JP2005143428A - Utilization of dna encoding kynurenine oxidase gene of silkworm - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for utilizing DNA encoding kynurenine oxidase gene of silkworm as a selection marker for a transgenic silkworm. <P>SOLUTION: A new vector plasmid to which a promoter of cytoplasmic actin of silkworm is linked is built on the upstream zone of kynurenine oxidase cDNA prepared from a wild silkworm in a vector derived from transposon piggyBac, and the vector plasmid is introduced into the egg of a white egg line silkworm obtained by making a silkworm of a first white egg mutation line (w-1) cross with a silkworm of a colored nondormant line (pnd). As a result, color of eyes of an adult moth or egg of a second-generation moth into which the vector is introduced is changed to blackish brown. The color of the skin of larva individual into which the gene is introduced is further changed to blackish brown and the change can be identified by naked eyes. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to the use of DNA encoding the silkworm kynurenine oxidase gene.

  Known methods for producing recombinant proteins using genetic engineering techniques include methods using E. coli and yeast, mammalian cultured cells, plants and animal individuals. As for recombinant protein production systems using insects, methods using lepidopteran insect nuclear polynuclear disease virus and methods using transgenic silkworms (Non-Patent Documents 1 to 7) have been developed. The former uses a DNA-type virus as a vector. Therefore, it is possible to produce a recombinant virus relatively easily and to infect larvae such as silkworms. After infection, the virus grows in the insect individual, and as a result, a recombinant protein is synthesized from the gene introduced into the virus. In this case, the larvae after the virus infection die after a few days, and the silkworm body fluid just before death is collected to purify the protein. Therefore, in many cases, the amount of protein that can be produced is 1 mg or less per 1 ml of body fluid, and there is a problem that a lot of labor is required for purification. Regarding transgenic silkworms, new methods for producing transgenic silkworms using transposons and marker genes have recently been developed (Non-Patent Documents 1 to 7). In this method, a DNA-type transposon piggyBac is used as a vector, and a green fluorescent protein (GFP) gene is used as a marker gene. Since this method can efficiently produce recombinants, it can be used in many practical aspects, such as the production of useful proteins, the production of new fibers, and the production of varieties with virus resistance. Some have already begun.

  However, to detect GFP used as a marker for screening recombinants, it is necessary to use an expensive fluorescent stereomicroscope, and in addition, this gene has been patented by others. Nevertheless, so far, no marker gene has been developed that does not require a special device and has no restrictions on its use.

Prior art document information related to the present invention will be described below.
Tamura Toshiki (1999) A method for producing transgenic silkworms using transposons. Abstracts of the 7th Insect Function Research Meeting, p10-22. Horn, C., B. Jaunich and EA Wimmer, (2000) Highly sensitive, fluorescent transformation marker for Drosophila transgenesis. Dev Genes Evol 210: 623-629. Horn, C., and EA Wimmer, (2000) A versatile vector set for animal transgenesis. Dev Genes Evol 210: 630-637. Thomas, JL, M. Da Rocha, A. Besse, B. Mauchamp and G. Chavancy, 2002 3xP3-EGFP marker facilitates screening for transgenic silkworm Bombyx mori L. from the embryonic stage onwards.Insect Biochem Mol Biol 32: 247-253 . Tamura, T., Thibert, C., Royer, C., Kanda, T., Abraham, E., Kamba, M., Komoto, N., Thomas, J.-L., Mauchamp, B., Chavancy, G., Shirk, P., Fraser, M., Prudhomme, J.-C. and Couble, P. (2000) A piggyBac element-derived vector efficiently promotes germ-line transformation in the silkworm Bombyx mori L. Nature Biotechnology 18 , 81-84. Berghammer, AJ, M. Klingler and EA Wimmer, (1999) A universal marker for transgenic insects. Nature 402: 370-371. Masahiro Tomita, Tsutomu Sato, Takayasu Adachi, Hiroto Munazuna, Toshiki Tamura, Toshio Kanda, Masaru Yoshizato (2001) Creation of transgenic silkworms incorporating human collagen genes. Abstract of the 24th Annual Meeting of the Molecular Biology Society of Japan. Tamura, Toshiki (2000) Transgenic silkworms: Current status and future prospects. Toshiki Tamura (2000) Introduction of useful genes in silkworm development. Report on 21st Basic Breeding Symposium: Development of developmental engineering methods in animal and plant molecular breeding. p23-29. Tamura, T., Quan, GX, Kanda, T., and Kuwabara, N. (2001) Transgenic silkworm research in Japan: Recent progress and future. Proceeding of Joint International Symposium of Insect COE Research Program and Insect Factory Research Project. -82. Tomita M, MH, Sato T, Adachi T, Hino R, Hayashi M, Shimizu K, Nakamura N, Tamura T, Yoshizato K., (2003) Transgenic silkworms produce recombinant human type III procollagen in cocoons. Nat Biotechnol 21, 52- 56.

