JP2003088274A - Method for introducing polynucleotide to silkworm producing dormant egg - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for introducing a polynucleotide to a silkworm having property delivering dormant eggs and to provide a method for preparing a transgenic silkworm having the property delivering the dormant eggs by using the method. SOLUTION: This invention provides a method for making an adult insect to deliver nondormant eggs by culturing a dormant egg at 18-25 deg.C or feeding a larva produced from the dormant egg in full light in commercial varieties of silkworms such as Gunma and 200. A method for preparing the transgenic silkworm by introducing a plasmid DNA having a transposon as the vector to the obtained nondormant eggs is provided. The method for preparing the transgenic silkworm has a practical meaning such as high productivity of silk, robustness or the same and is expected as a method for producing a new transgenic silkworm having a newly introduced extraneous character to be used in various applications in farm field and drug industry.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for introducing a polynucleotide into a silkworm, which has the property of laying dormant eggs.

[0002]

2. Description of the Related Art It has not been possible for a long time to introduce a foreign gene into silkworm (Bombyx mori) to make a transgenic silkworm. Recently, Tamura et al.
A method for producing transgenic silkworms was established by injecting a plasmid DNA having a transposon as a vector into silkworm eggs in early development (Tamura, T.,
Thibert, C., Royer, C., Kanda, T., Abraham, E., Kamb
a, M., Komoto, N., Thomas, J.-L., Mauchamp, B., Ch
avancy, G., Shirk, P., Fraser, M., Prudhomme, J.-
C., and Couble, P. (2000). A piggyBac-derived vect
or efficiently promotes germ-line transformation i
n the silkworm Bombyx mori L. Nature Biotech., 18,
81-84.). As a result, it has become possible to produce silkworms that have genes of other organisms that silkworms did not have, and it has become possible to develop new products using silkworms. However, because practical silkworms bred by ordinary farmers have the property of laying dormant eggs, transgenic individuals could not be produced. Dormant eggs of silkworms can be released from diapause by treating them with hydrochloric acid 24 hours after spawning. Therefore, silkworms that have been soaked in acid can be used by ordinary farmers and practical varieties will lay dormant eggs. Nature was not a problem. But,
DNA for eggs to make transgenic silkworms
Since the injection must be performed within 6 hours after spawning, it is difficult to release the dormancy by the acid treatment, and such a technique has not been developed yet. Moreover, when used as a dormant egg, it takes at least several months until hatching, and it cannot be used for research to make transgenic silkworms. In addition, there is no report on the production of transgenic silkworms using dormant eggs as they are. Therefore, a non-diapause egg line that hatches immediately after spawning without any special treatment has been used. However, since non-diapause strains have hardly improved practical traits such as silk production efficiency and robustness, it was impossible to put the foreign silkworm-introduced silkworms to practical use.

If a method for producing a transgenic silkworm of a practical variety can be developed, the silkworm variety currently bred by a farmer can have an epoch-making new trait. Therefore, development of a method for introducing a polynucleotide into a practical silkworm cultivar having a property of laying dormant eggs has been demanded, but there have been no reports to date of the development of these methods.

[0004]

The present invention has been made in view of such circumstances, and an object thereof is to provide a method for introducing a polynucleotide into a silkworm having a property of producing dormant eggs. It is in.

[0005]

The present inventors cultivated dormant eggs at 18 ° C. or 25 ° C. in Gunma and 200, which are practical varieties of silkworms having the property of laying dormant eggs, or from dormant eggs. It was found for the first time that the grown larvae produce non-diapause eggs by rearing the resulting larvae under light conditions. Furthermore, a plasmid DN using the transposon as a vector to the obtained non-diapause silkworm eggs
By introducing A, it was revealed that transgenic silkworms are produced. These results indicate that a method for introducing a polynucleotide into a silkworm having the property of producing dormant eggs and a method for producing a transgenic silkworm for the silkworm having the property of producing dormant eggs have been developed. . By applying the newly developed transgenic silkworm production method to practical varieties, it has practical characteristics such as high productivity and robustness of silk, virus resistance, and spider-derived silk materials and It is possible to prepare a novel transgenic silkworm having a new gene introduced from the foreign such as a useful protein, and the method of the present invention is highly expected to be used in the fields of agricultural industry, pharmaceutical manufacturing, etc. It

