JP7184233B2 - Gene Constructs for Recombinant Protein Expression Containing a Lettuce Ubiquitin Promoter - Google Patents

Description

TECHNICAL FIELD The present invention relates to genetic recombination technology using plant cells, and more particularly to technology for producing recombinant proteins using plant cells.

In the production of useful proteins using genetically modified organisms, it is necessary to stably maintain the host that produces the proteins and to expand the production scale to a commercial production scale. Strict control of these processes is important in the manufacture of pharmaceuticals. When a plant is used as a host, once a genetically modified plant is produced, it can be stored for a long period of time in the form of a seed, and It is considered possible to expand the scale of production. Therefore, in the seed-mediated production system, it is required that the recombinant gene is stably expressed even in the progeny and that the seed of the recombinant plant is stably obtained.

Conventionally, when expressing foreign genes in plants, the 35S promoter derived from cauliflower mosaic virus (CaMV) has been widely used.
It has been reported that gene silencing (decrease in target protein production) occurs in progeny when the 35S promoter is used (Non-Patent Documents 1 to 3). Therefore, in order to stably express a foreign gene using progeny seeds, a technique for avoiding silencing was required.
In addition, when a gene expression cassette containing a 35S promoter was incorporated into transgenic lettuce, it was found that the amount of progeny seeds produced was significantly reduced. there were.
Furthermore, it has been reported that expression decreases under high light intensity conditions when the 35S promoter is used (Non-Patent Document 4). In general, high light intensity promotes the growth of plants, so construction of an expression system that does not reduce expression even under high light intensity conditions has been a challenge.

Furthermore, Non-Patent Document 2 discloses a gene expression cassette containing a lettuce ubiquitin promoter and a ubiquitin terminator. and the amount of mature seeds of progeny seeds were not examined.

Pniewski et al., Plant Cell Rep. 2012, 31:585-95 Hirai et al., Plant Cell Rep. 2011, 30:2255-65 Dubois et al., J. Exp. Bot. 2005 56: 2379-88 Elliott et al., Plant Cell 1989, 1:691-8

As described above, when a conventional promoter such as the 35S promoter is used, there are problems such as a decrease in the seed yield of progeny seeds, silencing, and decreased expression under high light intensity conditions.
Therefore, the present invention is to efficiently collect mature seeds of genetically modified plants, avoid gene silencing, and construct an expression system that does not reduce the expression of the target foreign gene even at high light intensity. is the subject.

The present inventors first conducted extensive studies with the aim of constructing an expression system that is resistant to gene silencing. Then, a gene construct (gene expression cassette) containing a lettuce ubiquitin promoter and an Arabidopsis heat shock protein 18.2 (HSP18.2) terminator was prepared, and the target gene was inserted and expressed in lettuce. We found that the target gene can be expressed efficiently. At that time, as a completely unexpected effect, it was found that the number of seeds of recombinant lettuce was improved, and seeds could be stably collected over generations. Furthermore, when using the gene expression cassette, the inventors have also found the effect that the expression of the target gene does not decrease even at high light intensity, and have completed the present invention.

The present invention provides the following.
[1] A gene construct for recombinant protein expression comprising a lettuce ubiquitin promoter and an Arabidopsis heat shock protein 18.2 (HSP18.2) terminator.
[2] The lettuce ubiquitin promoter hybridizes under stringent conditions with a DNA containing the nucleotide sequence of SEQ ID NO: 1 or a polynucleotide having a sequence complementary to the nucleotide sequence of SEQ ID NO: 1, and functions as a promoter in plant cells. The genetic construct of [1], which is a DNA that
[3] Arabidopsis thaliana HSP18.2 terminator hybridizes under stringent conditions with a DNA containing the nucleotide sequence of SEQ ID NO: 2 or a polynucleotide having a sequence complementary to the nucleotide sequence of SEQ ID NO: 2, and the terminator in plant cells The gene construct of [1] or [2], which is a DNA that functions as
[4] The gene construct according to any one of [1] to [3], further comprising a translation enhancer sequence.
[5] A gene construct according to any one of [1] to [4], which contains a DNA encoding a target protein.
[6] The gene construct of [5], wherein the target protein is a bacterial toxin protein and/or a virus-derived protein.
[7] The genetic construct of [5] or [6], wherein the target protein is a fusion protein of two or more proteins.
[8] The gene construct of [7], wherein the fusion protein is a fusion protein in which two or more proteins are linked by a peptide linker.
[9] The gene construct of any one of [5] to [8], wherein the target protein contains a secretory signal peptide.
[10] The gene construct of any one of [5]-[9], wherein the target protein contains a peptide tag.
[11] The gene construct of any one of [5] to [10], wherein the target protein contains an endoplasmic reticulum retention signal peptide.
[12] A vector comprising the gene construct according to any one of [1] to [11].
[13] A transgenic plant transformed with the gene construct of any one of [1] to [11] or the vector of [12].
[14] The transgenic plant of [13], which is lettuce.
[15] A seed obtained from the transgenic plant of [13] or [14].
[16] A method for producing a target protein, which comprises the step of cultivating the transgenic plant of [13] or [14], which expresses the target protein.
[17] The method for producing a target protein according to [16], wherein the cultivation is performed under a light intensity of 300-600 PPFD.
[18] A step of cultivating, under a light intensity of 300 to 600 PPFD, a transgenic plant that has been transformed with a gene construct containing a DNA encoding a target protein expressably linked to a lettuce ubiquitin promoter and that expresses the target protein. A method for producing a protein of interest, comprising:
[19] A method for increasing the amount of mature seeds obtained from a transgenic plant expressing a target protein, the gene comprising a DNA encoding the target protein expressively linked to a lettuce ubiquitin promoter. A method comprising transforming a plant with the construct and cultivating the transformed plant.
[20] The method of [19], wherein the transformed plant is cultivated by hydroponics.
[21] The method according to [19] or [20], wherein the amount of mature seeds obtained from the plant is the number of mature seeds per strain (individual) of the plant.
[22] The method of [19] or [20], wherein the amount of mature seeds obtained from the plant is the number of mature seeds per cluster of flowers of the plant.