  The present invention has been made in view of such a situation, and an object thereof is to develop a new marker gene for screening a silkworm recombinant. That is, an object of the present invention is to provide a method of using a DNA encoding a silkworm kynurenine oxidase as a selection marker for transgenic silkworms.

  There are many mutant strains in silkworms, and the causative gene of the first white egg mutation w-1, one of them, is kynurenine oxidase.・ It has been shown that 10 exons are deleted (Quan, G.-X., Kim, I. Komoto, N., Sezutsu, H. Ote, M. Shimada, T., Kanda T. , Mita, K., Kobayashi, M., and Tamura, T. (2002) Characterization of the kynurenine 3-monooxygenase gene corresponding to the white egg 1 mutant in the silkworm, Bombyx mori. Mol. Genet. Genomics 267, 1- 9). By introducing a wild type gene into a silkworm of a mutant strain, the mutant trait can be returned to the wild type. The present inventors considered that a wild type gene can be used as a marker gene for detecting a recombinant by using a mutant strain as a host for producing a recombinant. From this point of view, in the present invention, first, a new vector plasmid was constructed in which a silkworm actin promoter of silkworm was linked upstream of a kynurenine oxidase cDNA prepared from a wild-type silkworm to a vector derived from transposon piggyBac. Using the constructed vector, the actin promoter was added to silkworm silkworm eggs obtained by mating silkworms of the first white egg mutant line (w-1) and silkworms of the colored non-dormant egg line (pnd). When we introduced kynurenine oxidase cDNA with, we found that the eyes of adult pupae and the next-generation egg color with this gene turned brown. Surprisingly, it was found that the skin of the larvae into which this gene was introduced also turned brown and could be identified with the naked eye.

That is, the present invention provides the following [1] to [19].
[1] A method for changing the color of a silkworm, comprising introducing a DNA encoding a silkworm kynurenine oxidase into a silkworm egg having a mutation in a gene region encoding the kynurenine oxidase.
[2] The method of [1], wherein the silkworm skin, eyes, or eggs are discolored.
[3] Transgenic silkworm screening for DNA encoding kynurenine oxidase, including the step of introducing a DNA encoding silkworm kynurenine oxidase into a silkworm egg having a mutation in the gene region encoding kynurenine oxidase Method used as a marker.
[4] A method for producing a transgenic silkworm, comprising the following steps (a) and (b):
(A) a step of introducing a DNA encoding a silkworm kynurenine oxidase into a silkworm egg having a mutation in the gene region encoding the kynurenine oxidase (b) a silkworm generated from the egg into which the DNA has been introduced A step of selecting a transgenic silkworm [5] The silkworm having a mutation in the gene region encoding kynurenine oxidase is a silkworm of the first white egg mutant line [1] to [4] The method described.
[6] Silkworms having a mutation in the gene region encoding kynurenine oxidase are silkworms of the white egg line obtained by crossing silkworms of the first white egg mutant line and silkworms of the colored non-dormant egg line. [1] The method according to any one of [4].
[7] A transgenic silkworm, wherein a silkworm having a mutation in a gene region encoding kynurenine oxidase is introduced with a DNA encoding a silkworm kynurenine oxidase.
[8] The transgenic silkworm according to [7], wherein skin, eyes, or eggs are discolored.
[9] The transgenic silkworm according to [7] or [8], wherein the silkworm having a mutation in the gene region encoding kynurenine oxidase is a silkworm of the first white egg mutant line.
[10] Silkworms with mutations in the gene region encoding kynurenine oxidase are silkworms of the white egg line obtained by crossing silkworms of the first white egg mutant line and silkworms of the colored non-dormant egg line. [7] or the transgenic silkworm according to [8].
[11] A vector having a transposon inserted with a DNA functionally linked to a silkworm kynurenine oxidase DNA downstream of a silkworm cytoplasmic actin promoter region.
[12] A vector in which a DNA in which a DNA encoding a silkworm kynurenine oxidase is functionally linked is inserted downstream of a silkworm cytoplasmic actin promoter region between transposons' inverted terminal repeats.
[13] A kit comprising the vector according to [11] or [12] and a vector having a DNA encoding a transposon transferase.
[14] A drug for discoloring a silkworm having a mutation in a gene region encoding kynurenine oxidase, which contains, as an active ingredient, a DNA encoding a silkworm kynurenine oxidase.
[15] A drug for discoloring a silkworm having a mutation in a gene region encoding kynurenine oxidase, comprising the vector according to [11] or [12] as an active ingredient.
[16] A drug for discoloring a silkworm having a mutation in a gene region encoding kynurenine oxidase, comprising the kit according to [13] as an active ingredient.
[17] The drug according to any one of [14] to [16], for discoloring silkworm skin, eyes, or eggs.
[18] The drug according to any one of [14] to [17], wherein the silkworm having a mutation in the gene region encoding kynurenine oxidase is a silkworm of the first white egg mutant line.
[19] Silkworms with mutations in the gene region encoding kynurenine oxidase are silkworms of the white egg line obtained by crossing silkworms of the first white egg mutant line and silkworms of the colored non-dormant egg line. [14] The drug according to any one of [17].