That is, the present invention provides a method for introducing a polynucleotide into a silkworm having the property of producing dormant eggs, a method for producing a transgenic silkworm having the property of producing dormant eggs using the method, And a method for obtaining a non-diapause egg from a silkworm having a property of yielding a dormant egg, more specifically, [1] a method for introducing a polynucleotide into a silkworm having a property of yielding a dormant egg, comprising: , (A) Under the condition that the grown adult lays non-diapause eggs, the dormant eggs of the silkworm, which has the property of laying dormant eggs, are cultivated and the larvae are bred, and the grown adults produce non-diapause eggs. [2] a method comprising the steps of: (b) introducing a polynucleotide into the non-diapausing egg produced in step (a), [2] the method according to [1], which is a practical variety, [3] ] Practical varieties [4] The method according to [2], wherein the number of practical varieties is 200, [5] The silkworm having the property of yielding dormant eggs of step (a) is a non-diapause egg. The condition for giving birth to dormant eggs is 15 ° C.
The method according to [3], which is a condition of culturing at -21 ° C.
[6] In the step (a), the silkworm having the property of producing dormant eggs produces non-diapause eggs under the conditions of 15 ° C to 2 ° C.
The polynucleotide is introduced into the silkworm by the method described in [4] or the method described in any one of [1] to [6], which is a condition of culturing at 1 ° C. or a condition in which larvae are raised in total light. A method for producing a transgenic silkworm having a property of producing dormant eggs, the transgenic silkworm produced by the method according to [8] or [7],

[9] A method for obtaining non-diapause eggs of Gunma, comprising the steps of producing dormant eggs of Gunma at 15 ° C.
A method comprising culturing at 21 ° C. and producing non-diapause eggs from grown adults, [10] A method for obtaining 200 non-diapause eggs, wherein 200 dormant eggs are produced at 15 ° C. to 2 ° C.
A method characterized by culturing at 1 ° C. or raising larvae under all-light conditions, and producing non-diapause eggs from grown adults.
[11]

A non-diapause egg obtained by the method according to [9] or [10].

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention provides a method for introducing a polynucleotide into a silkworm, which has the property of laying dormant eggs. In this method, first, under the condition that the grown adult lays a non-diapause egg, the dormant egg of the silkworm, which has the property of producing a diapause egg, is cultured and the larvae are bred. Give birth to eggs.

The silkworm in the present invention usually refers to a silkworm having a property of producing dormant eggs, but the kind of silkworm is not particularly limited. Specific examples of silkworms that give rise to dormant eggs include Gunma, 200, Haruning, Kanetsuki, Nishiki, Kanewa and the like. In the present invention, a dormant egg refers to an egg in which post-spawning embryo development temporarily stops, and a non-diapause egg refers to an egg in which post-spawning embryo development does not stop and a larva hatches.
The conditions under which the grown adult of the present invention yields non-diapause eggs include:
It includes culture conditions for dormant eggs of the silkworm, which has the property of producing dormant eggs, and rearing conditions for larvae. The temperature condition is a particularly important factor under the culturing conditions of the silkworm dormant eggs, and the day length condition is the important factor under the larval rearing conditions. For example, in Gunma, there can be mentioned conditions for culturing dormant eggs at 15 ° C to 21 ° C, preferably conditions for culturing dormant eggs at 16 ° C to 20 ° C, and more preferably conditions for culturing dormant eggs at 18 ° C. . Most preferably, the dormant eggs are cultured at 18 ° C. and the larvae are reared in total light. Also, 200
In, the conditions for culturing dormant eggs at 15 ℃ ~ 21 ℃, preferably conditions for culturing dormant eggs at 16 ℃ ~ 20 ℃, more preferably conditions for culturing dormant eggs at 18 ℃, or larvae in total light The conditions for breeding can be mentioned. Most preferably,
It is a condition in which dormant eggs are cultivated at 25 ° C and larvae are bred in a clear light.