By using the gene construct of the present invention, a target gene can be expressed with high efficiency using a plant as a host, and the yield of protein production can be improved. A plant transformed with the gene construct of the present invention does not show reduced gene expression even in its progeny, making it possible to construct a stable seed-mediated production system. In addition, since gene expression does not decrease even at high light intensity, efficient recombinant protein production is possible under high light intensity conditions. Furthermore, since it has become possible to stably obtain a large amount of mature seeds of transgenic plants, it has become possible to store them and expand the scale of production.

FIG. 1 shows gene constructs for expressing recombinant proteins used in Examples. Abbreviations are as follows. NOST: A.tumefaciens nopaline synthase gene terminator NPTII: Selection marker NOS pro.: A.tumefaciens nopaline synthase gene promoter NtADHmod 5′-UTR: Modified 5′-untranslated region of tobacco alcohol dehydrogenase gene SP: Tobacco β-D-glucan exohydrolase-derived secretory signal peptide HA: HA peptide tag HDEL: endoplasmic reticulum retention signal peptide MCS: multiple cloning site FIG. 4 shows the expression levels of recombinant proteins in each generation of 35S::Stx2eB-Stx2eB-introduced lettuce lines. FIG. 2 shows the expression levels of recombinant proteins in the progeny of two UBQ::Stx2eB-Stx2eB-introduced lettuce lines (A: 18-1, B: 5-1). The figure which shows the number of mature seeds harvested in each lettuce system. For each line, seeds were collected from multiple strains (individuals) over the progeny (T5 generation), and the average and standard deviation of the number of mature seeds collected are shown. It indicates that there is no significant difference (p<0.05) between the same alphabets. For comparison, wild-type (WT) lettuce was also harvested under the same conditions. 35S::Stx2eB-Stx2eB-introduced lettuce seed morphology (photograph). Mature seeds on the left, immature seeds on the right. A diagram showing the number of mature seeds per aggregate flower in each line. It indicates that there is no significant difference (p<0.05) between the same alphabets. For comparison, wild-type (WT) lettuce was also harvested under the same conditions and measured. FIG. 10 is a diagram showing the number of mature seeds collected in various fusion protein gene-introduced lettuce. Several T0 generation lines of each expression cassette-introduced lettuce were collected. The number of mature seeds per line (individual) was counted, and the average value and standard deviation between lines of the same expression cassette are shown.

Modes of the present invention will be described below.

<Lettuce ubiquitin promoter>
The lettuce ubiquitin promoter is present in the lettuce genome sequence upstream of the transcription start point of the lettuce ubiquitin gene and means a sequence that induces the expression of the lettuce ubiquitin gene. GenBank, a base sequence database provided by Biotechnology Information, Inc., accession No. AB500086.1, or a DNA containing a part thereof.
Also included is a DNA comprising the base sequence of SEQ ID NO: 1. As long as the DNA has promoter activity in plants, one or more bases in the base sequence of SEQ ID NO: 1 are substituted, deleted, inserted and/or Alternatively, it may be DNA having an added base sequence. Here, "one or more bases" means, for example, 1 to 50, 1 to 30, 1 to 10, or 1 to 5 bases. Such DNAs include DNAs that hybridize under stringent conditions with a polynucleotide having a sequence complementary to the nucleotide sequence of SEQ ID NO: 1 and that have promoter activity in plants.

Here, "stringent conditions" refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, two DNAs with high identity , two DNAs with identity of 90 % or more, particularly preferably 95% or more, hybridize, but two DNAs with lower identity do not hybridize. is mentioned. For example, at a salt concentration and temperature corresponding to 60 ° C., 0.1×SSC, 0.1% SDS, more preferably 68° C., 0.1×SSC, 0.1% SDS, wash once, preferably 2-3 times. can be mentioned. That is, the lettuce ubiquitin promoter may have a base sequence having 90% or more, particularly preferably 95% or more identity with the base sequence of SEQ ID NO: 1, as long as it has promoter activity.

