Method for producing transgenic silkworm using dormant egg

Abstract

To develop and provide a technique for producing a genetically modified silkworm of a practical line in a short period of time with less labor.SOLUTION: The present invention provides a method for producing a transgenic silkworm, comprising a step of introducing a target nucleic acid into an egg and then a step of breaking dormancy of a dormant egg by non-thermal equilibrium plasma treatment.

Description

[Technical Field]

[0001] The present invention relates to a method for producing transgenic silkworms using diapause eggs. [Background technology]

[0002] In recent years, silkworms (Bombyx mori) have emerged as a suitable host organism for mass production of proteins. Silkworms are insects that have long been used industrially to produce silk. The cocoons made by the larvae can be used to produce a large amount of silk thread in a short period of time. Genetic engineering technology has made it possible to mass-produce useful proteins other than silk, Some of these drugs have been marketed as animal testing drugs, and the development of drug substance production technology for human pharmaceuticals is progressing. It is being done.

[0003] To create transgenic silkworms, the desired gene is injected into eggs using a transposon. The microinjection method is used to inject silkworm eggs into the ovipositor (Non-Patent Document 1). Fertilization occurs within two hours after fertilization, and then a naked nucleus without a cell membrane, called a syncytium, separates. During this period, specifically between 2 and 8 hours after collection of the fertilized eggs, The desired gene must be introduced into the egg during this period. This is because the efficiency of transformants emergence is significantly reduced (Non-Patent Document 2). To produce transgenic silkworms using this method, the eggs do not stop developing, and specific It is important to introduce the target gene within this time frame.

[0004] By the way, most silkworms are univoltine, meaning that adults usually emerge once a year. Silkworms of this strain lay diapause eggs, which then enter a state of diapause. Since the gene is maintained even after injection, transformants can be obtained even if the gene is introduced into diapause eggs. Therefore, the microinjection method is not suitable for non-diapause eggs, which do not become diapause. However, many of the non-diapause strains that lay non-diapause eggs have poor silk productivity. Therefore, conventionally, the strains obtained from non-dormant strains were experimental strains and unsuitable for industrial use. After performing genetic recombination using non-diapause eggs by microinjection, the obtained Crossbreeding transgenic silkworms with a diapause strain to develop a practical strain suitable for industrial use. There was a need.

[0005] In order to solve the above problem, dormant eggs that do not enter a diapause state are extracted from practical dormant strains. or non-diapause eggs (in this specification, these are often collectively referred to as "non-diapause eggs, etc.") For example, methods have been developed to obtain diapause eggs by external physical or chemical stimulation. Maintain embryonic development by avoiding the transition to diapause, or awaken dormant eggs from diapause Breaking diapause by inducing embryonic development and treating parent silkworms of diapause strains to produce non-diapause eggs There is a method for spawning non-diapause eggs.

[0006] Non-patent document 3 describes the dormancy-breaking method, which is an acid treatment, which does not enter the dormancy state. A method for microinjection into dormant eggs has been disclosed. However, this method does not involve the use of acid. In addition to the chemical stimulation by microinjection, a physical stimulation by microinjection is given to the eggs. As a result, the hatching rate subsequently dropped significantly, reaching only 3.4-4.6%.

[0007] In the low-temperature dark incubation method, parent eggs are incubated under low-temperature dark conditions, and the hatched parent silkworms lay non-diapause eggs. However, the low-temperature dark incubation method is a method There is a significant difference in the number of non-diapause eggs laid by the strain of the oyster, and sometimes no non-diapause eggs are laid at all. Furthermore, there is a problem that the non-diapausing eggs obtained by this method cannot be used for the purpose of microorganism breeding (Patent Document 1). When injection is used, there is the same problem as with the acid immersion method in that the hatching rate drops dramatically. It was.

[0008] As mentioned above, although it is possible to prepare non-diapause eggs from diapause strain silkworms using conventional methods, There was a problem that the hatching rate significantly decreased after the microinjection treatment. Since genetic modification only occurs in some of the microinjected eggs, If the hatching rate after microinjection is low, the production of transgenic silkworms will inevitably be hindered. Therefore, in order to efficiently produce transgenic silkworms, We have developed a technology that can not only obtain non-diapause eggs but also maintain the hatch rate after microinjection. It was needed.

[0009] In order to solve the above problem, Patent Document 2 discloses a method for producing a silkworm diapause strain by using a diapause hormone antibody. This method involves microinjecting non-diapausing eggs obtained from the It has been found that if the larvae are microinjected, the hatching rate after microinjection will increase regardless of the silkworm strain. However, this method also requires the preparation of antibodies and two injections. However, it was not easy in terms of the amount of work required.

[0010] Non-patent document 6 describes the use of microinjection in eggs that have been treated with corona discharge to break diapause. This method allows processing under a gas atmosphere and is The process of breaking diapause by ion discharge can be completed in a short time. Furthermore, a high hatching rate of over 95% can be achieved. This is an excellent method, but there are problems with this method. As such, microinjection must be performed into eggs 2 to 8 hours after oviposition, when embryonic development is progressing. If the eggs are laid, the efficiency of the subsequent emergence of transformants will be significantly reduced. It is necessary to break the dormancy process within the limited time before injection. In general corona discharge devices, the electric field discharge range is very localized, and it is difficult to apply it to many eggs at once. Therefore, the number of eggs that can break diapause within the time limit is limited, resulting in a decrease in the number of genetically modified eggs. The problem of low silkworm production efficiency remains.

[0011] Furthermore, the transgenic silkworms obtained by the conventional microinjection method Even if the target gene introduced is the same, the genome composition of each transformant will differ. This is because the transferase activity derived from transposons is utilized to create gene sequences. When recombination is performed, the insertion of the target gene occurs at a random position on the genome. This is because strains are maintained as genetically heterogeneous populations through interbreeding.

[0012] Additionally, silkworms are generally stored in the egg state, which can last up to a year. Therefore, in order to maintain the lineage, they must be raised and bred every year to obtain eggs for the next generation. Repeated breeding over a long period of time can ensure genetic stability even within the same lineage. It gets difficult.

[0013] Therefore, in conventional techniques, the expression level and quality of the target gene vary depending on the lineage of the transformant, The amount of protein produced varies from individual to individual, resulting in inconsistent quality and production. This is because raw materials for human medicines, which require high quality, are genetically modified. This is a particularly big problem when manufacturing in mass production systems using ECO.

[0014] As mentioned above, the production of transgenic silkworms and the maintenance of their lineage require a great deal of time and effort. Furthermore, even if time and effort are spent on the genetic modification, Silkworms have the problem of genetic instability.

