Nicotiana plant production method and target protein manufacturing method

Abstract

Provided is a nicotiana plant production method that requires less electricity and that improves production efficiency of a target protein.　This nicotiana plant production method comprises: a step for cultivating a host nicotiana plant; a step for further cultivating the host nicotiana plant while irradiating the middle portion and the lower portion of the above-ground part of the host nicotiana plant with light (first irradiation step); a step for introducing the gene of a target protein into the host nicotiana plant (introduction step); and a step for further cultivating the nicotiana plant that has been introduced with the gene of the target protein while irradiating the middle portion and the lower portion of the above-ground part of the host nicotiana plant with light (second irradiation step).

Description

Method for producing Nicotiana plants and method for producing target proteins

[0001] The present invention relates to a method for producing Nicotiana plants and a method for producing a target protein.

[0002] In recent years, attention has been focused on plant-based protein production, in which genes for target proteins are introduced into plants and expressed.

[0003] For example, Non-Patent Document 1 discloses the advantages of using plants, recent trends in practical application and research and development, an overview of plant biotechnology, and the importance of environmental control in plant factories regarding the production of pharmaceutical proteins using plant factories.

[0004] On the other hand, as shown in Non-Patent Document 2, the project "Development of Bio-derived Product Production Technology to Accelerate the Realization of Carbon Recycling" aims to efficiently produce products such as pharmaceuticals while satisfying the requirements of rationalizing energy use and reducing carbon dioxide emissions.

[0005] Therefore, in plant-based protein production, there is a need to further improve the production efficiency of target proteins while satisfying the above-mentioned requirements of energy conservation and reduction of carbon dioxide emissions.

[0006] "Production of pharmaceutical proteins using plant factories," Seibutsu to Kisetsu 22:58-68, 2022; "Development of bio-based product production technology to accelerate the realization of carbon recycling," New Energy and Industrial Technology Development Organization, National Research and Development Agency, [online], Internet: https: / / www.nedo.go.jp / activities / ZZJP_100170.html (accessed June 14, 2023)

[0007] However, there is still room for improvement in the efficiency of plant-based protein production.

[0008] The present invention aims to improve the above situation, and its object is to provide a target protein efficiently with low power consumption, with the goal of saving energy and reducing carbon dioxide emissions.

[0009] The present invention that achieves the above object is as follows.

[0010] Aspect 1 is a method for producing a Nicotiana plant, comprising the steps of: cultivating a host Nicotiana plant; further cultivating the host Nicotiana plant while irradiating the middle and lower above-ground parts of the plant with light (first irradiation step); introducing a gene for a target protein into the host Nicotiana plant (introduction step); and further cultivating the host Nicotiana plant into which the gene for the target protein has been introduced while irradiating the middle and lower above-ground parts of the plant with light (second irradiation step). Aspect 2 is a method for cultivating the host Nicotiana plant at a density of 400 to 1200 plants / m ２The method for producing a Nicotiana plant according to Aspect 1, wherein the host Nicotiana plant is cultivated in a humid atmosphere. Aspect 3: The method for producing a Nicotiana plant according to Aspect 1 or 2, wherein the first irradiation step is carried out after at least a portion of adjacent plants of the host Nicotiana plant have come into contact with each other and before the introducing step. Aspect 4: The method for producing a Nicotiana plant according to any one of Aspects 1 to 3, wherein the host Nicotiana plant is Nicotiana benthamiana or Nicotiana tabacum. Aspect 5: The method for producing a Nicotiana plant according to any one of Aspects 1 to 4, wherein the first irradiation step and the second irradiation step are each independently carried out for a period of 1 day to 14 days. Aspect 6: The method for producing a Nicotiana plant according to any one of Aspects 1 to 5, wherein the first irradiation step and the second irradiation step are each independently carried out for a period of 3 days to 10 days. <Aspect 7> The method for producing a Nicotiana plant according to any one of Aspects 1 to 6, further comprising irradiating the host Nicotiana plant with light from above. <Aspect 8> The method for producing a Nicotiana plant according to any one of Aspects 1 to 7, wherein the introducing step involves introducing a gene for the target protein into the host Nicotiana plant using a vacuum infiltration method. <Aspect 9> The method for producing a Nicotiana plant according to any one of Aspects 1 to 8, wherein the target protein comprises at least one selected from the group consisting of hemagglutinin, green fluorescent protein, and human anti-C-reactive protein antibody. <Aspect 10> The method for producing a Nicotiana plant according to any one of Aspects 1 to 9, wherein the efficiency of production of the target protein is improved. <Aspect 11> A method for producing a target protein, comprising: producing a Nicotiana plant using the method for producing a Nicotiana plant according to any one of Aspects 1 to 10; and extracting the target protein from the Nicotiana plant produced by the above step. Aspect 12: The method for producing a target protein according to Aspect 11, wherein the target protein comprises at least one selected from the group consisting of hemagglutinin, green fluorescent protein, and human anti-C-reactive protein antibody.