Promoter activity can be detected by ligating a gene of interest to the 3' side of the promoter sequence, introducing it into host cells, and examining the expression of the gene of interest.

The lettuce ubiquitin promoter can be obtained by a technique such as PCR using the lettuce genome as a template. In addition, introduction of substitution or deletion into a promoter can be performed by a known site-directed mutagenesis method or the like.

<Arabidopsis thaliana HSP18.2 terminator>
The Arabidopsis thaliana HSP18.2 terminator is located downstream of the termination codon of the HSP18.2 gene in the Arabidopsis thaliana genome sequence and means a sequence that terminates the transcription of the HSP18.2 gene. DNA can be mentioned. However, as long as the DNA has terminator activity in plants, it may be a DNA having a base sequence in which one or more bases are substituted, deleted, inserted and/or added to the base sequence of SEQ ID NO:2. Here, "one or more bases" means, for example, 1 to 50, 1 to 30, 1 to 10, or 1 to 5 bases. Such DNAs include DNAs that hybridize under stringent conditions with a polynucleotide having a sequence complementary to the base sequence of SEQ ID NO: 2 and that have terminator activity in plants. The stringent conditions are as described above. The HSP18.2 terminator is presumed to contain a nuclear matrix attachment region (MAR) and is thought to efficiently terminate transcription.

The Arabidopsis thaliana HSP18.2 terminator can be obtained by a technique such as PCR using the Arabidopsis thaliana genome as a template. In addition, introduction of substitution or deletion into a terminator can be performed by a known site-directed mutagenesis method or the like.

The gene construct of the present invention may contain the lettuce ubiquitin (UBQ) promoter and the Arabidopsis thaliana HSP18.2 terminator downstream (3′ side) thereof. In order to place it between the promoter and the HSP18.2 terminator, it is preferable to include a multiple cloning site (site containing multiple restriction enzyme recognition sequences) between the promoter and the terminator.

A gene encoding a target protein (target gene) is ligated between the promoter and the terminator in an expressible state, and the target protein is expressed in the host cell by introducing the target protein into the host cell using a vector or the like. can be done. That is, the gene construct of the present invention may contain the target gene.

<Target protein>
The type of target protein is not particularly limited as long as it can be expressed in host cells, and includes growth factors, hormones, cytokines, blood proteins, enzymes, antigens, antibodies, transcription factors, receptors, partial peptides thereof, and the like. . The origin of the protein is not particularly limited, and may be derived from mammals such as humans, from plants, or from bacteria or yeast.

Enzymes include, for example, lipases, proteases, steroidogenic enzymes, kinases, phosphatases, methylases, demethylases, oxidases, reductases, cellulases, aromatase, collagenases, transglutaminases, glycosidases, and chitinases.

Growth factors include, for example, epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor (TGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), vascular endothelial cell proliferation factor (VEGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), fibroblast proliferation factor (FGF), hepatocyte growth factor (HGF).

Hormones include, for example, insulin, glucagon, somatostatin, growth hormone, parathyroid hormone, prolactin, leptin, calcitonin.

Cytokines include, for example, interleukins, interferons (IFNα, IFNβ, IFNγ), tumor necrosis factor (TNF).

Blood proteins include, for example, thrombin, serum albumin, factor VII, factor VIII, factor IX, factor X, tissue plasminogen activator.

Antibodies include, for example, whole antibodies, Fab, F(ab'), F(ab') ₂ , Fc, Fc fusion proteins, heavy chain (H chain), light chain (L chain), single chain Fv (scFv) , sc(Fv) ₂ , disulfide-bonded Fv (sdFv), Diabody.

The antigen protein used as a vaccine is not particularly limited as long as it can elicit an immune response in the subject of administration, and may be appropriately selected according to the target of the assumed immune response. and proteins derived from pathogenic viruses.

Proteins derived from pathogenic viruses include, but are not limited to, parvovirus capsid protein VP2, feline immunodeficiency virus envelope protein gp120, swine epidemic diarrhea virus spike protein, or rotavirus capsid protein VP7. (International Publication No. WO 2017/094793 pamphlet), or a partial sequence thereof. It may also be the extraviral domain (ectGP5) of Glycoprotein 5 (GP5) of porcine reproductive and respiratory syndrome (PRRS) virus (International Publication WO2016/021276 pamphlet).

Examples of pathogenic bacterium-derived proteins include, but are not limited to, Escherichia coli thermostable toxin (ST)-derived peptides, Shiga toxin-derived peptides, E. coli heat-labile toxin-derived peptides, and cholera toxin-derived peptides.

