JP2014193153A - Method for introducing gene into euglena - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for introducing a gene into Euglena for stable maintenance of a heterogenous gene.SOLUTION: A method for introducing a DNA fragment containing a sequence encoding a protein into a Euglena cell comprises the steps of: preparing a binary vector containing the DNA fragment; preparing a linear gene fragment from the binary vector, where the linear gene fragment comprises a T-DNA region containing the DNA fragment; and directly introducing the linear gene fragment into a Euglena cell. The step of direct DNA introduction comprises the steps of: coating a micro-carrier with the linear gene fragment; and delivering the micro-carrier into Euglena cells by using a particle gun method.

Description

  The present invention relates to a gene introduction method for Euglena, in which a foreign gene is introduced into Euglena for transformation.

Euglena (genus name: Euglena, Japanese name: Euglena) is promising for use as food, feed, fuel, etc.
For example, Euglena has 59 kinds of nutrients that correspond to most of the nutrients necessary for human life, such as vitamins, minerals, amino acids, and unsaturated fatty acids, and supplements for taking a balanced intake of many kinds of nutrients. The possibility of use as a food supply source in poor areas where the necessary nutrients cannot be ingested has been proposed.
Furthermore, Euglena is expected to be used as feed for livestock and farmed fish because of its high protein and high nutritional value.

Euglena produces oil and fat when it grows with carbon dioxide fixed by photosynthesis, which can be used as a source of biofuel.
Unlike fossil fuels such as oil, biofuels do not have to worry about running out of resources. In addition, when fossil fuel is used as a fuel, carbon dioxide is newly discharged. However, biofuels fix carbon dioxide when plants and algae as raw materials grow and discharge it as fuel. Accordingly, if viewed as a whole, the amount of carbon dioxide emission will not increase, and it is considered that it is effective in preventing global warming.
Furthermore, biofuels such as corn that are used as a raw material for food additives compete for use as biofuels and foods, and use as biofuels may cause food shortages and price increases. Currently, there is no consumption as an eating part, so there is no competition with food use.

As described above, Euglena has been attracting attention for a long time because its use as food, feed and fuel is promising. However, Euglena is located at the bottom of the food chain and has been successfully mass-cultured because it is preyed by predators and the culture conditions such as light, temperature conditions, and stirring speed are difficult compared to other microorganisms. There are very few examples, and only the inventors have succeeded.
Furthermore, various attempts have been made to increase the yield of Euglena and to provide a stable supply of Euglena in a rare plant for mass cultivation of Euglena. For example, transformation of Euglena by gene transfer is expected as one means to achieve an increase in Euglena yield, but no example of successful transformation that increases the yield is known.

On the other hand, chloroplasts vary greatly in form and function depending on the state of differentiation of the organism, and the types of proteins constituting them also differ. Chloroplast differentiation occurs when various genes encoded in the nucleus and chloroplast genome are subjected to coordinated and stepwise expression control according to the differentiation stage.
The regulation of gene expression in such chloroplast differentiation is hardly clarified, and even if a gene sequence is successfully introduced into a plant, the gene sequence is introduced into another plant. The expectation that it can be done is very thin. Further, even if the gene transfer is successful, the effect of the introduced gene is not necessarily increased.
Therefore, successful examples of gene transfer into higher plants and algae are not necessarily a guide for the direction of gene transfer into Euglena cells.

In particular, Euglena cells have unique properties that make it difficult to study gene transfer. For example, it is known that the DNA amount of Euglena cells reaches 50 to 100 times that of algae and fungi and is close to that of mammals. Euglena's genome is considered to be a microbial genome in that it is not considered that all such large genomes are read and become protein genes, and at least the genome is thought to be formed from many repetitive DNAs. Is considered characteristic.
Euglena has also been believed to be a genetically extremely stable organism because it has no sex and does not undergo meiosis. Various attempts have been made to introduce genes into Euglena. Not only is gene introduction difficult, but there is a characteristic that genes can be eliminated immediately after introduction, and foreign genes are stably maintained in Euglena cells. There has been no report.
Although an example of an auxotrophic mutant derived from Euglena nuclei has been reported, it has not been confirmed and no other mutants related to nuclear or mitochondrial genes are known (Non-patent Document 1). Not changed.

Shozaburo Kitaoka, “Euglena Physiology and Biochemistry” Society Publishing Center, December 10, 1989, first edition, 2nd page

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a gene introduction method into Euglena that can stably maintain a foreign gene.