[0011] According to the present invention, it is possible to provide a method for producing Nicotiana plants that requires low power consumption and has improved efficiency in producing a target protein.

[0012] Furthermore, the present invention can provide a method for producing a target protein with low power consumption and improved production efficiency.

[0013] FIG. 1 is a process diagram showing one embodiment of the method for producing Nicotiana plants of the present invention. FIG. 2 is a schematic diagram showing the configuration of a host Nicotiana plant and a light source according to one embodiment of the first irradiation step. FIG. 3 is a schematic diagram showing the configuration of the cultivation shelf and the side supplemental light treatment used in the examples. FIG. 4 is a diagram showing the results of "HA production amount / power consumption" for each of Example 1 and Comparative Examples 1 to 3. FIG. 5 is a diagram showing the results of "anti-CRP antibody production amount / power consumption" for each of Example 2 and Comparative Example 4.

[0014] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail below with reference to the preferred embodiments. However, the present invention is not limited to the following embodiments, and various modifications can be made within the scope of the present invention.

[0015] <<Method for Producing Nicotiana Plants>> The method for producing Nicotiana plants of the present invention is a production method comprising the steps of: cultivating a host Nicotiana plant; further cultivating the host Nicotiana plant while irradiating the middle and lower above-ground parts of the plant with light (first irradiation step); introducing a gene for a target protein into the host Nicotiana plant (introduction step); and further cultivating the Nicotiana plant into which the gene for the target protein has been introduced while irradiating the middle and lower above-ground parts of the plant with light (second irradiation step).

[0016] The Nicotiana tabacum plant production method of the present invention achieves low power consumption and improved target protein production efficiency compared to conventional methods. Without being limited by theory, this effect is believed to be primarily due to the two irradiation steps (i.e., the first and second irradiation steps) performed before and after the step of introducing the target protein gene into the host Nicotiana tabacum plant (the introduction step). Surprisingly, the first and second irradiation steps not only increased biomass (e.g., dry matter amount of leaves, stems, etc.), but also increased the production of the introduced target protein, particularly relative to the amount of power consumed. Even more surprisingly, the effects of the present invention were found to be exerted not only on a specific target protein, but also on the introduction of various target proteins, such as medical proteins and industrial proteins, as described below.

[0017] The present invention will be described in detail below by taking as an example each step included in the method for producing Nicotiana plants of the present invention shown in FIG.

[0018] FIG. 1 is a flow chart showing one embodiment of the method for producing Nicotiana plants of the present invention.

[0019] As shown in FIG. 1 , the method for producing a Nicotiana plant of the present invention includes the steps of cultivating a host Nicotiana plant (S1), further cultivating the host Nicotiana plant while irradiating the middle and lower above-ground parts of the plant with light (first irradiation step) (S2), introducing a gene for a target protein into the host Nicotiana plant (introduction step) (S3), and further cultivating the Nicotiana plant into which the gene for the target protein has been introduced while irradiating the middle and lower above-ground parts of the plant with light (second irradiation step) (S4).

[0020] <Step S1> In step S1, a host Nicotiana plant is cultivated.

[0021] In the present invention, the term "host" refers to a host for expressing a target protein, and may also be referred to as an "expression host." The host plant is not particularly limited as long as it can express the target protein and accumulate it within the plant.

[0022] In the present invention, the host Nicotiana plant is not particularly limited, and examples thereof include Nicotiana benthamiana and Nicotiana tabacum.