[0015] Therefore, we are aiming to develop a method to produce genetically modified silkworms in a short period of time with minimal effort. New technologies are needed to reduce the costs of gene replication. There is also a need for techniques to maintain genetic stability of transgenic silkworms. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] Japanese Patent Application Laid-Open No. 2003-88274 [Patent Document 2] Patent No. 6765803 [Non-patent literature]

[0017] [Non-Patent Document 1] Tamura T., et al. (2000) Nat. Biotechnol.18, 81-84. [Non-patent document 2] Tamura, T. (2007) Development and Use of Methods for Producing Transgenic Silkworms (Series: 21st Century Agriculture: Genetic Engineering Research of Animals and Microorganisms, edited by the Agricultural Society of Japan), pp. 57-76. Yokendo, Tokyo. [Non-patent document 3] Zhao AC, et al., 2012, Insect Science, 19: 172-182 [Non-patent document 4] Eiichi Ozegawa et al., 2000, Japanese Journal of Sericulture, 69(6): 369-375 [Non-patent document 5] Shimizu, Isamu, 1991, Odokon, 35: 81-91 [Non-patent document 6] Zhang Yu-Li, et al., 2022, Frontiers in Bioengineering and Biotechnology, 10, Article853543 Summary of the Invention [Problem to be solved by the invention]

[0018] Genetically modified silkworms suitable for industrial use can be produced in a relatively short period of time with little effort. To develop and provide the technology to produce transgenic silkworms, and to genetically stabilize the produced transgenic silkworms. The goal is to develop and provide the technology to maintain the quality of transgenic silkworms. The objective is to reduce the costs required for production and system maintenance. [Means for solving the problem]

[0019] In order to solve the above problems, the present inventors have conducted extensive research and have developed a method for producing diapause eggs by microinjecting them with ATP. This is different from the conventional method, which involves breaking dormancy by performing a non-thermal plasma treatment after the treatment. We came up with the opposite method. In the technical field, there are cases where transgenic silkworms are produced using diapause eggs. In this case, it is common technical knowledge to perform microinjection after breaking dormancy. There is no idea of ​​breaking the diapause after injection. However, with this method, After 2 to 8 hours, the eggs are microinjected and then treated to break diapause. This allows for the processing of a large number of eggs without the time constraints of conventional methods. In fact, the hatching rate of eggs obtained by this method was higher than that of eggs that had undergone nucleic acid transfer after diapause breaking. The results also revealed that the positive rate of transformants was more than double that of the control group.

[0020] The present invention is based on this new finding and provides the following. (1) A method for producing transgenic silkworms, in which fertilized eggs are collected from individuals of a dormant silkworm strain. a step of collecting fertilized eggs by microinjection of a nucleic acid of interest into the fertilized eggs; a nucleic acid introduction step for introducing a nucleic acid into the fertilized egg, a non-thermal equilibrium plasma treatment step for breaking the diapause of the fertilized egg, and A recombination step of selecting genetically modified silkworms from the next generation silkworms hatched from the fertilized eggs. The production method further comprises a step of selecting a fly. (2) The method according to (1), wherein the step of breaking dormancy is carried out after the step of introducing nucleic acid. (3) The method according to (1) or (2), wherein the nucleic acid introduction step is carried out within 8 hours after collection of the fertilized egg. Manufacturing method. (4) A method for producing transgenic silkworms, comprising the steps of: (1) culturing unfertilized eggs from individuals of a parthenogenetic silkworm line; an unfertilized egg collection step of collecting the above-mentioned unfertilized eggs, and inducing parthenogenesis in the unfertilized eggs collected in the unfertilized egg collection step. a parthenogenesis induction step of injecting the desired cells into the unfertilized eggs by microinjection; a nucleic acid introduction step of introducing the nucleic acid of the above-mentioned unfertilized egg into the oocyte, and a step of breaking the diapause of the unfertilized egg by non-thermal equilibrium plasma treatment. and a step of breaking the diapause by carrying out the step of producing a genetically modified silkworm from the next generation silkworms hatched from the unfertilized eggs. The method for producing the recombinant vector further comprises a step of selecting a recombinant. (5) The method according to (4), wherein the step of breaking dormancy is carried out after the step of introducing nucleic acid. (6) The parthenogenesis induction treatment is a high temperature treatment in which unfertilized eggs are exposed to 45°C to 50°C for 15 to 20 minutes. The method for producing the compound according to (4) or (5), (7) Any of (4) to (6), wherein the nucleic acid introduction step is carried out within 24 hours after the parthenogenetic induction treatment. The method for producing the same is described above. (8) The non-thermal equilibrium plasma is generated by corona discharge or dielectric barrier discharge. The method for producing the present invention according to any one of (1) to (7), wherein the (9) The method according to (8), wherein the voltage in the corona discharge is 1 kV to 50 kV. (10) Any of (1) to (9), wherein a marker gene is further introduced in the nucleic acid introduction step. The preparation method described. (11) The selection in the recombinant selection step is based on the expression of the marker gene. The production method described in [Effects of the Invention]

[0021] According to the method for producing transgenic silkworms of the present invention, it is possible to produce practical strains of transgenic silkworms suitable for industrial use. It is possible to produce transgenic silkworms or genetically modified cloned silkworms in a relatively short period of time with little effort. We can provide the technology to manufacture such products. [Brief explanation of the drawings]

[0022] [Figure 1] This is a flow diagram for producing genetically modified silkworms using fertilized eggs of the present invention. This diagram illustrates a flow in which a fertilized egg collection step (S0101) is followed by a nucleic acid introduction step (S0102) and then a diapause-breaking step (S0103). However, the order of the nucleic acid introduction step (S0102) and the diapause-breaking step (S0103) can be arbitrary, and the nucleic acid introduction step (S0102) may be performed after the diapause-breaking step (S0103). [Figure 2]This is a flow diagram for producing genetically modified silkworms using unfertilized eggs of the present invention. This diagram illustrates a flow in which the parthenogenesis induction step (S0106) is followed by the nucleic acid introduction step (S0102), and then the diapause-breaking step (S0103). However, the nucleic acid introduction step (S0102) and the diapause-breaking step (S0103) can be performed in any order, and the nucleic acid introduction step (S0102) may be performed after the diapause-breaking step (S0103). [Figure 3] 1 is a conceptual diagram of a discharge device used in non-thermal equilibrium plasma treatment generated by corona discharge in Example 1. FIG. [Figure 4] FIG. 1 is a conceptual diagram of a plasma device used for non-thermal equilibrium plasma processing generated by dielectric barrier discharge in Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0023] 1. Method for producing genetically modified silkworms Overview The present invention relates to a method for producing transgenic silkworms. In the method of the present invention, the following is added to diapause eggs: Prevent diapause in dormant eggs by introducing the target nucleic acid and breaking diapause through non-thermal equilibrium plasma treatment According to the present invention, the method is characterized in that the microorganism is activated or awakened from a dormant state. In a relatively short period of time, we will be able to produce practical strains of genetically modified silkworms or genetically modified cloned silkworms. And it can be produced with little effort.

[0024] 1-2.Definition As used herein, the following terms are defined. "Genetic modification" refers to the artificial modification of the natural genetic information of a host organism. The modification of genetic information referred to here includes addition, deletion, substitution, etc. of genetic information. Artificial modification of information can be achieved by existing genetic engineering techniques, such as plasmids. A method of adding genetic information that is not possessed by the host organism using vectors or transposons such as or a method for destroying the genetic information possessed by the host organism. So, genome editing technology, which modifies the genomic information of a host organism through genome editing, is also considered a genetic engineering technology in the broad sense. Included as a child recombination.

