JP2021153454A - Method for producing heteroprotein - Google Patents

Abstract

To provide a method for producing heteroprotein in a plant, the method capable of being conducted at relatively low cost and conveniently.SOLUTION: A method for producing heteroprotein in a plant includes the step of: expressing heteroprotein under the light with a photosynthetic photon flux density less than 30 μmol m-2 s-1 in a plant transformed with a gene encoding heteroprotein.SELECTED DRAWING: None

Description

The present invention relates to a method for producing a heterologous protein in a plant.

Escherichia coli, yeast, insect cells, plant cells, animal cells and the like are known as hosts for producing heterologous proteins. Plant cells have the advantages of being able to express relatively complex proteins, and the obtained proteins are also easy to separate and purify, and are highly safe.

As a method for introducing a heterologous gene into a plant, there is an Agrobacterium method using an infectious system of Agrobacterium, which is a soil bacterium. So far, many cultivation methods suitable for producing heterologous proteins in plants infected with Agrobacterium have been reported.

For example, Patent Document 1 discloses a method for cultivating a plant and a method for treating a plant in an infection step with Agrobacterium. Patent Document 1 further describes that the photosynthetic effective photon flux density (PPFD) is usually 30 μmol · m ⁻² · s ^-1 or more under light irradiation conditions.

Further, Patent Document 2 discloses a technique for improving the expression efficiency and the expression level of a protein by using a plant cultivated under a condition containing a certain percentage or more of red light for transient expression of the protein.

Japanese Unexamined Patent Publication No. 2015-154769 International Publication No. 2015/034042

However, in the prior art described in Patent Document 1 and Patent Document 2, the conditions for expressing a heterologous protein in a plant at a lower cost and with higher efficiency are not yet sufficient.

Therefore, an object of the present invention is to provide a relatively inexpensive and simple production method using a plant as a production method for a heterologous protein.

As a result of diligent studies, the present inventors surprisingly produced heterologous proteins in plants even if they were less than ^{30 μmol · m -2} · s ^-1 , which was conventionally regarded as the lower limit of the effective photosynthetic flux density. We found that it was possible and completed the present invention.

That is, the present invention includes the following inventions.

[1] In a plant transformed with a gene encoding a heterologous protein, the plant comprises a step of expressing the heterologous protein under light having a photosynthetic effective quantum bundle density of less than ^{30 μmol · m −2} · s ^-1. A method for producing a heterologous protein.
[2] The method according to [1], wherein the expression step is carried out under a photoperiod in which a light period and a dark period are alternately repeated under light having a photosynthetic effective quantum flux density.
[3] The method according to [1] or [2], wherein the photosynthetic effective quantum flux density is 0.1 to 20 μmol · m ^-2 · s ^-1.
[4] The method according to any one of [1] to [3], wherein the photoperiod is adjusted using an irradiation device in a dark room.
[5] The method according to any one of [1] to [4], wherein the light period is 2 to 20 hours.
[6] The method according to any one of [1] to [5], wherein the plant belongs to the genus Nicotiana.
[7] A step of growing a plant before transformation under an alternating light cycle of light and dark periods with a photosynthetic effective quantum bundle density of 30 μmol · m ^-2 · s ^{-1 or more.} The method according to any one of [1] to [6], further comprising.
[8] The method according to any one of [1] to [7], wherein the light period before transformation is longer than the light period after transformation.
[9] The method according to any one of [1] to [8], wherein the transformation is carried out using the Agrobacterium method.
[10] The method according to any one of [1] to [9], wherein the plant is cultivated by soil or hydroponics.

According to the present invention, in the process of expressing a heterologous protein, the heterologous protein can be expressed with high efficiency in a plant by a simple means of reducing the photosynthetic effective quantum bundle density at the time of light irradiation. Therefore, the present invention greatly contributes to cost reduction in producing a heterologous protein.