The present inventors fill a gap between the future prospects of Euglena as food, feed, fuel, and the like, and the technical barrier for realizing gene transfer into Euglena, which is unlikely to mutate and has a stable gene structure. Therefore, many gene transfer methods and transgenes were tried.
As a result, when a circular plasmid vector is used, it is difficult to introduce a gene into Euglena by various gene introduction methods such as the Agrobacterium method and electroporation method, and even if the gene is introduced into Euglena. In contrast to the transient expression, when a linear gene fragment was used, it was surprising that the gene was successfully transferred by the direct gene transfer method, and at the same time, stably transferred in Euglena cells. The inventors have found that the gene is maintained and have completed the present invention.
That is, the subject is a method for introducing a DNA fragment comprising a nucleotide sequence encoding a protein into Euglena cells according to the method for introducing a gene into Euglena according to claim 1, wherein the binary vector comprises the DNA fragment. A step of obtaining a linear gene fragment comprising a T-DNA region containing the DNA fragment of the binary vector, and direct introduction of the linear gene fragment directly into the Euglena cells. And a gene introduction step.
Thus, since it comprises a direct gene transfer step for directly introducing the linear gene fragment into the Euglena cells, conventionally, no successful example has been known and it has been believed to be difficult to Euglena. Gene transfer becomes possible.

At this time, the direct gene introduction step includes a step of coating a microcarrier with the linear gene fragment, and a particle that shoots the microcarrier coated with the linear gene fragment into the Euglena cells by a particle gun method. And a gun step.
In this way, since it has a particle gun process in which microcarriers coated with linear gene fragments are shot into the Euglena cells by a particle gun method, Euglena has been believed to have been difficult and difficult to achieve. Stable gene transfer into can be achieved.

In this case, the DNA fragment may be a DNA fragment containing a base sequence encoding a protein having fructose-1,6-bisphosphatase activity / sedheptulose-1,7-bisphosphatase activity derived from cyanobacteria.
As described above, since a DNA fragment containing a base sequence encoding a protein having fructose-1,6-bisphosphatase activity / sedheptulose-1,7-bisphosphatase activity derived from cyanobacteria is used, The believed Euglena transformant can be obtained.
The obtained Euglena transformed strain has improved Euglena cell proliferation, cell size, chlorophyll content, photosynthetic activity and respiratory activity.
As a result, the yield of Euglena itself during culture can be improved, and the yield of useful components such as nutrient components of Euglena and the function of Euglena can be improved.
Moreover, it is possible to improve the yield and function of Euglena, which is technically difficult to mass culture, and it can be expected to open the way to the realization of mass supply of Euglena for uses such as food, feed and fuel.

At this time, the microcarrier may be gold fine particles having a diameter of 0.26 μm or less.
When the microcarrier diameter is 0.26 μm or less, gene fragments are most stably introduced.

At this time, the number of proliferating cells, cell size, chlorophyll amount, photosynthetic activity and respiratory activity of the Euglena may be improved.
At this time, the Euglena may be Euglena gracilis.
Due to such a configuration, a Euglena transformant suitable for food, feed, fuel and the like can be obtained.

At this time, the base sequence may encode a protein having an amino acid sequence shown in the following (a) or (b).
(A) Among the amino acid sequences shown in SEQ ID NO: 2 in the sequence listing, the amino acid sequence represented by amino acid numbers 1-356 (b) Activity as fructose-1,6-bisphosphatase / sedheptulose-1,7-bisphosphatase An amino acid sequence in which a part of the amino acid sequence of (a) is deleted, substituted or added

At this time, the base sequence may be a base sequence shown in the following (c) or (d).
(C) Among the nucleotide sequences shown in SEQ ID NO: 1 in the Sequence Listing, the nucleotide sequence represented by nucleotide numbers 181-1251 (d) activity as fructose-1,6-bisphosphatase / sedheptulose-1,7-bisphosphatase Euglena transformant that has been believed to be very difficult so far because of the base sequence in which a part of the base sequence of (c) is deleted, substituted, or added. Can be obtained.

According to the present invention, since a direct gene transfer step of directly introducing a linear gene fragment into Euglena cells is provided, no successful example has been known so far and it has been believed to be difficult to Euglena. Stable gene transfer is possible.
Moreover, since a Euglena cell uses a DNA fragment containing a base sequence encoding a protein having fructose-1,6-bisphosphatase activity / sedheptulose-1,7-bisphosphatase activity derived from cyanobacteria, It is possible to obtain a transformed strain of Euglena that was believed to be difficult.
The obtained Euglena transformed strain has improved Euglena cell proliferation, cell size, chlorophyll content, photosynthetic activity and respiratory activity.
As a result, the yield of Euglena itself during culture can be improved, and the yield of useful components such as nutrient components of Euglena and the function of Euglena can be improved.
Moreover, it is possible to improve the yield and function of Euglena, which is technically difficult to mass culture, and it can be expected to open the way to the realization of mass supply of Euglena for uses such as food, feed and fuel.

