research Institute qui peuvent être trouvées sur leur site internet https://www.kazusa.or.jp/codon. One can rely on the information given in Nakamura et al (Plant J. 2007 Jan; 49 (1): 128-34. 2006), Liu et al (J Genet. 2005 Apr; 84 (1): 55-62) or Zhang et al (Journal of Integrative Plant Biology 2007, 49 (2): 246-254). In particular, the databases of the Kazusa DNA Research Institute are used which can be found on their website https://www.kazusa.or.jp/codon.
Les séquences SEQ ID NO: 1 à SEQ ID NO: 5 sont ainsi des séquences optimisées pour une expression dans les chloroplastes. On peut utiliser ces
séquences, ou des séquences présentant au moins 90% d’identité, de préférence au moins 95% d’identité, de préférence au moins 98% d’identité, de façon plus préférée au moins 99% d’identité avec ces séquences et qui codent pour les protéines SEQ ID NO: 20 à SEQ ID NO: 24 respectivement. The sequences SEQ ID NO: 1 to SEQ ID NO: 5 are thus sequences optimized for expression in chloroplasts. We can use these sequences, or sequences having at least 90% identity, preferably at least 95% identity, preferably at least 98% identity, more preferably at least 99% identity with these sequences and which encode the proteins SEQ ID NO: 20 to SEQ ID NO: 24 respectively.
De préférence, les gènes optimisés le sont pour obtenir un taux de GC compris entre 35% et 40%. On choisit aussi les codons préférentiellement exprimés dans les chloroplastes notamment en tenant compte des données de Liu et al. Preferably, the genes are optimized to obtain a GC level of between 35% and 40%. The codons preferentially expressed in the chloroplasts are also chosen, in particular by taking into account the data of Liu et al.
Organisme Organization
Le système utilisé est un système issu de champignons (fungus). En particulier, on préfère utiliser le système d’enzymes issu d’un champignon choisi parmi Neonothopanus nambi, Neonothopanus gardneri, ou Omphalotus olearius. The system used is a system derived from fungi (fungus). In particular, it is preferred to use the enzyme system derived from a fungus selected from Neonothopanus nambi, Neonothopanus gardneri, or Omphalotus olearius.
De préférence, on préfère lorsque l’ensemble des enzymes utilisées (HispS, H3H, Luz, et CPH) provient du même organisme, de préférence Neonothopanus nambi. L’utilisation de séquences provenant de Neonothopanus gardneri , qui sont très similaires à celles de Neonothopanus nambi, est également envisagée. On peut aussi utiliser à la fois certaines séquences provenant de Neonothopanus nambi et d’autres provenant de Neonothopanus gardneri. Preferably, it is preferred when all of the enzymes used (HispS, H3H, Luz, and CPH) originate from the same organism, preferably Neonothopanus nambi. The use of sequences from Neonothopanus gardneri, which are very similar to those from Neonothopanus nambi, is also envisaged. It is also possible to use both certain sequences from Neonothopanus nambi and others from Neonothopanus gardneri.
Il est rappelé que le gène npgA (4'-phosphopantetheinyl transferase) provient d’Aspergillus nidulans. It is recalled that the npgA (4'-phosphopantetheinyl transferase) gene comes from Aspergillus nidulans.
Promoteurs et séquences codantes Promoters and coding sequences
Opéron Operon
Dans un premier mode de réalisation, on exprime les différents gènes sous le contrôle d’un unique promoteur, sous la forme d’un opéron, ainsi qu’il est connu dans l’art, pour des gènes impliqués dans une même voie métabolique (cf Saxena et al., 2014 J Biosci. 2014 Mar;39(1):33-41 ; Kumar et al, 2012 Metab Eng. 2012 Jan;14(1):19-28). In a first embodiment, the different genes are expressed under the control of a single promoter, in the form of an operon, as is known in the art, for genes involved in the same metabolic pathway ( cf Saxena et al., 2014 J Biosci. 2014 Mar; 39 (1): 33-41; Kumar et al, 2012 Metab Eng. 2012 Jan; 14 (1): 19-28).
Dans ce mode de réalisation, on peut utiliser un promoteur choisi parmi les promoteurs Patpl, Prrn, PrbcL ou PpsbA (en particulier ceux décrits par les séquences SEQ ID NO : 8 à SEQ ID NO: 12). In this embodiment, it is possible to use a promoter chosen from the Patp1, Prrn, PrbcL or PpsbA promoters (in particular those described by the sequences SEQ ID NO: 8 to SEQ ID NO: 12).
Ce mode de réalisation n’est toutefois pas préféré. This embodiment, however, is not preferred.
Promoteurs individuels
Dans un autre mode de réalisation, chaque transgène introduit dans le chloroplaste de la plante est sous le contrôle de son propre promoteur (d’un promoteur qui lui est propre, étant entendu que l’on peut utiliser le même promoteur pour deux gènes, mais qu’il est préféré quant au moins deux transgènes sont sous le contrôle de deux promoteurs différents). Dans ce mode de réalisation, les transgènes ne sont donc pas exprimés au sein d’un opéron. Ce mode de réalisation est préféré. En effet, le fait d’attribuer, à chaque gène, un système de régulation (promoteurs et terminateurs) optimal qui lui est propre, permet d’augmenter les niveaux d’expression, par rapport à l’utilisation d’un système basé sur un opéron. Ce mode d’opération va ainsi à l’encontre des modes généralement observés dans l’art, résumés dans Boehm et Bock (Plant Physiol. 2019 Mar;179(3):794-802) qui se rapporte aux recherches récentes pour l’expression optimale de plusieurs protéines dans les chloroplastes. Ce document montre que, dès que l’on souhaite l’expression simultanée de deux protéines ou plus, on arrange les gènes sous forme d’opéron comme par exemple pour l’expression des : polyhydroxybutyrate (Bohmert-Tatarev et al., Plant Physiol. 2011 Apr;155(4):1690-708), des protéines d’insecte (De Cosa et al., Nat Biotechnol. 2001 Jan;19(1):71-4)7, ou de caroténoïdes (Hasunuma et al., Plant J. 2008 Sep;55(5):857-68), mais aussi pour l’expression de voies métaboliques entière telles que celles de : la vitamine E (Lu et al., Proc Natl Acad Soi U S A. 2013 Feb 19;110(8):E623-32), de l’acide artémisinique (Saxena et al., J Biosci. 2014 Mar;39(1):33-41, Fuentes ét al., eLife. 2016; 5: e13664), du mévalonate (Kumar et al., Metab Eng. 2012 Jan;14(1):19-28, Saxena et al., op. cit.) et de dhurrin (Gnanasekaran et al., J Exp Bot. 2016 Apr;67(8):2495-506). Individual promoters In another embodiment, each transgene introduced into the chloroplast of the plant is under the control of its own promoter (a promoter of its own, it being understood that the same promoter can be used for two genes, but whereas it is preferred that at least two transgenes are under the control of two different promoters). In this embodiment, the transgenes are therefore not expressed within an operon. This embodiment is preferred. Indeed, the fact of attributing, to each gene, an optimal regulatory system (promoters and terminators) which is specific to it, makes it possible to increase the levels of expression, compared to the use of a system based on an operon. This mode of operation thus goes against the modes generally observed in the art, summarized in Boehm and Bock (Plant Physiol. 2019 Mar; 179 (3): 794-802) which relates to recent research for the optimal expression of several proteins in chloroplasts. This document shows that, as soon as one wishes the simultaneous expression of two or more proteins, the genes are arranged in the form of an operon, for example for the expression of: polyhydroxybutyrate (Bohmert-Tatarev et al., Plant Physiol . 2011 Apr; 155 (4): 1690-708), insect proteins (De Cosa et al., Nat Biotechnol. 2001 Jan; 19 (1): 71-4) 7, or carotenoids (Hasunuma et al. ., Plant J. 2008 Sep; 55 (5): 857-68), but also for the expression of entire metabolic pathways such as those of: vitamin E (Lu et al., Proc Natl Acad Soi US A. 2013 Feb 19; 110 (8): E623-32), artemisinic acid (Saxena et al., J Biosci. 2014 Mar; 39 (1): 33-41, Fuentes et al., ELife. 2016; 5: e13664), mevalonate (Kumar et al., Metab Eng. 2012 Jan; 14 (1): 19-28, Saxena et al., op. cit.) and dhurrin (Gnanasekaran et al., J Exp Bot. 2016 Apr; 67 (8): 2495-506).
