CN104994731A - Euryphagous sericin silkworm strain and method for producing same - Google Patents

Abstract

The object of the present invention is to provide a method for establishing a polyphagous sericin silkworm strain which can be reared at the whole larval stage even with a low-cost artificial diet containing almost no mulberry leaves among the sericin silkworm strains. Provided is a method for establishing a polyphagous silkworm strain, the method comprising mating the polyphagous silkworm strain with the sericin silkworm strain and mating the obtained hybrid individual with the polyphagous silkworm strain at least twice, the sericin silkworm strain There is a gene mutation involved in the silk protein synthesis pathway; then, based on the characteristics of the sericin silkworm strain and the characteristics of the polyphagous silkworm strain, the obtained strain is mated more than 5 times.

Description

Polyphagous sericin silkworm strain and its establishment method

technical field

The invention relates to a polyphagous sericin silkworm strain and a method for establishing the same. The polyphagous sericin silkworm strain can be fed at the whole larval stage even with artificial feed with a low mulberry leaf content.

Background technique

Many silkworms (Bombyx mori) are easy to raise, and compared with bacteria such as Escherichia coli, they can synthesize a large amount of protein in a short period of time, so they are useful organisms as hosts for the production of recombinant proteins and other substances. In the case of producing a target recombinant protein using silkworms, a method of synthesizing it in silk glands producing silk is known (Non-Patent Documents 1-3). The silk gland is easy to remove due to its large size, and the synthesized protein is stored in the lumen of the middle silk gland, so it is also suitable for recovering the target recombinant protein. However, a large amount of fibroin, which is a fibrous protein constituting silk, is usually accumulated in the middle silk gland. Since this fibroin has viscoelasticity, it becomes an inhibitory factor when recovering a recombinant protein (Non-Patent Document 4). Therefore, the sericin silkworm strain is suitable for recombinant protein production. Sericin is one of the proteins constituting silk together with fibroin, and is a gelatin-like protein that coats fibroin in a layer. Usually the cocoon of the silkworm is composed of about 75% by weight of silk protein and about 25% by weight of sericin. The aforementioned sericin silkworm strain is a mutant that can normally synthesize sericin but can hardly synthesize silk protein due to a mutation in the synthesis pathway of silk protein. Therefore, sericin silkworm strains usually fail to form cocoons and pupate in the naked pupal state. Among such sericin silkworm strains, strains attributed to three different genes are known so far. The above-mentioned problems based on silk protein can be solved by using this strain.

However, when the silkworm is used as a host for recombinant protein production, the most expected substances in the recombinant protein are pharmaceuticals. In the case of producing pharmaceuticals from silkworms, a breeding process that ensures stable quality is established, and thorough breeding management and hygiene management are required. Therefore, breeding using sterile artificial feed is indispensable. In addition, in order to reduce the manufacturing cost of pharmaceuticals, it is necessary to use low-cost artificial feed for the 4th to 5th instar larvae that require a large amount of feed. Generally, the lower the mulberry leaf content, the cheaper the commercially available artificial feed.

However, all of the above-mentioned sericin silkworm strains have a big problem of poor feedability of artificial diets with a low mulberry leaf content (Non-Patent Documents 5 and 6).

Prior art literature

patent documents

Patent Document 1: Japanese Patent No. 3374177

Non-Patent Literature

Non-Patent Document 1: Ogawa S., et al., 2007, J Biotechnol, 128:531-544.

Non-Patent Document 2: Tomita M., et al., 2003, Nat Biotechnol, 21:52-56.

Non-Patent Document 3: Tomita M., et al., 2007, Transgenic Res 16:449-465.

Non-Patent Document 4: Kataoka K. & Uematsu I., 1977, Kobunshi Ronbunshu (Japanese), 34; 7-13.

Non-Patent Document 5: Yamamoto T., et al., 2003. Bio Industry, Vol.20, No.2PP13-18.

Non-Patent Document 6: Mase K., et al., 2006, Insect Biotechnology and Sericology75, 85-88.

Contents of the invention

The problem to be solved by the invention

The object of the present invention is to provide a method for establishing a polyphagous silkworm strain and a polyphagous sericin silkworm strain which can be reared at the whole larval stage with an artificial feed with a low mulberry leaf content in a sericin silkworm strain.

Technical solutions for solving problems

In order to solve the above-mentioned problems, the inventors of the present invention tried to use one of the sericin silkworm strains, the cocooning sericin silkworm strain (Sericin-hope silkworm strain) (patent No. 3374177, non-patent literature 2 and 3) to establish a mulberry leaf A wide-feeding sericin silkworm strain that can also be raised in artificial diets with low content. However, the Sericin-hope silkworm strain is a silkworm strain that has a special feature of being able to form cocoons among the sericin silkworm strains that cannot grow cocoons originally. Therefore, even if the polyphagous property can be obtained by breaking the genetic balance by mating with the polyphagous silkworm strain, new problems such as loss of cocooning property and pupation property will arise. The present inventors mated the Sericin-hope silkworm strain with the polyphagous silkworm strain, and then backcrossed the Sericin-hope silkworm strain. As a result, they successfully established a polyphagous sericin silkworm strain that maintains cocooning ability and high pupation rate. In existing breeding methods, where a trait of interest (here polyphagia) is introduced into a base line, the method of mating with a line with a trait not suited to that purpose is accompanied by the risk of destabilizing the trait of interest acquired, so , cannot be performed as technical common sense in this field. However, in the present invention, the cocooning polyphagous sericin silkworm strain was successfully and efficiently established by using the cocooning sericin-hope silkworm strain (Sericin-hope strain) for mating against this technical common sense. The present invention is completed based on this establishment method, and provides the following contents.

(1) A method for establishing a polyphagous silkworm strain, which includes: (a) the first mating process, combining a sericin silkworm strain having a mutation in at least one gene involved in the silk protein synthesis pathway with a polyphagous silkworm strain Strain mating; (b) the second mating process, the hybrid individual after the first mating process is mated with the said or other polyphagous silkworm strains at least twice; and (c) the polyphagous sericin silkworm selection process is carried out more than 5 times Mating within the same strain, select individuals with the characteristics of the sericin silkworm strain and the characteristics of the polyphagous silkworm strain.

(2) The establishment method according to (1), wherein the mutation of the gene is a dominant mutation.

(3) The establishment method according to (2), wherein the gene is Nd, Nd-s or Nd-s ^D .

(4) The establishment method according to any one of (1) to (3), wherein the sericin silkworm strain is a cocooning sericin silkworm strain having a silk yield of 50 mg or more per head.

(5) The establishment method as described in (4), which includes: (d) a cocooning mating process, mating the hybrid individual after the second mating process with said or other cocooning sericin silkworm strains for at least 1 time; and (e) a selection process of cocooning polyphagous sericin silkworms, further selecting cocooning individuals in the polyphagous sericin silkworm selection process after the cocooning mating process.

(6) The establishment method according to any one of (1)-(5), further comprising: (f) a step of selecting homozygotes of the mutant gene.

(7) A polyphagous sericin silkworm strain and its progeny, which have a mutation in at least one gene involved in the silk protein synthesis pathway.

(8) The polyphagous sericin silkworm strain and its progeny as described in (7), which are obtained by combining a sericin silkworm strain having a mutation in at least one gene involved in the silk protein synthesis pathway with a polyphagous silkworm strain The hybrid individuals obtained by mating the silkworm strains are mated with the said or other polyphagous silkworm strains at least twice; further, based on the characteristics of the sericin silkworm strains and the characteristics of the polyphagous silkworm strains, more than 5 intra-strain matings are carried out.