[0023] In the present invention, the cultivation density of the host Nicotiana plant is not particularly limited, but high-density cultivation is preferred from the viewpoint of reducing power consumption. ２ This refers to cultivating plants at a density of at least 1000 m².

[0024] In the present invention, host Nicotiana plants are grown at a density of 400 to 1200 plants / m ２ More specifically, the host Nicotiana plant can be cultivated at a density of 400 plants / m ２ Above, 450 plants / m ２ Above, 500 plants / m ２ Above, 550 plants / m ２ Above, 600 plants / m ２ Above, 650 plants / m ２ More than 700 plants / m ２ Above, 750 plants / m ２ or more than 800 plants / m ２ It can be cultivated at a density of 1200 plants / m or more. ２ Below, 1150 plants / m ２ Below, 1100 plants / m ２ Below, 1050 plants / m ２ Below, 1000 plants / m ２ Below, 950 plants / m ２ Below, 900 plants / m ２ Below, 850 plants / m ２ Below, 800 plants / m ２ Below, 750 plants / m ２ Below, 700 plants / m ２ Below, 650 plants / m ２ Below, 600 plants / m ２ Below, 550 plants / m ２ or less, or 500 plants / m ２ It may be cultivated in the following conditions:

[0025] In the present invention, the seeds of the host Nicotiana plant may be sown at the cultivation density range of the host Nicotiana plant described above, or the cultivation density of the host Nicotiana plant may be adjusted to within the above-mentioned range by sowing at a density higher than the above-mentioned density range and then thinning out the buds. Alternatively, the cultivation density of the host Nicotiana plant may be adjusted to within the above-mentioned range by thinning out the buds until immediately before performing step S2 (first irradiation step) described below.

[0026] In the present invention, from step S1, a host Nicotiana plant may be cultivated in a cultivation chamber. Alternatively, an artificial light-type seedling growing system or the like may be used as the cultivation chamber. The cultivation chamber may be equipped with a light source capable of irradiating the host Nicotiana plant to be cultivated, for example, an overhead light source capable of irradiating the Nicotiana plant from above.

[0027] In the present invention, the host Nicotiana plant may be cultivated according to a conventional plant cultivation method, which may be appropriately determined depending on the plant species, etc. For example, a method may be used in which the seeds are germinated in soil, rock wool, sponge cubes, etc., using trays or pots, and then cultivated using water, preferably water and a culture solution. The culture solution is not particularly limited and may contain nutrients such as nitrogen, phosphate, potassium, calcium, magnesium, etc., and may further contain trace amounts of iron, copper, manganese, zinc, molybdenum, boron, etc.

[0028] The method for producing Nicotiana plants of the present invention may include irradiating the host Nicotiana plant with light from above in at least one of steps S1 to S3 described below, preferably in all steps S1 to S3.

[0029] In each step of the present invention, the means for irradiating light is not particularly limited, and for example, sunlight or artificial light may be used. Examples of artificial light include, but are not limited to, fluorescent lights, LEDs, fluorescent lamps, and inorganic / organic ELs.

[0030] Furthermore, light intensity can be evaluated by measuring the photosynthetically effective photon flux density (PPFD) or the like. PPFD is expressed as the number of photons per unit time and per unit area of ​​light in the visible range of 400 to 700 nm that is effective for photosynthesis, and can be measured using a photon meter or the like. In the present invention, PPFD is, for example, 30 to 600 μmol m －２ ・s －１ More specifically, the PPFD may be adjusted within a range of, for example, 30 μmol m －２ ・s －１ Above, 50 μmol・m －２ ・s －１ Above, 100μmol・m －２ ・s －１ Above, 150μmol・m －２ ・s －１ or more, or 200 μmol m －２ ・s －１ or more, and －２ ・s －１ Below, 550 μmol・m －２ ・s －１ Below, 500μmol・m －２ ・s －１ Below, 450 μmol・m －２ ・s －１ or less, or 400 μmol m －２ ・s －１ In the present invention, the PPFD of the irradiation light in each step may be the same or different.

[0031] In the present invention, the light irradiation time is not particularly limited and may be, for example, 6 hours or more, 10 hours or more, 12 hours or more, or 16 hours or more per day, or may be less than 24 hours, 18 hours or less, 16 hours or less, or 12 hours or less. Here, the light irradiation time may be the total time in the case of continuous irradiation, or the total time in the case of discontinuous irradiation. Furthermore, the light irradiation time in each step may be the same or different.