  The present inventors use a silkworm having a mutation in the gene region encoding kynurenine oxidase as a host for producing a recombinant, whereby a DNA encoding a silkworm kynurenine oxidase using a silkworm actin as a promoter. Was found to be effective as a marker gene for detecting recombinants. Compared with the GFP gene used so far, this marker gene does not require a device such as a fluorescence microscope for detection, and can distinguish the recombinant with the naked eye. Therefore, a large number of individuals can be rapidly screened. In silkworms, recombinants into which DNA encoding kynurenine oxidase has been introduced have not been produced, and the effects of introduction and expression in individuals have not been investigated. Furthermore, it is considered to be effective for producing recombinant silkworms introduced with several kinds of genes in combination with fluorescent protein genes that have been used so far.

  The present inventors have developed kynurenine oxidation of silkworms to eggs of silkworms of white eggs obtained by mating silkworms of the first white egg mutant line (w-1) and silkworms of the colored non-dormant egg line (pnd). By introducing a vector plasmid linked to the silkworm actin promoter of silkworm upstream of the enzyme-encoding DNA, the skin of the larvae into which this gene has been introduced, the eyes of adult moths, and the color of the next-generation egg color change. I found out. The present invention is based on this finding.

  The present invention provides a method for changing the color of a silkworm, which comprises the step of introducing a DNA encoding a silkworm kynurenine oxidase into a silkworm egg having a mutation in the gene region encoding the kynurenine oxidase. Examples of the color change target by introduction of the gene include silkworm skin, eyes (compound eyes or monocular) or silkworm eggs, more preferably skin, compound eyes or eggs, and still more preferably skin of larvae. . The hue of the color after the discoloration is preferably a hue in the range of brown to red beans, more preferably brown or red beans, but is not limited thereto. Further, the density (lightness) and saturation of the color after modification are not particularly limited.

  Silkworm kynurenine oxidase has an enzymatic activity to convert kynurenine into 3-hydroxykynurenine. The activity is measured by the method of Ogawa et al. (Ogawa, H., and Hasegawa, K, (1975) Kynurenine 3-hydoroxylase activity and follicle development in the silkworm, Bombyx mori. Insect Biochem., 5, 119-134). be able to. The base sequence of the DNA encoding the silkworm kynurenine oxidase is shown in SEQ ID NO: 1, and the amino acid sequence of the silkworm kynurenine oxidase is shown in SEQ ID NO: 2.

  The DNA encoding the silkworm kynurenine oxidase of the present invention is not limited to the nucleotide sequence of SEQ ID NO: 1, but also includes a DNA encoding a protein functionally equivalent to the silkworm kynurenine oxidase. “Functionally equivalent” means having the activity to oxidize kynurenine to 3-hydroxykynurenine, and the presence or absence of this activity is a conventional method for measuring kynurenine oxidase activity (for example, Ogawa, H., and Hasegawa , K, (1975) Kynurenine 3-hydoroxylase activity and follicle development in the silkworm, Bombyx mori. Insect Biochem., 5, 119-134).

  The protein functionally equivalent to the protein includes a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence shown in SEQ ID NO: 2. In order to prepare DNA encoding a protein functionally equivalent to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, other methods well known to those skilled in the art include high-performance under stringent conditions. Hybridization technology (Southern EM: J Mol Biol 98: 503, 1975) and polymerase chain reaction (PCR) technology (Saiki RK, et al: Science 230: 1350, 1985, Saiki RK, et al: Science 239: 487, 1988) The method of using is mentioned.

  In the present invention, stringent hybridization conditions refer to hybridization conditions of 6M urea, 0.4% SDS, 0.5 × SSC or equivalent stringency. It is expected that DNA with higher homology can be isolated under conditions of higher stringency, for example, 6M urea, 0.4% SDS, 0.1 × SSC. High homology refers to sequence identity of at least 50% or more, preferably 70% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more of the entire amino acid sequence. In the mutant, the number of amino acids to be mutated is usually within 30 amino acids, preferably within 15 amino acids, more preferably within 5 amino acids, still more preferably within 3 amino acids, and even more preferably 2 amino acids. Is within. The identity of the amino acid sequence and base sequence is determined by the algorithm BLAST (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA 90: 5873, 1993). Can be determined.

  In addition, as a DNA encoding a silkworm kynurenine oxidase introduced into a silkworm egg in the present invention, a DNA functionally linked to a silkworm kynurenine oxidase DNA downstream of the silkworm cytoplasmic actin promoter region Is preferred. The present invention also provides such DNA.

  Here, “functionally bound” means that the transcription factor binds to the promoter region, so that expression of a DNA encoding a silkworm kynurenine oxidase existing downstream of the promoter region is induced. It means that the promoter and the DNA are bound. Therefore, even when the DNA is bound to another gene and forms a fusion protein with another gene product, the expression of the fusion protein is induced by the transcriptional regulatory factor binding to the promoter. Is included in the meaning of “functionally coupled”.