(9) The polyphagous sericin silkworm strain and its progeny as described in (8), wherein the mutation of the gene is a dominant mutation.

(10) The polyphagous sericin silkworm strain and its progeny as described in (9), wherein the gene is Nd, Nd-s or Nd-s ^D .

(11) The polyphagous sericin silkworm strain and its progeny as described in any one of (7)-(10), wherein the sericin silkworm strain is a cocoon-forming silkworm with a silk yield of 50 mg or more per one Sexual sericin silkworm strain.

(12) The polyphagous sericin silkworm strain and its progeny as described in (11), after mating the hybrid individual with the said or other polyphagous silkworm strain at least 2 times, with the said or other polyphagous silkworm strain Sexual sericin silkworm strain mated at least once, and has cocooning ability.

(13) The polyphagous sericin silkworm strain and its progeny as described in any one of (7)-(12), which are homozygous for the mutant gene.

(14) A method of using the polyphagous sericin silkworm strain described in any one of (7)-(13) or its progeny as a host for producing substances to produce a recombinant peptide in the silk gland of the silkworm strain .

This specification includes the contents described in the specification and/or drawings of Japanese Patent Application No. 2012-235259 on which the priority of the present application is based.

Invention effect

According to the method for establishing a polyphagous silkworm strain of the present invention, a polyphagous sericin silkworm strain having characteristics of both the sericin silkworm strain and the polyphagous silkworm strain can be established.

According to the polyphagous sericin silkworm strain of the present invention, it is possible to provide a kind of polyphagous sericin silkworm strain that can be raised with the artificial feed with low mulberry leaf content rate in the whole larval stage.

According to the method for producing a recombinant peptide of the present invention, a large amount of recombinant protein can be produced in a short period of time by using the polyphagous sericin silkworm strain of the present invention as a host for producing substances.

Description of drawings

Fig. 1 is a graph showing the change of the feeding rate of the mulberry leaf low-content artificial feed in the polyphagous sericin silkworm strain individual obtained by mating the cocooning property sericin silkworm strain and the polyphagous silkworm strain by the establishment method of the present invention picture;

Fig. 2 is a graph showing the change of the cocooning rate in the individual of the polyphagous sericin silkworm strain obtained by crossing the cocoon-producing sericin silkworm strain and the polyphagous silkworm strain through the establishment method of the present invention;

Fig. 3 is a graph showing the change in the pupation rate in the individual of the polyphagous sericin silkworm strain obtained by mating the cocooning sericin silkworm strain and the polyphagous silkworm strain by the establishment method of the present invention;

Fig. 4 is the establishment process of the cocoon-eating property and wide-eating sericin silkworm strain MNS300 that has used the establishment method of the present invention;

Fig. 5 is the establishment process of the cocoon-eating and polyphagous sericin silkworm strain MCS300 using the establishment method of the present invention;

Fig. 6 is a graph showing the expression level of EGFP per individual when the recombinant peptide EGFP is produced using the polyphagous sericin silkworm strain of the present invention. A represents MNS300×(Ser1-Gal4×UAS-EGFP), B represents (MNS300×MCS300)×(Ser1-Gal4×UAS-EGFP), and C represents Sericin-hope×(Ser1-Gal4×UAS-EGFP).

Detailed ways

Next, specific embodiments of the present invention will be described.

1. The establishment method of polyphagous sericin silkworm strain

1-1. Overview

The first embodiment of the present invention is a method for establishing a polyphagous sericin silkworm strain. According to the establishment method of the present invention, the polyphagous sericin silkworm strain having the characteristics of both the sericin silkworm strain and the polyphagous silkworm strain can be established.

1-2. Establishment method

The establishment method of the present invention includes the first to second mating processes and the selection process of the polyphagous sericin silkworm as essential processes, and further, may include the cocoon-forming mating process, the cocoon-forming polyphagous sericin silkworm selection process, and homozygous selection process as an optional process. Next, each step will be specifically described.

(1) The first mating process

The "first mating process" is an essential process for mating the polyphagous silkworm strain and the sericin silkworm strain.

In the present specification, a "silkworm strain" refers to a mutant species of silkworm having specific characteristics.

In this specification, "wide-feeding (silkworm strain)" refers not only to artificial diets containing leaves of the genus Morus (hereinafter referred to as "mulberry leaves" in this specification), but also to foods that do not contain mulberry leaves. Artificial feed or artificial feed with a low mulberry leaf content can also be ingested, and silkworm strains in the entire larval stage can be raised.

It should be noted that with regard to food intake, wild-type silkworm strains and commonly raised silkworm strains are generally unable to ingest artificial diets that do not contain mulberry leaves or artificial diets that contain low mulberry leaves. Therefore, polyphagous silkworm strains are not suitable.

The "artificial feed for silkworms containing mulberry leaves" in this specification refers to the artificial feed for silkworms containing mulberry leaves prepared for the development of larvae of silkworms. In addition, the "artificial feed with a low mulberry leaf content" means, for example, an artificial feed with a mulberry leaf content of 5% by weight or less, 3% by weight or less, or 1% by weight or less. Concrete examples of the artificial feed containing mulberry leaves, for example, can be enumerated: Horie Yasuhiro, Watanabe Kijiro (1969) Japan Sericulture Journal, 30:377-385; ) Silk Experiment Station Report 30:569-588; Yanagawa Hiroaki, Watanabe Kijiro, Suzuki Kiyoshi (1991) Silk Insect Research Report 3:57-75, etc. The artificial feed recorded. The artificial feed containing mulberry leaves can be a commercially available artificial feed. For example, SilkMate L4M (Nippon Nosan Kogyo) with a low mulberry leaf content and 1 to 3 instar silkworm eggs with a high mulberry leaf content (Nippon Nosan Kogyo) are listed.

In this specification, an artificial feed for silkworms not containing mulberry leaves, or a synthetic artificial feed for silkworms containing no leaves of plants other than mulberry leaves, and the like correspond to the "artificial feed not containing mulberry leaves".

The polyphagous silkworm strain in the present invention can be used as a parental strain in the establishment of the polyphagous sericin silkworm strain. The polyphagous silkworm strain is preferably a polyphagous silkworm strain with a large silk mass, that is, a polyphagous silkworm strain. Specific examples of polyphagous multifilament silkworm strains include: TN29 silkworm strain, TN38 silkworm strain, Japanese silkworm strain No. 603, Japanese silkworm strain No. 604, or NS2 silkworm strain, or Chinese silkworm strain Silkworm strain TCS40, Zhong604 silkworm strain, or CS83 silkworm strain. These silkworm strains are all preserved in the Institute of Agricultural Bioresources (2-1-2 Kannondai, Tsukuba City, Ibaraki Prefecture, Japan), and can be sold separately through prescribed procedures.

"Sericin silkworm strain" means that there is a mutation in at least one gene involved in the synthesis pathway of silk protein (complex composed of silk protein H chain, silk protein L chain and p25 protein), so that the synthesis amount of silk protein is the same as that of wild silkworm Type silkworm strains were significantly reduced compared to silkworm strains. Specific examples of such genes include Nd gene involved in silk protein H chain synthesis, Nd-s gene or Nd-s ^D gene involved in silk protein L chain synthesis. Although the mutation of the above-mentioned genes may be a dominant mutation or a recessive mutation, it is preferred that a dominant mutation appearing in the next generation is characteristic in consideration of post-mating selection. Dominant negative is further preferred.