Par ailleurs, Boehm et Bock (op. cit.) rappellent également les dernières avancées pour améliorer l’efficacité des opérons synthétiques ; par ajout d’éléments d’expression intercistroniques (IEE) (Zhou et al., Plant J. 2007 Dec; 52(5): 961-972), ou par stabilisation des ARN messagers avec des protéines qui se lient à ces derniers (les PRR), (Legen et al., Plant J. 2018 Apr;94(1):8-21). Plus récemment encore, ils rappellent que Fuentes et al. (op. cit.) ont montré que la complexité et le nombre de voies accessibles ont été étendus grâce à une astuce qui combine la transformation de chloroplaste à une transformation nucléaire (« combinatorial supertransformation of transplastomic récipient Unes (COSTREL) »). Ces auteurs ont ainsi transformé classiquement les chloroplastes de plant de Tabac avec la voie métabolique de l’acide artémisinique (sous forme
d’opéron) et le noyau de ces plantes une fois transformées avec cinq gènes (CYB5, ADH1, ALDH1, DBR2, DXR). Avec cette approche, les auteurs ont amélioré de 77 fois la production de l’acide artémisinique. In addition, Boehm and Bock (op. Cit.) Also recall the latest advances to improve the efficiency of synthetic operons; by adding intercistronic expression elements (IEE) (Zhou et al., Plant J. 2007 Dec; 52 (5): 961-972), or by stabilizing messenger RNAs with proteins which bind to them ( PRRs), (Legen et al., Plant J. 2018 Apr; 94 (1): 8-21). More recently still, they recall that Fuentes et al. (op. cit.) have shown that the complexity and the number of accessible pathways have been extended thanks to a trick that combines chloroplast transformation with nuclear transformation (“combinatorial supertransformation of transplastomic receptacle Unes (COSTREL)”). These authors thus classically transformed the chloroplasts of tobacco plants with the metabolic pathway of artemisinic acid (in the form of operon) and the nucleus of these plants once transformed with five genes (CYB5, ADH1, ALDH1, DBR2, DXR). With this approach, the authors improved the production of artemisinic acid by 77 times.
Ainsi, l’art propose plutôt d’utiliser des opérons et de les optimiser (fabrication d’opérons synthétiques), lorsque l’on souhaite exprimer plusieurs gènes dans les chloroplastes, en particulier lorsque les gènes considérés doivent coopérer les uns avec les autres dans une voie métabolique donnée. Thus, the art proposes rather to use operons and to optimize them (manufacture of synthetic operons), when it is desired to express several genes in chloroplasts, in particular when the genes considered must cooperate with each other in a given metabolic pathway.
On peut notamment choisir les promoteurs Patpl, Prrn, PrbcL ou PpsbA. It is in particular possible to choose the Patpl, Prrn, PrbcL or PpsbA promoters.
De préférence, le gène H3h est sous le contrôle du promoteur PpsbA, en particulier la partie spécifiée dans SEQ ID NO: 9. Preferably, the H3h gene is under the control of the PpsbA promoter, in particular the part specified in SEQ ID NO: 9.
De préférence, le gène Luz est sous le contrôle du promoteur PpsbA, en particulier la partie spécifiée dans SEQ ID NO: 9. Preferably, the Luz gene is under the control of the PpsbA promoter, in particular the part specified in SEQ ID NO: 9.
De préférence, le gène Cph est sous le contrôle du promoteur PrbcL, en particulier la partie spécifiée SEQ ID NO: 10. Preferably, the Cph gene is under the control of the PrbcL promoter, in particular the part specified SEQ ID NO: 10.
De préférence, le gène HispS est sous le contrôle du promoteur Prrn, en particulier la partie spécifiée dans SEQ ID NO: 11. Preferably, the HispS gene is under the control of the Prrn promoter, in particular the part specified in SEQ ID NO: 11.
De préférence le gène npgA est sous le contrôle du promoteur Patpl, en particulier la partie spécifiée dans SEQ ID NO: 12. Preferably, the npgA gene is under the control of the Patp1 promoter, in particular the part specified in SEQ ID NO: 12.
Dans un mode de réalisation particulier, on optimise les séquences, et en particulier les promoteurs en choisissant des séquences 5’UTR particulières, afin d’optimiser l’expression des gènes dans les chloroplastes (De Costa et al. Genes Genet Syst. 2001 Dec;76(6):363-71) ; Drechsel et Bock Nucleic Acids Res. 2011 Mar;39(4): 1427-38) ; Shinozaki et Sugiura Gene. 1982 Nov 20(1) 91-102 et Nucleic Acids Res. 1982 Aug 25;10(16):4923-34; Kuroda and Maliga, Plant Physiol. 2001 Jan;125(1):430-6). In a particular embodiment, the sequences are optimized, and in particular the promoters by choosing particular 5′UTR sequences, in order to optimize the expression of the genes in the chloroplasts (De Costa et al. Genes Genet Syst. 2001 Dec. ; 76 (6): 363-71); Drechsel and Bock Nucleic Acids Res. 2011 Mar; 39 (4): 1427-38); Shinozaki and Sugiura Gene. 1982 Nov 20 (1) 91-102 and Nucleic Acids Res. 1982 Aug 25; 10 (16): 4923-34; Kuroda and Maliga, Plant Physiol. 2001 Jan; 125 (1): 430-6).
Les séquences SEQ ID NO: 9 à SEQ ID NO: 12 représentent de tels promoteurs optimisés avec des 5’UTR ajoutés pour améliorer l’expression. The sequences SEQ ID NO: 9 to SEQ ID NO: 12 represent such optimized promoters with added 5′UTRs to improve expression.
Ainsi, de préférence, le gène H3h est sous le contrôle du promoteur optimisé décrit par SEQ ID NO: 9. Thus, preferably, the H3h gene is under the control of the optimized promoter described by SEQ ID NO: 9.
De préférence, le gène Luz est sous le contrôle du promoteur PpsbA optimisé décrit par SEQ ID NO: 9. Preferably, the Luz gene is under the control of the optimized PpsbA promoter described by SEQ ID NO: 9.
De préférence, le gène Cph est sous le contrôle du promoteur PrbcL optimisé décrit par SEQ ID NO: 10.
De préférence, le gène HispS est sous le contrôle du promoteur Prrn optimisé décrit par SEQ ID NO: 11. Preferably, the Cph gene is under the control of the optimized PrbcL promoter described by SEQ ID NO: 10. Preferably, the HispS gene is under the control of the optimized Prrn promoter described by SEQ ID NO: 11.
De préférence le gène npgA est sous le contrôle du promoteur Patpl optimisé décrit par SEQ ID NO: 12. Preferably, the npgA gene is under the control of the optimized Patpl promoter described by SEQ ID NO: 12.