  The silkworm having a mutation in the gene region encoding the kynurenine oxidase in the present invention (including the coding region, the promoter region, and the untranslated region) is preferably a white egg as a result of the displacement at the w-1 locus. A silkworm of the first white egg mutant line (w-1), more preferably a non-white egg line obtained by crossing a silkworm of the first white egg mutant line and a silkworm of the colored non-dormant egg line. It is a dormant silkworm. The silkworm of the colored non-dormant egg line is preferably pnd or pnd-2, more preferably pnd, but is not limited thereto. The white egg strain silkworm obtained by mating the first white egg mutant silkworm and the non-dormant egg silkworm is preferably w-1, pnd, but is not limited to this, and other combinations are also possible. Is possible. Here, the non-dormant egg line means a mutant line having a property of producing a non-dormant egg. Further, a non-dormant egg refers to an egg that does not stop embryogenesis after laying but larvae hatch, and a dormant egg refers to an egg that temporarily stops embryo development after laying.

  In the present invention, a non-dormant egg can also be laid by a silkworm having the property of laying a dormant egg having a mutation in the gene region encoding kynurenine oxidase. That is, silkworms having a mutation in the gene region encoding kynurenine oxidase and silkworms having the property of producing dormant eggs (for example, the practical varieties Gunma, 200, Shunchu, Kangetsu, Nishikiaki, Kanwa, etc.) ), A non-dormant egg can be laid by a silkworm having a mutation in the gene region encoding kynurenine oxidase. The reason for the production of non-dormant eggs is that when a foreign gene is introduced as it is dormant eggs, it takes at least several months to hatch, and it is practically impossible to produce transgenic silkworms. As a method for producing non-dormant eggs, for example, in a gumma having a mutation in a gene region encoding kynurenine oxidase, culturing the dormant eggs at 15 ° C. to 21 ° C. to develop adults generated from the dormant eggs A method for producing a non-diapause egg, preferably a method for allowing a non-diapause egg to lay on an adult produced from the dormant egg by culturing the dormant egg at 16 ° C. to 20 ° C., more preferably a dormant egg at 18 ° C. A method of producing non-dormant eggs by allowing the adults generated from the dormant eggs to cultivate, most preferably culturing the dormant eggs at 18 ° C. to raise and grow the larvae generated from the dormant eggs. A method for allowing adults to lay non-dormant eggs can be mentioned. In addition, in 200 having a mutation in the gene region encoding kynurenine oxidase, a method of causing non-dormant eggs to lay on adults produced from dormant eggs by culturing dormant eggs at 15 ° C to 21 ° C, preferably A method of allowing non-dormant eggs to lay on adults produced from dormant eggs by culturing dormant eggs at 16 ° C. to 20 ° C., more preferably adults produced from dormant eggs by culturing dormant eggs at 18 ° C. By laying non-dormant eggs in the larvae, or by rearing the larvae generated from the dormant eggs in full light and allowing the grown adults to lay the non-dormant eggs, most preferably by culturing the dormant eggs at 25 ° C. Examples include a method in which the larvae produced from the dormant eggs are bred in full light and the grown adults lay non-dormant eggs.

  Egg culturing can be performed, for example, by placing it in an incubator at 18 ° C. to 25 ° C. or in a constant temperature room, and raising larvae can be performed using artificial feed in a breeding room at 20 ° C. to 29 ° C. .

  In the present invention, the day length condition means a daily light-dark cycle during egg culture or larval rearing. Such conditions include a light condition and a dark condition. In particular, the all-light condition means a 24-hour light condition without darkness. The day length condition can be changed according to the variety.

  Those skilled in the art can culture the dormant egg of the present invention according to a general silkworm egg culture method. For example, the culture is performed according to the method described in “Ministry of Education (1978) Soybean Manufacture.pp193, Jikkyo Publishing, Tokyo”. In addition, breeding of silkworm larvae in the present invention can be performed by those skilled in the art by a well-known method. For example, breeding is performed according to the method described in “Ministry of Education (1978) Soybean Manufacture.pp193, Jikkyo Publishing, Tokyo”.

  Regardless of whether the mutation is artificial or not, any silkworm having a mutation in the gene region encoding kynurenine oxidase is included in the silkworm of the present invention. For example, by using double-stranded RNA, it is possible to produce so-called transgenic silkworms that lack the functions of certain silkworm genes (Quan, GX, Kanda, T. and Tamura, T. (2002): Induction. of the white egg 3 mutant phenotype by injection of the double-stranded RNA of the silkworm white gene. Insect Molecular Biology 11217-222). In addition, so-called transgenic silkworms that lack the functions of certain genes in silkworms can also be produced by using viruses as vectors (Yamao, M., Katayama, N., Nakazawa, H., Yamakawa, M ., Hayashi, Y., Hara, S., Kamei, K., & Mori, H. (1999). Gene targeting in the silkworm by use of a baculovirus. Genes Dev, 13 (5), 511-516.) .

  Whether or not a mutation has occurred in the gene region encoding kynurenine oxidase in the silkworm thus produced can be determined by the color of the silkworm skin, eyes, eggs, or the like. If the skin, eyes, eggs, etc. of silkworm are discolored, it can be determined that a mutation has occurred in the gene region encoding kynurenine oxidase.