As mentioned above, the silkworm's cocoon is generally composed of about 75% by weight of silk protein and about 25% by weight of sericin. However, the sericin silkworm strain has mutations in the genes involved in the silk protein synthesis pathway, so only 35% by weight or less of the silk protein is synthesized in the cocoon silk, and the rest is all sericin. For example, the Nd gene mutant sericin silkworm strain (hereinafter referred to as "Nd silkworm strain" in this specification) having a dominant mutation in the Nd gene exemplified above is hardly able to synthesize silk even if the Nd mutation is heterologous. protein strains. In addition, an Nd-s gene mutant sericin silkworm strain having a dominant mutation in the Nd-s gene (hereinafter referred to as "Nd-s silkworm strain" in this specification) and a Nd-s ^D gene mutant All of the Nd-s ^D gene mutant sericin silkworm strains (hereinafter referred to as "Nd-s ^D silkworm strain" in this specification) are strains in which the silk protein content in cocoon silk is about 30% by weight or less. Therefore, sericin silkworm strains usually cannot spin all the silk, so they cannot cocoon (Nakano Yoshio, 1950, Japanese Journal of Sericulture, 20:232-248; Horiuchi Binming et al. 1963, Japanese Journal of Sericulture, 33:195-196; Gamo Takumo, 1979, Japanese Silkworm Kanto Lectures, 30:26). These sericin silkworm strains are registered in the gene bank of the Ministry of Agriculture, Forestry and Fisheries, and can be accepted for separate sale through prescribed procedures.

The sericin silkworm strain can be used as another parental strain in the establishment method of the polyphagous sericin silkworm strain of the present invention. Sericin silkworm strains usually cannot spin silk as mentioned above, so they cannot cocoon and become naked or half-pupated. However, there is also known a "cocooning sericin silkworm strain" which exceptionally spins silk and has the characteristic of being capable of cocooning. Such a cocooning sericin silkworm strain has a silk yield of 50 mg or more, preferably 60 mg or more, more preferably 70 mg or more, or 60% or more, preferably 70% or more, more preferably 80% or more, and even more preferably 90% per one silkworm strain. The above camp cocoon rate. The "amount of silk produced" per one (individual) refers to the amount of silk produced by one individual during cocooning. The amount of spinning silk has the same meaning as the yield of only cocoons obtained from each individual (cocoon layer weight). In addition, the "cocooning rate" refers to the ratio of the number of individuals capable of cocooning to the total number of hybrid individuals obtained in this step. The cocooning sericin silkworm strain is particularly preferable as the parent strain of the polyphagous sericin silkworm strain of the present invention. The silk produced by the cocooning sericin silkworm strain consists of 35% by weight or less of silk protein and other sericin. Thus, a cocoon is formed, but becomes a "sericin cocoon" different from the usual wild-type cocoon. In addition, in addition to cocooning, in order to obtain adults of the first generation of hybridization (hereinafter, generally referred to as "F1"), a silkworm strain with a high pupation rate (pupation rate) is further preferably used as the polyphagous sericin silkworm of the present invention. The parental line of the line. As specific examples of silkworm strains that have cocooning properties, high pupation rate, and sericin silkworm strains, for example, the Sericin-hope silkworm strain or the Sericin-hope silkworm strain described in Japanese Patent No. 3374177 or the above-mentioned non-patent documents 2 and 3 A hybrid of the hope silkworm strain. "Sericin-hope silkworm strain" is a silkworm strain obtained by mating an Nd silkworm strain with a robust multifilament quantitative strain.

The mating method of the polyphagous silkworm strain and the sericin silkworm strain is not particularly limited as long as it is performed by a method known in the art. Just put the male and female of each silkworm strain into the same container to make them mate naturally. Any silkworm strain can be used as the male. In this case, the sericin silkworm strain is preferably homozygous for the causative mutation gene. After mating, eggs are collected by a method known in the art, and F1 are raised by a known method.

It should be noted that, in the selection of polyphagia, only the feed for polyphagous silkworm strains with low mulberry leaf content (for example, SilkMate L4M) should be given only from the first instar in the rearing of F1 larvae. Individuals that can develop after the second age can be identified as individuals that have acquired the characteristics of polyphagia. On the other hand, the selection of the characteristics of the sericin silkworm strain is not particularly limited as long as the mutation of the gene involved in the silk protein synthesis pathway can be confirmed, and all methods known in the art can be used. For example, it only needs to be carried out based on molecular markers and characteristic markers such as SNP and RFLP (Restriction Fragment Length Polymorphism). In addition, if the mutation of the causative gene (Nd gene) of the Sericin-hope silkworm strain is a dominant mutation cocooning sericin silkworm strain, it can be realized by selecting individuals that form sericin cocoons.

(2) Second mating process

The "second mating process" is an essential process for mating the hybrid individual after the first mating process with the above or other polyphagous silkworm strains. This step can be performed for the purpose of further introducing the characteristic of polyphagia into the sericin silkworm strain.

In this step, the "hybrid individual after the first mating process" refers to the F1 individual obtained after the above-mentioned first mating process, or the hybrid individual (equivalent to F2) that has undergone intra-line mating 1 to 4 times for its F1 individual. ~F5). Intra-strain mating will be described later.

In this step, "the aforementioned polyphagous silkworm strain" refers to the same polyphagous silkworm strain as the polyphagous silkworm strain used as a parent strain in the first mating step. For example, in the first mating step, when the polyphagous silkworm strain uses the TN38 silkworm strain, the polyphagous silkworm strain in this step is the TN38 silkworm strain. At this time, the mating performed in the second mating step is a backcross of the polyphagous silkworm strain. On the other hand, in this step, "another polyphagous silkworm strain" refers to a polyphagous silkworm strain different from the polyphagous silkworm strain used as a parent strain in the first mating step. For example, in the first mating process, when the polyphagous silkworm strain uses the TN38 silkworm strain, in this process as the polyphagous silkworm strain, the situation of using the TCS40 silkworm strain and the situation of using the F1 individual of the TN38 silkworm strain and the TCS40 silkworm strain .

The mating method of the hybrid individual obtained in the above-mentioned first mating step and the polyphagous silkworm strain only needs to be carried out according to the first mating step. The sexes of the two strains were not considered when mating. Just set one as male and the other as female.

In this step, the times of mating the hybrid individuals obtained in the first mating step with the above or other polyphagous silkworm strains are at least 2 times. The in-line mating of the polyphagous sericin silkworm selection step described later can be carried out 1 to 4 times between each time. In each time, the selection was made based on the characteristics of the sericin silkworm strain and the characteristics of the polyphagous silkworm strain. The selection method of each characteristic should just be performed according to the 1st mating process.

(3) Selection process of polyphagous sericin silkworm

The "polyphagous sericin silkworm selection process" is an essential process for selecting individuals with the characteristics of the sericin silkworm strain and the characteristics of the polyphagous silkworm strain, and stabilizing the target characteristics. This step can be realized by performing intra-line mating among hybrid individuals after the first mating step and the second mating step.

In the present specification, "intra-strain mating" (sib mating) refers to mating between individuals having the same target characteristics (more specifically, genes that are responsible for the characteristics). For example, if it is intra-strain mating of a polyphagous sericin silkworm strain, both males and females to be mated are polyphagous and belong to the sericin strain. In addition, intra-strain mating also includes sibling mating with the same characteristics as the object of the same-abdominal male and female.

In this step, selection is performed based on the characteristics of the sericin silkworm strain and the characteristics of the polyphagous silkworm strain after mating within the strain. Each selection may be performed according to the selection method described in the above-mentioned first mating step.