On utilise également des terminateurs positionnés en 3’ des séquences nucléiques codantes. On peut utiliser les terminateurs représentés par les séquences SEQ ID NO: 13 à SEQ ID NO: 18. Terminators positioned at 3 'of the coding nucleic sequences are also used. The terminators represented by the sequences SEQ ID NO: 13 to SEQ ID NO: 18 can be used.
Dans un mode de réalisation préféré, on utilise un construit (5’-3’) SEQ ID NO:In a preferred embodiment, a construct (5'-3 ') SEQ ID NO is used:
9- SEQ ID NO: 1- SEQ ID NO:14 pour exprimer Luz. 9- SEQ ID NO: 1- SEQ ID NO: 14 to express Luz.
Dans un mode de réalisation préféré, on utilise un construit (5’-3’) SEQ ID NO:In a preferred embodiment, a construct (5'-3 ') SEQ ID NO is used:
9- SEQ ID NO: 2- SEQ ID NO:15 pour exprimer H3H. 9- SEQ ID NO: 2- SEQ ID NO: 15 for expressing H3H.
Dans un mode de réalisation préféré, on utilise un construit (5’-3’) SEQ ID NO:In a preferred embodiment, a construct (5'-3 ') SEQ ID NO is used:
10- SEQ ID NO: 3- SEQ ID NO:16 pour exprimer Cph. 10- SEQ ID NO: 3- SEQ ID NO: 16 to express Cph.
Dans un mode de réalisation préféré, on utilise un construit (5’-3’) SEQ ID NO:In a preferred embodiment, a construct (5'-3 ') SEQ ID NO is used:
11- SEQ ID NO: 4- SEQ ID NO:14 pour exprimer HispS. 11- SEQ ID NO: 4- SEQ ID NO: 14 for expressing HispS.
Dans un mode de réalisation préféré, on utilise un construit (5’-3’) SEQ ID NO:In a preferred embodiment, a construct (5'-3 ') SEQ ID NO is used:
12- SEQ ID NO: 5- SEQ ID NO:18 pour exprimer NpgA. 12- SEQ ID NO: 5- SEQ ID NO: 18 for expressing NpgA.
Dans un mode de réalisation préféré, on utilise un construit (5’-3’) SEQ ID NO: 8- SEQ ID NO: 19- SEQ ID NO:17 pour exprimer le gène de sélection aadA. Intégration de la cassette d’expression au sein des chloroplastes Ainsi que vu plus haut, dans un mode de réalisation particulier, l’ensemble des gènes présents dans les chloroplastes forme une cassette d’expression, c’est-à- dire que ces gènes sont présents les uns à la suite des autres sur un fragment d’ADN. Ainsi, on transforme les chloroplastes avec cette cassette d’expression, afin d’obtenir l’expression des gènes codées dans cette cassette d’expression. In a preferred embodiment, a construct (5′-3 ′) SEQ ID NO: 8-SEQ ID NO: 19-SEQ ID NO: 17 is used to express the aadA selection gene. Integration of the Expression Cassette into the Chloroplasts As seen above, in a particular embodiment, all of the genes present in the chloroplasts form an expression cassette, that is to say that these genes are present one after the other on a DNA fragment. Thus, the chloroplasts are transformed with this expression cassette, in order to obtain the expression of the genes encoded in this expression cassette.
Il est préféré lorsque les gènes sont intégrés dans le génome des chloroplastes. En particulier, on utilise la recombinaison homologue pour introduire les gènes à une localisation sélectionnée dans le génome du chloroplaste. It is preferred when the genes are integrated into the genome of chloroplasts. In particular, homologous recombination is used to introduce the genes to a selected location in the genome of the chloroplast.
De nombreux sites d’insertions sont possibles, dans le génome du chloroplaste. On choisit préférentiellement d’intégrer la cassette d’expression dans une région non codante du génome du chloroplaste. On peut ainsi utiliser des séquences inter-géniques comprises entre deux séquences codant pour les ARN de transfert du chloroplaste.
En particulier, on choisit les sites trnl (SEQ ID NO: 6) et trnA (SEQ ID NO: 7), codant pour les ARN de transfert de l’isoleucine et l’alanine. On peut ainsi utiliser les séquences SEQ ID NO: 6 ou SEQ ID NO: 7, ou des séquences contenant ces séquences. On peut aussi utiliser des séquences incluses dans SEQ ID NO: 6 ou SEQ ID NO: 7. Toutefois, dans ce cas, on préfère utiliser des séquences présentant au moins 1000 bases, de préférence au moins 1300 bases, de préférences au moins 1500 bases, de préférence au moins 1700 bases de SEQ ID NO: 6 ou SEQ ID NO: 7. De fait, afin d’augmenter les chances de recombinaison homologue, il est préférable d’utiliser des séquences les plus longues possibles. Many insertion sites are possible in the genome of the chloroplast. It is preferentially chosen to integrate the expression cassette into a non-coding region of the genome of the chloroplast. It is thus possible to use inter-gene sequences comprised between two sequences encoding the chloroplast transfer RNAs. In particular, the trnl (SEQ ID NO: 6) and trnA (SEQ ID NO: 7) sites are chosen, encoding the transfer RNAs of isoleucine and alanine. It is thus possible to use the sequences SEQ ID NO: 6 or SEQ ID NO: 7, or sequences containing these sequences. It is also possible to use sequences included in SEQ ID NO: 6 or SEQ ID NO: 7. However, in this case, it is preferred to use sequences having at least 1000 bases, preferably at least 1300 bases, preferably at least 1500 bases. , preferably at least 1700 bases of SEQ ID NO: 6 or SEQ ID NO: 7. In fact, in order to increase the chances of homologous recombination, it is preferable to use the longest possible sequences.
Les séquences SEQ ID NO: 6 et SEQ ID NO: 7 sont issues du tabac (Nicotiana benthamiana). Elles sont donc particulièrement adaptées pour une intégration par recombinaison homologue dans les chloroplastes du tabac. Elles peuvent toutefois être utilisées pour d’autres plantes, en raison de la grande homologie existant entre ces séquences et les séquences trnl et trnA de chloroplastes d’autres plantes. Ainsi, on peut utiliser des séquences présentant au moins 99% d’identité, de façon plus préférée au moins 99.45% d’identité, de façon plus préférée au moins 99.5% d’identité, de façon plus préférée au moins 99.7% d’identité avec SEQ ID NO: 6 ou SEQ ID NO: 7. The sequences SEQ ID NO: 6 and SEQ ID NO: 7 are obtained from tobacco (Nicotiana benthamiana). They are therefore particularly suitable for integration by homologous recombination in tobacco chloroplasts. However, they can be used for other plants, due to the high homology between these sequences and the trnl and trnA sequences of chloroplasts from other plants. Thus, one can use sequences having at least 99% identity, more preferably at least 99.45% identity, more preferably at least 99.5% identity, more preferably at least 99.7% identity. identity with SEQ ID NO: 6 or SEQ ID NO: 7.