It is a schematic diagram which shows the DNA fragment of the linear gene fragment for introduction | transduction to Euglena. It is a figure which shows the confirmation of the transgene and expression protein in Euglena. It is a figure which shows the number of proliferating cells when a Euglena transformant and a wild species are cultured. It is a photograph which shows the external appearance of the transformed strain of Euglena and wild species on the 8th day. It is a figure which shows the amount of chlorophylls of a transformed strain of Euglena and wild species. It is a figure which shows the photosynthetic activity and the respiratory activity of a transformed strain of Euglena and wild species. It is a figure which shows the number of proliferating cells when a Euglena transformant and a wild species are cultured. It is a graph which shows contrast of the carbohydrate content in the culture solution of a wild strain and a transformant. It is the photograph which contrasted the accumulation state of musilage when a transformed strain of Euglena and wild species were cultured.

Hereinafter, the Euglena gene introduction method according to an embodiment of the present invention will be described in detail.
The gene transfer method to Euglena according to the present embodiment includes a base sequence encoding a protein having fructose-1,6-bisphosphatase activity / sedheptulose-1,7-bisphosphatase activity derived from cyanobacteria in Euglena cells. The base sequence is introduced into Euglena by bombarding Euglena cells with a microcarrier coated with a linear gene fragment by a particle gun method.

(Euglena to introduce the gene)
Euglena that can be used in the present invention is widely distributed in fresh water such as ponds and swamps, and may be used separately from these, or any Euglena that has already been isolated may be used. .
As Euglena into which a gene is introduced by the method of the present embodiment, species such as Euglena gracilis, Euglena gracilis krebs, Euglena gracilis barbatillas, etc. included in the genus Euglena are used. In particular, Euglena gracilis (Euglena gracilis) ) Z strain, its mutant SM-ZK strain (chloroplast-deficient strain) and its variant var. Bacillaris are preferably used. Alternatively, gene mutants such as chloroplast mutants of these species may be used.

(Linear gene fragment used for gene transfer)
For the gene introduction of the present invention, a DNA fragment encoding a protein having activity as fructose-1,6-bisphosphatase / sedheptulose-1,7-bisphosphatase derived from cyanobacteria, or a DNA fragment having homology thereto A linear gene fragment comprising a transgene comprising:
Examples of the DNA fragment encoding a protein having activity as fructose-1,6-bisphosphatase / sedheptulose-1,7-bisphosphatase derived from cyanobacteria include, for example, fructose-1, isolated from cyanobacteria Synechococcus PCC 7942 A gene encoding 6-bisphosphatase (hereinafter referred to as FBPase) / sedheptulose-1,7-bisphosphatase (hereinafter referred to as SBPase) can be used.

(Protein having FBPase / SBPase activity)
Cyanobacterial FBPase / SBPase used in the present invention is a protein that can be a rate-limiting enzyme of the Calvin cycle.

  Examples of the protein showing FBPase / SBPase activity include the amino acid sequence of fructose-1,6-bisphosphatase / sedheptulose-1,7-bisphosphatase derived from the cyanobacterium Synechococcus PCC 7942 gene represented by SEQ ID NO: 2. .

This protein is an enzyme widely distributed in cyanobacteria, a prokaryotic algae, and has a primary structure and enzymological properties different from those of higher plant chloroplasts, FBPase and SBPase. One protein is a bifunctional enzyme having two enzyme activities, FBPase and SBPase.
Examples of the protein having FBPase / SBPase activity include a protein having the amino acid sequence represented by SEQ ID NO: 2. In addition, since the 1st-356th amino acid sequence is a part which has FBPase / SBPase activity among the amino acid sequences shown by sequence number 2, if it has the 1st-356th amino acid sequence, Good.

The protein having FBPase / SBPase activity has an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 2 have been deleted, substituted, added or inserted Also included are proteins having
The protein having FBPase / SBPase activity is a protein having at least 60% homology with the first to 356th amino acid sequences of the amino acid sequence shown in SEQ ID NO: 2, preferably 80% or more homology. More preferably, a protein having a homology of 90% or more, more preferably a protein having a homology of 95% or more, and a protein having FBPase / SBPase activity is also included.

