METHOD FOR PRODUCING BIOLUMINESCENT PLANTS

Abstract

The present invention relates to a method of making bioluminescent plants by introducing luciferase genes and luciferin biosynthesis genes into plant chloroplasts.

Claims

1. A plant in which at least one chloroplast of a cell contains comprises genes encoding fungal proteins hispidin-3-hydroxylase (H3H) and luciferase (Luz) under control of promoters active in the chloroplast.

2. The plant according to claim 1, wherein the chloroplast also comprises genes encoding the proteins caffeylpyruvate hydrolase (CPH), hispidin synthase (HispS) and phosphopantetheinyl transferase (NpgA) under the control of the promoters active in the chloroplasts.

3. The plant according to claim 1, wherein a nucleic acid sequence of the genes has been optimized for expression in the chloroplasts (adaptation of chloroplast codon usages).

4. The plant according to claim 1, wherein all cells comprise, at least one chloroplast transformed by the genes coding for the H3H and the Luz proteins and optionally genes coding for CPH HispS and NpgA proteins.

5. The plant according to claim 1, wherein the promoters are selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

6. The plant according to claim 1, wherein the genes are integrated into a chloroplast genome at trnI and trnA sites, represented by SEQ ID NO: 6 and SEQ ID NO: 7 respectively.

7. The plant according to claim 1, wherein the genes have been integrated into a chloroplast genome by homologous recombination.

8. The plant according to claim 1, selected 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, Aloe vera, Aloe jucunda, Aloe juvenna, Orchidaceae, Dieffenbachi, and Livistona speciosa.

9. A method of producing the plant according to claim 1 comprising inserting transgenes into a genome of chloroplasts of plant cells, and regenerating a plant by callus culture.

10. The method according to claim 9, wherein the inserting of the transgenes is performed by bombarding plant leaves with a plasmid using a particle gun.

11. The method according to claim 9, wherein inserting of the transgenes is performed by destabilizing plasma membranes with polyethylene glycol (PEG).

12. The method according to claim 9, wherein the transgenes are integrated into a chloroplast genome by homologous recombination.

13. The method according to claim 9, wherein a resistance gene is also inserted into the genome of the chloroplasts and the callus culture is performed on a selective medium.

14. A light system comprising a plant according to claim 1.

15. A method of producing light comprising adding hispidin to a culture medium of the plant according to claim 1.

16. The plant according to claim 1, wherein all cells comprise at least one chloroplast transformed by genes coding for the H3H and the Luz proteins and optionally genes coding for CPH HispS and NpgA proteins.

17. The plant according to claim 16, wherein the promoters are selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

18. The plant according to claim 17, wherein the genes are integrated into a chloroplast genome at trnI and trnA sites, represented by SEQ ID NO: 6 and SEQ ID NO: 7 respectively.

19. The plant according to claim 18, wherein the genes have been integrated into the chloroplast genome by homologous recombination.

20. The plant according to claim 19 selected 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, Aloe vera, Aloe jucunda, Aloe juvenna, Orchidaceae, Dieffenbachi, and Livistona speciosa.

Description

EXAMPLES

[0112] The following examples and figures describe a particular embodiment of the invention.

Example 1. Preparation of the Plasmid Containing the H3H and Luz Genes and the Sequences Allowing the Integration of These Genes Into the Chloroplast Genome and Their Expression

[0113] The H3H and Luz gene sequences were adapted with chloroplast usage codons and then synthesized (SEQ ID NO: 2 and SEQ ID NO: 1). The promoters were selected from the set of promoters present in the chloroplast genome and modified in their 5UTR sequence so as to maximize the expression of genes under their controls. These modified promoters were then synthesized. The terminators were selected from the set of terminators present in the chloroplast genome and some were optimized to be as short as possible while keeping their functions. They were also synthesized. The trnI and trnA sequences (SEQ ID NO: 6 and SEQ ID NO: 7) were chosen to allow the integration of the H3H and Luz genes in the chloroplast genome. They were amplified by PCR (Polymerase Chain Reaction) from chloroplast DNA of Nicotiana benthamiana. The spectinomycin/streptomycin resistance gene named aadA was amplified by PCR from a plasmid containing it (SEQ ID NO: 19).

[0114] To elaborate the plasmid vector containing all these sequences, the plasmid pUC19 was used. The promoters, genes and terminators were amplified by PCR and each part of the trio was ligated together: promoter, gene and terminator with the In-fusion method from Takara. 50 ng or 100 ng of DNA was incubated at 50 C. for 1 h with the ligation enzymes from the In-fusion kit. The ligation products were then amplified by PCR.

[0115] 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 linearized pUC19 plasmid by PCR, following the NEBuilder protocol.

[0116] The NEBuilder is based on the Gibson assembly cloning strategy. The primers were designed so that the fragments have a 25bp sequence overlap with each other and with the sequence of the pUC19 plasmid insertion site.