<Stx2eB>
Shiga toxin (Stx) is a protein toxin produced by enterohemorrhagic Escherichia coli (EHEC, STEC) that causes edema, and is divided into type 1 (Stx1) and type 2 (Stx2). Stx1 is classified into subclasses a to d, and Stx2 is classified into subclasses a to g. It is a holotoxin consisting of one toxic A subunit molecule and five B subunit molecules that bind to the intestinal mucosa. It acts on the ribosomes of eukaryotic cells and inhibits protein synthesis.
Among these, the B subunit of Stx2e (Stx2eB) is preferred, and Stx2eB is represented by, for example, the amino acid sequence of SEQ ID NO:4, and the nucleotide sequence encoding Stx2eB is represented by SEQ ID NO:3. Stx2eB may also be a mutant in which Asn73 (that is, the Asn residue at position 55 in the amino acid sequence of SEQ ID NO: 4) is replaced with a Ser residue. Wild-type Stx2eB undergoes N-linked glycosylation at this Asn residue, but this mutant does not undergo N-linked glycosylation.
In addition, Stx2eB has preferably 85% or more, more preferably 90% or more, more preferably 95% or more identity with the amino acid sequence of SEQ ID NO: 4, and is administered to animals such as pigs and chickens. It may also be capable of eliciting an immune response.

<LTB>
Escherichia coli diarrhea is caused by the proteinaceous toxin LT produced by enterotoxigenic Escherichia coli (ETEC), also called E. coli heat-labile toxin. LT is a holotoxin consisting of one toxic A subunit molecule and five B subunit molecules. The A subunit (LTA) of LT enters the cytoplasm, increases intracellular cAMP concentration, and activates the plasma membrane chloride channel, causing water leakage into the intestinal tract, namely diarrhea. The B subunit of LT (LTB) is nontoxic and is involved in the adhesion of LT toxin to intestinal cells.
Among these, LTB is preferred, and LTB is represented, for example, by the amino acid sequence of SEQ ID NO:6, and the nucleotide sequence encoding LTB is represented by SEQ ID NO:5. In addition, LTB has 85% or more, preferably 90% or more, more preferably 95% or more identity with the amino acid sequence represented by SEQ ID NO: 6, and is administered to animals such as pigs and chickens. It may also be capable of eliciting an immune response.
Sugar chains may be added to LTB. For example, an N-linked sugar chain is added to the Asn residue at position 90 of LTB (ie, position 90 of SEQ ID NO:6). On the other hand, mutant LTB in which position 90 of SEQ ID NO: 6 is replaced with a Ser residue does not undergo N-linked glycosylation.

<CTB>
The cholera toxin (CT) protein consists of one A subunit (CTA), which is the toxic entity, and five B subunits (CTB), which are involved in intestinal mucosal penetration.
Among these, CTB is preferred, and CTB is represented by, for example, the amino acid sequence of SEQ ID NO:8, and the nucleotide sequence encoding CTB is represented by SEQ ID NO:7. In addition, CTB has 85% or more, preferably 90% or more, more preferably 95% or more identity with the amino acid sequence represented by SEQ ID NO: 8, and is administered to animals such as pigs and chickens. It may also be capable of eliciting an immune response.

Proteins such as the bacterial toxin proteins and virus-derived proteins described above may be fusion proteins in which two or more proteins are bound. 2 or more is preferably 2 to 5, more preferably 2 to 3, and still more preferably 2.
A fusion protein of two or more proteins may be a fusion protein of two or more heterologous proteins or a fusion protein of two or more homologous proteins.
For example, a fusion protein of two or more toxin B subunits selected from the B subunit of Shiga toxin 2e (Stx2eB), the B subunit of E. coli heat-labile toxin (LTB) and the B subunit of cholera toxin (CTB). . A fusion protein of two or more heterologous proteins selected from these toxin proteins can be used as a vaccine against multiple toxins.
Fusion proteins of these bacterial toxin proteins and the above-mentioned virus-derived proteins may also be used. A fusion protein of a toxin protein and a virus-derived protein can be used as a vaccine against bacterial toxins and viruses.

In a preferred embodiment of the fusion protein, each protein making up the fusion protein is linked in tandem via a peptide linker.
The number of amino acids in the peptide linker is, for example, 5-30, preferably 10-25, more preferably 10-22, more preferably 12-22. Also, the proline content in the peptide linker is preferably 20-27%, more preferably 20-25%. In the peptide linker, the prolines are preferably placed every other or every third (flanked by 2 or 3 other amino acids). Amino acids placed between prolines are preferably selected from glycine, serine, arginine. However, 5 or less, preferably 4 or less amino acids other than proline may be added to one or both ends of the peptide. Such preferred peptide linkers are described, for example, in International Publication WO2009/133882. Peptide linkers described in International Publication WO 2017/115853 can also be preferably used.

In the present invention, the peptide linker is preferably a peptide (PG12) consisting of the amino acid sequence represented by SEQ ID NO: 10 or a peptide (PG12-20v7) consisting of the amino acid sequence represented by SEQ ID NO: 12. The peptide linker may preferably be a peptide having 80% or more, preferably 90% or more identity with the peptide consisting of the amino acid sequence represented by SEQ ID NO: 10 or 12.