In the present specification, the term “homologous” for amino acid sequences is used to mean the degree of coincidence of amino acid residues constituting each sequence by comparing the primary structure of proteins.
In addition, regarding the amino acid sequence, when “one or several (about 2 to 6) amino acids are deleted, substituted, added or inserted”, by a well-known technical method such as site-directed mutagenesis, It means that the number of naturally occurring numbers has been deleted, substituted, added or inserted.

(Transgenic gene)
Examples of the DNA fragment containing a base sequence encoding a protein having FBPase / SBPase activity used in the present invention include a gene consisting of the base sequence represented by SEQ ID NO: 1. In addition, since the structural gene part which expresses an enzyme is the 181st-1251st base sequence among the base sequences shown by sequence number 1, what is necessary is just to have the base sequence of this part.
In the DNA fragment encoding the protein having FBPase / SBPase activity used in the present invention, one or several bases are deleted, substituted, added or inserted in the DNA sequence shown in SEQ ID NO: 1. DNA encoding a protein having a base sequence and having FBPase / SBPase activity is included. Regarding the nucleotide sequence, when “one or several bases are deleted, substituted, added or inserted”, it is a number that can be naturally generated by a well-known technical method such as site-directed mutagenesis (1 It means that (~ several) bases have been deleted, substituted, added or inserted.

In addition, the DNA fragment encoding the protein having FBPase / SBPase activity used in the present invention is hybridized under stringent conditions with the DNA sequence shown in SEQ ID NO: 1 and DNA each consisting of a complementary base sequence. DNA comprising a base sequence encoding a protein that is soybean and has FBPase / SBPase activity is included.
DNA that can hybridize under stringent conditions means DNA obtained by using the above-mentioned DNA as a probe and using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. The stringent conditions are an SSC solution having a salt concentration of about 0.1 to 2 times (the composition of the SSC solution having a concentration of 1 is 150 mM sodium chloride and 15 mM sodium citrate), and a temperature of about 65 ° C. Hybridization conditions in terms of degree.

Furthermore, the DNA fragment encoding the protein having FBPase / SBPase activity used in the present invention has at least 60% homology with the DNA sequence shown in SEQ ID NO: 1, and has FBPase / SBPase activity. DNA comprising a base sequence encoding a protein is included.
The DNA having homology is a DNA having at least about 60% homology, preferably about 80% or more homology, more preferably about 90% or more homology under highly stringent conditions. DNA having sex, more preferably DNA having homology of about 95% or more.
The high stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C., preferably about 60 to 65 ° C. Say. In particular, the most preferable conditions are when the sodium concentration is about 19 mM and the temperature is about 65 ° C.

(Linear gene fragment)
The linear gene fragment used for gene introduction in the present invention is schematically shown in FIG. 1 as an example, and includes an expression cassette for a DNA fragment encoding a protein having FBPase / SBPase activity derived from cyanobacteria.
The expression cassette preferably has a chloroplast transit peptide upstream of the translation start point of a gene encoding a protein having FBPase / SBPase activity derived from cyanobacteria.
As the chloroplast transit peptide, rbcS-TP, which is a transport peptide derived from a plant ribulose-1,5-diphosphate carboxylase small subunit (RbcS), may be used.

The expression cassette preferably further has a translation enhancer region, which is a sequence that promotes translation, upstream of the chloroplast transit peptide.
As the translation enhancer region, for example, a 5 ′ untranslated region (5′-UTR) derived from an ADH (Alcohol Dehydrogenase) gene can be preferably used.

The expression cassette preferably further has a promoter for gene expression in plants upstream of the translation enhancer region.
The promoter may be adjacent to the translation enhancer region as long as it is upstream of the translation enhancer region, or may be about 1 to 30 bases upstream.
Examples of the promoter include an elongation factor 1α gene promoter (EF1α promoter), 35S promoter, psbA promoter, PPDK promoter, PsPAL1 promoter, PAL promoter, UBIZM1 ubiquitin promoter, rrn promoter, and the like. Among them, the cauliflower mosaic virus (CaMV) 35S promoter and the like can be particularly preferably used.