Comparaison de séquences / détermination du pourcentage d’identitéComparison of sequences / determination of the percentage of identity
Afin d’évaluer l’identité entre deux séquences nucléiques, on utilise le logiciel Blastn ( nucléotide blast) développé à partir de Altschul et al, (1997), Nucleic Acids Res. 25:3389-3402; Altschul et al, (2005) FEBS J. 272:5101-5109, disponible sur le site du NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) en utilisant les paramètres suivants, indiqués en anglais : In order to assess the identity between two nucleic acid sequences, the Blastn software (nucleotide blast) developed from Altschul et al, (1997), Nucleic Acids Res. 25: 3389-3402; Altschul et al, (2005) FEBS J. 272: 5101-5109, available on the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using the following parameters, shown in English :
Max target sequences: 100 Max target sequences: 100
Select the maximum number of aligned sequences to display Select the maximum number of aligned sequences to display
Short queries: Automatically adjust parameters for short input sequencesShort queries: Automatically adjust parameters for short input sequences
Expect threshold: 10 Expect threshold: 10
Word size: 28 Word size: 28
Max matches in a query range: 0 Scoring Parameters Match/M ismatch Scores: 1,-2 Gap Costs: Linear Filters and Masking
Filter : Low complexity régions filter : on Max matches in a query range: 0 Scoring Parameters Match / M ismatch Scores: 1, -2 Gap Costs: Linear Filters and Masking Filter: Low complexity regions filter: on
Mask : Mask for lookup table only : on Mask: Mask for lookup table only: on
Afin d’évaluer l’identité entre deux séquences protéiques, on utilise le logiciel Blastp ( protein blast) développé à partir de Altschul et al, (1997), Nucleic Acids Res. 25:3389-3402; Altschul et al, (2005) FEBS J. 272:5101-5109, disponible sur le site du NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) en utilisant les paramètres suivants, indiqués en anglais : In order to assess the identity between two protein sequences, the Blastp (protein blast) software developed from Altschul et al, (1997), Nucleic Acids Res. 25: 3389-3402; Altschul et al, (2005) FEBS J. 272: 5101-5109, available on the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using the following parameters, shown in English :
Expected threshold : 10 Expected threshold: 10
Word size : 3 Word size: 3
Max matches in a query range: 0 Max matches in a query range: 0
Matrix: BLOSUM62 Matrix: BLOSUM62
Gap Costs: Existence 11, Extension 1. Gap Costs: Existence 11, Extension 1.
Compositional adjustments: Conditional compositional score matrix adjustment Compositional adjustments: Conditional compositional score matrix adjustment
No filter for low complexity régions No filter for low complexity regions
Plante Plant
La plante selon l’invention est préférentiellement une plante ornementale. Elle est préférentiellement choisie dans le groupe constitué de Hedera hélix, Pétunia axillaris subsp. axillaris, Nicotiana benthamiana, Ficus benjamina, ficus elastica, Ficus microcarpa, Chlorophytum comosum, Monstera deliciosa, Sansevieria socotrana, Pélargonium x hortorum, Spathiphyllum wallisii, Dracaena draco, Dracaena angustifolia, Yucca aloifolia, Beaucarnea recurvata, Syngonium podophyllum, Fittonia verschaffeltii, Aloe vera, Aloe Aloe jucunda, Aloe juvenna, Dieffenbachia. Livistona speciosa, Orchidaceae. En particulier, la plante est Nicotiana benthamiana. Dans un autre mode de réalisation, la plante est le pétunia (Pétunia axillaris subsp. Axillaris). Dans un autre mode de réalisation, la plante est le lierre ( Hedera hélix). The plant according to the invention is preferably an ornamental plant. It is preferably chosen from the group consisting of Hedera helix, Petunia axillaris subsp. axillaris, Nicotiana benthamiana, Ficus benjamina, ficus elastica, Ficus microcarpa, Chlorophytum comosum, Monstera deliciosa, Sansevieria socotrana, Pelargonium x hortorum, Spathiphyllum wallisii, Dracaena draco, Dracaena angustoifolia, Wormittonia almataarnonia, Dracaena angustoifolia, Yuccaellonium venaittonia, Dracaena angustoifolia, Synoellolia, Dracaena, Dracaena angustoifolia, Spathiphyllum wallisii, Dracaena draco, Dracaena angusteaifolia, Recoellolia, Synoellolia, Dracaena, Dracaena angusteaifolia, Spathiphyllum wallisii, Dracaittonia almittonia, Dracaena angusteaifolia, Synittonia, Dracaittonium, Dracaittonium wallisii, Dracaittonium draco, Dracaena angusteaifolia, Dracaittonia, Dracaittonium, Dracaittonia, Dracaittonium, Dracaittonium , Aloe Aloe jucunda, Aloe juvenna, Dieffenbachia. Livistona speciosa, Orchidaceae. In particular, the plant is Nicotiana benthamiana. In another embodiment, the plant is petunia (Petunia axillaris subsp. Axillaris). In another embodiment, the plant is ivy (Hedera helix).
Méthode de production Method of production
L’invention se rapporte également à une méthode de production d’une plante telle que décrite ci-dessus, comprenant une étape d’insertion des transgènes tels que décrits plus haut dans le génome de chloroplastes de cellules de plantes. La
méthode comprend aussi préférentiellement l’étape de régénération d’une plante par culture de cals. The invention also relates to a method for producing a plant as described above, comprising a step of inserting the transgenes as described above into the genome of chloroplasts of plant cells. The method also preferably comprises the step of regenerating a plant by culturing callus.
Dans un premier mode de réalisation, la méthode comprend l’insertion des transgènes codant pour les enzymes H3H et Luz dans le génome de chloroplastes de cellules de plantes. In a first embodiment, the method comprises inserting the transgenes encoding the enzymes H3H and Luz into the genome of chloroplasts of plant cells.
Dans un autre mode de réalisation, la méthode comprend l’insertion des transgènes codant pour les enzymes Cph, H3H et Luz dans le génome de chloroplastes de cellules de plantes. In another embodiment, the method comprises inserting the transgenes encoding the enzymes Cph, H3H and Luz into the genome of chloroplasts of plant cells.
Dans un autre mode de réalisation, la méthode comprend l’insertion des transgènes codant pour les enzymes Cph, HispS, H3H et Luz dans le génome de chloroplastes de cellules de plantes. In another embodiment, the method comprises inserting the transgenes encoding the enzymes Cph, HispS, H3H and Luz into the genome of chloroplasts of plant cells.
Dans un autre mode de réalisation, la méthode comprend l’insertion des transgènes codant pour les enzymes Cph, HispS, H3H et Luz dans le génome de chloroplastes de cellules de plantes, ainsi qu’un transgène codant pour NpgA. In another embodiment, the method comprises inserting transgenes encoding the enzymes Cph, HispS, H3H and Luz into the genome of chloroplasts of plant cells, as well as a transgene encoding NpgA.
Dans un mode de réalisation, l’intégration des transgènes est effectuée par bombardement de feuilles de plantes avec un plasmide à l’aide d’un canon à particules. Pour ce faire, on prépare la cassette d’expression (fragment d’ADN portant les transgènes que l’on souhaite intégrer dans le génome du chloroplaste), et on recouvre des microbilles de métal (préférentiellement en or, mais pouvant également être en tungstène) qui sont ensuite projetées sur les cellules végétales. In one embodiment, integration of the transgenes is accomplished by bombarding plant leaves with a plasmid using a particle gun. To do this, we prepare the expression cassette (DNA fragment carrying the transgenes that we want to integrate into the genome of the chloroplast), and we cover metal microbeads (preferably gold, but can also be tungsten. ) which are then projected onto plant cells.
Dans un autre mode de réalisation, les cellules végétales sont mise en contact avec du polyéthylène glycol (PEG), qui déstabilise les membranes plasmiques et permet l’entrée des fragments d’ADN portant les transgènes à intégrer dans le génome du plaste. In another embodiment, the plant cells are contacted with polyethylene glycol (PEG), which destabilizes the plasma membranes and allows the entry of DNA fragments carrying the transgenes to integrate into the plastid genome.