[0032] In the present invention, the cultivation environment of the host Nicotiana plant is not particularly limited. For example, the carbon dioxide concentration in the cultivation environment is not particularly limited, and is, for example, 200 μmol mol －１ Above, 300μmol・mol －１ or more, or 400 μmol·mol －１ or more, and －１ Below, 1500μmol・mol －１ Below, 1000μmol・mol －１ or less, or 800 μmol·mol －１ or less. The carbon dioxide concentration in each step may be the same or different. The temperature in the cultivation environment is not particularly limited and may be, for example, 10°C or higher, 15°C or higher, 18°C ​​or higher, 20°C or higher, 21°C or higher, or 22°C or higher, or may be 30°C or lower, 28°C or lower, 25°C or lower, or 22°C or lower. The temperatures in each step may be the same or different. The temperatures before and after the introduction step (step S3) (i.e., the temperatures in steps S2 and S4) may be adjusted appropriately depending on the type of protein of interest. The humidity in the cultivation environment is not particularly limited and may be, for example, 40% or higher, 45% or higher, 50% or higher, 55% or higher, or 60% or higher, or 100% or lower, or 95% or lower. The humidity in each step may be the same or different.

[0033] In the present invention, the period of step S1, i.e., the period from sowing of the host Nicotiana plant until performing step S2 described below, is not particularly limited and may be set appropriately depending on the plant species, sowing density, etc. Furthermore, from the viewpoint of further improving production efficiency, the period of step S1 can preferably be set, for example, as the period from sowing of the host Nicotiana plant until at least a portion of adjacent plants come into contact with each other. More specifically, the period of step S1 may be, for example, 1 day or more, 7 days or more, 14 days or more, or 21 days or more, and may be 30 days or less, 28 days or less, 21 days or less, or 14 days or less.

[0034] In step S2, the host Nicotiana plant is further cultivated while being irradiated with light to the middle and lower above-ground parts. In the present invention, this step S2 is also referred to as a "first irradiation step."

[0035] In the present invention, the definitions of "the middle and lower aerial parts of a host Nicotiana plant" and "the middle and lower aerial parts of a Nicotiana plant into which a gene for a target protein has been introduced" described below can be based on the following: the plant individual is divided vertically into thirds, and these are defined as the "lower aerial part," "middle aerial part," and "upper aerial part" of the plant, in that order from the medium side.

[0036] Furthermore, in the present invention, irradiating the "middle and lower above-ground parts of a host Nicotiana plant" and the "middle and lower above-ground parts of a Nicotiana plant into which a gene for a target protein has been introduced" with light, as described below, refers to exposing the middle and lower above-ground parts of each of these plant individuals to light over, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% of the total surface area of ​​the middle and lower above-ground parts. Of course, the light may or may not reach the upper above-ground parts of each plant individual. Furthermore, the light source can be placed on the side of these plant individuals. There may be one light source or multiple light sources. In the case of a single light source, it may be placed on the side of any one or multiple plant individuals (e.g., one row) and adjusted so that the light reaches the "middle and lower above-ground parts" of the other plant individuals. For a general explanation of light irradiation, please refer to the above-mentioned section "Step S1" as appropriate.

[0037] FIG. 2 is a schematic diagram showing the configuration of a host Nicotiana plant and a light source according to one embodiment of the first irradiation step.

[0038] As shown in Figure 2, host Nicotiana plants A, B, and C are cultivated while being irradiated with light onto the middle and lower above-ground parts. More specifically, light source 10 is placed on the side of host Nicotiana plant A, and irradiates light onto middle and lower above-ground parts 1b and 1c of host Nicotiana plant A. Furthermore, the light from light source 10 is adjusted so that it can also irradiate the middle and lower above-ground parts of host Nicotiana plants B and C.

[0039] In the present invention, step S2 may be performed, for example, after at least a portion of adjacent host Nicotiana plants obtained in step S1 contact each other and before the step of introducing a gene for a target protein into the host Nicotiana plant (the introduction step). For example, as shown in Figure 2, light irradiation of the middle and lower above-ground parts of the host Nicotiana plant in step S2 may begin when at least a portion of adjacent plants (e.g., host Nicotiana plant A and host Nicotiana plant B, or host Nicotiana plant B and host Nicotiana plant C) contact each other.