Cultivation of eggs can be carried out, for example, by placing them in an incubator at 18 ° C to 25 ° C or in a room at a constant temperature, and raising larvae is carried out in a breeding room at 20 to 29 ° C using artificial feed. be able to.

In the present invention, the day length condition means egg culture,
Alternatively, it means a light-dark cycle every day during rearing of larvae. Such conditions include bright conditions and dark conditions, and all-bright conditions mean 24-hour light conditions without darkness. The day length condition can be changed according to the variety.

Cultivation of the dormant eggs of the present invention can be performed by those skilled in the art according to a general method for culturing silkworm eggs. For example, culturing is performed according to the method described in “Ministry of Education (1978) Silkworm Seed Production. The breeding of silkworm larvae in the present invention can be carried out by a method well known to those skilled in the art. For example,
"Ministry of Education (1978) Silkworm Seed Production. Pp193, Education Publisher, Tokyo"
Breeding is performed according to the method described in.

In the present invention, whether or not the laid eggs are non-diapause eggs can be determined by the color of the eggs. It is generally known that dormant eggs are colored dark brown and non-dormant eggs are yellowish white. Therefore, in the present invention,
It is judged that the eggs laid are non-diapause eggs because they are not dark brown, more preferably yellowish white.

In the method for introducing a polynucleotide of the present invention, the polynucleotide is then introduced into the non-diapausing eggs that have been laid.

Examples of the above-mentioned polynucleotide in the present invention include gene DNA and RN which is a transcription product of a gene.
A, vector containing antisense RNA / DNA, gene DNA,
Examples thereof include a vector containing a transposon.

The introduction of the polynucleotide into the silkworm is carried out, for example, by the method of Kanda and Tamura (Toshio Kanda and Toshiki Tamura (1991).
Microinjection method into early stage silkworm embryos using air pressure. Silkworm insect research report, 3, 31-46.). As a method for examining whether or not the DNA has been introduced into the silkworm egg, for example, a method in which the injected DNA is extracted again from the egg and measured (Nagaraju,
J., Kanda, T., Yukuhiro, K., Chavancy, G., Tamur
a, T., & Couble, P. (1996). Attempt of transgenesi
s of the silkworm (Bombyx mori L) by egg-injection
of foreign DNA. Appl. Entomol. Zool., 31, 589-59
8) and a method of observing the expression of DNA as an injected gene in eggs (Tamura, T., Kanda, T., Takiya, S., Okano,
K., & Maekawa, H. (1990). Transient expression of
chimeric CAT genes injected into early embryos of
the domesticated silkworm, Bombyxmori. Jpn. J. Gen
et., 65, 401-410) and the like.

In the present invention, the use of the above-mentioned method for introducing a polynucleotide has enabled the production of transgenic silkworms having a characteristic of producing dormant eggs. Therefore, the present invention provides a method for producing a transgenic silkworm for a silkworm having a property of laying dormant eggs, which comprises introducing the polynucleotide into the silkworm by the method for introducing a polynucleotide.

The transgenic silkworms of the present invention are produced by culturing dormant eggs and rearing larvae under the conditions that the silkworms having the property of producing dormant eggs produce non-diapausing eggs. It is carried out by introducing a polynucleotide into a non-diapausing egg that has been laid. The selection of transgenic silkworms from the silkworms generated from the non-diapausing eggs into which the polynucleotide has been introduced can be performed using, for example, a selectable marker. As the selection marker in the present invention, a marker generally used by those skilled in the art, for example, a fluorescent protein such as GFP or oil silkworm that makes skin transparent, a wild type corresponding to a mutation such as a white egg whose egg color becomes white. And the like. Silkworms having these markers can be selected using, for example, fluorescence, body color, egg color and the like as indices. In addition, transgenic silkworms can also be produced by using a virus as a vector (Yamao,
M., Katayama, N., Nakazawa, H., Yamakawa, M., Haya
shi, Y., Hara, S., Kamei, K., & Mori, H. (1999). G
ene targeting in the silkworm by use of a baculovi
rus. Genes Dev, 13 (5), 511-516.). The polynucleotide of the above method of the present invention includes, for example, antisense RNA.
Silkworms in which the expression of a desired gene is controlled can be produced by using or DNA. Also, double-stranded RNA
It is also possible to produce so-called transgenic silkworms in which the function of a certain gene of the silkworm is defective by using
(Quan, GX, Kanda, T. and Tamura, T .: Induction
of the white egg 3 mutant phenotype by injection o
f the double-stranded RNA of the silkworm white ge
(Posted to ne. Insect Molecular Biology).