Gibson Reaction and Bacterial Transformation

[0117] 1. 40 to 75 ng of DNA from each PCR product is used with NEBuilder HiFi DNA assembly 2 buffer and incubated for 1 h at 50 C. 2. 10-alpha NEB competent bacteria are transformed with the ligation products by heat shock, incubated for 1 h at 37 C. and then plated on LB agar plate with antibiotic and incubated overnight at 37 C. 3. Plasmid DNA from about 15 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 of Tobacco Plants with the Obtained Plasmids

[0118] Coating the Gold Beads with Plasmid DNA

[0119] Materials required: 1. 100% ethanol. 2. Sterile gold beads (Biorad). 3. 2.5 M CaCl2. 4. 0.1 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 the concentration of 1 mg/mL. 7. Spectinomycin at 500 mg/L.

[0120] The gold beads are prepared following the Biorad protocol provided with the beads.

[0121] The plasmid DNA is then precipitated onto the gold beads (for 5 samples): 1. Vortex 50 L of gold beads for 1 minute. 2. Add 10 L of plasmid DNA (at 1 g/L) and vortex the mixture. 3. Add 50 L of 2.5 M Cacl2 and vortex the mixture. 4. Add 20 L of 0.1 M spermidine and vortex the mixture. The beads are then washed with 100% ethanol and resuspended in 40 l of 100% ethanol

Bombardment of Nicotiana benthamiana Leaves with Gold Beads

[0122] Preparation of the bombardment chamber: 1. wash the chamber and the grids with 70% ethanol 2. Place the gold beads coated with plasmid DNA on the grid provided. 3. Place the intact sheet on Whatman No. 1 filter paper placed on antibiotic-free medium. 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 release the pressure and open the chamber. 6. Incubate the bombarded samples on the box for 2 days in the dark. On the third day, cut the explants 3-5 mm square 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 spectinomycin. 3. Transgenic stems appear after 3 to 5 weeks of transformation. Cut the leaves of the emerged transgenic stems into small 2 mm squares and place them in new selection medium, to achieve homoplasmy. Regenerate plants according to known methods.

[0123] It can be verified that plant cells produce light when grown on a medium containing hispidine.

Example 2. Preparation of the Plasmid Containing the H3H, Luz, CPH, HispS, NpgA Genes and the Sequences Allowing the Integration of These Genes Into the Chloroplast Genome and Their Expression

[0124] The sequences of Luz, H3H, CPH, HispS, NpgA genes were adapted with chloroplast usage codons and then synthesized (SEQ ID NO: 1 to SEQ ID NO: 5 respectively). The selected promoters 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).

[0125] The trnI and trnA sequences (SEQ ID NO: 6 and SEQ ID NO: 7) were chosen to allow the integration of the H3H and Luz genes into the chloroplast genome. They were amplified by PCR (Polymerase Chain Reaction) from chloroplast DNA of Nicotiana benthamiana. The spectinomycin/streptomycin resistance gene named aadA was amplified by PCR from a plasmid containing it (SEQ ID NO: 19).

[0126] An expression cassette was prepared as described above and integrated into a plasmid.

[0127] Chloroplast transformation was performed by biolistics on tobacco leaves, as described above.

[0128] Samples were recovered, cultured (several times to achieve homoplasmy) on medium containing spectinomycin.

[0129] One can thus verify that the cells produce light without the addition of an external compound.

Example 3 Obtaining Bioluminescent Chloroplasts (From Plant Cells Containing Bioluminescent Chloroplasts

[0130] Chloroplasts of Nicotiana benthamiana leaves from shoots were visualized 15 weeks after transformation by bombardment.

[0131] For this purpose, several independent transformations were performed on different shoots of Nicotiana benthamiana. On two of them, leaf pieces were taken, which were mounted between slide and coverslip in a drop of sterile miliQ water. As soon as the mounting was obtained, the samples were immediately visualized by microscopy.

[0132] Imaging was performed with a Nikon Eclipse Ti microscope with a 1001.49 NA immersion objective. The 405 nm laser (cw, Oxxius) was used to image the chloroplasts (10 W/cm.sup.2). The emission from the samples was spectrally filtered using a dichroic mirror (Di01-R488-2536, Semrock) and then imaged on a Hamamatsu EM-CCD camera (ImagEM). To detect bioluminescence, the 405 nm laser was blocked with a mechanical shutter. An additional lens was used to obtain a final magnification of 150 corresponding to a pixel size of 106.67 nm. The acquisition time was 1 s.

[0133] The results obtained show that chloroplasts exhibit construct-related bioluminescence.

[0134] Taking into account the volume of a chloroplast (measurements made with the ImageJ software) which is on average 20 M.sup.2 and that a pixel has a surface of 0.01 M.sup.2, we can evaluate the global emission of a chloroplast. It is found that the number of photons per second per chloroplast is between 1600 and 3200.