On préfère lorsque l’intégration des transgènes dans le génome des chloroplastes est effectuée par recombinaison homologue. Ainsi, les transgènes sont flanqués de séquences homologues à des séquences du chromosome du chloroplaste. L’intégration des transgènes est donc effectuée par la machinerie de l’organite par recombinaison homologue au site It is preferred when the integration of transgenes into the chloroplast genome is accomplished by homologous recombination. Thus, the transgenes are flanked by sequences homologous to sequences of the chromosome of the chloroplast. The integration of transgenes is therefore carried out by the machinery of the organism by homologous recombination at the site
Dans l’un et l’autre cas, on cultive les cellules transformées dans les conditions permettant d’obtenir des cals, que l’on cultive, puis à partir desquels on régénère une plante par des méthodes connues dans l’art. In either case, the transformed cells are cultured under callus conditions, which are cultured, and from which a plant is regenerated by methods known in the art.
Dans un mode de réalisation préféré, la culture des cals est effectuée sur un milieu sélectif. Un milieu sélectif est un milieu contenant un élément sélectif (souvent un antibiotique ou un herbicide) sur lequel ne peuvent pousser que les
cellules contenant un gène de résistance à l’élément sélectif, alors que les cellules ne contenant pas ce gène ne peuvent pousser ou poussent de façon ralentie. In a preferred embodiment, the culture of the calli is carried out on a selective medium. A selective medium is a medium containing a selective element (often an antibiotic or a herbicide) on which only plants can grow. cells containing a gene for resistance to the selective element, while cells without this gene cannot grow or grow sluggishly.
Parmi les éléments sélectifs, on peut citer les antibiotiques neomycine/kanamycine et nptll (aminoglycoside 3'-phosphotransférase), betain et badh (betain aldéhyde deshydrogenase), hygromycine B et hph (hygromycine B phosphotransférase), spectinomycine / streptomycine et aadA (aminoglycoside 3'- adenyltransferase), chloramphenicol et cat (chloramphénicol acétyltransférase), amikacin et aphA6 (3'-aminoglycoside phosphotransférase), blasticidine S et bsr (blasticidine S deaminase), sulfonamides et sull (dihydropteorate synthase DHPS), gentamycine et aacC1 (gentamycine acetyltransferase) ou les herbicides bialophos / phosphinotricine / glufosinate et pat (phosphinotricine acteyltransferase), glyphosate et gox (glyphosate oxydoreductase) ou epsp (5 eonylpyruvyl shikimate- 3-phosphate synthase), bromoxynil et bxn (bromosynile nitrilase), sulfonylureas / imidazolines / triazolopyrimidines / pyrimidylbenzoates et als (acetolactate synthase). Among the selective elements, mention may be made of the antibiotics neomycin / kanamycin and nptll (aminoglycoside 3'-phosphotransferase), betain and badh (betain aldehyde dehydrogenase), hygromycin B and hph (hygromycin B phosphotransferase), spectinomycin / alycosadosAlycin 3 (aminoglycoside 3'-phosphotransferase) and '- adenyltransferase), chloramphenicol and cat (chloramphenicol acetyltransferase), amikacin and aphA6 (3'-aminoglycoside phosphotransferase), blasticidin S and bsr (blasticidin S deaminase), sulfonamides and sull (dihydropteorate acetaminate Camycamyferase and gentcamyferase) or the herbicides bialophos / phosphinotricin / glufosinate and pat (phosphinotricin acteyltransferase), glyphosate and gox (glyphosate oxidoreductase) or epsp (5 eonylpyruvyl shikimate- 3-phosphate synthase), bromoxynase and bxnenzo / nitro-azimurazinylates / tri-zimolazimrilines / tri-rosimidylazin / tri-rosimidylines / tri-rosimidylsimolazimidylines / tri-rosimidylsimolazimrilines (bromosidoreductase) et al (acetolactate synthase).
On utilise en particulier le gène aadA (aminoglycoside 3'-adenyltransferase) qui confère la résistance à la spectinomycine, dont une séquence codante est représentée par SEQ ID NO: 19. In particular, the aadA (aminoglycoside 3'-adenyltransferase) gene is used which confers resistance to spectinomycin, a coding sequence of which is represented by SEQ ID NO: 19.
Le gène de résistance est introduit dans la cassette d’expression contenant les transgènes d’intérêt, sous le contrôle d’un promoteur actif dans les chloroplastes. On peut également mettre en œuvre un système dans lequel le gène de résistance peut être excisé après transformation, notamment en suivant l’enseignement de Scutt et al (Biochimie 84 (2002) 1119-1126) ou Lantham et al (Nature Biotechnology, 2000, (18), 1172-76). The resistance gene is introduced into the expression cassette containing the transgenes of interest, under the control of a promoter active in chloroplasts. It is also possible to implement a system in which the resistance gene can be excised after transformation, in particular by following the teaching of Scutt et al (Biochimie 84 (2002) 1119-1126) or Lantham et al (Nature Biotechnology, 2000, (18), 1172-76).
L’invention se rapporte également à un système lumineux comprenant une plante émettant de la bioluminescence telle que décrite ci-dessus, c’est-à-dire dont au moins une cellule contient au moins un chloroplaste contenant les gènes cités ci-dessus, et qui émet de la bioluminescence par oxydation de la luciférine par l’enzyme Luz. The invention also relates to a light system comprising a plant emitting bioluminescence as described above, that is to say in which at least one cell contains at least one chloroplast containing the genes mentioned above, and which emits bioluminescence by oxidation of luciferin by the enzyme Luz.
De préférence, le système contient une plante dont au moins 50% des chloroplastes contient le système enzymatique mentionné ci-dessus. Preferably, the system contains a plant of which at least 50% of the chloroplasts contains the enzymatic system mentioned above.
L’invention se rapporte également à un procédé de production de lumière, comprenant l’étape d’ajout d’hispidine dans le milieu de culture d’une plante telle que décrite ci-dessus, et dont au moins un chloroplaste contient au moins les
gènes codant pour les enzymes H3H et Luz (de préférence intégrés dans son génome). The invention also relates to a method for producing light, comprising the step of adding hispidin to the culture medium of a plant as described above, and of which at least one chloroplast contains at least the genes encoding the enzymes H3H and Luz (preferably integrated into its genome).
L’invention se rapporte également à un procédé de production de lumière (par une plante), comprenant l’étape d’ajout d’acide caféique dans le milieu de culture d’une plante telle que décrite ci-dessus, et dont au moins un chloroplaste contient au moins les gènes codant pour les enzymes HispS, H3H et Luz (de préférence intégrés dans son génome). The invention also relates to a method for producing light (by a plant), comprising the step of adding caffeic acid to the culture medium of a plant as described above, and of which at least a chloroplast contains at least the genes encoding the enzymes HispS, H3H and Luz (preferably integrated into its genome).
L’invention se rapporte également à un procédé de production de lumière (par une plante), comprenant l’étape d’ajout d’acide caféique dans le milieu de culture d’une plante telle que décrite ci-dessus, et dont au moins un chloroplaste contient les gènes codant pour les enzymes HispS, H3H, Luz, Cph (de préférence intégrés dans son génome). The invention also relates to a method for producing light (by a plant), comprising the step of adding caffeic acid to the culture medium of a plant as described above, and of which at least a chloroplast contains the genes encoding the enzymes HispS, H3H, Luz, Cph (preferably integrated into its genome).
Préférentiellement le chloroplaste contient également et le gène codant pour NpgA, dans les procédés ci-dessus. Preferably, the chloroplast also contains and the gene encoding NpgA, in the above methods.
EXEMPLES EXAMPLES
Les exemples ci-dessous et figures décrivent un mode de réalisation particulier de l’invention. The examples and figures below describe a particular embodiment of the invention.