The protein of interest may be appended with a secretory signal peptide that functions in plant cells for secretory production. For example, plants belonging to Solanaceae , Rosaceae , Brassicaceae , Asteraceae , more preferably Nicotiana , Arabidopsis , Fragaria , Lactuca , etc., preferably Nicotiana tabacum , Arabidopsis thaliana, Fragaria x ananassa , Lactuca sativa , and the like.
More specifically, the secretory signal peptide is derived from tobacco β-D-glucan exohydrolase and includes a peptide having the amino acid sequence represented by SEQ ID NO:14. The nucleotide sequence of DNA encoding tobacco β-D glucan exohydrolase is represented by SEQ ID NO: 13, for example.

Furthermore, the target protein may be added with a transport signal peptide such as an endoplasmic reticulum retention signal peptide, a vacuolar translocation signal peptide, etc., in order to express it in a specific cell compartment.
A preferred endoplasmic reticulum retention signal peptide is described, for example, in WO2009/004842 and WO2009/133882, but the HDEL sequence (SEQ ID NO: 15) can be used.
Vacuolar localization signal peptides are described, for example, in International Publication WO2009/004842 and International Publication WO2009/133882.
Chloroplast localization signal peptides are described, for example, in International Publication WO2009/004842 and International Publication WO2009/133882.

Also, the target protein may have a tag sequence such as a His tag, HA tag (SEQ ID NO: 20), FLAG tag, or GST tag added for the purpose of detection or purification.

A DNA encoding a target protein (target gene) can be obtained, for example, by a general genetic engineering technique based on a known base sequence. In addition, codons representing protein-constituting amino acids may be appropriately modified in the DNA encoding the target protein so that the amount of translation of the protein increases depending on the host cell in which the protein is produced. As a method of codon modification, for example, the method of Kang et al. (Protein Expr Purif. 2004 Nov;38(1):129-35.) can be referred to. Other methods include selecting codons with high frequency of use in host cells, selecting codons with high GC content, and selecting codons with high frequency of use in housekeeping genes of host cells.

In addition, the gene construct may be used by linking a translation enhancer sequence or the like that functions in the host cell to the promoter in order to improve gene expression in the host cell. Examples of translation enhancers include Kozak sequences and the 5'-untranslated region (5'-UTR) of plant-derived alcohol dehydrogenase genes.
As the 5'-untranslated region of the alcohol dehydrogenase gene, for example, the 5'-untranslated region (NtADH5'UTR) (SEQ ID NO: 16) of the alcohol dehydrogenase gene derived from tobacco (Nicotiana tabacum) can be used. Further high translation can be expected by using the NtADH 5'UTR region (NtADHmod 5'UTR) (SEQ ID NO: 17) in which the 3 nucleotides upstream of the dot are modified.

The gene construct of the present invention can be obtained and produced by general genetic engineering techniques. For example, a DNA encoding a promoter and a terminator is obtained from lettuce genomic DNA or the like by a technique such as PCR, and the DNA encoding the useful protein of interest is ligated using PCR or DNA ligase to obtain the gene construct of the present invention. Obtainable.

The recombinant vector of the present invention contains the gene construct of the present invention and is used to express the gene of interest in host cells. The recombinant vector of the present invention preferably contains the target gene as described above. Moreover, the recombinant vector of the present invention preferably contains a selection marker such as a drug resistance gene.

The expression vector used to prepare the recombinant vector of the present invention is not particularly limited as long as it can replicate and grow in host cells, and includes plasmid vectors, shuttle vectors, virus vectors and the like.

For expression in plant cells or plants, chromosomal insertion vectors and viral vectors such as tobacco mosaic virus can be used. In the case of transformation using bacteria of the genus Agrobacterium, plasmid vectors replicable in bacteria of the genus Agrobacterium, such as pBI121, pBI221, pBI101, pIG121Hm, pRI909 and pRI910, can also be used.

The transformed plant (genetically modified plant) of the present invention is characterized by being transformed with the genetic construct or recombinant vector. Plant cells used for transformation are not particularly limited, but examples include plants belonging to Solanaceae , Rosaceae , Brassicaceae , Asteraceae , Chenopodiaceae , and the like. Examples include cells of sunflower, tomato, strawberry, lettuce, white clover, rapeseed, cotton, rapeseed, poplar, Arabidopsis thaliana, rice, corn, potato, tobacco, and soybean. Plant cells include cultured cells as well as cells in plants. Also included are protoplasts, adventitious embryos, shoot primordia, polyblasts, and hairy roots.