  For example, DNA can be introduced into silkworm eggs by injecting a transposon piggyBac into a silkworm early egg as a vector (Tamura, T., Thibert, C., Royer, C., Kanda, T., Abraham, E ., Kamba, M., Komoto, N., Thomas, J.-L., Mauchamp, B., Chavancy, G., Shirk, P., Fraser, M., Prudhomme, J.-C. and Couble, P., 2000, Nature Biotechnology 18, 81-84).

  For example, a transposon inverted terminal repeat (Handler AM, McCombs SD, Fraser MJ, Saul SH. (1998) Proc. Natl. Acad. Sci. USA 95 (13): 7520-5) A vector (helper vector) having a DNA encoding a transposon transferase is introduced into a silkworm egg together with a vector in which a DNA functionally linked to a DNA encoding a silkworm kynurenine oxidase is inserted downstream of the promoter region. Helper vectors include pHA3PIG (Tamura, T., Thibert, C., Royer, C., Kanda, T., Abraham, E., Kamba, M., Komoto, N., Thomas, J.-L., Mauchamp, B., Chavancy, G., Shirk, P., Fraser, M., Prudhomme, J.-C. and Couble, P., 2000, Nature Biotechnology 18, 81-84). It is not limited.

  The transposon in the present invention is preferably piggyBac, but is not limited to this, and mariner, minos, etc. can also be used (Shimizu, K., Kamba, M., Sonobe, H ., Kanda, T., Klinakis, AG, Savakis, C. and Tamura, T. (2000) Insect Mol. Biol., 9, 277-281; Wang W, Swevers L, Iatrou K. (2000) Insect Mol Biol 9 (2): 145-55).

  In the present invention, transgenic silkworms can also be produced by using baculovirus vectors (Yamao, M., N. Katayama, H. Nakazawa, M. Yamakawa, Y. Hayashi et al., 1999, Genes Dev 13: 511-516).

  Hereinafter, specific examples of the method for introducing DNA into silkworm eggs will be described, but the method for introducing DNA into silkworm eggs in the present invention is not limited to this method. For example, although it is possible to introduce DNA directly into the egg using a tube for DNA injection into a silkworm egg, in a preferred embodiment, a hole is made in the eggshell physically or chemically in advance, and from the hole, Introduce DNA. At this time, the DNA injection tube can be inserted into the egg through the hole so that the insertion angle is substantially perpendicular to the ventral side surface of the egg.

  In the present invention, as a method for physically making a hole in an eggshell, for example, a method for making a hole using a needle, a micro laser, or the like can be mentioned. Preferably, the eggshell can be pierced by a method using a needle. As long as the needle can make a hole in a silkworm eggshell, the material, strength, and the like of the needle are not particularly limited. The needle in the present invention usually refers to a rod-like needle having a sharp tip, but is not limited to this shape, and the overall shape is not particularly limited as long as it can make a hole in the eggshell. For example, a pyramidal material with a sharp tip or a triangular pyramid shaped material with a sharp tip is also included in the “needle” of the present invention. In the present invention, a tungsten needle can be preferably used. The thickness (diameter) of the needle of the present invention may be a thickness that can open a hole through which a capillary described later can be made, and is usually 2 to 20 μm, preferably 5 to 10 μm. On the other hand, as a method of chemically making a hole in an eggshell, for example, a method of making a hole using a chemical (such as hypochlorous acid) can be mentioned.

  In the present invention, the position for making a hole is not particularly limited as long as the insertion angle with respect to the side surface on the ventral side of the egg when a tube for DNA injection is inserted from the hole, but is not particularly limited, preferably It is the ventral side or the opposite side, more preferably the ventral side, and even more preferably the middle part of the egg on the ventral side slightly from birth.

  In the present invention, “substantially vertical” means 70 ° to 120 °, preferably 80 ° to 90 °. In the present invention, the “position to become a germ cell in the future” is usually a position close to the egg surface on the ventral side of the egg (usually a position of 0.01 mm to 0.05 mm from the egg table), preferably an egg This is a position near the egg surface in the middle of the ventral side, and a position slightly rearward.

  The DNA injection tube of the present invention is not particularly limited in the material, strength, inner diameter, etc. of the tube, but when inserting a hole in the eggshell physically or chemically before inserting the DNA injection tube, The thickness (outer diameter) is preferably such that it can pass through the vacated hole. Examples of the DNA injection tube of the present invention include a glass capillary.

  In the DNA introduction method of the present invention, as a preferred embodiment, a hole is physically or chemically formed in the silkworm egg, and a DNA injection tube is inserted at an angle substantially perpendicular to the ventral side surface of the egg. The step of inserting into the egg through the hole and injecting DNA is performed using a manipulator in which the needle and the tube for DNA injection are integrated. In general, the present invention is preferably implemented using an apparatus having the manipulator as one of the components.

