Process for transferring DNA into plant green pigment to produce protein

Abstract

The invention relates to a process for transferring DNA (Deoxyribose Nucleic Acid) into a plant green pigment (chloroplast) to produce protein, which specifically comprises the following steps of: cloning a gene to be transferred after a chloroplast specific promoter, embedding the artificial gene and an antibiotic resistance gene into a transformation vector, the cloning site is located between two adjacent DNA sequences in a plant pigment genome to be transformed, then in a deionized protoplast liquid culture medium with the osmotic pressure identical to that in plant cells, cell walls of the plant cells are digested with appropriate cell wall degrading enzymes (cellulase, polygalacturonase and the like), protoplast spheres and protoplast are obtained, and the plant pigment genome is transformed. The method comprises the following steps: preparing a protoplast culture medium containing enzyme, mixing plasmid DNA carrying coding genes with the protoplast culture medium containing no enzyme, adding the DNA into the protoplast, putting the mixture into an electroporation cuvette, impacting with current pulse, and finally transferring cells with resistance to selective antibiotics into the selective culture medium to proliferate, thereby regenerating the whole plant.

Description

[0001] The subject of the present invention is a method for transferring DNA into plant green pigments (chloroplasts) to produce proteins, which method is suitable for mass production of various proteins, such as antibodies, protein hormones, and enzymes.

[0002] To date, there are two methods for transferring exogenous DNA into plant green chromatin: one is the so-called biodegradation method, which is to bind to the surface of gold or tungsten particles and inject them into cells under high pressure; the other is the polyethylene glycol (PEG)-mediated DNA cell uptake method.

[0003] Procedures have been developed in the past to transform bacterial, yeast, and mammalian cells by opening the cell membrane through various impulses, allowing DNA to be taken up by the cell through the temporary pores. Such procedures usually involve the introduction of DNA that is either able to replicate on its own, is not integrated into the cell's genome, remains as an extrachromosomal element (e.g., a plasmid), or is integrated into one of the cell's chromosomes.

[0004] It has not yet been discovered that exogenous DNA can be transferred and embedded into organelles such as chloroplasts and mitochondria through electric current (electroporation).

[0005] The advantage of electroporation transformation over biodegradation is that the DNA-coated gold / tungsten particles can only hit a portion of the cells to be transformed, which may or may not hit the chromoplasts, while the current pulse can hit all the cells to be transformed and all the chromoplasts therein, so the transformation efficiency is higher.

[0006] Our goal is mainly to use the process described in this invention to produce plants where large quantities of any protein are required, such as antibodies, protein hormones, enzymes.

[0007] The process can also produce plants that glow in the dark, providing ample lighting for outdoor and indoor use after dark, which can be used to decorate apartments, courtyards, outdoor parks and even roads. It is known from practice that there are naturally luminous bacteria, worms, fungi, and luminous bacteria that live in symbiosis with animals, but there are no luminous plants in nature.

[0008] The process described in the present invention is used to demonstrate the production of such luminous plants that can glow in the dark. If the transformation is successful, the results can be observed with the naked eye in a dark room. The plants produced are mainly ornamental plants for indoor or outdoor decorative lighting.

[0009] In the course of the present invention, we realized that if at a certain stage of the process a mixture of protoplasts and DNA is electroporated for 0.005-0.01 seconds (electric field strength 5-50 V / cm, 75-150 times) at room temperature, the cells can not only absorb exogenous DNA from their environment, but also the DNA that has entered the cells can pass through the membranes of organelles (including green pigments) and be embedded in the genome of the organelles through homologous recombination of appropriately designed DNA.

[0010] Therefore, the subject of the present invention is a process for transferring DNA into plant green pigment-chloroplast to produce protein, and the specific method is as follows:

[0011] —The gene to be transferred is cloned behind the chloroplast-specific promoter, and then the artificial gene and the antibiotic resistance gene are embedded in a plasmid transformation vector with a copy number of 300-500. The vector is large and easy to purify, and the cloning site is located between two adjacent DNA sequences in the pigment genome of the plant to be transformed, ensuring that the cloned artificial gene and the antibiotic resistance gene to be selected are embedded in the pigment genome through homologous recombination.

[0012] - In a deionized protoplast liquid medium having the same osmotic pressure as that inside plant cells, the cell walls of plant cells are digested with appropriate cell wall degrading enzymes (cellulase, polygalacturonase, etc.) to obtain spheroplasts and protoplasts. The specific method is as follows:

[0013] - Mix the DNA with protoplast medium without enzymes and add the DNA to the protoplasts,

[0014] -Then the mixture of protoplasts and DNA was transferred into the electroporation cuvette and subjected to a 50-500 V current pulse for 0.005-0.01 seconds for 75-150 times at room temperature and an electric field strength of 5-50 V / cm, with an interval of 0.005-0.01 seconds between two current pulses, and then

[0015] - The electroporated protoplasts are transferred in a known manner to a nutrient solution containing salts, metabolizable sugars and cell division stimulating hormones, and then transferred to a selective medium in which cells resistant to the selective antibiotic grow, thereby regenerating the entire plant.

[0016] The implementation method of the present invention can be found in the dependent claims.