The transformed plant of the present invention can be produced by introducing the recombinant vector of the present invention into host cells using general genetic engineering techniques. For example, the electroporation method (Tada, et al., 1990, Theor. Appl. Genet, 80:475), the protoplast method (Gene, 39, 281-286 (1985)), the polyethylene glycol method (Lazzeri, et al. , 1991, Theor. Appl. Genet. 81: 437), introduction method using Agrobacterium (Hood, et al., 1993, Transgenic, Res. 2: 218, Hiei, et al., 1994 Plant J. 6 : 271), particle gun method (Sanford, et al., 1987, J. Part. Sci. tech. 5: 27), polycation method (Ohtsuki, et al., FEBS Lett. 1998 May 29;428(3) :235-40.) can be used.
After introduction of the recombinant vector of the present invention into host cells, transformants can be selected using the phenotype of the selectable marker as an indicator. Plant bodies can be regenerated by culturing the selected plant cells according to a conventional method (eg, Toki et al. (1995) Plant Physiol. 100:1503-1507).

By cultivating a plant obtained by transformation, the target protein can be accumulated inside the plant cell or outside the cell membrane of the plant cell, and the target protein can be produced. Cultivation conditions can be appropriately selected according to the species of the transformed plant, but hydroponics is preferred. In addition, in order to cultivate genetically modified plants, it is preferable to cultivate them in a closed system such as a plant factory. A plant factory is preferable because the temperature, light intensity, light irradiation time, etc. can be controlled. Cultivation period is not particularly limited as long as the period is sufficient for accumulating the target protein.

Plants transformed with the gene construct of the present invention are also suitable for cultivation under high light intensity because the expression of the target gene does not easily decrease even under high light intensity. Here, the high light intensity is preferably 300-600 PPFD, more preferably 300-400 PPFD. The light irradiation time per day is, for example, 10 to 16 hours.
The effect that the expression of the target gene is less likely to decrease even under high light intensity can also be exhibited by the lettuce ubiquitin promoter alone, so cultivation is performed under high light intensity (for example, 300 to 600 PPFD) to produce the target protein. In an embodiment, the genetic construct used to obtain the transformed plant may be a genetic construct comprising DNA encoding a protein of interest expressably linked to a lettuce ubiquitin promoter, and the genetic construct is Arabidopsis thaliana HSP18.2. A terminator other than a terminator may be included.

The accumulated target protein can be separated and purified according to methods well known to those skilled in the art. For example, known suitable methods such as salting out, ethanol precipitation, ultrafiltration, gel filtration chromatography, ion exchange column chromatography, affinity chromatography, medium and high pressure liquid chromatography, reverse phase chromatography, hydrophobic chromatography, etc. or by combining them. However, purification of the target protein is not necessary when directly using the plant body in which the target protein has been accumulated.

Mature seeds can be obtained from the plant body of the present invention. Plants containing the gene construct of the present invention can be stably maintained and preserved by using the seeds. Since the plant of the present invention yields a large amount of mature seeds, the plant of the present invention can be cultivated and self-pollinated to produce a large amount of recombinant seeds.
The effect of increasing the yield of mature seeds of transgenic plants can also be exhibited by the lettuce ubiquitin promoter alone. Any gene construct containing a DNA encoding a target protein expressively linked to a lettuce ubiquitin promoter may be used, and the gene construct may contain a terminator other than the Arabidopsis thaliana HSP18.2 terminator, or may not contain a terminator. . That is, in one aspect of the present invention, in a transgenic plant expressing a target protein, a method for increasing the amount of mature seeds obtained from the plant, comprising: A method is provided comprising transforming a plant with a genetic construct comprising DNA encoding for and cultivating the transformed plant. Here, as indicators of the seed yield of mature seeds, the number of mature seeds per plant strain (individual) (number of mature seeds/strain (individual)), the number of mature seeds per cluster flower of a plant (number of mature seeds / Aggregate flowers).

Examples of the present invention will be described below, but the present invention is not limited to these examples.

(1) Construction of expression vector Using the cauliflower mosaic virus 35S RNA promoter (CaMV 35S pro.) or the lettuce ubiquitin promoter (LsUBQ pro.), bacterial toxin proteins were linked together, or bacterial toxin proteins and virus-derived proteins were linked with peptide tags. A gene construct for fusion protein expression was generated.
Fig. 1 shows its construction diagram.

35S::Stx2eB-Stx2eB used the cauliflower mosaic virus 35S RNA promoter. For the protein to be expressed, the non-toxic B subunit of Shiga toxin (Stx2eB) was linked via a PG12 linker. This gene expression cassette is the same as 2BH in the non-patent document (Matsui et al., Transgenic Res. 2011 Aug;20(4):735-48.), and was constructed by the method described in this document. The Arabidopsis heat shock protein 18.2 gene terminator of 250 bp was used (HSPT250).