The linear gene fragment preferably has a selection marker gene for identifying a gene recombinant. The selectable marker gene is not particularly limited, and a known marker gene may be used.
Examples of such genes include various drug resistance genes (aadA) and genes that complement the auxotrophy of the host. More specifically, for example, an ampicillin resistance gene, neomycin resistance gene (G418 resistance), chloramphenicol resistance gene, kanamycin resistance gene, spectinomycin resistance gene, URA3 gene and the like can be mentioned. In particular, a kanamycin resistance gene (NPTII gene (kanr gene)) expressing an APH (3 ′) II (NPTII) protein that inactivates aminoglycoside antibiotics is preferably used.

In addition, a promoter for recognizing the gene and a terminator of the gene are preferably arranged upstream and downstream of the gene, respectively. As the promoter and terminator, the above plant-derived promoter and terminator can be preferably used, but the nopaline of the soil bacterium Agrobacterium tumefaciens (Agrobacterium)
The NOS promoter (P-NOS) and NOS terminator (T-NOS) derived from the synthase (NOS) gene are particularly suitable.

The linear gene fragment of the present invention is prepared by treating a binary vector having the sequence of this linear gene fragment with a restriction enzyme to obtain only the T-DNA region sandwiched between LB and RB. The
In the present specification, the linear gene fragment means a DNA fragment having a free 5 ′ end and a 3 ′ end, and means not a circular DNA. Further, the actual shape of the linear gene fragment is not necessarily linear, but may be curved or twisted. Although the form of the linear gene DNA fragment at the time of gene introduction of this embodiment may be double-stranded or single-stranded, double-stranded is preferable.

(Flow of gene transfer)
The gene transfer method to Euglena is performed as follows.
First, pre-culture of Euglena is performed.
Subsequently, gene introduction for introducing the above-mentioned linear gene fragment into Euglena is performed by a known particle gun method.
For gene transfer, a known gun powder type (shot gun) type, arc discharge type, nitrogen gas pressure type, compressed air thickness (air gun) type, helium type particle gun device can be used, but DNA coating installed in the cartridge It is preferable to use a helium system device in which helium gas is blown directly onto the particles for firing.
Moreover, the gene introduction carried out in the present invention is not limited to the particle gun method, and any gene can be used as long as it directly introduces the above-mentioned linear gene fragment. In particular, it is preferable to use a method called a direct gene transfer method in which a foreign gene is directly introduced into a cell by applying mechanical force. For example, an electroporation method, a microinjection method, a PEG method (polyethylene glycol method) Etc. can be used.

A T-DNA region sandwiched between LB and RB is amplified by PCR from the binary vector to obtain a linear gene fragment.
A microcarrier made of fine particles of metal such as gold or tungsten is coated with a linear gene fragment amplified by a known dry particle method, and gene introduction is performed by a particle gun method using a helium system.
After the introduction, static culture is performed for 24 hours, and the culture medium is replaced with a CM medium containing an antibiotic corresponding to the selection marker gene contained in the linear gene fragment, and selected for several weeks.

  The appearing antibiotic-resistant strain is suspended in CM medium and plated on the selection medium. The colonies that appeared after one week are streaked and further selected. PCR and Western blot analysis are performed using the antibiotic resistant strain obtained.