This step consists of 5 or more, preferably 7 or more, more preferably 9 or more in-line matings. The upper limit of the number of matings within a strain is not particularly limited, but usually 15 or less are sufficient. Intra-line mating does not need to be performed consecutively, and in the establishment method of this embodiment, it is only necessary to select a total of 5 or more times for the target characteristic. Therefore, this process can be distributed between other processes in terms of mating units within the strain. For example, as long as after the second mating process, intra-line mating is performed twice in the selection process of polyphagous sericin silkworms before the cocooning mating process described later, the selection process of polyphagous sericin silkworms after the cocooning mating process (At this time, it becomes the cocoon-forming polyphagous silkworm selection process described later), the intra-strain mating is performed more than 3 times, and then, in each time, the characteristics of the sericin silkworm strain and the characteristics of the polyphagous silkworm strain are selected. That's it.

(4) cocooning mating process

The "cocooning mating process" is a process of mating the hybrid individual after the second mating process with the cocooning sericin silkworm strain.

When the sericin silkworm strain used for the parent strain in the first mating step is a cocooning property sericin silkworm strain, this step is a step to be performed as necessary.

In the case of using a cocooning sericin silkworm strain as a parental line in the first mating process, even if a polyphagous sericin silkworm strain is obtained after the second mating process, the cocooning property and pupation rate will be accompanied by a decrease or loss of such issues. This step is characterized in that the sericin silkworm strain that has cocoon-forming but not polyphagous properties is crossed against the ultimate goal of introducing a polyphagous characteristic into the sericin silkworm strain. Such a mating method is a method of line establishment that defies existing breeding methods established in the field.

The cocooning sericin silkworm strain used for mating in this process is the above-mentioned or other cocooning sericin silkworm strains.

In this process, "the above-mentioned cocooning sericin silkworm strain" means the same silkworm strain as the cocooning sericin silkworm strain used as a parent strain in the first mating step. For example, in the first mating step, when the Sericin-hope silkworm strain is used as the cocooning sericin silkworm strain, the Sericin-hope silkworm strain is also used in this step. At this time, the mating carried out in this process is equivalent to the backcross of the cocooning property sericin silkworm strain.

On the other hand, in this process, "other cocooning sericin silkworm strain" refers to a cocooning sericin silkworm strain different from the cocooning sericin silkworm strain used as the parent strain in the first mating process . For example, in the first mating process, when the Sericin-hope silkworm strain is used for the cocooning sericin silkworm strain, F1 individuals of the Sericin-hope silkworm strain and the Japanese silkworm strain No. 603 are suitable.

In this step, the hybrid individual after the second mating step is mated with the cocoon-making sericin silkworm strain at least once. As shown in Figure 5, this process can also be carried out between the second mating process, but this process will be carried out again after the last second mating process at this time. The mating method only needs to be carried out according to the first mating procedure. As in the first mating procedure, the male and female of the two lines mated were not considered.

(5) Selection process of cocooning and polyphagous sericin silkworm

The "cocooning polyphagous sericin silkworm selection process" is a form of the polyphagous sericin silkworm selection process performed after the cocooning mating process, and is an accompanying process accompanying the cocooning mating process. The above-mentioned polyphagous silkworm selection process selects individuals with two characteristics, namely, the characteristics of the sericin silkworm strain and the characteristics of the polyphagous silkworm strain. In contrast, in this process, it is characterized in that three characteristics are selected. Characteristics, that is, in addition to the characteristics of sericin silkworm strains and the characteristics of polyphagous silkworm strains, there are also individuals with cocooning characteristics.

In this process, the selection method about the individual which has the characteristic of a sericin silkworm strain and the characteristic of a polyphagous silkworm strain follows the said polyphagous sericin silkworm selection process. That is, regarding the selection of the characteristics of the sericin silkworm strain, it is only necessary to confirm the mutation of the gene involved in the silk protein synthesis pathway by a known method. For example, it can be performed based on molecular markers and characteristic markers such as SNP and RFLP. In the selection of the characteristics of the polyphagous silkworm strain, it is only necessary to feed only the polyphagous silkworm strain with a low mulberry leaf content in the breeding of F1 larvae from the first instar. The developable individuals after the 2nd age can be identified as the individuals who acquired the characteristics of polyphagia. In addition, as for the selection method of individuals having the characteristics of the cocooning silkworm strain, it is only necessary to select based on the cocooning rate or the amount of silk produced per individual. In selection, the cocoon formation rate should just be 60% or more, Preferably it is 70% or more, More preferably, it is 80% or more, More preferably, it may be 90% or more. Moreover, what is necessary is just to select the individual whose spinning amount per one head is 50 mg or more, Preferably it is 60 mg or more, More preferably, it is 70 mg or more. If the mutation of the gene (Nd gene) that is the cause of the Sericin-hope silkworm strain is a cocooning property sericin silkworm line with a dominant mutation, then the cocooning property silkworm line and the cocooning property silkworm line can be selected simultaneously by selecting individuals who have formed sericin cocoons. Characteristics of both sericin silkworm strains.

This process is a form of the selection process of polyphagous sericin silkworm. Therefore, as long as the intra-strain mating is carried out more than 5 times, preferably more than 7 times, more preferably more than 9 times, after each time, aim at the target characteristic, that is, the sericin silkworm strain The characteristics of the characteristics, the characteristics of the polyphagous silkworm strain and the characteristics of cocooning can be selected.

(6) Homozygous selection process

The "homozygote selection step" is a step of selecting a homozygote for a mutant gene involved in the silk protein synthesis pathway possessed by the sericin silkworm strain used in the first mating step.

This process is a selection process performed as needed in the polyphagous sericin silkworm selection process (including the cocoon-forming polyphagous sericin silkworm selection process). If the homozygous selection method is a method that can detect whether the mutant gene involved in the silk protein synthesis pathway is homologous or heterologous to individuals after the selection process of the polyphagous sericin silkworm, any method known in the art can be used. For example, based on the known base mutation information, the method of typing the homology and heterology of the mutation site by DNA sequencing and base sequence information by TaqMan (registered trademark) PCR method, etc., and using SSCP (Single -Strand Conformation Polymorphism) method and RFLP method analysis methods, etc. These methods are known, for example, what is necessary is just to refer to the method described in Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press. In addition, if the mutation of the causative gene (Nd gene, etc.) of the Sericin-hope silkworm strain is a cocooning sericin silkworm strain with a dominant mutation, it is possible to repeat intra-line mating to the cocooning polyphagous sericin silkworm selection Individuals produced in the process all produce sericin cocoons to obtain candidate individuals. In the case that the above-mentioned genes involved in the silk protein synthesis pathway are homologous individuals with the wild type and the larvae obtained by mating the above-mentioned candidate individuals all form sericin cocoons, as long as the candidate individual is selected as a mutation for the gene that is the cause Homozygous can be.

2. Polyphagous sericin silkworm strain

The second embodiment of the present invention is a polyphagous sericin silkworm strain and its progeny. According to the polyphagous silkworm strain and its progeny of the present invention, a silkworm strain having the characteristics of both the sericin silkworm strain and the polyphagous silkworm strain can be provided.

The polyphagous sericin silkworm strain of this embodiment has a mutation in at least one gene involved in the silk protein synthesis pathway, and can be fed to the whole larval stage with an artificial feed that does not contain mulberry leaves or an artificial feed with a low mulberry leaf content. Silkworm strains. The polyphagous sericin silkworm strain of this embodiment may be eggs, larvae, pupae or adults.