  Such devices include a dissecting microscope, an illumination device, a movable stage, a coarse manipulator fixed to the microscope with metal fittings, a micromanipulator attached to this manipulator, and air pressure for injecting DNA. Consists of injectors to be adjusted. The pressure used for the injector is supplied from a nitrogen cylinder, and the pressure switch can be turned on by a foot switch. Injection is performed on an egg fixed on a substrate such as a glass slide, and the position of the egg is determined by a movable stage. The glass capillaries of the micromanipulator are operated by an operation unit connected by four tubes. The actual procedure is to determine the position of the tungsten needle relative to the egg with a coarse manipulator, and move the egg horizontally with the stage lever to make a hole. Subsequently, the lever of the operation part of the micromanipulator is operated to guide the tip of the glass capillary to the position of the hole, and the capillary is again inserted into the egg by the lever of the stage. In this case, it is necessary to insert a glass capillary perpendicular to the ventral side surface of the egg. Turn on the foot switch to inject DNA, and operate the lever to pull the capillary from the egg. The hole is covered with an instant adhesive and protected with an incubator at a constant temperature and constant humidity. The apparatus used in the present invention preferably includes the apparatus described in Japanese Patent No. 1654050 or an apparatus obtained by improving the apparatus.

  In the embodiment of the present invention, it is preferable that a silkworm egg used for DNA introduction is fixed to a substrate. As the substrate of the present invention, for example, a slide glass, a plastic plate or the like can be used, but is not particularly limited thereto. In the above embodiment of the present invention, it is desirable to fix the eggs in the same direction in order to accurately inject DNA into a position that will become a germ cell in the silkworm egg in the future. Moreover, in the said aspect, there is no restriction | limiting in particular in the number of the silkworm eggs fixed to a board | substrate. When a plurality of silkworm eggs are used, the directionality for fixing the silkworm eggs to the substrate is preferably such that the orientation of the dorsal belly is constant. The silkworm egg is fixed to the substrate of the present invention by, for example, laying eggs on a commercially available mount (rose seed mount) pre-applied with aqueous glue, adding water to the mount to peel off the egg, and then getting wet The eggs are aligned on a substrate and air-dried. The eggs are preferably fixed on the slide glass with the direction of the eggs aligned. Also, the egg can be fixed to the base by using a double-sided tape or an adhesive.

  As a method for examining whether or not DNA has been introduced into a silkworm egg, for example, a method of extracting and measuring the injected DNA again from the egg (Nagaraju, J., Kanda, T., Yukuhiro, K., Chavancy, G., Tamura, T., & Couble, P. (1996). Attempt of transgenesis of the silkworm (Bombyx mori L) by egg-injection of foreign DNA. Appl. Entomol. Zool., 31, 589-598) Transformal expression of chimeric CAT genes injected (Omura, T., Kanda, T., Takiya, S., Okano, K., & Maekawa, H. (1990). Transient expression of chimeric CAT genes injected) to early embryos of the domesticated silkworm, Bombyx mori. Jpn. J. Genet., 65, 401-410).

  Moreover, it becomes possible to select transgenic silkworms by using the above-described method. That is, the present invention also includes a method of using a silkworm kynurenine oxidase-encoding DNA as a selection marker for transgenic silkworms, which comprises the step of introducing a DNA encoding a silkworm kynurenine oxidase into a silkworm egg having a mutation in the gene region encoding kynurenine oxidase. provide.

  In the present invention, the “selection marker” means a marker that can be used as an index for selecting a transgenic silkworm that has changed color and a transgenic silkworm that has not changed color using the naked eye or a microscope.

  The present invention also includes a step of introducing a silkworm kynurenine oxidase-encoding DNA into a silkworm egg having a mutation in the gene region encoding the kynurenine oxidase, and the egg into which the DNA was introduced. Provided is a method for producing a transgenic silkworm, comprising a step of selecting a transgenic silkworm from silkworms. The above-described method can be used for the step of introducing the DNA into silkworm eggs. Moreover, as described above, transgenic silkworms can be selected using the silkworm color as an index.

  The present invention provides a transgenic silkworm, wherein a silkworm having a mutation in a gene region encoding kynurenine oxidase is introduced with a DNA encoding a silkworm kynurenine oxidase. Such silkworms include transgenic silkworms with discolored skin, eyes, or eggs. Preferably, the silkworm having a mutation in the gene region encoding kynurenine oxidase is a transgenic silkworm that is a silkworm of the first white egg mutant line, more preferably, a silkworm of the first white egg mutant line. It is a transgenic silkworm of a white egg line obtained by crossing a silkworm and a silkworm of a colored non-dormant egg line. The state of the transgenic silkworm is not particularly limited, and may be, for example, an egg state.