Exemple 1. Préparation du plasmide contenant les gènes H3H et Luz et les séquences permettant l’intégration de ces gènes dans le génome du chloroplaste ainsi que leur expression Example 1. Preparation of the plasmid containing the H3H and Luz genes and the sequences allowing the integration of these genes into the genome of the chloroplast as well as their expression
Les séquences des gènes H3H et Luz ont été adaptées avec des codons d’usage des chloroplastes puis synthétisées (SEQ ID NO: 2 et SEQ ID NO: 1). Les promoteurs ont été choisis parmi l’ensemble des promoteurs présents dans le génome du chloroplaste et modifiés dans leur séquence 5’UTR de sorte de maximiser l’expression des gènes sous leurs contrôles. Ces promoteurs modifiés ont alors été synthétisés. Les terminateurs ont été choisis parmi l’ensemble des terminateurs présents dans le génome du chloroplaste et certains ont été optimisés pour être les plus courts possibles tout en gardant leurs fonctions. Ils ont aussi été synthétisés. Les séquences trnl et trnA (SEQ ID NO: 6 et SEQ ID NO: 7) ont été choisies pour permettre l’intégration des gènes H3H et Luz dans le génome du chloroplaste. Elles ont été amplifiées par PCR (Polymerase Chain Reaction) à partir d’ADN chloroplastique de Nicotiana benthamiana. Le gène de résistance à la spectinomycine / streptomycine nommé aadA a été amplifié par PCR à partir d’un plasmide le contenant (SEQ ID NO: 19).
Pour élaborer le vecteur plasmidique contenant l’ensemble de ces séquences, le plasmide pUC19 a été utilisé. Les promoteurs, gènes et terminateurs ont été amplifiés par PCR et chaque partie du trio ont été liguées entre elles : promoteur, gène et terminateur avec la méthode ln-fusion de chez Takara. 50 ng ou 100ng d’ADN ont été mis à incuber à 50°C pendant 1h avec les enzymes de ligation du kit ln-fusion. Les produits de ligation ont alors été amplifiés par PCR. The sequences of the H3H and Luz genes were adapted with codons for use by chloroplasts and then synthesized (SEQ ID NO: 2 and SEQ ID NO: 1). The promoters were chosen from among all the promoters present in the genome of the chloroplast and modified in their 5′UTR sequence so as to maximize the expression of the genes under their controls. These modified promoters were then synthesized. The terminators were chosen from among all the terminators present in the chloroplast genome and some have been optimized to be as short as possible while keeping their functions. They have also been synthesized. The trnl and trnA sequences (SEQ ID NO: 6 and SEQ ID NO: 7) were chosen to allow integration of the H3H and Luz genes into the genome of the chloroplast. They were amplified by PCR (Polymerase Chain Reaction) from chloroplast DNA of Nicotiana benthamiana. The spectinomycin / streptomycin resistance gene called aadA was amplified by PCR from a plasmid containing it (SEQ ID NO: 19). To develop the plasmid vector containing all of these sequences, the plasmid pUC19 was used. The promoters, genes and terminators were amplified by PCR and each part of the trio were ligated together: promoter, gene and terminator with the ln-fusion method from Takara. 50 ng or 100ng of DNA were incubated at 50 ° C. for 1 hour with the ligation enzymes from the ln-fusion kit. The ligation products were then amplified by PCR.
Les trois gènes (aadA, H3H, et Luz) ainsi fusionnés avec leurs promoteurs et terminateurs respectifs, et les séquences trnl et trnA ont alors été clonés dans le plasmide pUC19 linéarisé par PCR, en suivant le protocole de NEBuilder. The three genes (aadA, H3H, and Luz) thus fused with their respective promoters and terminators, and the trnI and trnA sequences were then cloned into the plasmid pUC19 linearized by PCR, following the NEBuilder protocol.
Le NEBuilder se base sur la stratégie de clonage par la méthode de « Gibson assembly. » Les primers ont été dessinés de sorte que les fragments présentent un chevauchement de séquence de 25pb entre eux et avec la séquence du site d’insertion du plasmide pUC19. The NEBuilder is based on the “Gibson assembly. The primers were designed so that the fragments show a 25bp sequence overlap with each other and with the insertion site sequence of the plasmid pUC19.
Gibson Reaction and Bacterial Transformation Gibson Reaction and Bacterial Transformation
1. 40 à 75 ng d’ADN de chaque produit de PCR sont utilisés avec du tampon NEBuilder® HiFi DNA assembly 2X et incubés 1h à 50°C. 2. Les bactéries compétentes NEB 10-alpha sont transformées avec les produits de ligation par choc thermique, incubées 1h à 37°C puis étalées sur boite LB agar avec antibiotique et mises à incuber sur la nuit à 37°C. 3. L’ADN plasmidique d’une quinzaine de clones est extrait et analysé par séquençage. 4. Les clones positifs sont alors amplifiés dans un plus grand volume de LB + antibiotique (100ml) et leur ADN plasmidique est extrait et analysé par séquençage. 1. 40 to 75 ng of DNA of each PCR product are used with NEBuilder® HiFi DNA assembly 2X buffer and incubated for 1 hour at 50 ° C. 2. The competent NEB 10-alpha bacteria are transformed with the ligation products by heat shock, incubated for 1 hour at 37 ° C. then spread on an LB agar dish with antibiotic and incubated overnight at 37 ° C. 3. The plasmid DNA of about fifteen clones is extracted and analyzed by sequencing. 4. The positive clones are then amplified in a larger volume of LB + antibiotic (100 ml) and their plasmid DNA is extracted and analyzed by sequencing.
Transformation des plants de Tabac avec les plasmides obtenusTransformation of the Tobacco plants with the plasmids obtained
Recouvrir les billes d’or avec l’ADN plasmidique Cover the gold beads with plasmid DNA
Matériel nécessaire : 1. Ethanol 100%. 2. Billes d’or stériles (Biorad). 3. CaCI2 2.5M. 4. 0.1 M de spermidine. 5. Milieu de croissance des plantes in vitro : MS avec vitamines supplémenté de sucrose à 3%. 6. Hormones 6-benzyl aminopurine (BAP), indole-3-acetic acid (IAA), indole3-butyric acid (IBA), à la concentration de 1 mg/mL. 7. Spectinomycine à 500 mg/L. Materials needed: 1. 100% ethanol. 2. Sterile gold balls (Biorad). 3. 2.5M CaCl2. 4.1 M spermidine. 5. In vitro plant growth medium: MS with vitamins supplemented with 3% sucrose. 6. Hormones 6-benzyl aminopurine (BAP), indole-3-acetic acid (IAA), indole3-butyric acid (IBA), at a concentration of 1 mg / mL. 7. Spectinomycin at 500 mg / L.
Les billes d’or sont préparées en suivant le protocole de Biorad fourni avec les billes. The gold beads are prepared following the Biorad protocol supplied with the beads.