Western blot analysis results of heterologous proteins of plants cultivated under each light intensity condition. From the left, each lane shows Comparative Examples 1, 2, and 3, and Examples 1, 2, and 3. Changes in the degree of growth of plants cultivated under each light intensity condition. The vertical axis shows the total leaf weight (g) per plant, and the horizontal axis shows the number of days elapsed after infection (dpi = day post inoculation). Changes in the degree of growth of plants cultivated under each light intensity condition. The vertical axis shows the dry matter weight (%) per strain, and the horizontal axis shows the number of days elapsed after infection (dpi = day post inoculation).

(Manufacturing method of heterologous protein)
<Expression process>
The method for producing a heterologous protein according to the present embodiment is a method for producing a heterologous protein in a plant transformed with a gene encoding the heterologous protein under light having a photosynthetic effective quantum bundle density of less than ^{30 μmol · m −2} · s ^-1. It includes a step of expressing a protein (hereinafter, also simply referred to as an “expression step”). In the expression step, the light period (hereinafter, also referred to as “irradiation step”) and the dark period may be performed in a fixed cycle, or the heterologous protein may be expressed only in the light period. A photoperiod in which the light period and the dark period are alternately repeated is preferable, and a photoperiod in which the light period and the dark period are set at a predetermined time and repeated is more preferable.

The plant is not particularly limited as long as it can produce a heterologous protein, but a plant infected with Agrobacterium is preferable. Examples of such plants include dicotyledonous plants and monocotyledonous plants. Specifically, among dicotyledonous plants, the genus Tobacco, the genus Cynara such as potatoes and tomatoes are used as dicotyledonous plants, and the genus Cynara such as Luccola, the genus Cynara such as Mizuna and Karasina, and the genus Cynara are used as plants of the Abrana family. The genus is Chicory, Endive, etc., Cynara, Artichoke, etc., and Cynara, Alfalfa, etc., Dicotyledon, Ryokuto, etc., and Soybean, etc. Examples of plants include Cynara, Fudansou such as Tensai, Dicotyledons such as Chicotyledon and Basil, and Dicotyledons such as Chicotyledon. Examples of monocotyledonous plants include the genus Oryza, the genus Wheat, the genus Hordeum, and the genus Corn as plants of the family Gramineae, and the genus Wata and the like as plants of the Malvaceae family. Solanaceae plants are preferred, with the genus Nicotiana being more preferred.

The genus of tobacco includes tobacco (Nicotiana tabacum), Bensamiana tobacco (Nicotiana benthamiana), Hana tobacco (Nicotiana alata), Kidachi tobacco (Nicotiana glauca), Nagabana tobacco (Nicotiana lanico) anacia lanico ana nicaria Nicotiana rustica), Nicotiana silvestris (Nicotiana silvestris) and the like. Preferably, it is Bensamianaa tobacco.

Examples of the light source used in the irradiation step include artificial light, for example, light from a fluorescent lamp, LED, cold cathode fluorescent lamp (CCFL, HEFL), inorganic / organic EL, or the like. When expressing heterologous proteins indoors, especially in a dark room, the light source is preferably artificial light derived from an irradiation device such as a fluorescent lamp, an LED, or a cold cathode fluorescent lamp. As used herein, "darkroom" refers to a space that can block outdoor sunlight. LEDs are preferable because they have high light conversion efficiency and power saving as compared with incandescent lamps and HID lamps. LEDs are also preferable because they emit a small amount of heat rays that cause leaf burning damage to plants.

In the expression step, the photosynthetic effective quantum bundle density of the light irradiated to the plant is appropriately adjusted in the range of less than ^{30 μmol · m −2} · s ^-1. As used herein, "photosynthetic photon flux density" (hereinafter simply referred to as "PPFD") is a unit time included in the wavelength of 400 to 700 nm required for photosynthesis. , Means the number of photons per unit area. When sunlight is converted into a photosynthetic photon flux density, it is said to be ^{50 to 200 μmol · m −2} · s ^-1.

Preferably photosynthetically active quantum flux density in the developing process is 0.1μmol · m ^-2 · s ^-1 or more in a range of less than ^{30μmol · m -2 · s -1,} 0.1~20μmol · m -2 · The range of s ^-1 is more preferable, and the range of 0.1 to 10 μmol · m- ² · s ^-1 is even more preferable. A person skilled in the art can appropriately adjust the effective quantum flux density for photosynthesis using a photon flux density meter. For example, when the expression step is carried out indoors, particularly in a dark room, the photosynthetic effective quantum flux density can be adjusted within a desired range by adjusting the amount of light of the irradiation device. In this specification, when the photosynthetic effective quantum flux density is described as, for example, 0.1 to 20, it means that it is 0.1 or more and 20 or less.