  The present invention provides a vector and kit for use in the above-described method. That is, a vector in which a DNA in which a DNA encoding a silkworm kynurenine oxidase is functionally linked downstream of the silkworm cytoplasmic actin promoter region is inserted into the transposon, more preferably a silkworm between the inverted terminal repeats of the transposon. A vector in which a DNA functionally linked to a DNA encoding a silkworm kynurenine oxidase is inserted downstream of the cytoplasmic actin promoter region is provided. Such vectors include the upstream region of the cytoplasmic actin gene between the inverted terminal repeats of the transposon of the piggyBac-derived vector (Tamura, T., et al., 2000, Nature Biotechnology 18, 81-84). Examples include a vector in which a DNA (polynucleotide sequence corresponding to SEQ ID NO: 3) in which a DNA encoding a silkworm kynurenine oxidase is functionally linked is inserted downstream of intron 675 bp. Also provided is a kit comprising the above vector and a vector (helper vector) having a DNA encoding a transposon transferase. Examples of the helper vector include pHA3PIG (Tamura, T., et al., 2000, Nature Biotechnology 18, 81-84). Furthermore, the present invention also provides use of the DNA, vector and kit of the present invention. In other words, it also provides use as a drug for discoloring silkworms with mutations in the gene region encoding kynurenine oxidase, which contains DNA, vectors, and / or kits encoding silkworm kynurenine oxidase as active ingredients can do.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
[Example 1]
The first white egg mutant line of silkworm (w-1) and the colored non-dormant egg line (pnd) for facilitating DNA injection into the early embryo of this line are obtained from the National Institute of Agrobiological Sciences did. By crossing these two lines and selecting a white egg line, a w-1, pnd line was produced. The eyes and eggs of the silkworm of the first white egg strain are white, the skin is brown immediately after hatching, and becomes white at the end of the first year. Note that the eyes and eggs of wild-type silkworms are brown and the skin is white after the third day of age.

  Wild-type kynurenine oxidase cDNA was prepared by Quan et al. (2002). Upstream of this cDNA, approximately 450 bp upstream of the silkworm cytoplasmic actin gene was ligated by PCR. In addition, the SV40 polyA signal was inserted downstream. The obtained gene was inserted into a vector pA3GFP derived from a transposon piggyBac to create a vector in which both GFP and kynurenine oxidase were inserted (FIG. 1).

[Example 2]
Next, the w-1 and pnd strain eggs used as the host strain were simultaneously injected with the newly prepared vector plasmid and a helper plasmid that produces a transferase that facilitates gene transfer. More specifically, a vector plasmid and a helper plasmid pHA3PIG (Tamura, T., et al., 2000, Nature Biotechnology 18, 81-84) are mixed, and 0.5 mM phosphorous containing 5 mM KCL at a concentration of 0.2 mg / ml, respectively. It was dissolved in an acid buffer and injected into eggs 3-6 hours after laying. DNA injection was performed by the method described in Tamura, T., et al., 2000, Nature Biotechnology 18, 81-84.

  Individuals hatched from the injected eggs were bred with artificial feed (Nippon Nosan Kogyo Co., Ltd.). As a result of injecting DNA into about 700 eggs and screening the next generation larvae of about 50 3, three target recombinant silkworms were obtained.

  In order to systematize these silkworms, w-1 and pnd, which are host strains, were backcrossed to the recombinant silkworms, and the next-generation silkworms hatched from the silkworms obtained were visually inspected for the first three days of larvae. As a result of observation with a fluorescent microscope, the skin of the larvae was brownish compared to that without gene insertion (FIG. 2). The separation ratio from the host-type skin larvae that did not turn brown was 1: 1, as can be seen from the figure. In order to confirm whether these silkworms are really recombinant silkworms, we examined the expression of GFP, another marker gene present in the vector, and as a result, all those whose skin color had changed expressed GFP. It was. Furthermore, as a result of investigating the color of these strains and the color of eggs in the next generation, as shown in FIG. However, the degree of brownishness was high in the recombinant line 129 in both the eye color and egg color, and the other two lines tended to be similar and slightly lower.

Example 3
Next, it was investigated whether or not the introduced gene was expressed in silkworms into which kynurenine oxidase was introduced. Silk glands were removed from individuals whose morphology had changed, DNA was purified, and Southern blotting confirmed that the gene had been inserted. In addition, gene expression was examined by RTPCR.

  As a result, as shown in FIG. 4, a band derived from the introduced gene was observed in any strain, and it was found that the gene was expressed in each tissue. The expression level was strong in line 129 and the other lines tended to be slightly lower. This result is in good agreement with the eye color and egg color results, and the degree of brownishness depends on the expression level of the introduced gene.

  Furthermore, in order to examine the gene introduction position in these strains, both sides of the insertion site were amplified by PCR, and the nucleotide sequence was determined by an autosequencer. As a result, the lines 50 and 66 were found to have the same sequence as shown in FIG.

  Based on the above results, the wild-type silkworm kynurenine oxidase gene that uses the cytoplasmic actin of silkworm as a promoter has the ability to change silkworm skin, eyes, and egg color to brown, and can be used as a marker gene for screening recombinants. It was revealed.