L’ADN plasmidique est alors précipité sur les billes d’or (pour 5 échantillons) : 1. Vortexer 50 pL de billes d’or pendant 1 minute. 2. Ajouter 10 pL d’ADN
plasmidique (à 1 pg/pL) et vortexer le mélange. 3. Ajouter 50mI de Cacl2 2,5 M et vortexer le mélange. 4. Ajouter 20 mI_ de spermidine à 0,1 M et vortexer le mélange. Les billes sont ensuite lavées avec de l’éthanol à 100% puis resuspendues dans 40mI d’éthanol 100% The plasmid DNA is then precipitated on the gold beads (for 5 samples): 1. Vortex 50 μL of gold beads for 1 minute. 2. Add 10 µL of DNA plasmid (at 1 pg / pL) and vortex the mixture. 3. Add 50mI of 2.5M Cacl2 and vortex the mixture. 4. Add 20 ml of 0.1 M spermidine and vortex the mixture. The beads are then washed with 100% ethanol and then resuspended in 40 ml of 100% ethanol
Bombardement des feuilles de Nicotiana benthamiana avec les billes d’orBombing the leaves of Nicotiana benthamiana with the gold balls
Préparation de la chambre à bombardement : 1. Laver la chambre et les grilles à l’éthanol 70%. 2. Placer les billes d’or recouvertes avec l’ADN plasmidique sur la grille prévue à cet effet. 3. Placer la feuille intacte sur papier filtre Whatman No. 1 placée lui-même sur milieu sans antibiotiques. Placer l’échantillon et fermer la chambre de bombardement. 4. Allumer la pompe pour atteindre la pression attendue et presser le bouton pour tirer. 5. Arrêter la pompe pour enlever la pression et ouvrir la chambre. 6. Incuber les échantillons bombardés sur boite pendant 2 jours dans le noir. Le troisième jour, couper les expiants de 3-5mm de côté et les placer sur milieu de sélection (MS supplémenté de sucrose 3% et d’hormones : 1 mg/L BAP, et 0.1 mg/IAA, avec 500 mg/L de spectinomycine. 3. Les tiges transgéniques apparaissent après 3 à 5 semaines de transformation. Couper les feuilles des tiges transgéniques apparues en petits carrés de 2 mm de côté et les placer dans un nouveau milieu de sélection, pour atteindre l’homoplasmie. Régénérer des plantes selon les méthodes connues. Preparation of the bombardment chamber: 1. Wash the chamber and screens in 70% ethanol. 2. Place the gold beads coated with the plasmid DNA on the grid provided. 3. Place the intact sheet on Whatman No. 1 filter paper placed itself on medium without antibiotics. Place the sample and close the bombardment chamber. 4. Turn on the pump to reach the expected pressure and press the button to fire. 5. Stop the pump to relieve pressure and open the chamber. 6. Incubate the bombarded plate samples for 2 days in the dark. On the third day, cut the explants 3-5mm aside and place them on selection medium (MS supplemented with 3% sucrose and hormones: 1 mg / L BAP, and 0.1 mg / IAA, with 500 mg / L of spectinomycin 3. Transgenic stems appear after 3 to 5 weeks of transformation Cut the leaves of the transgenic stems that have appeared into small squares 2 mm apart and place them in a new selection medium, to achieve homoplasmy Regenerate plants according to known methods.
On peut vérifier que les cellules des plantes produisent de la lumière lorsqu’elles sont cultivées sur un milieu contenant de l’hispidine. Plant cells can be verified to produce light when grown on medium containing hispidin.
Exemple 2. Préparation du plasmide contenant les gènes H3H, Luz, CPH, HispS, NpgA et les séquences permettant l’intégration de ces gènes dans le génome du chloroplaste ainsi que leur expression Example 2. Preparation of the plasmid containing the genes H3H, Luz, CPH, HispS, NpgA and the sequences allowing the integration of these genes into the genome of the chloroplast as well as their expression
Les séquences des gènes Luz, H3H, CPH, HispS, NpgA ont été adaptées avec des codons d’usage des chloroplastes puis synthétisées (SEQ ID NO: 1 à SEQ ID NO: 5 respectivement). Les promoteurs choisis sont SEQ ID NO: 9 à SEQ ID NO: 12 respectivement et les terminateurs SEQ ID NO: 14 (Luz et HispS), SEQ ID NO :15 (H3H), SEQ ID NO: 16 (CPH) et SEQ ID NO: 18 (NpgA). The sequences of the Luz, H3H, CPH, HispS, NpgA genes were adapted with codons for use by chloroplasts and then synthesized (SEQ ID NO: 1 to SEQ ID NO: 5 respectively). The promoters chosen are SEQ ID NO: 9 to SEQ ID NO: 12 respectively and the terminators SEQ ID NO: 14 (Luz and HispS), SEQ ID NO: 15 (H3H), SEQ ID NO: 16 (CPH) and SEQ ID NO: 18 (NpgA).
Les séquences trnl et trnA (SEQ ID NO: 6 et SEQ ID NO: 7) ont été choisies pour permettre l’intégration des gènes H3H et Luz dans le génome du chloroplaste. Elles ont été amplifiées par PCR (Polymerase Chain Reaction) à partir d’ADN chloroplastique de Nicotiana benthamiana. Le gène de résistance à la
spectinomycine / streptomycine nommé aadA a été amplifié par PCR à partir d’un plasmide le contenant (SEQ ID NO: 19). The trnl and trnA sequences (SEQ ID NO: 6 and SEQ ID NO: 7) were chosen to allow integration of the H3H and Luz genes into the genome of the chloroplast. They were amplified by PCR (Polymerase Chain Reaction) from chloroplast DNA of Nicotiana benthamiana. The resistance gene spectinomycin / streptomycin named aadA was amplified by PCR from a plasmid containing it (SEQ ID NO: 19).
Une cassette d’expression a été préparée ainsi que décrit plus haut et intégrée dans un plasmide. An expression cassette was prepared as described above and integrated into a plasmid.
La transformation de chloroplastes a été réalisée par biolistique sur des feuilles de tabac, ainsi que décrit plus haut. The transformation of chloroplasts was carried out biolistically on tobacco leaves, as described above.
Les échantillons ont été récupérés, cultivés (plusieurs fois pour atteindre l’homoplasmie) sur milieu contenant de la spectinomycine. Samples were collected, cultured (several times to achieve homoplasmia) on medium containing spectinomycin.
On peut ainsi vérifier que les cellules produisent de la lumière sans ajout de composé externe. It is thus possible to verify that the cells produce light without adding any external compound.
Exemple 3 Obtention de chloroplastes bioluminescents (de cellules végétales contenant des chloroplastes bioluminescents Example 3 Obtaining bioluminescent chloroplasts (from plant cells containing bioluminescent chloroplasts
Les chloroplastes de feuilles de Nicotiana benthamiana issues de pousses ont été visualisés 15 semaines après transformation par bombardement. The chloroplasts of Nicotiana benthamiana leaves originating from shoots were visualized 15 weeks after transformation by bombardment.
Pour cela, plusieurs transformations indépendantes ont été réalisées sur différentes pousses de Nicotiana benthamiana. Sur deux de celles-ci, des morceaux de feuille ont été prélevés, qui ont été montés entre lame et lamelle dans une goutte d’eau miliQ stérile. Dès le montage obtenu, les échantillons ont été immédiatement visualisés par microscopie. For this, several independent transformations were carried out on different shoots of Nicotiana benthamiana. From two of these, pieces of leaf were taken, which were mounted between slide and coverslip in a drop of sterile miliQ water. As soon as the assembly was obtained, the samples were immediately visualized by microscopy.
L’imagerie a été performée avec un microscope Nikon Eclipse Ti avec un objectif à immersion 100x 1.49 NA. Le laser à 405 nm (cw, Oxxius) a été utilisé pour visualiser les chloroplastes (10 W/cm2). L’émission des échantillons a été spectralement filtrée à l’aide d’un miroir dichroïque (Di01-R488-25x36, Semrock) et ensuite imagée sur une caméra EM-CCD de Hamamatsu (ImagEM). Pour détecter la bioluminescence, le laser à 405 nm a été bloqué avec un obturateur mécanique. Une lentille additionnelle a été utilisée pour obtenir un grossissement final de 150X correspondant à une taille de pixel de 106.67 nm. Le temps d’acquisition était de 1s. Imaging was performed with a Nikon Eclipse Ti microscope with a 100x 1.49 NA immersion objective. The 405 nm laser (cw, Oxxius) was used to visualize the chloroplasts (10 W / cm 2 ). The emission of the samples was spectrally filtered using a dichroic mirror (Di01-R488-25x36, Semrock) and then imaged on a Hamamatsu EM-CCD camera (ImagEM). To detect bioluminescence, the 405nm laser was blocked with a mechanical shutter. An additional lens was used to obtain a final magnification of 150X corresponding to a pixel size of 106.67 nm. The acquisition time was 1s.