The present invention further provides a transgenic silkworm produced by the above method. The genotype or phenotype of the transgenic silkworm of the present invention is not particularly limited, and any silkworm with a modified gene is included in the transgenic silkworm of the present invention. Specific examples include a silkworm in which an exogenous polynucleotide is inserted into the chromosome and modified so that the polynucleotide is expressed as a gene, a silkworm in which the function of the target gene is deficient by the interference of double-stranded RNA, and the like. be able to.

The egg of the transgenic silkworm of the present invention is very useful because it can be stored as a dormant egg for a long period of time.
Further, the transgenic silkworm of the present invention, for example,
When a practical silkworm cultivar having practical traits such as high productivity and robustness of silk is produced, in addition to these traits, as a new silkworm with a new trait introduced from an alien, It is highly expected to be useful in the field of pharmaceutical manufacturing.

The present invention is also a method for obtaining non-diapause eggs of Gunma, characterized by culturing the dormant eggs of Gunma at 15 ° C to 21 ° C and producing non-diapause eggs from grown adults. And a method for obtaining 200 non-diapausing eggs, which comprises culturing 200 dormant eggs at 15 ° C to 21 ° C or rearing larvae under total light conditions, and producing non-diapausing eggs from grown adults. Is provided. In the method, dormant eggs are cultivated and larvae are bred under the conditions described above. Furthermore, the non-diapause eggs obtained by the above method of the present invention are also included in the present invention.

[0021]

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

[Example 1] Practical varieties are known to lay dormant eggs under normal breeding conditions. First, we attempted to develop a new method for obtaining non-diapause eggs for silkworms that have the property of producing dormant eggs (Fig. 1). It is already known that whether or not a practical variety lays non-diapause eggs depends on environmental conditions such as temperature and light at each development stage. Therefore, it was examined whether the grown adults produce non-diapause eggs by changing the growth conditions of dormant eggs and larvae of the practical silkworm variety. In general, the dormant eggs are colored dark brown and the non-diapause eggs are yellowish white, so that they can be easily distinguished (Fig. 2). Incubation at 25 ° C to see if the non-stained eggs were non-diapausing eggs revealed that all the non-stained eggs hatched within 2 weeks.

[0023] Next, the original species of the hybrid "Gunma x 200" bred by the Gunma silkworm test, the Japanese "Gunma" and the Chinese "20".
It was investigated whether the property of producing dormant eggs could be changed depending on the environmental conditions. In this case, silkworms can be bred throughout the year, and it is preferable to eliminate restrictions on the timing of their implementation. As a result of investigating the dormancy of the eggs laid by the adults in each group by changing the egg blueing temperature and the light and dark conditions during rearing of the larvae, the Japanese species "Gunma" incubated the eggs at 18 ° C It was revealed that the adult lays non-diapausing eggs when raised under light conditions (Table 1). The Chinese practical breed "200" is capable of producing non-diapause eggs by adults when the eggs are incubated at 18 ° C or the eggs are incubated at 25 ° C and the larvae are reared under all-light conditions. Was found (Table 1).

[0024]

[Table 1]

[Example 2] Next, the obtained non-diapause eggs were injected with a plasmid DNA having a transposon as a vector to try to produce a transgenic silkworm of a practical variety (Fig. 1).