[0025] "Genetically modified silkworms" (transgenic silkworms) are silkworms that are produced using genetic engineering technology. The term "genetically modified silkworms" as used herein refers to the genetically modified silkworms or their progeny produced by the above method. The method is not limited to the above, but is obtained by introducing foreign DNA into silkworm eggs by microinjection. It refers to a genetically modified organism that can be produced.

[0026] "Clone silkworms" are genetically identical silkworms that have the same genome composition and gene composition. It refers to silkworm individuals that make up a homogeneous population. Generally, they are derived from parthenogenetic silkworms, as described below. The resulting unfertilized eggs are then treated to induce parthenogenesis, resulting in a clonal population of silkworms. Equivalent to an equalizer.

[0027] As used herein, the term "genetically modified cloned silkworms" refers to genetically modified cloned silkworms. In the case of genetically modified cloned silkworms, the foreign DNA introduced is also cloned. It is identical among individuals in the population. Therefore, each individual of the transgenic cloned silkworm In principle, the quality and expression level of the protein encoded by the foreign DNA produced in the

[0028] As used herein, a "lineage" refers to individuals that share specific genetic traits within the same species. A population of individuals, roughly the same concept as a "strain." A mutant with a specific gene mutation In this specification, the term "line" also includes groups and varieties that share common morphology or characteristics. Therefore, unless otherwise specified, the biological species in this specification The term "lineage" refers to "silkworm lineage," and silkworm individuals belonging to each lineage are called "lineage silkworms." For example, the diapause strain described below is a diapause strain of silkworm, i.e., "diapause silkworm" Silkworms that belong to this dormant silkworm lineage are called "dormant silkworm lineages." When focusing on different genetic traits, one individual may belong to multiple lineages. For example, the dormant single Parthenogenetic lines have two genetic traits: dormancy and parthenogenesis. belongs to the dormant lineage, and if we focus on parthenogenesis, it belongs to the parthenogenetic lineage.

[0029] In this specification, "dormancy" refers to a state in which an organism ceases to develop or grow at a specific time in its life cycle. or to temporarily stop activities and enter a dormant state.

[0030] In this specification, "avoiding dormancy" means that a cell that originally had the property of being able to enter a dormant state Diapausing eggs continue to develop without being put into a dormant state by artificial treatment such as breaking the diapause, which will be described later. To continue or be able to continue.

[0031] In this specification, "awakening from diapause" refers to the process by which diapause eggs enter a diapause state and are awakened as described below. This refers to the process of awakening a plant from its dormant state and resuming development through artificial processes such as breaking dormancy. cormorant.

[0032] As used herein, the term "diapause (silkworm) strain" refers to a strain that is related to a specific genetic trait called egg diapause. Silkworms are divided into univoltine, bivoltine and multivoltine strains. Among these, many of the univoltine lineages become diapause silkworm lineages that lay diapause eggs. "Univoltine" refers to a strain in which adults emerge once a year when reared under natural conditions. A "bivoltine lineage" is a lineage in which adults emerge twice a year, and a "multivoltine lineage" is a lineage in which adults emerge twice a year. This is a lineage in which adults emerge multiple times a year. Generally, the lineage has an ancestral lineage in temperate regions where winter exists. Most of the living lineages are univoltine. However, even diapause silkworm lineages can be classified as non-diapause silkworm lineages, which will be described later. Diapause egg-laying treatment may cause the silkworm to lay non-diapause eggs. Examples include, but are not limited to, Daizo, Nihon No. 137, Shi No. 146, Nihon No. 603, Nihon No. 604, and Chubu No. No. 604, No. 605, No. 514, No. 515, No. 9.0, Sun 9.0, Shunrei, Kanetsuki, Hitachi, Nishiki, Sun 50 These include No. 2, China No. 146, China No. 122, Special China No. 2, Europe No. 7, Special Europe No. 4, and Jiang China.

[0033] As used herein, the term "diapause eggs" refers to eggs obtained from diapause strain silkworms, The term "eggs" refers to eggs that have the property of transitioning to a diapause state, regardless of whether they actually transition to a diapause state. Usually, diapause eggs are produced by allowing female adult silkworms of diapause strains to lay eggs under general rearing conditions. Diapause eggs are eggs that stop embryonic development at the embryonic stage about two days after spawning, and are low in They enter a dormant state with temperature tolerance (Yaginuma Toshinobu, 2015, Silkworm and Insect Biotech, 84: 100) This is thought to be one of the life cycle control phenomena based on environmental responses acquired by silkworms for overwintering. Therefore, in general, derived lineages with ancestral lineages in temperate regions where winter is present, Many of them are dormant strains.

[0034] As used herein, the term "non-diapause (silkworm) strain" refers to a non-diapause silkworm strain that does not enter a diapause state during the egg stage. It refers to a population of individuals that lay eggs. Generally, they have ancestral lineages in subtropical or tropical regions where there is no winter. Many of the derived strains are polyvoltine silkworm strains that repeat multiple generations per year, and these are non-diapause strains. It is a non-diapause silkworm strain that lays eggs. Specific examples of non-diapause silkworm strains include, but are not limited to: However, some examples include Mysore, Nistari, Pure Mysore, Annan, and Ringetsu.

[0035] In this specification, "breaking diapause" refers to the process of breaking diapause eggs by subjecting them to various artificial treatments. This means preventing the transition of eggs to a diapause state or awakening diapause eggs that are in a diapause state. As mentioned above, the diapause eggs laid by the diapause strain silkworms usually undergo early fertilization around the second day after laying. Embryonic development stops at the embryonic stage and enters a state of diapause. However, by breaking diapause, the diapause eggs remain in a state of diapause. In addition, dormant eggs have already entered a dormant state and stopped embryonic development. If the cells have been in a dormant state, they will be awakened and stopped by breaking the dormancy through non-equilibrium plasma treatment, etc. The embryonic development that had been stopped begins to resume.

[0036] As used herein, the term "non-diapause eggs" refers to silkworm eggs that do not have the property of entering a diapause state. Non-diapause eggs obtained from multivoltine strains, non-diapause eggs obtained from diapause silkworm strains by non-diapause egg-laying treatment Non-diapause eggs are exemplified by non-diapause eggs obtained by the method described in Japanese Patent No. 6765803. The diapause eggs that have escaped diapause by breaking the diapause tend to return to a diapause state, so in this specification they are not Distinguish from diapausing eggs.

[0037] As used herein, the term "non-diapause egg-laying treatment" refers to the treatment of diapause silkworm strains that normally lay diapause eggs. It refers to the process of making individuals lay non-diapause eggs by subjecting them to a specific treatment. As a specific example of the treatment, low-temperature dark blueing treatment described in the method disclosed in JP 2017-085958 A is used. Examples include:

[0038] As used herein, the term "parthenogenetic (silkworm) line" refers to a line in which parthenogenesis is induced with high efficiency. In parthenogenetic silkworm strains, the unfertilized eggs are stimulated physically or chemically to produce the silkworms. Parthenogenesis is induced. Generally, most silkworm strains that are capable of parthenogenesis are diapause-prone. Therefore, in this specification, parthenogenetic silkworm strains are referred to as "diapause parthenogenetic (C. Although not limited to parthenogenetic silkworm strains, for example, PK1 strain, P 14 lines, and a hybrid of Camboge x Nihon 106.