To adjust the photosynthetic effective quantum bundle density, increase the photosynthetic effective quantum bundle density when you want to increase the plant growth amount, and lower it when you want to decrease the growth amount, while checking the plant growth amount at any stage. It can be done as appropriate. An example of the amount of growth is the total leaf weight.

Irradiation in the expression step is preferably carried out daily for a time equal to or less than the light period required for plant cultivation. For example, the irradiation period per day is appropriately adjusted in the range of 2 to 20 hours, preferably 4 to 16 hours according to the desired expression level of the heterologous protein, and is not the time obtained by integrating the intermittent irradiation times. It is preferably in the light period of continuous irradiation time.

The number of days required for producing a heterologous protein is not particularly limited because it varies depending on the type of heterologous protein, the desired expression level thereof, the type and growth state of the host plant, the cultivation method, and the like. Taking the case where Nicotiana benthamiana tobacco is cultivated in an artificial environment in a nutrient solution instead of soil cultivation, the number of days required for producing a heterologous protein is 1 day or more, for example 10 days or more, preferably 10 days or more. It is 15 days or more, more preferably 20 days or more, for example, about 35 days.

As used herein, "heterologous protein" means a protein derived from a heterologous organism other than a plant used as a host, and examples thereof include proteins used for medical purposes and other industries. As used herein, "heterologous protein" is understood as a concept that also includes peptides. A gene encoding a heterologous protein is called a heterologous gene. As the heterologous protein, a medical protein is preferable.

Examples of medical proteins include antibodies, enzymes, hormones, and more specifically, proteins, cytokines, monoclonal antibodies, fragments thereof, and the like used as vaccines.

Proteins used as vaccines preferably include constituent proteins of virus-like particles (VLPs). The constituent proteins of the VLP may be a single protein or may contain two or more proteins.

Other industrial proteins are proteins or peptides thereof used in foods, feeds, cosmetics, fibers, detergents, chemicals and the like, and examples thereof include enzymes and functional proteins. Specifically, such proteins include proteases, lipases, cellulases, amylases, peptidases, luciferases, lactamases, collagens, gelatins, lactoferrins, jellyfish green fluorescent proteins (GFPs) and the like.

The method for introducing a heterologous gene into a plant is not particularly limited. Those skilled in the art can appropriately select from known methods such as the Agrobacterium method, the particle gun method, and the whisker method, depending on the type of host plant and the like.

The Agrobacterium method is a method that utilizes a Ti plasmid contained in Agrobacterium tumefaciens. The Agrobacterium method includes an intermediate vector method and a binary vector method.

The method for producing a heterologous protein includes an arbitrary step other than the expression step, for example, a step of growing a plant before transformation (growth step), a step of transforming a plant (transformation step), and the like.

In the case of the Agrobacterium method, the transformation step is also called an infection step. In the infection step, the plant obtained in the growth step is infected with Agrobacterium having a polynucleotide encoding a heterologous protein by pressure cycle treatment. The pressure cycle treatment includes pressurization or depressurization treatment. When the pressure treatment is performed at a pressure higher than the atmospheric pressure (usually 1 atm = 101.325 kPa = about 0.1 MPa), at least 1.1 atm (112 kPa), 1.5 atm (152 kPa), 2 Atmospheric pressure (203 kPa), 2.5 atm (253 kPa), 3 atm (304 kPa), 4 atm (405 kPa) or 5 atm (507 kPa), or any intermediate value or any value exceeding this is processed. It is preferably in the range of 1.7 atm (172 kPa) to 10 atm (1013 kPa), and more preferably in the range of 4 atm (405 kPa) to 8 atm. When performing decompression treatment at a pressure lower than atmospheric pressure, the range is preferably 0.005 atm (0.5 kPa) to 0.3 atm (30 kPa), and 0.01 atm (1.0 kPa) to 0.1. The range of atmospheric pressure (10.1 kPa) is more preferable, and the range of 0.02 atmospheric pressure (2.0 kPa) to 0.06 atmospheric pressure (6.1 kPa) is even more preferable. If the depressurizing pressure is too high, the infiltration of the infectious solution will be insufficient, which is not preferable. On the other hand, if the pressure is too low, the liquid reaches the boiling point and evaporates significantly, the liquid decreases and infiltration becomes insufficient, and the manufacturing process and equipment become large-scale, which is also not preferable. Therefore, the time for these pressurization treatments or decompression treatments can be appropriately set according to the type of plant and the tissue to be treated, but is 10 seconds to 10 minutes, preferably 20 seconds to 5 minutes. More preferably, it is about 30 seconds to 3 minutes.