In this specification, the "progeny of the polyphagous sericin silkworm strain" refers to the progeny of the polyphagous sericin silkworm strain maintaining the characteristics of the sericin silkworm strain and the characteristics of the polyphagous silkworm strain. It can be the offspring obtained by mating the same strain, or it can be the offspring obtained by mating different polyphagous sericin silkworm strains.

The polyphagous sericin silkworm strain of this embodiment can be obtained, for example, by the establishment method of the polyphagous sericin silkworm strain described in the said 1st embodiment. Specifically, for example, F1 individuals obtained by crossing a sericin silkworm strain with a polyphagous silkworm strain can be mated with the above-mentioned or other polyphagous silkworm strains at least twice, and further, based on the characteristics of the sericin silkworm strain and the polyphagous silkworm strain The characteristics of the sericin silkworm are obtained by crossing more than 5 times within the strain, and the sericin silkworm strain has a mutation in at least one gene involved in the silk protein synthesis pathway.

Examples of genes involved in the silk protein synthesis pathway include: Nd, Nd-s or Nd-s ^D genes, and mutations in these genes are preferably dominant mutations. In addition, the polyphagous sericin silkworm strain of the present embodiment and its progeny are preferably homozygous for the above-mentioned mutant gene.

In addition, the above-mentioned sericin silkworm strain used as a parent strain may have a silk yield of 50 mg or more, preferably 60 mg, more preferably 70 mg, or a cocooning rate of 60% or more, preferably 70% or more, or more per one. Preferably more than 80%, more preferably more than 90% of cocooning sericin silkworm strains. At this time, the cocooning and polyphagous silkworm strain can be obtained by the following method: F1 individuals obtained by mating the cocooning and polyphagous silkworm strain with the polyphagous silkworm strain are mixed with the above-mentioned or other polyphagous silkworm strains and the above-mentioned or other nutrition. Cocooning sericin silkworm strains were mated at least once to select for cocooning, that is, whether to form cocoons, and then, based on the characteristics of sericin silkworm strains and the characteristics of polyphagous silkworm strains, more than 5 intra-strain matings were carried out. In addition, the offspring of the cocooning polyphagous sericin silkworm strain can also be obtained by mating cocooning polyphagous sericin silkworm strains of the same strain or different strains. For example, if it is the progeny of two cocooning polyphagous sericin silkworm strains MNS300 silkworm strain and MCS300 silkworm strain described in the examples described later, the same strains of MNS300 silkworm strains or MCS300 silkworm strains can be mentioned. The offspring obtained by mating among different strains of the silkworm strain MNS300 and the silkworm strain MCS300, and the offspring obtained by mating the same strain or the offspring obtained by crossing between different strains with TN29, TN38 , Day 603, Day 604, CS40 or Zhong 604 such as the offspring obtained by mating silkworm strains such as Bombyx mori polyphagia and multifilament quantity.

According to the polyphagous sericin silkworm strain of this embodiment, it is possible to provide a sericin silkworm strain which is suitable for the production of substances such as useful recombinant proteins and which can be raised at the whole larval stage even with low-cost artificial feed. In particular, according to the cocooning polyphagous sericin silkworm strain of this embodiment, the recombinant protein synthesized in the body of the silkworm strain can be recovered from the cocoon.

3. Recombinant peptide manufacturing method

The third embodiment of the present invention is a method for producing a recombinant peptide using the polyphagous sericin silkworm strain of the above-mentioned second embodiment as a host for producing substances.

In the present embodiment, "recombinant peptide" refers to a peptide that can be synthesized in silk glands of silkworms using gene recombination technology. For example, it refers to a peptide having a specific biological activity in vivo or in cells, such as enzyme activity, catalytic activity, substrate function, or biological inhibition or promotion function (such as cell damage activity). Specifically, for example, antibodies (including humanized antibodies, chimeric antibodies, multispecific antibodies such as bispecific antibodies, single-chain antibodies such as scFv, etc.) and fragments thereof, enzymes, and antigenic peptides can be mentioned. Recombinant peptides can be natural or non-natural. Natural recombinant peptides refer to (poly)peptides that occur in nature. On the other hand, a non-natural recombinant peptide is based on the amino acid sequence of a natural recombinant peptide, and an appropriate mutation (addition, deletion, Altered (poly)peptides. The origin of the biological species is not considered as long as the recombinant peptide does not affect the host, the silkworm. For example, it may be a (poly)peptide derived from humans.

In the method for producing the recombinant peptide of the present embodiment, first, the gene encoding the above-mentioned recombinant peptide is placed under the control of a promoter functioning in the silk gland of the silkworm, etc., and an expression unit containing it is prepared to be introduced into the second embodiment. The recombinant silkworm obtained from the wide-feeding sericin silkworm strain. The "expression unit" referred to here refers to a gene expression system including at least one set of control factors required for the expression of the gene, such as a promoter, a poly A signal, and a terminator, in addition to the target gene. Specific examples of promoters that function in silk glands of silkworms include, for example, the sericin 1 promoter that functions in middle silk glands. These methods are well known in the art, for example as described in Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press; Tamura T, et al., 2007, J Insect Biotechnol Sericol 76:155-159.

Next, the recombinant peptide synthesized in the silk gland was recovered from the host silkworm. The recovery method is not particularly limited. As mentioned above, sericin silkworm strains are almost unable to synthesize silk protein due to mutations in the silk protein synthesis pathway, and usually cannot spin silk. Therefore, most of the recombinant peptides synthesized in the silk gland directly accumulate in the lumen of the middle silk gland. In this case, it is only necessary to dissect the host silkworm and remove the silk gland, or to inject the external silkworm into the middle silk gland of the host silkworm to recover the target recombinant peptide accumulated in the middle silk gland. In addition, when the polyphagous sericin silkworm strain of the second embodiment is a cocoon-forming polyphagous sericin silkworm strain, the recombinant peptide synthesized in the silk gland can be spit out together with the sericin, so it can be directly recovered from the cocoon .

According to the method for producing a recombinant peptide of the present invention, a large amount of recombinant peptide can be synthesized in a short period of time by using a polyphagous sericin silkworm strain as a host for producing a substance. In addition, it is also possible to produce recombinant peptides as pharmaceuticals in silkworms by feeding a sterile artificial feed to a polyphagous sericin silkworm strain in a sterile environment.

Example

The present invention will be specifically described by the following examples, but the technical scope of the present invention is not limited to the following examples. In addition, in the following examples, "silkworm strain" is often omitted.

<Example 1>

(Purpose)

The changes in the feeding rate, cocooning rate and pupation rate of the artificial diet with low mulberry leaf content in the polyphagous sericin silkworm strain individuals obtained by mating the cocooning sericin silkworm strain and the polyphagous silkworm strain were verified.

(method)

1. Mating

The cocooning sericin silkworm strain Sericin-hope (Japanese Patent No. 3374177) and the polyphagous multifilament silkworm strain (TN38, TCS40) were mated. Specifically, the female Sericin-hope was mated with the male polyphagous silkworm silkworm strain. Afterwards, intra-line mating was carried out for each hybrid individual, and the mating was repeated until the sixth generation (F6) of the hybrid.

2. Incubation and transfer of eggs and rearing

The incubation and transfer of eggs is to mix the eggs laid by 4 to 10 females of the same line to hatch them. The hatched larvae are moved from the egg cardboard to the container used as the silkworm basket using a duster. In the rearing of silkworm larvae from the 1st to 3rd instar, use plastic lunch boxes (DX-HS8: 222×143×35mm; or DX-HS10: 256×189×35mm; both are Chuo Chemical) or styrene lunch boxes. A container with anti-drying paper (paraffin processed paper) inside the container. As for the bait, SilkMate L4M (Nippon Agricultural Industry Co., Ltd.), an artificial feed for the polyphagous silkworm with low cost and low mulberry leaf content, was arranged on the dry-proof paper and given. For the exchange of bait, in principle, it is performed once at the 1st to 2nd age, and 1 to 3 times at the 3rd age, but it is appropriate to exchange when the bait is insufficient and dry. The old bait is removed when there is a lot of leftovers, but it is kept when there is a little bit, and new artificial bait is added.