UBQ::Stx2eB-Stx2eB uses LsUBQ pro. board. UBQ-F primer (5'- TCTAGA GGCGCGCCAAGCTTCTCGAGGAAACAAGTG-3' (SEQ ID NO: 18), underlined XbaI site) and UBQ-R (5'- GGTACC AACCATAATTAAAACATATTAA-3' (SEQ ID NO: 19), underlined KpnI site) primers was used to amplify LsUBQ pro. After inserting the obtained fragment into pCR2.1-TOPO (Thermo Fisher Scientific Inc.) and confirming the base sequence, a Stx2eB-Stx2eB expression plasmid containing the 35S promoter and HSPT878 (Matsui et al., Plant Biotechnol. 2014) to replace the promoter.

The genes (UBQ::Stx2eB-LTB and UBQ::LTB-Stx2eB) that express vaccines in which Stx2eB and E. coli heat-labile toxin (LTB) are linked in tandem via PG12 under the control of LsUBQ pro. It was constructed. In each of the E-L+ and L+E- plasmids (WO2015/080100) containing the 35S promoter and HSPT878, LsUBQ pro. was inserted using XbaI and KpnI instead of the 35S promoter.

A vaccine antigen expression cassette in which the PRRS neutralizing epitope ectGP5 region (neutralizing epitope of North American PRRS virus GP5: NAectG) was fused to LTB was constructed as follows. Based on the gene cassette for plant expression or yeast expression described in WO2016/021276, LsUBQ pro, the 5'-untranslated region of the alcohol dehydrogenase gene (NtADHmod
5′UTR), a secretory signal peptide (SP) and HSPT878 were fused to construct a gene expression cassette (UBQ::LTB-NAectG). In addition, a gene expression cassette was constructed by fusing UBQ::LTB-Stx2eB with NAectG and EUectG (neutralizing epitopes of European PRRS virus) (UBQ::LTB-Stx2eB-NAectG-EUectG).

A vaccine antigen expression cassette in which the neutralizing epitope (CP) of canine parvovirus coat protein VP2 was fused to LTB-Stx2eB was constructed as follows (UBQ::LTB-Stx2eB-CP). Based on the gene cassette for yeast expression described in WO2017/094793, LsUBQ pro, the 5'-untranslated region (NtADHmod 5'UTR) of the alcohol dehydrogenase gene with modified sequences near the translation initiation codon, and a secretory signal peptide (SP) and HSPT878 were fused to construct a gene expression cassette.

A tandem Stx2eB expression cassette linked by modified linkers was constructed as follows (UBQ: Stx2eB-Stx2eB(v7)). A protein (WO2017/217460) (Stx2eB-Stx2eB(v7)) in which two Stx2eBs were linked with a PG12-20v7 linker was fused to LsUBQ pro, NtADHmod 5'UTR, SP and HSPT878.

(2) Production of genetically modified lettuce Non-patent literature (Matsui et al., Biosci. Biotechnol. Biochem. 2009 Volume 73, Issue 7
Pages 1628-1634).

(3) Lettuce Seed Cultivation Conditions Hydroponics was performed using the Otsuka A recipe. A metal halide lamp was irradiated with a cycle of 16 hours light and 8 hours dark. Cultivation was carried out at a carbon dioxide concentration of 1000 ppm and a temperature of 22.5°C.

result
Progeny stable expression
The 2-ligated Stx2eB protein was expressed in recombinant lettuce using the 35S promoter. It has already been reported that vaccine proteins are highly accumulated in the lettuce (Matsui et al., Transgenic Res. 2011 Aug;20(4):735-48.). From the obtained recombinant lettuce, lines having a single copy of the recombinant gene were selected and homogenized in progeny. Figure 2 shows the amount of vaccine accumulated in each generation of the progeny of the superior line (21-5). As a result, in the recombinant lettuce using the 35S promoter, it was confirmed that the amount of vaccine accumulated decreased (silencing) as generations passed.

Stable expression in lettuce progeny has been reported using the lettuce ubiquitin promoter. An expression cassette (UBQ::Stx2eB-Stx2eB) incorporating a lettuce ubiquitin promoter was constructed to produce recombinant lettuce, and two single copy insertion lines were selected (18-1 line and 5-1 line). The strains (individuals) in which the inserted gene was homogenized in the T1 generation were selected, and their progeny seeds were obtained. Figure 3 shows the amount of vaccine accumulated in each generation. As a result, it was confirmed that the 18-1 line stably accumulated the vaccine until at least the T6 generation, and the 5-1 line stably accumulated the vaccine until at least the T3 generation.

Acquisition of progeny seeds
For two lines of 35S::Stx2eB-Stx2eB-introduced lettuce and two lines of UBQ::Stx2eB-Stx2eB-introduced lettuce, the number of mature seeds of progeny seeds was counted (Fig. 4). As a result, it was found that both the 35S::Stx2eB-Stx2eB-introduced lettuce lines yielded significantly less mature seeds than the wild type. On the other hand, two UBQ::Stx2eB-Stx2eB-introduced lettuce lines yielded the same amount of seeds as the wild type.