To overexpress a gene encoding a heterologous protein in a plant, the gene may be functionally linked downstream of an appropriate promoter and the resulting construct may be introduced into plant cells by the Agrobacterium method. Examples of the promoter include, but are not limited to, the 35S promoter of cauliflower mosaic virus (CaMV), the actin promoter of rice, the Elongation factor 1β promoter, and the ubiquitin promoter of maize.

When performing the Agrobacterium method, a binary vector or an intermediate vector containing T-DNA (transfer DNA) derived from Agrobacterium Ti plasmid or Ri plasmid can be used (Nucl. Acids Res. 12 (22): 8711-8721 (1984), plasmid, 7, 15-29 (1982)). Specific examples of the binary vector include a pBI-based vector (for example, pRiceFOX), a pPZP-based vector (Plant Molecular Biology 25 (6): 989-94. (1994)), a pCAMBIA-based vector (vector skeleton: pPZP vector), and a pSMA-based vector. Vectors (Plant Cell Reports 19: 448-453. (2000)) can be mentioned, but are not limited thereto.

In order to express the construct, a transient expression method using a vector as described above can be used (Journal of Virtual Methods, 105: 343-348 (2002)). Infection with Agrobacterium is preferably carried out under reduced pressure conditions, and more preferably the vacuum-based transient expression method described by Plant Science, 122, 1: 101-108 (1997)). .. By aggro inoculation or agroinfiltration, the construct-containing agrobacterium can be found in tissues such as leaves, parts of the above-ground structure of the plant (including stems, leaves and flowers), and other parts of the plant (stems, roots). , Flowers), or into the space between cells of the whole plant. After passing through the epidermis, Agrobacterium infects and transfers polynucleotides into cells.

<Growth process>
In the present specification, the step before the transformation step, that is, the step of sufficiently growing the plant until the transformation is carried out is particularly referred to as a growth step. The light irradiation conditions and other conditions in the growth step may be the same as or different from those in the expression step. However, from the viewpoint of shortening the growth period, the pre-transformed plants, 30μmol · m ^-2 · s ^-1 or more, for example 100~500μmol · m ^-2 · s ^-1, preferably 150~450μmol · m ^-2 -It is preferable to irradiate with light having a photosynthetic effective quantum flux density of s ^-1. The cycle of light irradiation may be composed of only the light period, or a dark period may be provided after the light period, and this may be repeated at a constant cycle. It is preferable to alternate between the light period and the dark period. The light period of the growth step is preferably longer than the light period of the expression step.

<Heterogeneous protein recovery process>
The heterologous protein expressed and accumulated in the plant is recovered from the plant by a known method and appropriately purified. Fractions containing the heterologous protein are obtained from the plant and the heterologous protein is purified by an appropriate method.

<Other processes>
The production of the heterologous protein may further include the steps of sowing, raising / temporary planting, transplanting, heart-stopping, harvesting, and drying. It is preferable to carry out the seedling raising step during the growing step. The period from sowing to the completion of expression of the heterologous protein may be collectively referred to as “cultivation step”.