[0040] In the present invention, the period of step S2 is not particularly limited and may be set appropriately depending on the plant species, sowing density, etc. More specifically, the period of step S2 may be, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more, or may be 14 days or less, 13 days or less, 12 days or less, 11 days or less, or 10 days or less.

[0041] <Step S3> In step S3, a gene encoding a target protein is introduced into a host Nicotiana plant. In the present invention, step S3 is also referred to as an "introduction step."

[0042] In the present invention, the target protein is not particularly limited and may be, for example, a protein used for medical or industrial purposes. Examples of medical proteins include, but are not limited to, vaccines, antibodies, peptides, enzymes, and hormones. Examples of industrial proteins include, but are not limited to, peptides, enzymes, and functional proteins.

[0043] More specifically, examples of vaccines include, but are not limited to, influenza hemagglutinin (HA) protein (also simply referred to as "hemagglutinin"). Examples of antibodies include, but are not limited to, human antibodies such as human anti-C-reactive protein (CRP) antibodies. Examples of industrial proteins include, but are not limited to, green fluorescent protein (GFP). Thus, in the present invention, the target protein may include at least one selected from the group consisting of hemagglutinin, green fluorescent protein, and human anti-C-reactive protein antibodies.

[0044] In step S3, the method for introducing the gene for the target protein into the host Nicotiana plant is not particularly limited, and for example, the "transient gene expression method" disclosed in Non-Patent Document 1 may be used. Specifically, for example, the agroinfection method or the agroinfiltration method may be used. Furthermore, the magnifection method may be used as the agroinfection method. More specifically, for example, the vacuum infiltration method or the syringe infiltration method may be used as a method for introducing the gene for the target protein into the host Nicotiana plant, and the vacuum infiltration method is preferred from the viewpoint of improving production efficiency.

[0045] For example, in one embodiment of the present invention, the introduction step can use vacuum infiltration to introduce a gene encoding a target protein into a host Nicotiana plant. This can be achieved by adjusting the pressure while at least a portion of the host Nicotiana plant obtained in step S2 is immersed in a suspension containing Agrobacterium. The entire host Nicotiana plant does not need to be immersed in the suspension; for example, a portion of the stem, roots, etc. may be exposed from the suspension. The pressure of the reduced pressure is not particularly limited and may be, for example, lower than atmospheric pressure (1 atmosphere = 101.325 kPa = approximately 0.1 MPa). More specifically, the pressure may be reduced to, for example, 0.9 atmospheres or less, 0.8 atmospheres or less, or 0.5 atmospheres or less. The lower limit of the reduced pressure is not particularly limited and may be, for example, 0.001 atmospheres or more, 0.005 atmospheres or more, 0.01 atmospheres or more, 0.05 atmospheres or more, or 0.1 atmospheres or more. The time for decompression is not particularly limited and may be, for example, 10 seconds or more, 20 seconds or more, 30 seconds or more, or 1 minute or more, or 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, or 3 minutes or less. The decompression operation may be carried out continuously, or decompression operation and pressure recovery operation may be repeated. The device used for the vacuum infiltration method is not particularly limited and may be, for example, a pressure-resistant container.

[0046] <Step S4> In step S4, the Nicotiana plant into which the gene for the target protein has been introduced is further cultivated while being irradiated with light to the middle and lower above-ground parts. In the present invention, this step S4 is also referred to as a "second irradiation step." In addition, in this step S4, the target protein can be expressed in the Nicotiana plant into which the gene for the target protein has been introduced.

[0047] Here, the explanation for step S2 above may be referred to as appropriate for the method of irradiating the middle and lower above-ground parts of a Nicotiana plant into which a gene for a target protein has been introduced with light.

[0048] In the present invention, the period of step S4 is not particularly limited and may be set appropriately depending on the plant species, sowing density, etc. More specifically, the period of step S4 may be, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more, or may be 14 days or less, 13 days or less, 12 days or less, 11 days or less, or 10 days or less. The period of step S4 may be the same as or different from the period of step S2 described above.