Next, we investigated the reasons for the low number of mature seeds. No abnormality was observed in plant growth and flowering in the 35S::Stx2eB-Stx2eB-introduced lettuce. In this lettuce cultivar (Green Wave), about 20 flowers gather to form a cluster, and a certain proportion of mature and immature seeds are formed (Fig. 5). It was confirmed that the 35S::Stx2eB-Stx2eB-introduced lettuce had fewer mature seeds per flower cluster. On the other hand, the UBQ::Stx2eB-Stx2eB-introduced lettuce had the same number of mature seeds as the wild type (Fig. 6).

The number of mature seeds collected was also confirmed for other vaccine expression cassettes using the UBQ promoter (Fig. 7). Several T0 generation lines of each expression cassette-introduced lettuce were collected. The T0 generation is the current generation that was recombined, and it is difficult to grow large because cultivation is started from strains (individuals) regenerated by aseptic culture, and the number of mature seeds collected is smaller than that of the progeny lettuce grown from seeds. Although there was a tendency, the number of seeds was larger than that of the aforementioned 35S::Stx2eB-Stx2eB-introduced lettuce. From this, it was confirmed that when UBQ pro was used, seeds could be collected stably regardless of the type of target protein to be produced.

Evaluation of expression levels of recombinant genes under high light intensity conditions It has been reported that lettuce weight increases under high light intensity conditions (Hata et al., Plant Environmental Engineering, 2011, 23 (4): 127). -36). On the other hand, when the conventional 35S promoter is used, it has been reported that the expression is reduced under high light intensity conditions (Elliott et al., Plant Cell 1989, 1:691-8). It was thought that efficient recombinant protein production would be possible by using a promoter whose activity does not decrease. Table 1 shows the results of examining the expression of the endogenous UBQ gene in lettuce grown under conditions of 160 PPFD and 400 PPFD using a next-generation sequencer (HiSeq2500, Illumina).

As a result, it was found that the incorporation of the UBQ promoter into the foreign gene expression cassette enabled efficient recombinant protein expression by cultivating under high light intensity.

Claims (20)

Lettuce ubiquitin promoter and Arabidopsis heat shock protein 18.2 (HSP18
. 2) Gene constructs for recombinant protein expression, including terminators. A DNA in which the lettuce ubiquitin promoter hybridizes under stringent conditions with a DNA containing the nucleotide sequence of SEQ ID NO: 1 or a polynucleotide having a sequence complementary to the nucleotide sequence of SEQ ID NO: 1, and functions as a promoter in plant cells. 2. The genetic construct of claim 1, wherein the construct is The Arabidopsis thaliana HSP18.2 terminator hybridizes under stringent conditions with a DNA containing the nucleotide sequence of SEQ ID NO: 2 or a polynucleotide having a sequence complementary to the nucleotide sequence of SEQ ID NO: 2, and functions as a terminator in plant cells. 3. The genetic construct of claim 1 or 2, which is DNA. The genetic construct of any one of claims 1-3, further comprising a translational enhancer sequence. A genetic construct according to any one of claims 1 to 4, comprising a DNA encoding a protein of interest. 6. The genetic construct of claim 5, wherein the protein of interest is a bacterial toxin protein and/or a virus-derived protein. 7. A genetic construct according to claim 5 or 6, wherein the protein of interest is a fusion protein of two or more proteins. 8. The genetic construct of claim 7, wherein the fusion protein is a fusion protein in which two or more proteins are linked by peptide linkers. A genetic construct according to any one of claims 5 to 8, wherein the protein of interest comprises a secretory signal peptide. The genetic construct of any one of claims 5-9, wherein the protein of interest comprises a peptide tag. A gene construct according to any one of claims 5 to 10, wherein the protein of interest comprises an endoplasmic reticulum retention signal peptide. A vector comprising a genetic construct according to any one of claims 1-11. A transgenic plant transformed with the genetic construct according to any one of claims 1 to 11 or the vector according to claim 12. 14. The genetically modified plant according to claim 13, which is lettuce. A seed obtained from a transgenic plant according to claims 13 or 14 and comprising a genetic construct according to any one of claims 1-11 . 15. A method for producing a target protein, which is the genetically modified plant according to claim 13 or 14, comprising the step of cultivating the genetically modified plant that expresses the target protein. 17. The method for producing a target protein according to claim 16, wherein cultivation is performed under a light intensity of 300-600 PPFD. Transgenic plants expressing the protein of interest transformed with a genetic construct comprising DNA encoding the protein of interest operably linked between the lettuce ubiquitin promoter and the Arabidopsis HSP18.2 terminator are exposed to a light intensity of 300-600 PPFD. A method for producing a protein of interest, comprising the step of cultivating under. A genetically modified plant that expresses a target protein, a method for stably harvesting seeds from the plant, wherein the DNA encoding the target protein is expressably linked between the lettuce ubiquitin promoter and the Arabidopsis thaliana HSP18.2 terminator. A method comprising transforming a plant with a genetic construct comprising and cultivating the transformed plant. 20. The method of claim 19, wherein said transformed plant is grown hydroponically.

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