Cultivation methods include forcing cultivation that utilizes the natural conditions of warm and cold regions, and open-field cultivation that cultivates crops in open-air cultivated land without using special equipment such as greenhouses and hotbeds, including controlled cultivation. Examples include greenhouse cultivation, factory cultivation, soil cultivation in which plants are cultivated in a plastic greenhouse made on the ground, and hydroponics such as hydroponic cultivation that does not use soil.

"Soil cultivation" generally means cultivation using the blessings of nature such as the sun, soil, and rainwater, but as used herein, it simply means cultivation using soil. Soil cultivation also includes hydroponic soil cultivation.

As used herein, "hydroponics" means hydroponics in which plants are cultivated using only water or nutrient solution that does not use soil. Hydroponics is said to supply sufficient oxygen to the roots by the flow of nutrient solution (liquid fertilizer) to promote nutrient absorption, stabilize the rhizosphere environment including the temperature and concentration of liquid fertilizer, and keep the cultivation environment constant. There are advantages.

In the cultivation process, transplantation may be carried out as many times as necessary. The time of transplantation is preferably 3 to 18 days after sowing (or after the previous transplantation).

It is also possible to increase the production efficiency of heterologous proteins by controlling the temperature, humidity, and carbon dioxide concentration in the cultivation process.

The temperature in the cultivation process is not particularly limited as much as possible for plant growth and production of heterologous proteins, and may be constant throughout the entire period, depending on the growth of the plant, day and night (light / dark period), and traits. The temperature may be changed before and after conversion. For example, the daytime temperature is preferably 2 ° C. or higher, more preferably 3 ° C. or higher, more preferably 4 ° C. or higher, still more preferably 5 ° C. or higher, and particularly preferably 8 ° C. or higher than the nighttime temperature. If this is the case, it is preferable because the expression efficiency of the heterologous protein is improved. The upper limit of the temperature difference between daytime and nighttime is preferably 30 ° C. or lower, more preferably 25 ° C. or lower, still more preferably 20 ° C. or lower, and in some cases preferably 15 ° C. or lower. Therefore, when the daytime temperature is set to 25 ° C., the nighttime temperature is preferably 5 to 24 ° C., more preferably 10 to 23 ° C. The temperature of daytime or nighttime does not have to be constant at all times and may be within the above range as an average value, but preferably within ± 5 ° C. within each period of daytime and nighttime. Is preferably adjusted within ± 2 ° C, more preferably within ± 1 ° C.

The carbon dioxide concentration in the cultivation process is not particularly limited as much as possible for plant growth and production of heterologous proteins, and may be constant throughout the entire period, depending on the growth of the plant, or day and night (light / dark period). , The carbon dioxide concentration may be changed before and after transformation. For example, the carbon dioxide concentration in the cultivation step is usually 300 ppm or more, preferably 400 ppm or more, and usually 1000 ppm or less, preferably 700 ppm or less. For example, the effect of the present invention may become remarkable by setting the carbon dioxide concentration in the daytime to be at least 100 ppm higher than the carbon dioxide concentration in the night time, preferably 500 ppm or more.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

1. 1. Preparation of plant biomass (1) Seeding The seedling raising pot (TS-10.5, Tokai Kasei Co., Ltd.) was filled with soil so that it could be fully ground, and 2-3 Bensamiana benthamiana seeds were sown. After that, a plurality of the seedling raising pots were arranged in a seedling raising box for medium seedlings (AZ-032, Safety Kogyo Co., Ltd.), bottom irrigated for 30 minutes, and covered with agricultural solid material (Paopao 90, Mitsubishi Chemical Agridream Co., Ltd.). bottom. The agricultural solid material was removed on days 5-7.

(2) Growth The plant after sowing was grown under cooling at room temperature of 22 ° C. for 14 days in a 16-hour daytime / 8-hour nighttime light cycle. After the grown plants germinated, they were thinned out to one plant per pot. After thinning, at the age of 2 weeks, the seedling raising box for middle seedlings was installed in the cultivation box. At this time, 32 pots were arranged per cultivation shelf. The environmental conditions and solvent conditions were adjusted as follows.
<Environmental conditions>
-Temperature: 22 ° C
-Relative humidity: 30-80%
-CO ₂ concentration: 400-700 ppm
-Light source: High output 4950 lm, HF fluorescent lamp, FHF32EX-N-H (Toshiba Lighting & Technology Corporation)
<Nutrition conditions>
The nutrient solution supplied to the cultivation shelf is a mixture ratio of fertilizer A solution (OAT House No. 1 (OAT Agrio Co., Ltd.) 150 g / L) and fertilizer B solution (OAT House No. 2 (OAT Agrio Co., Ltd.) 100 g / L). A mixture was used so as to have a ratio of 1: 1, electrical conductivity (EC): 1.8 mS / cm, and pH (6.0).