For the rearing of 4-5 instar silkworm larvae, use fish box B (model 01120; Sanka Co., Ltd.) with silkworm basket paper (kraft paper), anti-drying paper (paraffin paper) or both. container. Cover with anti-drying paper, acrylic or net depending on the humidity and the state of the container. After the 2nd day after the 4th instar, the number in each container was checked, and it was set to about 80 per container depending on the size of the container. The bait is the same as that of silkworm larvae, and SilkMate L4M (Nippon Agricultural Industry) is cut into strips or cut to give. The exchange of bait varies depending on the amount left over and the dry state of the bait, but in principle, it is performed once a day. When there is only a little leftover, the old bait is not removed, but the new artificial feed is directly cut and added. The raising temperature was set at 25 to 28° C. throughout the entire larval stage.

Feeding selection was carried out by administering the above-mentioned SilkMate L4M from early age. After feeding, observe at 27°C for 5-7 days, and select individuals who develop into 2nd instar larvae as polyphagous individuals. The feeding rate was calculated from the ratio of the number of the 2nd age to the number of the first age. In addition, cocooning selection is performed by selecting individuals that have formed cocoons. The cocooning rate was calculated from the ratio of the number of cocoons that could form cocoons to the number of 4-year-olds. Furthermore, selection for pupation is carried out by selecting normally pupated individuals. The pupation rate was calculated by the ratio of the number of normal pupation silkworms to the number of silkworms at the 4th instar. Because this gene mutation is a dominant mutation, the selection of the sericin silkworm strain, that is, the Nd mutation, is implemented by selecting the individual whose cocoon of the cocooning individual becomes a sericin cocoon.

(result)

Figure 1 shows the change in feeding rate, Figure 2 shows the change in cocooning rate, and Figure 3 shows the change in pupation rate.

As shown in Figure 1, the effect of feeding selection appeared earlier after mating, and the mating individuals of Sericin-hope and TN38 (hereinafter often referred to as "Sericin-hope × TN38"), and the mating individuals of Sericin-hope and TCS40 ( Hereinafter, it is often recorded as "Sericin-hope×TCS40") at F5, and almost all individuals ingest the artificial diet SilkMate L4M used by silkworms with low mulberry leaf content. That is, it shows that F5 becomes a polyphagous sericin silkworm strain.

On the other hand, Fig. 2 and Fig. 3 can also show that the cocooning rate and the pupation rate cannot obtain the respective selection effects, and the selection is carried out until F6 is destabilized. For example, the cocoon formation rate of Sericin-hope×TCS40 is 0% in F4, which is almost naked pupae. The results indicated that when the cocooning silkworm strain and the polyphagous silkworm strain were mated, and then the polyphagous, cocooning and pupating properties were selected at the same time, the sericin silkworm strain with polyphagous characteristics could be used in In F5, on the other hand, the cocooning and pupation properties originally possessed by the cocooning sericin silkworm strain were destabilized by mating with the polyphagous silkworm strain.

<Example 2>

(Purpose)

The method for establishing a polyphagous sericin silkworm strain of the present invention is used to establish a cocoon-forming polyphagous sericin silkworm strain (MNS300 silkworm strain).

(method)

1. Breeding

The flow chart of breeding is shown in FIG. 4 . First, the female cocooning sericin silkworm strain Sericin-hope was mated with the male polyphagous multifilament strain TN38, and the hatched larvae were reared with SilkMate L4M (Nippon Agricultural Industries) to select polyphagy ( first mating procedure). Simultaneously, among the individuals who cultivate cocoon silk gum cocoons, select individuals with more cocoon layer weight (only the weight of the cocoon: equivalent to the amount of silk spinning/only). Then, intra-line mating was performed for two generations, and TN38 was backcrossed twice (second mating step), and then intra-line mating was performed once. The artificial feed feeding ability of the individuals selected by this cross was increased, and as a result, they were mated with Sericin-hope to increase the cocooning rate (cocooning mating process), and then the intra-line mating was carried out for 9 generations, and the cocooning rate, cocoon layer amount, etc. were measured. The selection (cocooning property polyphagous sericin silkworm selection process).

2. Incubation and transfer, rearing and selection of eggs

The basic method is according to Example 1. Mix 4 to 10 moths to start breeding (mixed breeding), and start to raise multiple moth areas separately (single moth breeding). The hatched larvae were reared in a small plastic lunch box with a lid (DX-HS8: 222×143×35mm; Central Chemical), and only the individuals that normally developed to the 2nd instar were selected as polyphagia when feeding on SilkMate L4M. After the 4th age, about 80 animals/container were raised using a plastic container (fish box B 01120; Sanka Co., Ltd.). Basically, 1 zone is set to 2 containers (approximately 160 in total). Among the individuals who have formed sericin cocoons in their respective containers, the first 20 individuals are selected based on the weight of the cocoon layer, these lines are crossed, and eggs are collected to obtain the next generation. In the case of single moth breeding, comprehensively judge artificial feed intake rate, cocoon formation rate, pupation rate, cocoon layer quantity, etc., squeeze to the top 2-4 areas, then mate and collect eggs to obtain the next generation.

It should be noted that Sericin-hope is a limited common silkworm whose larvae are characterized by stripes and other characteristics only in females. Therefore, the stripes are shaped (common silkworm) in females and ji (sericin silkworm) in males. . On the other hand, TN38 was vegetarian in both male and female silkworms. Therefore, the hybrid individual of Sericin-hope female × TN38 male is a sex-limited silkworm. In this example, the MNS300 silkworm strain was set as a sex-limited common silkworm by selecting individuals that were female in common silkworms and male in plain silkworms.

<Example 3>

(Purpose)

A cocooning polyphagous sericin silkworm strain (MCS300 silkworm strain) was established by using the method for establishing a polyphagous sericin silkworm strain of the present invention in a process different from that in Example 2.

(method)

1. Breeding

The flow chart of breeding is shown in FIG. 5 . First, the female Sericin-hope was mated with the male polyphagous multifilament strain TCS40 (the first mating process). The obtained hybrid individuals were mated once within the strain (selection step of polyphagous sericin silkworm), backcrossed with TCS40 (second mating step), and then mated with Sericin-hope (cocooning mating step). Then, backcross TCS40 again (the second mating procedure), and perform intra-line mating once (the selection procedure of cocooning polyphagous sericin silkworm). Next, the hybrid individual was mated with a cocoon-forming polyphagous F1 individual obtained by mating Zhong 604 and Sericin-hope of the polyphagous multifilament line. (cocooning property mating process), and then, carry out 9 generations of in-line mating (cocooning property polyphagous sericin silkworm selection process).