[0039] A "marker gene" is a base sequence that encodes a marker protein, also known as a selection marker. It is a polynucleotide consisting of:

[0040] "Tagged protein" refers to a protein that confers new traits not present in the host silkworm by expressing a tagged gene. The type of labeled protein can be determined by a method known in the art. There are no particular limitations as long as the activity can be detected. These are less invasive labeling proteins that can be used in vivo. For example, fluorescent proteins, pigment-synthesizing proteins, Examples include photoproteins, exocrine proteins, and proteins that control external morphology. Fluorescent proteins, pigment-synthesizing proteins, luminescent proteins, and exocrine proteins are transformed The trait can be visually detected under certain conditions without changing the external morphology of the recombinant. It is particularly useful because it is very low in invasiveness to transformants and it is easy to distinguish and select transformants. It is suitable for.

[0041] As used herein, a "fluorescent protein" refers to a protein that emits light of a specific wavelength when irradiated with excitation light of a specific wavelength. It may be either a natural or non-natural type. The excitation wavelength and the fluorescence wavelength are not particularly limited. Specifically, for example, CFP, RFP, DsRed (3xP3- DsRed, YFP, PE, PerCP, APC, GFP (including derivatives such as EGFP and 3xP3-EGFP) (including)

[0042] As used herein, the term "pigment synthesis protein" refers to a protein involved in the biosynthesis of a pigment. The term "pigment" used here refers to a substance that can give a pigment to a transformant. It is a low molecular weight compound or peptide that can be used for the external color of the individual, regardless of its type. For example, melanin pigments (including dopamine melanin), omokro Examples of suitable dyes include rhodium-based dyes and pteridine-based dyes.

[0043] As used herein, the term "photoprotein" refers to a protein that emits light without the need for excitation light. It refers to a substrate protein capable of luminescence or an enzyme that catalyzes the luminescence of the substrate protein. For example, The substrate protein is luciferin or aequorin, and the enzyme is luciferase. It can be obtained.

[0044] As used herein, the term "exocrine protein" refers to a protein that is secreted outside the cell or outside the body. These include exocrine enzymes, as well as fibrous proteins such as fibroin and sericin. Exocrine enzymes contribute to the degradation or inactivation of drugs such as blasticidin, and In addition to enzymes that confer drug resistance, digestive enzymes also fall into this category.

[0045] In this specification, the term "non-thermal equilibrium plasma" is also referred to as non-equilibrium plasma or low-temperature plasma. It refers to a relatively low-temperature plasma generated by gas discharge under atmospheric pressure or low pressure. The thermal equilibrium plasma generated by the gas discharge below is in a state where the electron, ion, and atomic temperatures are in thermal equilibrium. In contrast, in non-thermal plasma, only the electron temperature is high, while the ions and atoms The temperature is about room temperature. There are no restrictions on the method of generating non-thermal plasma. For example, needle-to-plane current It is generated by corona discharge, which applies a high voltage to an electrode system that forms a polar or other significantly non-uniform electric field. The plasma generated by the dielectric barrier discharge is generated by applying an AC voltage between electrodes with a dielectric material. Therefore, plasma is generated.

[0046] Since the target of non-thermal plasma treatment is silkworm eggs, the temperature of the generated plasma is Non-limiting examples include 35°C to 80°C, 40°C to 75°C, 45°C to 70°C, 50°C to 65°C, and 55°C to 60°C. Preferably, the plasma is

[0047] In this specification, "corona discharge" refers to a discharge generated by a needle-to-plane electrode or other device that generates a significantly non-uniform electric field. When a voltage is applied to a system of electrodes, a local breakdown occurs in the gas between the electrodes, and a current flows. This refers to a gas discharge phenomenon that occurs when

[0048] In this specification, "dielectric barrier discharge" refers to a discharge that occurs when a voltage is applied between electrodes with a dielectric. The term "voltage" refers to a gas discharge phenomenon that occurs in the gas between electrodes. There are no limitations on the "voltage" used here. For example, commercial AC power sources, high frequency or microwave power sources, pulse power sources, and other power sources. Includes voltage from the source.

[0049] 1-3. Manufacturing method The flow of the method for producing transgenic silkworms of the present invention is shown in Figure 1. As shown in this figure, The method of the present invention includes a fertilized egg collection step (S0101), a nucleic acid introduction step (S0102), a dormancy breaking step ( S0103), recombinant selection process (S0104), unfertilized egg collection process (S0105), and parthenogenesis induction Includes step (S0106).

[0050] In the method for producing transgenic silkworms of the present invention, transgenic silkworms are produced using fertilized eggs. These methods can be broadly divided into two types: the production method using transgenic silkworms and the method using unfertilized eggs.

[0051] Among the above steps, the fertilized egg collection step (S0101), the nucleic acid introduction step (S0102), and the dormancy breaking step ( The four steps of the recombinant selection process (S0103), and recombinant selection process (S0104) are carried out by using fertilized eggs. This is a process used in the production method of the nucleic acid introduction process (S0102), the dormancy breaking process (S0103), ), recombinant selection step (S0104), unfertilized egg collection step (S0105), and parthenogenesis induction step ( The five steps in S0106) are used in the method for producing genetically modified silkworms using unfertilized eggs. In addition, there are methods for producing genetically modified silkworms using fertilized eggs and methods for producing genetically modified silkworms using unfertilized eggs. In all of the methods for producing transgenic silkworms, the nucleic acid introduction process (S0102) and the diapause breaking process are The order of steps (S0103) does not matter, and either step may be performed first. Each step in the method for producing genetically modified silkworms of the present invention will be specifically described below. do.

[0052] 1-3-1. Fertilized egg collection process The "fertilized egg collection process" (S0101) is a method for producing genetically modified silkworms using fertilized eggs. This is a selection process for collecting dormant fertilized eggs to be used in the nucleic acid transfer process (S0102). This process involves the use of fertilized eggs of G0 silkworms (nucleic acid-introduced generation) and the G1 silkworms (nucleic acid-introduced generation). This is done to obtain fertilized eggs of the first generation (post-transgenic silkworms). These are not cloned silkworms but ordinary genetically modified silkworms. This is only done on fertilized eggs of the G1 silkworms (acid-introduced generation), and the unfertilized egg collection process (S010) described below is not performed on the G1 silkworms. In some cases, unfertilized eggs are obtained by 5). In this case, the transgenic silkworms obtained are The resulting silkworms are cloned.

[0053] The preparation of dormant fertilized eggs can be carried out by allowing mated female adults to lay eggs naturally according to a known egg collection method. For female individuals to be used for egg collection, it is advisable to use a diapausing silkworm strain that lays diapause eggs. In the case of diapause silkworm strains, eggs laid by adult females after mating are, in principle, diapause fertilized eggs. is.