Hereinafter, the present invention will be described more specifically with reference to examples and test examples. However, the present invention is not limited to these.
(Example 1: Gene transfer to Euglena)
In accordance with the method described in the above embodiment, gene introduction into Euglena was performed.
First, pre-culture of Euglena was performed.
Euglena was added to Koren-Hutner medium (hereinafter referred to as KH medium, arginine hydrochloride: 0.5 g / L, aspartic acid: 0.3 g / L, glucose: 12 g / L, glutamic acid: 4 g / L, glycine 0.3 g / L). , Histidine hydrochloride 0.05 g / L, Malic acid: 6.5 g / L, Citric acid 3Na: 0.5 g / L, Succinic acid 2Na: 0.1 g / L, (NH ₄ ) 2 SO ₄ : 0.5 g / L L, NH ₄ HCO ₃ : 0.25 g / L, KH ₂ PO ₄ : 0.25 g / L, MgCO ₃ : 0.6 g / L, CaCO ₃ : 0.12 g / L, EDTA-Na ₂ : 50 mg / L , FeSO ₄ (NH ₄ ) ₂ SO ₄ .6H ₂ O: 50 mg / L, MnSO ₄ .H ₂ O: 18 mg / L, ZnSO ₄ .7H ₂ O: 25 mg / L, (NH ₄ ) ₆ Mo ₇ O ₂₄ · _4H 2 O: 4m _{/ L, CuSO 4: 1.2mg /} L, NH 4 VO 3: 0.5mg / L, CoSO 4 · 7H 2 O: 0.5mg / L, H 3 BO 3: 0.6mg / L, NiSO 4 · 6H ₂ O: 0.5 mg / L, Vitamin B ₁ : 2.5 mg / L, Vitamin B ₁₂ : 0.005 mg / L (pH 3.5)) Culturing for 5 days under continuous light irradiation conditions, or KH medium shading conditions Then, the cells were cultured for 1 day under continuous light irradiation conditions. The collected culture solution was centrifuged at 3,000 rpm at room temperature for 10 minutes, and sterilized water was added to the collected Euglena precipitate for washing, followed by centrifugation at 3,000 rpm for 10 minutes. Thereafter, 2 mL of a sample having a cell number of 2 × 10 ⁷ cells / mL was adsorbed to a membrane filter (MILLIPORE) while aspirating under reduced pressure. / L, (NH ₄ ) ₂ HPO ₄ : 1.0 g / L, KH ₂ PO ₄ : 1.0 g / L, MgSO ₄ · 7H ₂ O: 0.2 g / L, CaCl ₂ · 2H ₂ O: 0. 02 g / L, citric acid 3Na · 2H ₂ O: 0.8 g / L, Fe ₂ (SO ₂ ) ₃ · 7H ₂ O: 3 mg / L, MnCl ₂ · 4H ₂ O: 1.8 mg / L, CoSO ₄ · 7H ₂ O: 1.5 mg / L, ZnSO ₄ · 7H ₂ O: 0.4 mg / L, Na ₂ MoO ₄ · 2H ₂ O: 0.2 mg / L, CuSO ₄ · 5H ₂ O: 0.02 g / L , thiamine hydrochloride (vitamin _{B 1} : 0.1 mg / L, cyanocobalamin (vitamin _B 12): 0.0005mg / L every top of (pH 3.5)), the Petri dish from light with aluminum foil, for 24 hours standing culture at culture chamber.

In addition, a linear gene fragment to be introduced into Euglena was prepared.
A gene encoding a protein having FBPase / SBPase activity (fbp / sbp, a gene comprising a base sequence represented by SEQ ID NO: 1) at the multiple cloning site of the plant binary vector pRI101-35S (manufactured by Takara Bio Inc.) 1) and an antibiotic resistance gene (NPTII), and a chloroplast transit peptide rbcS-TP (obtained from Mr. Sugita, Nagoya University) is inserted upstream of the translation initiation point. -A binary vector pRI101-AN-35S (TP) fbp / sbp containing NDA was prepared.
Next, the region sandwiched between LB and RB in FIG. 1 was amplified by PCR to obtain a linear gene fragment.
Gold microparticles were coated with the amplified linear gene fragment by a known dry particle method. At this time, gold fine particles having a diameter of 0.26 μm were used.

Then, using a known helium system, gold particles were injected into the Euglena that had been pre-cultured on the membrane by the particle gun method (pressure: 900 psi, distance: 9 cm) to introduce linear gene fragments into Euglena cells. Went. Further, after light-shielding culture for 1 day, the cells were transferred to a CM agar medium containing the antibiotic kanamycin (50 μg / ml), replaced every 2 weeks, and transformants were selected. Kanamycin is an antibiotic corresponding to the selection marker kanamycin resistance gene (NPTII gene) contained in the linear gene fragment.
So far, various gene introduction conditions have been examined, but only the above-mentioned method has been successful. As a result of confirming the expression by PCR and Western blotting using the obtained antibiotic-resistant strain, it was revealed that the strain was transformed.

The antibiotic-resistant strain that appeared was suspended in CM medium and plated on the selection medium. Colonies that appeared after one week were streaked and further selected. When PCR was performed for each colony obtained by selection, a signal was observed at a position of 0.57 kbp as shown in FIG. This indicates that a base sequence encoding a protein having FBPase / SBPase activity was introduced into the 8 transformants in the nuclear genome. The eight transformants obtained were named PR2-1 to PR2-8 and subjected to Western blot analysis.
As a result, as shown in FIG. 2 (B), a 40 kDa signal indicating a protein having FBPase / SBPase activity derived from cyanobacteria was detected in at least three lines of PR2-1, PR2-2, and PR2-7. .