2. Incubation and transfer, rearing and selection of eggs

The basic method is according to Example 1. Mix 4 to 10 moths to hatch and transfer eggs. The hatched larvae were reared in a small plastic lunch box with a lid (DX-HS8: 222×143×35mm; Chuo Chemical), and only individuals that normally developed to the 2nd instar by feeding on SilkMate L4M (Nippon Agricultural Industries) were selected as polyphagous characteristic individuals. After the 4th age, about 80 animals/container were raised using a plastic container (fish box B 01120; Sanka Co., Ltd.). Zone 1 is set to 2 containers (about 160 in total). Individuals who formed sericin cocoons in their respective containers were selected as individuals characteristic of the sericin silkworm strain. Then, use the top 20 individuals to mate in the strain based on the weight of the cocoon layer, and collect eggs to obtain the next generation. The hatched larvae were fed with SilkMate L4M to select individuals that formed sericin cocoons, thereby selecting for the characteristics of both polyphagous and sericin silkworm strains. Then, backcross TCS40, a polyphagous multi-filament strain, among the obtained adults. After collecting eggs, for the hatched larvae, feed SilkMate L4M again and select sericin cocoons to form individuals. Both features are selected. Then, the obtained hybrid individuals were backcrossed to Sericin-hope. After the eggs were collected, the hatched larvae were fed with SilkMate L4M and sericin cocoons were selected to form individuals. Features are selected. Intra-line mating is carried out once between the individuals with the cocoon layer weight of the top 20 or so, and eggs are collected to obtain the next generation. This individual was mated with a cocooning polyphagous F1 individual obtained by mating Zhong 604 and Sericin-hope. After the eggs were collected, the hatched larvae were fed SilkMate L4M and selected sericin cocoon-forming individuals in the same manner as above, and the cocooning polyphagia and the three characteristics of the sericin silkworm strain were selected. The intra-strain mating was carried out for 9 generations, and in each generation, the cocoon-making, polyphagous and sericin silkworm strains were selected to obtain the target cocoon-forming polyphagous sericin silkworm strain.

It should be noted that, like Sericin-hope, TCS40 and Zhong 604 are sex-limited common silkworms. The stripes are shaped in females and jiji in males. They can distinguish between male and female in the larval stage, so they are separated in the 4th instar. Male and female, each with 80 animals.

<Example 4>

(Purpose)

The polyphagia of the cocoon-forming polyphagous sericin silkworm strain obtained in Examples 2 and 3 was verified.

(method)

The silkworm strains used in the experiment were MNS300×MCS300 obtained from the mating pair Sericin-hope, Ri 604 (wide-feeding multifilament strain) × Sericin-hope, MNS300, MCS300, MNS300 and MCS300 and the above-mentioned MNS300×MCS300 silkworm (MNS300×MCS300)×Day 604 obtained from strain and Day 604. The respective strains are divided into 2 zones. From the early age, they are divided into the zone given SilkMate L4M (Nippon Agricultural Industries) which is a low-cost and low-cost polyphagous silkworm artificial feed, and the zone given high mulberry-leaf content. The artificial feed for silkworms is SilkMate for the 1-3 instars of silkworm eggs (hereinafter referred to as "for the original silkworms") (Nippon Agricultural Industry). The hatched larvae were moved to a plastic rearing container, given each of the above-mentioned artificial diets, and observed at 27° C. for 5 to 7 days. The larvae that developed into the 2nd instar larvae were used as feeding individuals, and the feeding rate was calculated from the ratio of the number of 2nd instar larvae to the number of first instar larvae.

(result)

Table 1 shows the results of the SilkMate L4M-administered regions, and Table 2 the results of the SilkMate-administered regions for silkworms.

[Table 1]

Feeding SilkMate L4M (artificial feed for polyphagous silkworm with low mulberry leaf content)

Strain·Hybrid Feeding rate (%) Sericin-hope 0.0±0.0 Day 604 × Sericin-hope 10.8±7.9 MNS300 97.8±2.0

the MCS300 96.0±1.4 MNS300×MCS300 96.6±2.6 (MNS300×MCS300)×Day 604 97.4±2.0

[Table 2]

Feeding SilkMate silkworms for 1st to 3rd instars (artificial feed for silkworms with high mulberry leaf content)

Strain·Hybrid Feeding rate (%) Sericin-hope 37.1±6.5 Day 604 × Sericin-hope 92.0±2.9 MNS300 100.0±0.0 MCS300 99.8±0.2 MNS300×MCS300 99.9±0.2 (MNS300×MCS300)×Day 604 99.9±0.2

Sericin-hope does not ingest L4M artificial feed at all, even if the artificial feed is used for the original silkworm, it only ingests less than 40%. On the other hand, the F1 obtained by mating Sericin-hope with the polyphagous multifilament strain Ri 604 acquired polyphagy, and about 10% of the individuals ingested L4M. If it is used by the original silkworm, more than 90% of it will be fed. Furthermore, in the cocooning polyphagous sericin silkworm strain, the MNS300 described in Example 2, the MCS300 described in Example 3, and these F1 (MNS300×MCS300) and the F1 (MNS300×MCS300) and polyphagous The three-way hybrid individual (MNS300×MCS300)×Ni 604 obtained by mating the multi-filament strain Nichi 604 shows a very high feeding rate of 95% or more even for L4M, not to mention the original silkworm . The results showed that MNS300, MCS300, MNS300×MCS300 and (MNS300×MCS300)×Ri604 were all polyphagous sericin silkworm strains.

<Example 5>

(Purpose)

The production of the cocoons of the cocoon-growing polyphagous sericin silkworm strain obtained in Examples 2 and 3 was verified.

(method)

The basic method is according to Example 1. For silkworm strains, Sericin-hope, Hi 604 x Sericin-hope, MNS300, MCS300, and MNS300 x MCS300 were used. The respective strains were divided into 4 areas, respectively divided into the bait area (L4M) where SilkMate L4M was given to the whole larval stage, SilkMate L4M (Nippon Agricultural Industries) was given at the 1st to 4th instar, and SilkMate silkworms were given at the 5th instar The bait area (L4M→for the original silkworm) of (Nippon Agricultural Industry Industry Co., Ltd.), the bait area (for the original silkworm→L4M) given SilkMate for the original silkworm at the 1st to 4th instar and SilkMate L4M at the 5th instar, and the During the whole larval stage, SilkMate was given the bait area for the original silkworm (for the original silkworm) to feed, and after spinning the cocoon (cocoon spinning: moving the larvae to the cocoon), the cocoon yield of each individual was measured and the average value was calculated.

(result)

Table 3 shows the yield (g) of cocoons of the above five silkworm strains obtained in each bait area.

[table 3]

When administered to silkworms at the whole larval stage, the yields of MNS300, MCS300 and MNS300×MCS300 were 78-84 mg, which were higher than that of Sericin-hope (52 mg). MNS300, MCS300, MNS300×MCS300 can still feed normally even if the 5th instar silkworm is switched from the original silkworm to L4M, and the cocoon production is only slightly reduced compared with the case of feeding the original silkworm at the whole larval stage. In addition, when L4M was administered at the whole larval stage, even the smallest yield of MCS300 (57 mg) was higher than that of Sericin-hope (52 mg) administered to the original silkworm at the whole larval stage. From these results, MNS300, MCS300, and MNS300×MCS300 maintained a sufficient amount of multifilaments in the production of cocoons even if they were given SilkMate L4M, which is a low-cost artificial feed for polyphagous silkworms, at the whole larval stage.

<Example 6>

(Purpose)

The cocooning rate and pupation rate of the cocooning polyphagous sericin silkworm strains obtained in Examples 2 and 3 were verified.

(method)

The basic method is according to Example 1. In silkworm strains, Sericin-hope, MNS300, MCS300 and MNS300×MCS300 were used.