[0049] The production method for Nicotiana plants of the present invention, which is carried out by the above-mentioned steps S1 to S4, has improved the efficiency of target protein production compared to conventional methods.

[0050] <<Method for Producing a Target Protein>> The present invention also provides a method for producing a target protein, which comprises the steps of: producing a Nicotiana plant using the above-described method for producing a Nicotiana plant; and extracting the target protein from the Nicotiana plant produced by the above-described step.

[0051] The process for producing Nicotiana plants can be appropriately described in the above-mentioned "Method for producing Nicotiana plants of the present invention," and therefore a detailed description thereof will be omitted here.

[0052] The step of extracting the target protein from the produced Nicotiana plant is not particularly limited and can be carried out by a conventional method, for example, by collecting the Nicotiana plant produced in the step of producing the Nicotiana plant and treating it with a grinding or other process.

[0053] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0054] Example 1 and Comparative Examples 1 to 3 Example 1 and Comparative Examples 1 to 3 were carried out using HA (hemagglutinin H1N1) as the useful protein (target protein), and Example 2 and Comparative Example 4 were carried out using an anti-CRP antibody (human anti-C-reactive protein antibody) as the useful protein, depending on whether or not the steps shown in Table 1 below were performed.

[0055] For Example 1 and Comparative Example 1, four experiments (Experiments 1, 2, 3, and 4) were each conducted. For Comparative Examples 2 and 3, one experiment (Experiment 1) was each conducted. For Example 2 and Comparative Example 4, three experiments (Experiments 5, 6, and 7) were each conducted. Note that in Example 1 and Comparative Examples 1 to 3, in which HA was used as the useful protein, Experiments 1, 2, and 3 were conducted under the same conditions, with Experiment 4 being slightly different in the date of gene transfer and the measurement items; and in Example 2 and Comparative Example 4, in which an anti-CRP antibody was used as the useful protein, Experiments 6 and 7 were conducted under the same conditions, with Experiment 5 being slightly different in the date of gene transfer, details of which are as follows.

[0056] (Plant Materials and Cultivation Conditions) Nicotiana benthamiana was used as the test plant. The planting density was 800 plants / m －２ The cultivation medium used was a block-shaped rock wool medium (100 x 100 x 65 mm). Watering and fertilization were carried out using a hydroponic culture method in which the culture solution was flooded in a tray and allowed to penetrate the medium. A fluorescent white LED panel (440 x 580 mm) was used as the upper light source.

[0057] The environmental conditions in the cultivation room were set as follows: the light cycle was 16 h light / 8 h dark, and the light-phase PPFD on the top surface of the medium was 200 μmol m －２ ・s －１ , and 400 μmol m from the 28th day after sowing. －２ ・s －１ The temperature in the cultivation room was kept at 28 / 22°C (light / dark) before gene introduction (up to 35 days after sowing), and after gene introduction (35 days after sowing and thereafter), it was kept constant at 21°C in Example 1 and Comparative Examples 1 to 3, in which the useful protein was HA, and at 24°C in Example 2 and Comparative Example 4, in which the useful protein was anti-CRP antibody. The relative humidity in the cultivation room was 60-95%. CO ２ The concentration was 400 μmol·mol until the 21st day after seeding. －１ , and 800 μmol·mol from the 21st day after seeding. －１ The culture medium used was OTA House No. 1 and No. 2 (Otsuka Agritechno Co., Ltd.), and the EC (electrical conductivity) was 180 mS m －１, 270mS・m after 21st day after sowing －１ The pH was adjusted to 6.0 using an aqueous HCl solution.

[0058] On the seventh day after sowing, the cultivation density was 800 plants / m －２ The pressure was adjusted to 180 mS·m －１ The trays were flooded with culture medium once a day.

[0059] From the 21st day after sowing, CO ２ Concentration: 800 μmol / mol －１ , the culture medium EC was 270 mS·m －１ Gene transfer was performed on the 35th day after seeding (Experiments 1, 2, 3, and 5), the 36th day after seeding (Experiments 6 and 7), or the 37th day after seeding (Experiment 4). After gene transfer, the temperature was kept constant at 21°C for 24 hours in Example 1 and Comparative Examples 1 to 3, in which the useful protein was HA, and at 24°C for 24 hours in Example 2 and Comparative Example 4, in which the useful protein was an anti-CRP antibody.