(Example 2: Growth comparison between transformed strain and wild strain)
In accordance with the method described in the above embodiment, gene introduction into Euglena was performed.
Euglena preculture conditions were the same as in Example 1 except that continuous photoculture was performed for 5 days.
As linear gene fragments, two types of binary vectors pBI121-35S (manufactured by Takara Bio Inc.) and pRI101-35S similar to Example 1 were used, and FBPase / A gene encoding a protein having SBPase activity (fbp / sbp, a gene comprising the nucleotide sequence represented by SEQ ID NO: 1 cloned by the present inventors) and chloroplast translocation upstream of the translation start point Peptide rbcS-TP (obtained from Mr. Sugita, Nagoya University) was inserted to prepare pBI121-35S: fbp / sbp and pRI101-35S: fbp / sbp vectors.

After the region between LB and RB of these vectors was amplified by PCR to obtain a linear gene fragment, the gene was introduced into Euglena by the particle gun method under the same conditions as in Example 1.
After the introduction, selection was carried out by the same procedure as in Example 1, and 3 transformed strains were obtained from the obtained drug-resistant strain. Of the three obtained strains, one transformed strain (EpFS-1) was transferred to 1 L of CM medium with the same number of cells as the wild strain and the antibiotics removed, and the following growth comparison experiment was performed.

Two experiments were carried out in which the transformed strain (EpFS-1) and the wild strain were planted in a CM medium (1 L) containing no antibiotics in accordance with the number of cells (first experiment, second experiment in FIG. 3). Experiment). These samples were collected over time, and the number of cells, the observation of cell size, and the amount of chlorophyll were measured. In addition, photosynthetic activity and respiratory activity were measured using cells in the stationary phase (14 days after inoculation) using an oxygen electrode.
As a result, compared to the wild type, the transformed strain (EpFS-1) has a larger cell size as shown in FIG. 4, and the number of cells in the stationary phase is about 1.4 times as shown in FIG. Met. Moreover, as shown in FIG. 5, the amount of chlorophyll per volume and the number of cells of the transformed strain (EpFS-1) tended to increase about 1.5 times compared to the wild strain.
Furthermore, as shown in FIG. 6 described later in Example 3, both the photosynthetic activity and respiratory activity of the transformed strain (EpFS-1) tended to be higher than those of the wild strain.

(Contrast between average particle size and cell volume of wild type and transformed type)
In FIG. 4, a photograph of the cell size of the wild strain and the transformed strain (EpFS-1) is shown in contrast, but in order to compare not only visually but also quantitatively, the following culture is performed. The cell sizes of both strains were compared.
The culture conditions were as follows.
The transformed strain (EpFS-1) and wild strain obtained by gene transfer of Example 2 were continuously irradiated with a CM medium (pH 5.5) in a 50 mL test tube at a water temperature of 29 ° C. for 24 hours (200 μmol / m 2). ² / s), air alone and a gas in which 5% CO ₂ was mixed with air were aerated at a flow rate of 50 ml / min, and cultured for 6 days.
Euglena cells on day 6 of culture were collected, and the average particle size was measured with CDA-1000 (sysmex). The volume was calculated by substituting the average particle diameter as 4 / 3πr ³ as the diameter. The results are shown in Table 1.

As shown in Table 1, the average particle size of the transformed strain (EpFS-1) is larger than that of the wild strain regardless of whether only air or a gas in which 5% CO ₂ is mixed with air is vented. It was quantitatively shown that the volume calculated from the average particle size was also large.

(Comparison of carbohydrate content in culture solution of wild type and transformed type)
Moreover, it compared with respect to whether the carbohydrate content in the culture solution of Euglena has a difference between a wild strain and a transformed strain (EpFS-1).
The Euglena wild strain and the transformed strain (EpFS-1) were cultured for 6 days in the same manner as (the average particle diameter and cell volume comparison between the wild strain and the transformed strain).
The Euglena culture solution on the 6th day of culture was collected and heated at 95 ° C. for 1 hour. After heating, the mixture was centrifuged (4,000 rpm, 5 minutes), and only the upper layer was recovered. Since the upper layer does not contain Euglena cells, Euglena cells were removed by collecting only the upper layer in this way. Next, the carbohydrate content of the recovered upper layer was determined by the phenol / sulfuric acid method. This carbohydrate is a viscous polysaccharide secreted by Euglena out of the cell and is called musilage.
The quantitative results are shown in FIG.
When the amount of carbohydrate contained in the medium in which the wild strain and the transformed strain (EpFS-1) were cultured was compared, the amount of the medium in which the transformed strain (EpFS-1) was cultured was larger, and the transformation was more effective than the wild strain. In the strain (EpFS-1), a large amount of musilage was secreted.