[0054] There is no limitation on the method of egg collection. Usually, after mating, a mating female adult is given an egg-laying substrate, and the eggs are laid on the substrate. When mated female adults are given egg-laying mats, Spawning often begins within a few hours of application. After this step, dormant fertilized eggs can be obtained for use in the next nucleic acid transfer step.

[0055] 1-3-2. Nucleic acid introduction step The "nucleic acid introduction step" (S0102) is a step in which the target nucleic acid is introduced into the egg. The eggs used are fertilized or unfertilized eggs of G0 silkworms. In this process, nucleic acid is introduced into multiple eggs. It is desirable to do so.

[0056] In this process, the "nucleic acid of interest" refers to the nucleic acid that is used to produce the desired genetically modified silkworms. This is the nucleic acid to be introduced into silkworms. For example, DNA or RNA is applicable. More specific examples include: Examples include, but are not limited to, donor DNA / RNA, helper DNA / RNA, or guide DNA / RNA. Preferably, a gene of interest, an expression vector containing the gene, and a helper plasmid are used. When introducing a gene, the type of gene does not matter. For example, it may be a gene that encodes a protein or a functional nucleic acid (such as an RNAi molecule). In this case, the desired gene can be introduced into the target plant in the selection step described below. A marker gene may be introduced so that individuals into which the nucleic acid has been introduced can be easily selected. In this step, peptides (including proteins such as enzymes) and / or It is also possible to introduce low molecular weight compounds. In this case, the order of introduction with nucleic acids does not matter. The injection may be performed before the introduction of nucleic acid, after the introduction of nucleic acid, or simultaneously with the introduction of nucleic acid.

[0057] The gene to be introduced in this process can be of any biological origin. It may be derived from the host silkworm. Alternatively, genes derived from other organisms, such as humans, may be used. When producing antibodies using IgG, the foreign DNA contains the IgG antibody gene and promoter. The expression unit may include a gene expression regulatory region such as a nucleotide sequence or a terminator.

[0058] This step can be carried out by a method known in the art for introducing a foreign gene into silkworms. For example, if the expression vector contains the inverted terminal repeats (HTAs) of a transposon at both ends of the target DNA, ndler AM. et al., 1998, Proc. Natl. Acad. Sci. USA 95:7520-5) In the case of mido, the method of Tamura et al. (Tamura T. et al., 2000, Nature Biotechnology , 18, 81-84), and the method of Zhou et al. (Zhou W. et al., 2012, Insect Science, 19: 172-182) Specifically, water or a buffer solution can be used to adjust the concentration of the expression vector to an appropriate level. The injection is prepared by dissolving or diluting the drug in a solvent such as a fluoride. A helper plasmid containing DNA encoding a sposon transferase is added. An example of a per-plasmid is pHA3PIG.

[0059] The introduction of the target nucleic acid into the egg is generally, but not limited to, using a special injection device that utilizes air pressure. For example, the method described in Japanese Patent No. 1654050 or the method of Tamura et al. ., 2007, J Insect Biotechnol Sericol, 76: 155-159). The amount of acid is not particularly limited and may be determined appropriately depending on the type, properties, and purpose of the nucleic acid. It is 1nL to 5nL.

[0060] 1-3-3. Dormant breaking process The "dormancy breaking process" (S0103) is a process for breaking genetically modified corn sprouts using either fertilized or unfertilized eggs. This is an essential process that is also common to the method of producing egg yolks, and is the process of breaking the diapause of dormant eggs. Dormant eggs that have been artificially treated to break diapause, thereby avoiding diapause; or Diapausing eggs that have been awakened from a dormant state are prepared.

[0061] The resting eggs used in this step may be either fertilized or unfertilized. In the case of fertilized eggs, the eggs are from parthenogenetic silkworm strains and are collected through the unfertilized egg collection process (S0105) described below. The resting eggs used are those that have undergone the parthenogenesis induction process (S0106). The resting eggs used are those of G0 silkworms. Not only fertilized or unfertilized eggs, but also fertilized or unfertilized eggs of G1 silkworms are included.

[0062] As mentioned above, diapause eggs enter a diapause state after two days or more have passed since they were laid. If you use dormant eggs before they enter dormancy in the process, the time is 1 hour to 50 hours, 2 hours to 48 hours after laying. Between 3 and 42 hours, between 4 and 36 hours, between 6 and 30 hours, or between 8 and 24 hours It is desirable to carry out this procedure on eggs. In the case of using parthenogenetic silkworm eggs, the eggs can be extracted by dissection without going through egg laying. After parthenogenesis induction, unfertilized eggs can be collected directly from the ovaries. This process is carried out 144 hours (6 days) after parthenogenesis induction, because the cells will enter a dormant state after more than 144 hours. This test is carried out on eggs that are less than 126 hours old, 10 hours old, 90 hours old, or 72 hours old. It is desirable to do so.

[0063] On the other hand, if you use dormant eggs that have entered a dormant state in this process, they will be stored for 36 to 158 hours after laying. , 48 to 144 hours, 60 to 132 hours, 72 to 120 hours, or 84 to 96 hours, It is desirable to implement this measure.

[0064] The dormancy breaking treatment carried out in this step is a non-thermal equilibrium plasma treatment unless otherwise specified. As used herein, "non-thermal equilibrium plasma treatment" refers to a process in which diapause eggs of silkworms are treated under atmospheric or low pressure. This refers to exposing diapause eggs to non-thermal plasma. Non-thermal equilibrium plasma treatments include, but are not limited to, Plasma generated by corona discharge (corona discharge plasma) or dielectric barrier discharge This involves treatment with induced plasma (dielectric barrier discharge plasma).

[0065] The voltage in the corona discharge or dielectric barrier discharge is not limited to, but is 1 kV or more, 2 kV or more , 3kV or more, 4kV or more, 5kV or more, 6kV or more, 7kV or more, 8kV or more, 9kV or more, 10kV or more, 12kV or above, 13kV or above or 15kV or above, and 50kV or below, 45kV or below, 40kV or below, 35kV or below, 30kV or below The voltage is preferably 25 kV or less, or 20 kV or less. The range may be 0.45 mA, 0.15 mA to 0.4 mA, 0.2 mA to 0.35 mA, or 0.25 mA to 0.3 mA. The time is not limited, but can be set to 0.1 seconds to 20 minutes, 0.5 seconds to 18 minutes, 1 second to 15 minutes, 5 seconds to 14 minutes, or 10 seconds to 12 minutes. minutes, 30 seconds to 10 minutes, 45 seconds to 8 minutes, 1 minute to 6 minutes, 1 minute 30 seconds to 5 minutes, 2 minutes to 4 minutes, or 2 minutes 30 seconds to 3 minutes It's fine as long as it's within the range.

[0066] Non-thermal equilibrium plasma treatment can be performed on diapause eggs in the atmosphere. If necessary, the eggs may be fixed to a substrate such as paper with an adhesive.

[0067] This process prevents diapause of the diapause eggs or awakens them from the diapause state. The eggs may be incubated under suitable conditions, for example at 25°C, until they hatch.