[0060] (Side Light Compensation Treatment) The light irradiation in the "first irradiation step" and "second irradiation step" shown in Table 1 was performed by side light compensation treatment.

[0061] The amount of irradiance of the side light was adjusted so that the power consumption of the light source above was 50% of that of the light source above, and the plants were irradiated with the light. A white LED panel (95 x 580 mm) using phosphor was used as the light source for the side light. The LED panel for the side light was installed facing the plant canopy so that it irradiated the plant canopy horizontally from the side. Under these conditions, the average PPFD value from the center of the side light panel to the edge of the plant canopy was 365 ± 15 μmol m －２ ・s －１ It was.

[0062] The configuration of the cultivation shelf and the side supplemental light treatment described above are shown in Fig. 3. Although the illumination ranges of the side supplemental light sources 10a and 10b are not shown in Fig. 3, they were adjusted so that light reached the middle and lower parts of the above-ground parts of the plants.

[0063] The sowing dates and cultivation conditions are summarized in Table 2 below.

[0064]

[0065] (Gene Introduction and Production of Useful Proteins) In Example 1 and Comparative Examples 1 to 3, HA (hemagglutinin H1N1) derived from influenza virus (A / California / 07 / 2009 (H1N1)) was used as the useful protein. In Example 2 and Comparative Example 4, an anti-CRP antibody was used. For the production of useful proteins, a magnifection method using a magnICON vector (provided by Denka Co., Ltd.) was used as a transient gene expression method. Nicotiana benthamiana plants grown under the above-mentioned cultivation conditions on the 35th day after sowing (Experiments 1, 2, 3, and 5), the 36th day after sowing (Experiments 6 and 7), or the 37th day after sowing (Experiment 4) were used, and genes were introduced by vacuum infiltration.

[0066] <Measurements> Biomass production was measured before treatment (experiments 1, 2, 3, 5, 6, and 7 only), before gene introduction (experiments 1, 2, 3, 5, 6, and 7 only), and at harvest time: fresh weight, dry weight, and leaf area of ​​leaves and stems (experiments 1, 2, 3, 5, 6, and 7 only).

[0067] Only the leaf blade was considered as a leaf, and the stem and petiole were considered as a stem. For measuring the dry weight, the leaves and stems after measuring the fresh weight were dried at 100°C for 1 hour and then at 80°C for 3 days.

[0068] The HA content at harvest (Example 1 and Comparative Examples 1-3) and anti-CRP antibody content (Example 2 and Comparative Example 4) were measured as useful protein production. Leaves frozen and stored at -80°C immediately after measuring fresh weight were used as samples. The HA content was measured by sandwich enzyme-linked immunosorbent assay (hereinafter referred to as ELISA) using commercially available kits (HA: SEK001, Sino Biological Co., Ltd.; anti-CRP antibody: Human IgG ELISA Kit ab195215, Abcam).

[0069] The amount of power consumed by the lighting used during the cultivation period was calculated from the current and voltage values ​​of the DC stabilized power supply connected to the LED panel.

[0070] <Results> The above measurements showed that in the four experiments (Experiments 1 to 4) of Example 1, the HA production amount per power consumption was improved compared to the corresponding experiments (Experiments 1 to 4) of Comparative Example 1. Also, for example, the results of "HA production amount / power consumption" for Example 1 and Comparative Examples 1 to 3 in the case of Experiment 1 are shown in Figure 4. Furthermore, the results of Example 1 and Comparative Examples 2 and 3 obtained are shown in Table 3, with Comparative Example 1 being the standard ("1").

[0071]

[0072] Similarly, the above measurements showed that the amount of anti-CRP antibody produced per unit of power consumption was improved in each of the three experiments (Experiments 5 to 7) of Example 2 compared to the corresponding experiments (Experiments 5 to 7) of Comparative Example 2. Furthermore, the results of "anti-CRP antibody production amount / power consumption" for Example 2 and Comparative Example 4 in Experiment 6 are exemplarily shown in FIG. 5. Furthermore, the results of Example 2 obtained using Comparative Example 4 as the standard ("1") are shown in Table 4.