[0017] The specific implementation process of the process described in the present invention is shown in Example 1 below.

[0018] We clone the gene encoding the protein that provides antibiotic selection, promote its insertion into the chloroplast genome by homologous recombination, control the expression of the foreign gene and the enzyme to be produced (in this case, the enzyme responsible for the production of luciferin and its oxidation and luminescence), and then after cloning these genes into a specific promoter of the chloroplast, add the gene encoding this artificial operon and the aminoglycoside O-nucleotidyl transferase that confers resistance to the antibiotic spectinomycin to the plasmid DNA transformation vector, as shown in Appendix 1 Figure 1 As shown, the cloning site is located between two adjacent DNA sequences of the plant to be transformed, in this example the tobacco pigment genome.

[0019] In this process, a mixture of cell wall degrading enzymes (40 g / L cellulase R-10, 5 g / L macerate R-10) and a protoplast liquid culture medium (0.2 M saharose, 2 mM MES, pH = 5.7) is used to digest the cell walls of plant cells. The osmotic pressure is the same as that inside the plant cells (25°C, 48 hours), thereby obtaining spheroplasts and protoplasts.

[0020] Subsequently, the protoplasts were collected on the surface of a filter with a pore size of 0.45 μm, washed three times with a protoplast culture medium (0.2 M saharose, 2 mM MES, pH = 5.7), and then suspended in the same culture medium. Finally, 1 ml of the previously prepared DNA-containing concentrate was added to the protoplast suspension prepared according to the above point, and the protoplast-DNA mixture was placed in an electroporation cuvette and subjected to 99 current pulses of up to 500 V at a temperature of 25° C. and an electric field strength of 50 V / cm, each for 0.01 seconds, with an interval of 0.01 seconds between pulses. Subsequently, the electroporated protoplasts were transferred to a nutrient solution containing salts, metabolizable sugars and cell division stimulating hormones in a known manner, and then transferred to a selective culture medium, where cells resistant to selective antibiotics grow, and the whole plant can be regenerated.

[0021] In our experiments, when attempting to introduce a minigene encoding green fluorescent protein into chloroplasts, we observed fluorescent cells under UV light.

[0022] Furthermore, when the six genes required for bioluminescence were placed behind a chloroplast-specific promoter, light production could be observed in the bacteria. Based on these preliminary results, successful introduction of large gene constructs into chloroplasts resulted in bioluminescence.

[0023] The process described in the present invention achieves our purpose, and its advantages and scope of use are summarized as follows:

[0024] -The process described in the present invention is applicable to mass production of any protein in plants, such as antibodies, protein hormones, enzymes.

[0025] - The process described in the present invention is also suitable for producing luminescent plants that can glow in the dark. If the transformation is successful, the results can be observed with the naked eye in a dark room. The plants produced are mainly ornamental plants for indoor or outdoor decorative lighting.

Claims

1. The process of transferring DNA into plant green pigments (chloroplasts) to produce proteins is as follows: - The gene to be transferred is cloned behind the chloroplast-specific promoter, and then the artificial gene and the antibiotic resistance gene are embedded in a plasmid transformation vector with a copy number of 300-500. The cloning site is located between two adjacent DNA sequences in the genome of the plant pigment to be transformed, and then - In a protoplast liquid medium with the same osmotic pressure as that inside the plant cell, the cell wall of the plant cell is digested with an appropriate cell wall degrading enzyme mixture to obtain spheroplasts and protoplasts, characterized in that: - Mix the plasmid DNA carrying the coding gene with the protoplast medium without enzymes, then add the DNA to the protoplasts, and then put the protoplasts and DNA mixture into the electroporation cuvette, and shock with 50-500V current pulse for 0.005-0.01 seconds at room temperature and an electric field strength of 5-50V / cm for 75-150 times, and then —The electroporated protoplasts are transferred to a nutrient solution containing salts, metabolizable sugars, and cell division stimulating hormones, and then transferred to a selective medium in which cells resistant to the selective antibiotics grow, thereby regenerating the entire plant.

2. The method according to claim 1, characterized in that Tobacco (Nicotiana tabacum) was transformed with DNA.

3. The method according to claim 1, characterized in that DNA transformation of petunia (Petunia hybrida).

4. The method according to claim 1, characterized in that An antibiotic resistance gene was embedded into the transformation vector that provided selectability along with sequences derived from the pigment genome of tobacco or petunia plant species.

5. The method according to claim 4, characterized in that The genome sequence of tobacco chloroplasts (identifier number AP0119623 in the public GenBank database) is between nucleotides 99989-101248.

6. The method according to claim 4, characterized in that The genomic sequence of petunia chloroplasts (identifier number NC_040178 in the public GenBank database) is between nucleotides 100518-101804.

7. The method according to claim 4, characterized in that The genes encoding the flavin-dependent oxidoreductase, acyltransferase, long-chain fatty acyl thioester reductase, long-chain fatty acyl ligase, and NAD(P)H-flavin reductase protein α and β subunits of Photorhabditis elegans were inserted into the transformation vector behind the chloroplast-specific promoter.

8. The process according to claim 1, characterized in that In a deionized medium at pH 5.7, the cell walls of plant cells are digested by cell wall destroying enzymes - cellulase and melastase.

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