Mix 4 to 10 moths to hatch and transfer eggs. The hatched larvae are reared in small plastic containers with lids. It should be noted that, in this example, relative to any of the above-mentioned silkworm strains, the original silkworm was fed at the whole larval stage. After the 4th age, 80 animals/container were used for rearing in plastic containers. For cocoon spinning, mountain-shaped resin sacks (mannen sacks) are used.

(result)

The results are shown in Table 4. the

[Table 4]

Regarding the cocooning rate, Sericin-hope, MNS300, and MCS300 were approximately the same, but in MNS300×MCS300, an increase of about 5% was observed. For the pupation rate, MNS300×MCS300 was the highest, MNS300 was about 90%, and Sericin-hope and MCS300 were about 80%. This result proves that the cocoon-eating polyphagous sericin silkworm strain obtained in the method for establishing the polyphagous sericin silkworm strain of the present invention and its F1 maintenance and parent strain are the cocoon-eating property sericin silkworm strain (Sericin-hope silkworm strain). ) above the same level of cocooning and pupation rate.

<Example 7>

(Purpose)

The polyphagous sericin silkworm strain was used as a host for the production material, and the preparation of the recombinant peptide EGFP was carried out in this silkworm strain.

(method)

MNS300 and MNS300×MCS300 were used for the polyphagous sericin silkworm strain, and Sericin-hope was used as the control sericin silkworm strain. These silkworm strains were crossed with F1 (Ser1-Gal4×UAS-EGFP) obtained by mating the sericin promoter GAL4 silkworm strain and the UAS-EGFP silkworm strain. Here, in the Ser1-Gal4×UAS-EGFP silkworm strain, the GAL4 protein is expressed through the sericin promoter in the middle silk gland, and the expressed GAL4 protein binds to the recognition sequence UAS, thus, its downstream EGFP protein is expressed in the middle silk gland. Expressed specifically in silk glands.

For each hybrid of MNS300×(Ser1-Gal4×UAS-EGFP), (MNS300×MCS300)×(Ser1-Gal4×UAS-EGFP) and Sericin-hope×(Ser1-Gal4×UAS-EGFP), mix 4~ Eggs were hatched and transferred from 10 moths, and the entire larval stage was reared with SilkMate (Nippon Agricultural Industry Co., Ltd.). 80 animals/container were reared using plastic containers from the 4th age. Anesthetize silkworms at the 5th instar and 6th day before silking on ice, incise the dorsal side and remove them with tweezers without damaging the silk glands in the middle (refer to Yasushi Mori, edited by Kaiko ni Yoru New Biology 実験, Sanseido, 1970, pp.249-255), in 10 mL of extraction buffer (phosphate-buffered saline, pH 7.2, containing 1% Tween-20 and 0.05% sodium azide) with slow shaking at room temperature for 24 hours. Then, the EGFP-dissolved supernatant was collected by centrifugation at 2,000×g for 10 minutes. Quantification of EGFP was performed by ELISA (enzyme-linked immunosorbent assays, enzyme-linked immunosorbent assay) using Reacti-Bind Anti-GFP Coated Plates (Pierce, Rockford, IL, USA) according to the scientific experiment report. Specifically, each middle silk gland solution was injected into 3 wells of an ELISA plate and left at room temperature for 1 hour. Then, the wells were washed with phosphate-buffered saline buffer (pH 7.2) containing 0.05% Tween-20 buffer, and horseradish peroxidase-conjugated anti-GFP antibody (Rockland Immunochemicals, Gilbertsville, PA, USA) was added to make After reacting at room temperature for 1 hour, it was washed. Next, EGFP was developed using TMB Peroxidase EIA Substrate Kit (Bio-Rad, Hercules, CA, USA), and 1N H ₂ SO ₄ was added to stop the color development. Absorbance at 450 nm was measured using a plate reader (SpectraMax 250; Molecular Devices, Sunnyvale, CA, USA). A commercially available GFP protein (rGFP Protein; Takara Bio, Otsu, Japan) was used as a standard.

(result)

The results are shown in FIG. 6 . As shown in the figure, the EGFP expression level of MNS300×(Ser1-Gal4×UAS-EGFP) shown in A is almost the same as the EGFP expression level of Sericin-hope×(Ser1-Gal4×UAS-EGFP) shown in C. On the other hand, the EGFP expression level of (MNS300×MCS300)×(Ser1-Gal4×UAS-EGFP) shown in B is more than that of Sericin-hope×(Ser1-Gal4×UAS-EGFP) shown in C . This result shows that the polyphagous sericin silkworm strain of the present invention has usability as a host for producing substances equal to or higher than that of conventional sericin silkworm strains.

In addition, all the publications, patents, and patent applications cited in this specification are incorporated as a reference in this specification as it is.

Claims (14)

1. A method for establishing a wide-eating sericin silkworm strain, comprising: (a) the first mating process, mating the sericin silkworm strain having a mutation in at least one gene involved in the silk protein synthesis pathway with the polyphagous silkworm strain; (b) the second mating process, mating the hybrid individual after the first mating process with said or other polyphagous silkworm strains for at least 2 times; and (c) The polyphagous sericin silkworm selection step is performed 5 or more times of intra-strain mating, and individuals having the characteristics of the sericin silkworm strain and the characteristics of the polyphagous silkworm strain are selected. 2. The establishment method according to claim 1, wherein the mutation of the gene is a dominant mutation. 3. The establishment method according to claim 2, wherein the gene is Nd, Nd-s or Nd-s ^D . 4. The establishment method according to any one of claims 1-3, wherein the sericin silkworm strain is a cocooning sericin silkworm strain having a silk yield of 50 mg or more per one. 5. The establishment method of claim 4, comprising: (d) a cocooning mating process, mating the hybrid individual after the second mating process with the said or other cocooning sericin silkworm strain at least once; and (e) The selection process of cocooning polyphagous sericin silkworms, further selecting individuals with cocooning properties in the polyphagous sericin silkworm selection process after the cocooning mating process. 6. The establishment method according to any one of claims 1-5, further comprising: (f) A step of selecting homozygotes of the mutant gene. 7. A polyphagous sericin silkworm strain and its progeny, which have a mutation in at least one gene involved in the silk protein synthesis pathway. 8. The polyphagous silkworm strain and its progeny as claimed in claim 7, which are obtained by combining a sericin silkworm strain with a mutation in at least one gene involved in the silk protein synthesis pathway with a polyphagous silkworm strain The hybrid individuals obtained by mating the strains are mated with the said or other polyphagous silkworm strains at least twice; further, based on the characteristics of the sericin silkworm strains and the characteristics of the polyphagous silkworm strains, more than 5 intra-strain matings are carried out. 9. The polyphagous sericin silkworm strain and its progeny as claimed in claim 7 or 8, wherein the mutation of the gene is a dominant mutation. 10. The polyphagous sericin silkworm strain and its progeny as claimed in claim 9, wherein the gene is Nd, Nd-s or Nd-s ^D . 11. The polyphagous sericin silkworm strain and its progeny as claimed in any one of claims 7-10, wherein the sericin silkworm strain is a cocoon-forming sericin with a silk yield of 50 mg or more per one Silkworm strains. 12. The polyphagous sericin silkworm strain and its progeny as claimed in claim 11, after mating said hybrid individual with said or other polyphagous silkworm strains at least 2 times, with said or other cocoon-forming silkworm strains Sericin is obtained by mating silkworm strains at least once, and has cocooning properties. 13. The polyphagous sericin silkworm strain and its progeny according to any one of claims 7-12, which are homozygous for the mutant gene. 14. A method of using the polyphagous sericin silkworm strain according to any one of claims 7-13 or its progeny as a host for producing substances to produce recombinant peptides in silk glands of the silkworm strain.