[0068] 1-3-4. Recombinant selection process The "recombinant selection step" (S0104) is a step of selecting a recombinant strain by carrying out the nucleic acid introduction step (S0102) and the dormancy breaking step (S0 103), and then genetically modified silkworms are selected from the silkworms that have hatched from the eggs.

[0069] G0 silkworms that have undergone the diapause-breaking process (S0103) contain genetically modified somatic and germ cells. However, it is the genetic recombination that occurs in germ cells that is passed on to the next generation. Therefore, this process is carried out with G1 silkworms.

[0070] As shown in Figure 1, in the dormancy breaking step (S0103) after the nucleic acid introduction step (S0102), a non-thermal equilibrium process From the plasma-treated G0 silkworms of the diapause silkworm line, the fertilized eggs were collected (S0101). When the obtained G1 fertilized eggs are to be used in this step (S0104), the G1 fertilized eggs are The diapause breaking step (S0103) is carried out again. Silkworms obtained after this step are These are transgenic silkworms. Genes were introduced into the G1 fertilized eggs by microinjection. Therefore, the risk to the eggs can be reduced. The method is not limited to non-thermal equilibrium plasma treatment, and other known dormancy breaking methods can also be used. For example, DMSO treatment, acid treatment, centrifugation treatment, oxygen treatment, etc. may be mentioned.

[0071] As shown in Figure 2, in the dormancy breaking step (S0103) after the nucleic acid introduction step (S0102), The unfertilized egg collection process (S0105) was carried out from the plasma-treated parthenogenetic silkworm strain G0 silkworm. When using G1 unfertilized eggs obtained through this process, the G1 unfertilized eggs must be Then, a parthenogenesis induction step (S0106) is carried out, followed by a dormancy breaking step (S0103). This is performed on individuals that hatch after the breaking step (S0103). Each resulting silkworm will be an individual first generation (G1) transgenic cloned silkworm.

[0072] Furthermore, as an exceptional case, G0 chimeras can be generated from G0 fertilized eggs collected by crossbreeding, etc. In some cases, a female may have G1 unfertilized eggs that are likely to have a high rate of parthenogenesis. As shown in Figure 1, even if G0 silkworms were subjected to the fertilized egg collection step (S0101), they were not in diapause. After the breaking step (S0103), unfertilized eggs are collected from the G0 female individuals through an unfertilized egg collection step (S0105). The obtained G1 unfertilized eggs are subjected to a parthenogenesis induction step (S0106), and If necessary, this step can be carried out after the dormancy breaking step (S0103). In this case, each silkworm obtained after this step is a separate recombinant cloned silkworm No. 1. This is the generation (G1).

[0073] The selection of recombinants may be carried out by methods known in the art. Selection may be performed based on the traits brought about by the nucleic acid of interest introduced in step (S0102). For example, if the nucleic acid of interest is a foreign gene, the nucleic acid generated after the nucleic acid introduction step (S0102) Genomic DNA or mRNA is prepared from the silkworms and the presence or absence of the foreign gene is examined. Expression of the gene can be confirmed by PCR or the like.

[0074] Alternatively, if the expression vector used for nucleic acid transfer contains a marker gene, the marker gene The desired transgenic silkworms can be easily selected based on the expression of the offspring. The tagged protein produced by the expression of the offspring confers new traits that the host silkworm does not have. Based on the activity of this marker protein, it is possible to identify the trait of a cell that carries the introduced nucleic acid of interest. It is possible to easily distinguish transformants. Here, "based on activity" means that the activity can be detected. The activity detection is based on the results. The detection may be indirect or may be generated by the activity of a labeled protein such as a dye. Detection may be indirect via metabolites that react with the substance. detection (including reactive detection), physical detection (including behavioral detection), or sensory detection (including visual detection) by the detector. The detection may be by any of the senses (including detection by sense, touch, smell, hearing, or taste).

[0075] 1-3-5.Unfertilized egg collection process In principle, the "unfertilized egg collection process" (S0105) is carried out by collecting unfertilized eggs from individuals of parthenogenetic silkworm strains. This process involves collecting fertilized eggs. This process involves using a parthenogenetic silkworm strain as the parent strain to create a genetically modified silkworm. When producing recombinant cloned silkworms, this step is carried out together with the parthenogenesis induction step (S0106) described below. This process is used when producing genetically modified cloned silkworms from parthenogenetic silkworm strains. This is an essential process.

[0076] In addition, this process is carried out in such a manner that the G0 silkworms obtained through the fertilized egg collection process are not parthenogenetic silkworm strains. Even in this case, there are cases where G1 eggs obtained from G0 silkworms can be developed with a high probability by parthenogenesis induction treatment. In such cases, this may be carried out exceptionally.

[0077] Therefore, when this step and the next parthenogenesis induction step (S0106) are performed, the nucleic acid introduction step In principle, the resting eggs used in this process (S0102) are those obtained from individuals of parthenogenetic silkworm strains. However, as mentioned above, there are exceptions, such as the resting unfertilized eggs obtained from individuals that have undergone the fertilized egg collection process. The eggs may be dormant unfertilized eggs or non-dormant unfertilized eggs.

[0078] In this step, although there is no particular limitation, a state in which parthenogenesis is possible in vivo, i.e., a mature unfertilized egg It is preferable to obtain unfertilized eggs that have developed to the stage 1 from adult female individuals.

[0079] There are no limitations on the method of collecting unfertilized eggs. For example, they can be collected by dissection or by natural spawning. The method of collection includes:

[0080] Specifically, the method of collecting by dissection involves, for example, cutting the abdomen or tail of a female adult with a scalpel or dissection knife. After making an incision with dissection scissors, pressure is applied to the abdomen with the fingers to push the ovaries out through the incision. The clumped fallopian tubes can be loosened by immersing the removed ovaries in water. Next, place the ovaries on a fine stainless steel mesh or gauze and rub them with your fingers. This separates the oviduct tissue from the unfertilized eggs. Finally, the water is poured through a mesh to separate the oviduct tissue from the unfertilized eggs. The oviduct tissue floating on the surface is removed, and the unfertilized eggs remaining at the bottom are collected. This procedure can be repeated several times. This allows only unfertilized eggs to be collected.

[0081] Unfertilized eggs can also be collected by natural spawning, which involves allowing virgin females to lay eggs. For example, a mat is provided to the female individuals after hatching, and they are allowed to lay eggs on the mat. Specifically, the female individuals after emergence are kept at a low temperature of 0 to 10°C, preferably 5°C. After storing at that temperature for 1-2 days, or at most 1-7 days, the female silkworms are then transferred from the low temperature to room temperature ( The eggs were then transferred to a dark room (23-28°C) where they were provided with egg-laying mats and allowed to start laying eggs under dark conditions. It can be achieved.

[0082] 1-3-6. Parthenogenesis induction process The "parthenogenesis induction step" (S0106) is carried out by using the unfertilized eggs obtained in the unfertilized egg collection step (S0105). This is a step in which parthenogenesis induction treatment is performed on eggs. This step is performed in conjunction with the unfertilized egg collection step (S0105). This is a process carried out in pairs.