[0073]

[0074] As is clear from the results of Figure 4 and Table 3, Example 1, in which side illumination was performed, showed a significantly improved HA production amount per power consumption compared to Comparative Example 1, in which side illumination was not performed (i.e., the "first illumination step" and "second illumination step" shown in Table 1). Similarly, as is clear from the results of Figure 5 and Table 4, Example 2, in which side illumination was performed, showed a significantly improved anti-CRP antibody production amount per power consumption compared to Comparative Example 4, in which side illumination was not performed. In other words, it was found that the production efficiency of the target protein was improved by the production methods of Examples 1 and 4.

[0075] On the other hand, in Comparative Example 2, in which side lighting was performed only before the step of introducing the gene for the target protein into the host Nicotiana plant (i.e., the "first irradiation step" shown in Table 1), and Comparative Example 3, in which side lighting was performed only after the step of introducing the gene for the target protein into the host Nicotiana plant (i.e., the "second irradiation step" shown in Table 1), neither showed an improvement in HA production per unit of power consumption compared to Comparative Example 1, in which side lighting was not performed.

[0076] Although not shown in the figures or tables, the biomass production amount and HA production amount were both the highest in Example 1. Furthermore, the biomass production amount and anti-CRP antibody production amount in Example 2 were higher than those in Comparative Example 4.

[0077] It is believed that even if "green fluorescent protein" is used as the target protein instead of "HA" or "anti-CRP antibody," the same effect as in the case of "HA" or "anti-CRP antibody" can be obtained.

[0078] 1a Upper part of above-ground part of host Nicotiana plant A 1b Middle part of above-ground part of host Nicotiana plant A 1c Lower part of above-ground part of host Nicotiana plant A 10 Light source 10a, 10b Side supplementary light source 11 Top light source 100 Cultivation shelf 110 Top light source illumination range

Claims

1. A method for producing a Nicotiana plant, comprising the steps of: cultivating a host Nicotiana plant; further cultivating the host Nicotiana plant while irradiating light to the middle and lower above-ground parts of the plant (first irradiation step); introducing a gene for a target protein into the host Nicotiana plant (introduction step); and further cultivating the host Nicotiana plant into which the gene for the target protein has been introduced while irradiating light to the middle and lower above-ground parts of the plant (second irradiation step).

2. The host Nicotiana plant is grown at a density of 400 to 1,200 plants / m ２ The method for producing a Nicotiana plant according to claim 1, wherein the plant is cultivated under a temperature of 100° C. or higher.

3. The method for producing a Nicotiana plant according to claim 1, wherein the first irradiation step is carried out between the time when at least a portion of adjacent plants of the host Nicotiana plant come into contact with each other and before the introduction step.

4. The method for producing a Nicotiana plant according to claim 1, wherein the host Nicotiana plant is Nicotiana benthamiana or Nicotiana tabacum.

5. The method for producing a Nicotiana plant according to claim 1, wherein the first irradiation step and the second irradiation step are each independently carried out for a period of 1 day or more and 14 days or less.

6. The method for producing a Nicotiana plant according to claim 1, wherein the first irradiation step and the second irradiation step are each independently carried out for a period of 3 days or more and 10 days or less.

7. The method for producing a Nicotiana plant according to claim 1, further comprising irradiating light from above the host Nicotiana plant.

8. The method for producing a Nicotiana plant according to claim 1, wherein in the introduction step, the gene for the target protein is introduced into the host Nicotiana plant by vacuum infiltration.

9. The method for producing a Nicotiana plant according to claim 1, wherein the target protein comprises at least one selected from the group consisting of hemagglutinin, green fluorescent protein, and human anti-C-reactive protein antibody.

10. The method for producing a Nicotiana plant according to claim 1, wherein the efficiency of production of the target protein is improved.

11. A method for producing a target protein, comprising: producing a Nicotiana plant using the method for producing a Nicotiana plant described in any one of claims 1 to 10; and extracting the target protein from the Nicotiana plant produced by the above-mentioned step.

12. The method for producing a target protein according to claim 11, wherein the target protein comprises at least one selected from the group consisting of hemagglutinin, green fluorescent protein, and human anti-C-reactive protein antibody.

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