Les résultats obtenus montrent que les chloroplastes présentent une bioluminescence liée au construit. The results obtained show that the chloroplasts exhibit a bioluminescence linked to the construct.
En tenant compte du volume d’un chloroplaste (mesures faite avec le logiciel ImageJ) qui est d’en moyenne 20mM2 et qu’un pixel a une surface de 0.01 mM2, on peut évaluer l’émission globale d’un chloroplaste. On trouve ainsi que le nombre de photons par seconde par chloroplaste est compris entre 1600 et 3200.
By taking into account the volume of a chloroplast (measurements made with the ImageJ software) which is on average 20 mM 2 and that a pixel has a surface area of 0.01 mM 2 , we can evaluate the overall emission of a chloroplast. We thus find that the number of photons per second per chloroplast is between 1600 and 3200.
Claims
1. Plante dont au moins un chloroplaste d’une cellule contient les gènes codant pour les protéines hispidine-3-hydroxylase (H3H) et luciférase (Luz) de champignon sous le contrôle de promoteurs actifs dans le chloroplaste. 1. A plant in which at least one chloroplast of a cell contains the genes encoding the fungal hispidin-3-hydroxylase (H3H) and luciferase (Luz) proteins under the control of promoters active in the chloroplast.
2. Plante selon la revendication 1, caractérisée en ce que le chloroplaste contient également les gènes codant pour les protéines caffeylpyruvate hydrolase (CPH), hispidin synthase (HispS) et phosphopantetheinyl transferase (NpgA) sous le contrôles de promoteurs actifs dans les chloroplastes. 2. Plant according to claim 1, characterized in that the chloroplast also contains the genes encoding the proteins caffeylpyruvate hydrolase (CPH), hispidin synthase (HispS) and phosphopantetheinyl transferase (NpgA) under the control of promoters active in the chloroplasts.
3. Plante selon la revendication 1 ou 2, caractérisée en ce que la séquence nucléique des gènes a été optimisée pour expression dans les chloroplastes (adaptation des usages de codon des chloroplastes). 3. Plant according to claim 1 or 2, characterized in that the nucleic acid sequence of the genes has been optimized for expression in chloroplasts (adaptation of the codon uses of chloroplasts).
4. Plante selon l’une des revendications 1 à 3, caractérisée en l’ensemble de ses cellules contient au moins un chloroplaste transformé par les gènes codant pour les protéines H3H et Luz et optionnellement les gènes codant pour les protéines CPH HispS et NpgA. 4. Plant according to one of claims 1 to 3, characterized in all of its cells contains at least one chloroplast transformed by the genes encoding the H3H and Luz proteins and optionally the genes encoding the CPH HispS and NpgA proteins.
5. Plante selon l’une des revendications 1 à 4, caractérisé en ce que les promoteurs sont choisis parmi SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 et SEQ ID NO: 12. 5. Plant according to one of claims 1 to 4, characterized in that the promoters are chosen from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.
6. Plante selon l’une des revendications 1 à 5, caractérisée en ce que les gènes sont intégrés dans le génome du chloroplaste aux sites trnl et trnA, représentés par SEQ ID NO: 6 et SEQ ID NO: 7 respectivement. 6. Plant according to one of claims 1 to 5, characterized in that the genes are integrated into the genome of the chloroplast at the trnl and trnA sites, represented by SEQ ID NO: 6 and SEQ ID NO: 7 respectively.
7. Plante selon l’une des revendications 1 à 5, caractérisée en ce que les gènes ont été intégrés dans le génome du chloroplaste par recombinaison homologue. 7. Plant according to one of claims 1 to 5, characterized in that the genes have been integrated into the genome of the chloroplast by homologous recombination.
8. Plante selon l’une des revendications 1 à 7, caractérisée en ce qu’elle est choisie parmi Hedera hélix, Pétunia axillaris subsp. axillaris, Nicotiana benthamiana, Ficus benjamina, Ficus elastica, Ficus microcarpa, Chlorophytum
comosum, Monstera deliciosa, Sansevieria socotrana, Pélargonium x hortorum, Spathiphyllum wallisii, Dracaena draco, Dracaena angustifolia, Yucca aloifolia, Beaucarnea recurvata, Syngonium podophyllum, Fittonia verschaffeltii, Aloe vera, Aloe jucunda, Aloe juvenna, Orchidaceae, Dieffenbachi, et Livistona speciosa. 8. Plant according to one of claims 1 to 7, characterized in that it is chosen from Hedera helix, Petunia axillaris subsp. axillaris, Nicotiana benthamiana, Ficus benjamina, Ficus elastica, Ficus microcarpa, Chlorophytum comosum, Monstera deliciosa, Sansevieria socotrana, Pelargonium x hortorum, Spathiphyllum wallisii, Dracaena draco, Dracaena angustifolia, Yucca aloifolia, Beaucarnea recurvata, Syngonium podophyllum, Fittonia verschaffeltii, Aloebachibachibachi, Aloebachiospecacea jundabachi, Aloebachi jundabachi, Aloebachi junda jundallum, Fittonia verschaffeltii, Aloebacha junda junda, etabachi, Aloebachi.
9. Méthode de production d’une plante selon l’une des revendications 1 à 8, caractérisée en ce qu’elle comprend les étapes d’insertion des transgènes dans le génome des chloroplastes de cellules de plantes, et de régénération d’une plante par culture de cals. 9. Method for producing a plant according to one of claims 1 to 8, characterized in that it comprises the steps of inserting the transgenes into the genome of the chloroplasts of plant cells, and of regenerating a plant. by culture of calli.
10. Méthode selon la revendication 9, caractérisée en ce que l’intégration des transgènes est effectuée par bombardement de feuilles de plantes avec un plasmide à l’aide d’un canon à particules. 10. Method according to claim 9, characterized in that the integration of the transgenes is carried out by bombarding plant leaves with a plasmid using a particle gun.
11. Méthode selon la revendication 9, caractérisée en ce que l’intégration des transgènes est effectuée par déstabilisation des membranes plasmiques à l’aide de polyéthylène glycol (PEG). 11. Method according to claim 9, characterized in that the integration of the transgenes is carried out by destabilization of the plasma membranes using polyethylene glycol (PEG).
12. Méthode selon l’une des revendications 9 à 11, caractérisée en ce que les transgènes sont intégrés dans le génome des chloroplastes par recombinaison homologue. 12. Method according to one of claims 9 to 11, characterized in that the transgenes are integrated into the genome of chloroplasts by homologous recombination.
13. Méthode selon l’une des revendications 9 à 12, caractérisée en ce qu’un gène de résistance est également inséré dans le génome des chloroplastes et que la culture de cals est effectuée sur un milieu sélectif. 13. Method according to one of claims 9 to 12, characterized in that a resistance gene is also inserted into the genome of chloroplasts and that the callus culture is carried out on a selective medium.
14. Système lumineux comprenant une plante selon l’une des revendications 1 à 8. 14. A light system comprising a plant according to one of claims 1 to 8.
15. Procédé de production de lumière, comprenant l’étape d’ajout d’hispidine dans le milieu de culture d’une plante selon l’une des revendications 1 ou 3 à 7 dans leur dépendance à la revendication 1.
15. A method of producing light, comprising the step of adding hispidin to the growing medium of a plant according to one of claims 1 or 3 to 7 in dependence on claim 1.
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