[0083] The parthenogenetic induction treatment may be carried out according to a method known in the art, and is not particularly limited. For example, Sugai et al. (Sugai et al., 1983, Japanese Journal of Sericulture, Vol. 52, No. 1: 51-56) and Hirokawa ( Hirokawa, M., 1990, Fukushima Silkworm Research Institute Bulletin, 24: 1-6), and Kosegawa et al. (Kosegawa, E., et al. 2012, J Insect Biotechnol Sericology, 81: 37-44). Generally, parthenogenesis is induced by applying physical or chemical stimuli to unfertilized eggs. Specifically, high temperature treatment can be mentioned. The unfertilized eggs collected in the above process are treated with hot water at 45℃-50℃ or 46℃-48℃ for 15-20 minutes. Or, expose for 16 to 18 minutes. After that, keep the temperature at 12 to 18°C, 13 to 17°C, 14 to 16°C, or It is preferable to keep the mixture at 15°C for 2 to 6 days, or 3 to 5 days.

[0084] The dormant unfertilized eggs obtained after this process have already been induced to parthenogenize. These dormant eggs will not hatch. By carrying out the diapause breaking process (S0103) described above, the dormant eggs will be released from diapause. They avoid this and development continues, eventually leading to hatching. [Example]

[0085] <Example 1: Production of transgenic silkworms using diapause eggs> (the purpose) Hatching after microinjection by the method for producing genetically modified silkworms of the present invention, and We will also verify the acquisition of genetically modified silkworms. (Methods and Results) In each experimental area, the diapause silkworm strain "Ariake" was used. Silkworms were obtained from the National Agriculture and Food Research Organization. They were raised on diet (Nihon Nosan Co., Ltd.) at 28°C until the third instar, and then at 25°C under a 12-hour light and 12-hour dark cycle. were reared under standard conditions.

[0086] 1. Verification of hatching rate by non-thermal plasma treatment In this example, corona discharge was used for the non-thermal equilibrium plasma treatment. The treatment device shown in the figure is a corona discharge device and a high voltage current power supply (HAR-30P2, Matsu The corona discharge device is made up of two parts: an aluminum electrode plate and a The electrode needles are positively charged and the negatively charged The distance from the plate was adjusted in the range of 10 to 30 mm. After oviposition on the egg paper, the eggs were placed on an aluminum electrode plate with the egg paper still on it at 2, 4, and 24 hours. The eggs were then placed in a container and exposed to corona discharges at various intensities. After the treatment, the eggs are transferred to a moist plastic box and stored at 25°C for at least 14 days. The eggs were stored and the hatchability was measured. The control eggs were those not treated with corona discharge plasma. The results are shown in Table 1. show.

[0087] [Table 1]

[0088] From the results in Table 1, non-thermal equilibrium plasma treatment of dormant eggs can improve the survival rate at least after laying. It was found that eggs aged 2 to 24 hours can be hatched with a hatching rate of over 80%. When a high voltage exceeding V was applied, short-term and long-term treatments were not effective in breaking the diapause of diapause eggs. It was revealed that no significant differences were observed (data not shown).

[0089] 2. Generation of Transgenic Silkworms After microinjection, dormancy was broken by non-thermal plasma. Eggs laid on the surface of egg paper within 2 hours of being laid are immersed in water for 5-10 minutes to separate them from the paper and make them waterproof. The micrograph was fixed on a piece of acrylic art paper with instant adhesive. The art paper was placed on a glass slide and the micrograph was The vector plasmid and helper plasmid were injected into the eggs. Clonal injection was performed according to the method of Tamura et al. (Tamura T., et al. (2000) Nat. Biotechnol. The vector plasmid was pBac [3xP3DsRedafm] (Horn and Wimme r, 2000, Dev. Genes Evol., 210(12):630-637), and pHA3PIG (Tam (Ura T., et al., 2000, Nat. Biotechnol. 18: 81-84) was used, and the Qiagen plasmid Hispe The purified plasmid DNA was then purified using the ed Midi Kit (Qiagen). Each was dissolved in a phosphate buffer (0.5 mM phosphate pH 7.0 / 5 mM KCl) at a concentration of 200 μg / mL and injected into eggs. It was introduced.

[0090] After the microinjection, the art paper containing the egg was peeled off from the slide glass and Incubate at 25°C for 24 hours after oviposition until the injected eggs reach the developmental stage. The eggs were then treated with corona discharge plasma at 5 kV for 5 minutes.

[0091] As a comparison, we used eggs within 2 hours after spawning (after fertilized egg collection) in the same way as Zhang et al. (2022, mentioned above). After breaking dormancy by corona discharge plasma treatment, microinjection was performed. The corona discharge was performed under the same conditions as above, and the eggs were laid on the surface of the egg-laying paper within 2 hours after laying. After breaking the diapause, the eggs were soaked in water for 5 to 10 minutes to separate them from the egg laying paper, and then immediately The eggs were fixed to a glass slide with adhesive. The above conditions were applied to eggs within 6 hours of incubation.

[0092] The eggs were kept in a moist plastic container in an incubator at 25°C until they hatched. The hatched larvae were transferred to a Petri dish and fed an artificial diet at approximately 28°C for 2-3 days. The resulting G0 adults were then transferred to a plastic container for artificial diet and reared at 25-27°C until they reached adulthood. The insects were either sibling-mated or backcrossed to obtain the next generation of G1 eggs. To achieve this, 20 hours after egg laying, the eggs were soaked in 6N HCl at 25°C for 1 hour. Place eggs in a plastic container at 37°C and use them immediately before hatching to visualize the eggs under a microscope equipped with a DsRed filter. The fluorescence of DsRed was examined using a microscope. Larvae that hatched from the eggs were raised to adulthood. A transgenic line was established. Table 2 shows the hatching rate by injection, and Table Figure 3 shows the results of calculating the rate of transgenic lines (positive lines) obtained in G1.

[0093] [Table 2]

[0094] In the table, Experiment No. 1 shows the results after microinjection into diapause eggs within 6 hours after spawning. This shows the method of the present invention in which the treatment for breaking diapause was carried out when the eggs reached the developmental stage of 24 hours after egg laying. On the other hand, Experiment No. 2 was a comparative example, in which diapause-breaking treatment was performed on diapause eggs within 2 hours after spawning. A method of microinjecting DNA such as vector plasmids into eggs within 6 hours of hatching. show.

[0095] From Table 2, it is not important to perform the dormancy breaking treatment and microinjection in any order. It was found that almost the same hatching rate could be obtained regardless of the type of egg.

[0096] [Table 3]

[0097] From Table 3, in Experiment No. 1 (G1 positive lineage numbers 1-2) and Experiment No. 2 (G1 positive lineage numbers 3-6), In both cases, DsRed fluorescence expression was observed in the eggs. However, the frequency of positive embryos was relatively low. The comparative example, Experiment No. 2, was 1.1 to 5.2%, while Experiment No. 1, which is the method of the present invention, was 10 The positive rate was 0.8-22.9%, more than double that of the comparison example.