It is a figure which shows the structure of the vector used for recombinant silkworm production. SV40: SV40 poly A signal, GFP: green fluorescent protein gene, A3 and A3P: promoter region of silkworm cytoplasmic actin gene, KMO: wild-type silkworm kynurenine oxidase gene, R arm: right-hand inverted terminal of transposon piggyBac Repetitive sequence, L arm: transposon piggyBac left inverted terminal repeat, pUC18: plasmid pUC18 sequence It is a photograph showing a recombinant silkworm introduced with a kynurenine oxidase gene. Recombinant silkworms are indicated by arrows. It is a photograph which shows the color of the eye of an eagle and the egg color 3 days after egg-laying. w-1: The first white egg mutant line used as the host, 50: Recombinant silkworm strain 50 introduced with the kynurenine oxidase gene, 66: Recombinant silkworm strain 66 introduced with the kynurenine oxidase gene, 129: Kynurenin oxidation Recombinant silkworm strain 129 introduced with the enzyme gene, eye: eye of each eyelid, egg: egg color of each strain It is a photograph which shows the expression of the transgene in the larval stage of the recombinant strain into which the kynurenine oxidase gene was introduced. The gene expression level was examined by RTPCR by synthesizing cDNA from RNA extracted from each tissue. w-1: The first white egg mutant line used as the host, 50: Recombinant silkworm strain 50 introduced with the kynurenine oxidase gene, 66: Recombinant silkworm strain 66 introduced with the kynurenine oxidase gene, 129: Kynurenin oxidation Recombinant silkworm strain 129 introduced with the enzyme gene, KMO: expression of the introduced kynurenine oxidase gene, A3: expression of endogenous actin gene (control), 1: silk gland, 2: malbigy tube, 3: midgut, 4: Fat body. It is a figure which shows the insertion position of the gene in the recombinant silkworm strain | stump | stock which introduce | transduced kynurenine oxidase. The sequences on both sides of the transgene insertion site of each strain were amplified by PCR and examined with an autosequencer.

Claims (19)

A method for discoloring a silkworm, comprising introducing a DNA encoding a silkworm kynurenine oxidase into a silkworm egg having a mutation in a gene region encoding the kynurenine oxidase. 2. The method of claim 1, wherein the silkworm skin, eyes, or eggs are discolored. Using DNA encoding kynurenine oxidase as a selection marker for transgenic silkworms, including the step of introducing a DNA encoding silkworm kynurenine oxidase into silkworm eggs having a mutation in the gene region encoding kynurenine oxidase Method. A method for producing a transgenic silkworm, comprising the following steps (a) and (b):
(A) a step of introducing a DNA encoding a silkworm kynurenine oxidase into a silkworm egg having a mutation in the gene region encoding the kynurenine oxidase (b) a silkworm generated from the egg into which the DNA has been introduced Selecting a transgenic silkworm from The method according to any one of claims 1 to 4, wherein the silkworm having a mutation in the gene region encoding kynurenine oxidase is a silkworm of the first white egg mutant line. The silkworm having a mutation in a gene region encoding kynurenine oxidase is a silkworm of a white egg line obtained by crossing a silkworm of a first white egg mutant line and a silkworm of a colored non-dormant egg line. The method in any one of -4. A transgenic silkworm in which a silkworm having a mutation in a gene region encoding kynurenine oxidase is introduced with a DNA encoding a silkworm kynurenine oxidase. The transgenic silkworm according to claim 7, wherein the skin, eyes, or eggs are discolored. The transgenic silkworm according to claim 7 or 8, wherein the silkworm having a mutation in the gene region encoding kynurenine oxidase is a silkworm of the first white egg mutant line. The silkworm having a mutation in the gene region encoding kynurenine oxidase is a silkworm of a white egg line obtained by crossing a silkworm of the first white egg mutant line and a silkworm of a colored non-dormant egg line. Or the transgenic silkworm according to 8, A vector in which a transposon is inserted with a DNA functionally linked to a silkworm kynurenine oxidase downstream of the silkworm cytoplasmic actin promoter region. A vector in which a DNA functionally linked to a silkworm kynurenine oxidase DNA is inserted downstream of the silkworm cytoplasmic actin promoter region between transposons' inverted terminal repeats. A kit comprising the vector according to claim 11 or 12, and a vector having a DNA encoding a transposon transferase. A drug for discoloring silkworms having a mutation in the gene region encoding kynurenine oxidase, which contains DNA encoding silkworm kynurenine oxidase as an active ingredient. A drug for discoloring a silkworm having a mutation in a gene region encoding kynurenine oxidase, comprising the vector according to claim 11 or 12 as an active ingredient. A drug for discoloring a silkworm having a mutation in a gene region encoding kynurenine oxidase, comprising the kit according to claim 13 as an active ingredient. The agent according to any one of claims 14 to 16, for discoloring silkworm skin, eyes or eggs. The drug according to any one of claims 14 to 17, wherein the silkworm having a mutation in the gene region encoding kynurenine oxidase is a silkworm of the first white egg mutant line. The silkworm having a mutation in the gene region encoding kynurenine oxidase is a silkworm of the white egg line obtained by crossing a silkworm of the first white egg mutant line and a silkworm of the colored non-dormant egg line. The medicine in any one of -17.