2. Virus infiltration (infiltration)
(1) Culture of Agrobacterium The day before infiltration, 5 mL of LB liquid medium was placed in a test tube, and the final concentration of 20 mg / mL rifampicin solution was 50 μg / mL and that of 50 mg / mL kanamycin solution was 25 μg / mL. It was added so as to become. 100 μL of glycerol stock of Agrobacterium transformant was added, and the mixture was shake-cultured (230 rpm) at 28 ° C. for about 24 hours to obtain a culture solution.

(2) Preparation of infiltration buffer The culture solution of (1) above is collected, and Phosphate buffered saline (PBS) (10 × PBS Liquid Concentre, OmniPur®) is diluted 10-fold with purified water. It was diluted 10-fold with (stored in a refrigerator). With a simple optical density and optical density (OD) monitor (mini photo 518R, Titec Co., Ltd.), the OD value, which is the value of the light density measured under an absorbance of 660 mm, was measured with respect to the diluted solution, and the OD value was measured. It was confirmed that the value was 2.0 or more. To a wide-mouthed bucket, add 5.3 L of purified water, 0.5 M 2-morpholinoethanesulfonic acid (MES) (pH 5.4) 130 mL, 1 M sulfonyl ₄ 65 mL, and vacuum desiccator (300 G, 300 G, A wide-mouthed bucket was installed at AS ONE Co., Ltd.) and stirred. The final concentration was adjusted to 10 mM MES, 10 mM _{sulfonyl 4} , pH 5.4, and OD660 to 0.002, which was used as an infiltration buffer.

(3) Virus infiltration (infiltration)
Infiltration was performed on the plants aged 5 weeks by the following operations. The root of the plant was turned upward, and the above-ground part of the plant was immersed in the infiltration buffer of (2) above. A stick was passed to the mouth of the bucket to serve as a stopper so that the soil part would not be immersed in the infiltration buffer. The vacuum pump and the vacuum desiccator were connected by a hose, the vacuum pump was operated with the exhaust cock fully open, and suction was performed up to −0.092 MPaG. The exhaust cock was closed, the vacuum pump was removed from the vacuum desiccator, and the exhaust cock was opened at once. After recompression, the plant was removed from the infiltration buffer and the surface of the leaves was gently wiped dry. By setting the cooling chamber at 22 ° C. as a dark room, the effective photosynthetic quantum flux density in the room was adjusted to ^{5 μmol · m −2} · s ^-1. Plants were placed in this air-conditioning chamber and cultivated under a photoperiod in which a light period of 12 hours and a dark period of 12 hours were alternately repeated. The effective photosynthetic quantum flux density of the control was set to 450 μmol · m ^-2 · s ^-1 by installing 6 fluorescent lamps on the shelves in the cooling room and irradiating them. After cultivating each plant for a predetermined number of days after infection, the following measurements were carried out as appropriate.

3. 3. Measurement of expression level of heterologous protein (1) Measurement of growth state (i) Measurement of total leaf weight For infected leaves, harvested leaves stored at -80 ° C were designated as infected leaves. The stems of all plants were cut on the soil surface or near the planting board, the number of plants was counted, and then the weight of the plants was measured with a digital scale (total above-ground weight (g)). The true leaves and side buds of all plants were cut, and the weights of all leaves were measured with a digital scale (total leaf weight (g)).