[0098] The above results indicate that the method of the present invention can effectively break dormancy and prevent the growth of maize after breaking dormancy, as disclosed by Zhang et al. (2022, cited above). Compared to the conventional method using microinjection, the hatching rate was similar and the number of transgenic mice was more than doubled. This shows that it is possible to obtain a positive rate for the genic lineage.

[0099] To confirm the stability of the transgenic silkworms, silkworms hatched from G1 eggs were cultured as adults. The eggs were reared until G2 eggs were obtained. The expression of DsRed in the eggs and adults showed that the fluorescence expression was stable and It was confirmed that the trait is transmitted to the next generation, and that all established strains lay diapause eggs (De (data not shown).

[0100] <Example 2: Breaking dormancy by non-thermal equilibrium plasma treatment of dielectric barrier discharge> (the purpose) In Example 1, a non-thermal equilibrium plasma treatment was performed by directly irradiating corona discharge applied by a DC power source. This dormancy breaking was not caused by electrical stimulation of corona discharge, but by corona discharge. To confirm that the plasma was generated by a non-thermal equilibrium plasma, a dielectric barrier discharge (DBD) was performed. We will verify that dormancy can be broken even with non-thermal equilibrium plasma processing induced by electricity.

[0101] (Methods and Results) The dormant silkworm strains and rearing methods were basically the same as those in Example 1. For the dielectric barrier discharge, the treatment device shown in Figure 4 was used. This treatment device was operated at an alternating current of 10 to 12 kV. The current power supply, high voltage electrode for plasma generation, earth electrode, and nitrogen gas discharge device are integrated. The device is a handy type (HPJ-02A manufactured by Aqua Co., Ltd.). By applying a high voltage between the electrodes, a dielectric barrier discharge occurs between the electrodes. A non-thermal plasma is generated between the electrodes. By passing nitrogen gas at high speed, the nitrogen gas becomes plasma and turns into nitrogen ions (N 3- )gas This plasma gas is irradiated onto the surface of the diapausing eggs on the egg-laying paper, and then the plasma gas is irradiated onto the surface of the diapausing eggs. Equilibrium plasma treatment was performed. In this method, the electrical stimulation by the dielectric barrier discharge did not affect the eggs. The treatment is performed using only plasma gas.

[0102] To investigate the optimal conditions for plasma gas irradiation, eggs were 4 and 20 hours old. Plasma gas was irradiated to the eggs for 10, 20, 30, 60, and 180 seconds. The eggs were transferred to a plastic box and stored at 25°C for at least 14 days to check whether they had broken diapause by hatching. The controls were eggs that had not been exposed to plasma gas and were left 4 and 20 hours after oviposition.

[0103] (result) In the experimental area where plasma gas was irradiated, the time after spawning and the duration of plasma gas irradiation were not affected. Hatching was observed in all experimental areas, and the breaking of diapause in the dormant eggs was confirmed. From this result, it was concluded that the breaking of diapause was not caused by electrical stimulation but by a non-thermal equilibrium process. It has been proven that this occurs due to the action of plasma.

[0104] <Example 3: Breaking dormancy in parthenogenetic eggs by non-thermal equilibrium plasma treatment using corona discharge> (the purpose) In Example 1, the dormancy of dormant fertilized eggs was broken by corona discharge applied by a DC power source. In this example, the dormant oocytes collected from the parthenogenetic female parent were subjected to thermal equilibrium plasma treatment. We will verify whether the same treatment can break diapause in unfertilized eggs.

[0105] (Methods and Results) In each experimental area, the diapausing silkworm parthenogenetic strain pK1 (Kunashiri, Academy of Sciences of the Czech Republic) was used. After obtaining the silkworms, they were fed an artificial diet (Nihon Nosan Co., Ltd.) at 28°C. They were raised until the third instar, and then kept at 25°C under a 12-hour light and 12-hour dark cycle. The emerged female moths were stored at 4°C, and then unfertilized eggs were prepared according to the unfertilized egg collection step (S0105). Subsequently, parthenogenesis of the unfertilized eggs is induced in the parthenogenesis induction step (S0106), and then the unfertilized eggs are incubated for 72 hours. Stored at 5°C.

[0106] Corona discharge plasma treatment was performed at room temperature for 0, 24, and 72 hours after parthenogenetic induction. The corona discharge plasma treatment was carried out in accordance with the method of Example 1. The conditions for the treatment were 15 kV, 0.1 mA, and 5 or 10 minutes. The negative controls were untreated individuals and The positive control was an individual plant that had been subjected to acid treatment 72 hours after parthenogenesis induction.

[0107] (result) The results are shown in Table 4. [Table 4]

[0108] From the results in Table 4, even dormant unfertilized eggs collected from parthenogenetic female parents were able to induce parthenogenesis. If the eggs are bred after induction treatment, corona discharge plasma treatment can reduce the number of eggs bred within 0 to 72 hours by 30% or more. It was found that diapause could be broken with a hatching rate of less than 80%. When the eggs were treated for a short time (5 minutes) and a long time (10 minutes), the results were similar to those of dormant fertilized eggs. It was also revealed that there was no significant difference in the effectiveness of breaking dormancy.

Claims

1. A method for producing a genetically modified silkworm, comprising: a fertilized egg collection step of collecting fertilized eggs from individuals of the diapausing silkworm strain; a diapause-breaking step of breaking the fertilized eggs by non-thermal equilibrium plasma treatment; a nucleic acid introduction step of introducing a nucleic acid of interest into the fertilized egg after the diapause-breaking step by microinjection; and The production method further comprises a recombinant selection step of selecting genetically modified silkworms from next-generation silkworms hatched from the fertilized eggs.

2. The method according to claim 1, wherein the nucleic acid introduction step is carried out within 8 hours after collection of the fertilized egg.

3. A method for producing a genetically modified silkworm, comprising: an unfertilized egg collection step of collecting unfertilized eggs from individuals of the parthenogenetic silkworm strain; a parthenogenesis induction step in which the unfertilized eggs collected in the unfertilized egg collection step are subjected to parthenogenesis induction treatment; a diapause-breaking step of breaking the diapause of the unfertilized eggs by non-thermal equilibrium plasma treatment; a nucleic acid introduction step of introducing a nucleic acid of interest into the unfertilized egg after the diapause-breaking step by microinjection; a step of selecting recombinants from next-generation silkworms hatched from the unfertilized eggs; The method for producing the same.

4. 4. The method according to claim 3, wherein the parthenogenetic induction treatment is a high temperature treatment in which the unfertilized eggs are exposed to 45°C to 50°C for 15 to 20 minutes.

5. The method according to claim 3 or 4, wherein the nucleic acid introduction step is carried out within 24 hours after parthenogenesis induction treatment.

6. 4. The method according to claim 1, wherein the non-thermal equilibrium plasma is generated by corona discharge or dielectric barrier discharge.

7. 7. The method according to claim 6, wherein the voltage in the corona discharge is 1 kV to 50 kV.

8. The method according to claim 1 or 3, wherein a marker gene is further introduced in the nucleic acid introduction step.

9. The method according to claim 8 , wherein the selection in the recombinant selection step is based on the expression of the marker gene.

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