(Example 3: Culture after gene introduction)
After gene introduction in Example 2, Cramer-Myers medium (hereinafter, CM medium, (NH ₄ ) ₂ HPO ₄ : 1.0 g / L, KH ₂ PO ₄ : 1.0 g / L, MgSO ₄ · 7H ₂ O: 0.2 g / L, CaCl ₂ · 2H ₂ O: 0.02 g / L, citric acid 3Na · 2H ₂ O: 0.8 g / L, Fe ₂ (SO ₂ ) ₃ · 7H ₂ O: 3 mg / L, MnCl ₂ · 4H ₂ O: 1.8 mg / L, CoSO ₄ · 7H ₂ O: 1.5 mg / L, ZnSO ₄ · 7H ₂ O: 0.4 mg / L, Na ₂ MoO ₄ · 2H ₂ O: 0.2 mg / L, CuSO ₄ .5H ₂ O: 0.02 g / L, thiamine hydrochloride (vitamin B ₁ ): 0.1 mg / L, cyanocobalamin (vitamin B ₁₂ ): 0.0005 mg / L, (pH 3. 5)), the wild strain and the transformed strain of Example 2 (Ep FS-1) was planted in accordance with the number of cells.
The culture broth was collected over time, and the number of cells was measured. Further, photosynthetic activity and respiratory activity were measured using an oxygen electrode.
As shown in FIG. 7, the transformed strain (EpFS-1) reached a cell concentration 1.4 times that of the wild strain at the 13th day from the start of culture.
Moreover, as shown in FIG. 6, the transformed strain (EpFS-1) had higher photosynthesis and respiratory activity than the wild strain.
The above results functioned in Euglena cells into which a base sequence encoding a protein having FBPase / SBPase activity introduced into a transformed strain (EpFS-1) was introduced, and by enhancing the photosynthetic activity of Euglena, This suggests that the growth rate was improved compared to the wild type.
Moreover, as shown in FIG. 9, only with the culture solution of the right transformant (EpFS-1), musilage was observed as a dark floating substance. Therefore, in the transformed strain (EpFS-1), the production amount of carbohydrate as a photosynthesis product increased with the enhancement of photosynthesis, and a part of it was secreted extracellularly. As a result, as shown in FIG. It is thought that.

Claims (8)

A method of introducing a DNA fragment comprising a nucleotide sequence encoding a protein into Euglena cells, comprising:
Producing a binary vector containing the DNA fragment;
Obtaining a linear gene fragment consisting of a T-DNA region containing the DNA fragment of the binary vector;
A gene introduction method into Euglena, comprising a direct gene introduction step of directly introducing the linear gene fragment into the Euglena cells. The direct gene transfer step includes:
Coating a microcarrier with the linear gene fragment;
A method of introducing a gene into Euglena according to claim 1, further comprising a particle gun step of shooting a microcarrier coated with the linear gene fragment into the Euglena cells by a particle gun method.   The DNA fragment is a DNA fragment containing a base sequence encoding a protein having fructose-1,6-bisphosphatase activity / sedheptulose-1,7-bisphosphatase activity derived from cyanobacteria. Alternatively, the method for gene transfer into Euglena according to 2.   The method for introducing a gene into Euglena according to claim 2 or 3, wherein the microcarrier is a gold fine particle having a diameter of 0.26 µm or less.   The method for introducing a gene into Euglena according to any one of claims 1 to 4, wherein the number of proliferating cells, cell size, chlorophyll amount, photosynthetic activity and respiratory activity of the Euglena are improved.   6. The method for introducing a gene into Euglena according to claim 1, wherein the Euglena is Euglena gracilis. The method for introducing a gene into Euglena according to any one of claims 1 to 5, wherein the base sequence encodes a protein comprising the amino acid sequence shown in (a) or (b) below.
(A) Among the amino acid sequences shown in SEQ ID NO: 2 in the sequence listing, the amino acid sequence represented by amino acid numbers 1-356 (b) activity as fructose-1,6-bisphosphatase / sedheptulose-1,7-bisphosphatase An amino acid sequence in which a part of the amino acid sequence of (a) is deleted, substituted or added The method for introducing a gene into Euglena according to any one of claims 1 to 6, wherein the base sequence is a base sequence shown in (c) or (d) below.
(C) Among the nucleotide sequences shown in SEQ ID NO: 1 in the sequence listing, the nucleotide sequence represented by nucleotide numbers 181-1251 (d) activity as fructose-1,6-bisphosphatase / sedheptulose-1,7-bisphosphatase (C) a base sequence in which a part of the base sequence in (c) is deleted, substituted or added