(Ii) Measurement of dry matter weight ratio The weight of the cut sterilization roll was measured with an electronic balance (weight 1). Infected leaves for dry weight measurement were packed in a weighed sterile roll and sealed. After measuring the weight (weight 2) with an electronic balance, it was autoclaved at 121 ° C. for 20 minutes. After autoclaving, it was placed in a dry heat sterilizer (SI601, Yamato Scientific Co., Ltd.) set at 100 ° C. The next day, it was placed in a dry heat sterilizer (EYELA) set at 95 ° C. The next day, it was taken out from a dryer and allowed to stand at room temperature for about 10 minutes, and then the weight (weight 3) was measured with an electronic balance. The dry matter weight was calculated from the following formula.
Dry matter weight (%) = (weight 3-weight 1) / (weight 2-weight 1) x 100

(2) Quantitative analysis of the expression level of heterologous proteins (i) Preparation of crude extract The mortar and pestle are cooled in advance with liquid nitrogen, and the infected leaves stored at -80 ° C are added to the infected leaves until they become powdery. Grinded. The weight of the 1.5 mL tube (weight 4) was measured by measurement. The spatula was cooled with liquid nitrogen, an appropriate amount of the ground product was placed in a 1.5 mL tube, and the weight (weight 5) was measured. Add an extraction buffer (20 mM Tris-HCI to which Triton X-100 is added to a final concentration of 1 v / v% and store at room temperature), which is 3 times the weight of the ground product (weight 5-weight 4), and refrigerate (20 mM Tris-HCI). After allowing to stand at about 4 ° C. for 30 minutes, the mixture was centrifuged in a desktop centrifuge (Tomy Seiko Co., Ltd., Multi Spin) for 5 minutes. 100 μL of the supernatant was placed in a 1.5 mL tube and stored at −30 ° C. 75 μL of the supernatant was placed in a 1.5 mL tube, 25 μL of a sample buffer (20 μL of 1 M DTT mixed with 180 uL of 4 × Laemmli Sample buffer) was added, and the mixture was heated in a heat block set at 95 ° C. for 5 minutes.

(Ii) Polyacrylamide gel electrophoresis and Western blotting assay Polyacrylamide gel electrophoresis and Western blotting assay were performed according to a conventional method.

[Examples, comparative examples]
The irradiation step was performed under the conditions shown in Table 1 below.

The results are shown in FIGS.
From the results of FIG. 1, after the 11th day after Agrobacterium infection, the band of Example 3 (lane 6) was clearly darker than the band of Comparative Example 3 (lane 3). I understand. In addition, as can be seen from the relationship between the total leaf weight and the dry matter weight as shown in FIG. 2, the plants cultivated by irradiating with PPFD light of ^{5 μmol · m −2} · s ^{-1 as compared with the comparative example (implementation).} Examples 1 to 3), it can be seen that the total leaf weight decreases with each passing day, but the dry matter weight increases. This result suggests that the production of heterologous proteins became active within a predetermined low light intensity range.

Claims (10)

In plants transformed with a gene encoding a heterologous protein, the heterologous protein in the plant comprises the step of expressing the heterologous protein under light with a photosynthetic effective quantum bundle density of less than ^{30 μmol · m -2} · s- ^1. How to make. The method according to claim 1, wherein the expression step is carried out under a photoperiod in which a light period and a dark period are alternately repeated under light having a photosynthetic effective quantum flux density. The method according to claim 1 or 2, wherein the photosynthetic effective quantum flux density is 0.1 to 20 μmol · m ⁻² · s ^-1. The method according to any one of claims 1 to 3, wherein the photoperiod is adjusted using an irradiation device in a dark room. The method according to any one of claims 1 to 4, wherein the light period is 2 to 20 hours. The method according to any one of claims 1 to 5, wherein the plant belongs to the genus Nicotiana. It further includes a step of growing the untransformed plant under alternating light cycles of light and dark periods with a photosynthetic effective quantum bundle density of 30 μmol · m ^-2 · s ^{-1 or more.} The method according to any one of claims 1 to 6. The method according to any one of claims 1 to 7, wherein the light period before transformation is longer than the light period after transformation. The method according to any one of claims 1 to 8, wherein the transformation is carried out using the Agrobacterium method. The method according to any one of claims 1 to 9, wherein the plant is cultivated by soil cultivation or hydroponics.

Images