Specifically, it was examined whether or not it is possible to introduce a foreign gene into the non-diapause eggs obtained by the method of Example 1 from the original variety "200" of the practical variety to prepare transgenic silkworms. Green fluorescent protein (G) was generated between the helper plasmid DNA and piggyBac prepared using piggyBac, which is one of the transposons, in eggs within 6 hours after spawning.
FP) gene-inserted vector plasmid DNA (FIG. 3) and another foreign gene inserted into this vector were injected together into an egg immediately after egg laying. First, 1445 non-diapause "200" eggs were injected with these DNAs, and 489 hatched, yielding 107 moths of the next generation. This was cultivated for each moth, and two days after the start of culturing, the presence or absence of fluorescence of larvae was observed with a fluorescence microscope. As a result, larvae fluorescing GFP were observed from three moths (Table 2).

[0027]

[Table 2]

The frequency of appearance of fluorescent silkworms is No. 16
17 out of 319 in moth ward, 14 out of 433 in No. 50 moth ward
The number of heads was 22 out of 358 in the No. 94 moth plot (Table 3).

[0029]

[Table 3]

Next, GFP was applied to the silkworms emitting these fluorescences.
It was investigated whether the gene was inserted. Adult larvae that fluoresce were bred to adult worms, DNA was extracted from the adult worms after completion of egg collection, and the presence of the GFP gene was examined by Southern hybridization analysis. When the DNA extracted from adults is treated with restriction enzymes (Eco RV, Xmn I) that cut inside the transposon used as a vector, the size of approximately 3.5 Kb is obtained if only the GFP gene is inserted. When a DNA in which another gene was inserted from the GFP gene was inserted, the DNA was treated with a restriction enzyme having a size larger than 3.5 Kb, and Southern hybridization analysis was performed using the GFP gene as a probe. As a result, as shown in FIG. 4, a 3.5 Kb band and a larger band than 3.5 Kb were detected. From this, it was revealed that a transgenic silkworm in which another foreign gene and the GFP gene were inserted into the chromosome was obtained. Next, as a result of investigating the expression of the GFP gene in the next generation of silkworm in which the GFP gene was confirmed by Southern hybridization, it was revealed that the GFP-expressing silkworm appears in the next generation at the frequency predicted by Mendel's law. It was

From the above results, it was made possible to produce transgenic silkworms using a silkworm cultivar having a property of laying dormant eggs having excellent practical traits by temporarily changing the property of laying dormant eggs. . This method is considered to be effective for introducing a foreign gene having various useful properties into a practical variety and developing a new method of using silkworm.

[0032]

INDUSTRIAL APPLICABILITY By the present inventors, a novel method for obtaining non-diapause silkworm eggs, a method for introducing a polynucleotide into silkworms having the property of producing dormant eggs using the method, and the method were used. A method for producing a transgenic silkworm, and a transgenic silkworm produced by the method for producing a transgenic silkworm are provided. According to the present invention, traits found in practical varieties, for example,
It has practical characteristics such as high productivity and robustness of silk, and
It is possible to produce a novel transgenic silkworm having a new trait introduced from an alien. The present invention is highly expected to be applicable in the agricultural industry field, pharmaceutical manufacturing field, etc. as a novel method for producing a transgenic silkworm having a property of producing dormant eggs.

[Brief description of drawings]

FIG. 1 is a diagram showing an outline of a method for introducing a polynucleotide into a silkworm having a property of producing dormant eggs of the present invention.

[Figure 2] Light and dark reared larvae of "200"
(C) is a photograph showing laying eggs (4 days after spawning). Discolored eggs can be judged to have become non-diapause eggs due to bright breeding.

FIG. 3 shows the structure of the vector used.

FIG. 4 is a photograph showing the results of GFP gene detection by Southern hybridization.

Continued front page    (72) Inventor Toshiki Tamura             50-1-1 Hitachinohigashi, Ushiku City, Ibaraki Prefecture (72) Inventor Toshio Kanda             1711-3 Off Arakawa, Ami Town, Inashiki District, Ibaraki Prefecture (72) Inventors             1-16-13 Matsushiro Tsukuba City, Ibaraki Prefecture (72) Inventor Hisashi Mori             214-4 Nakajima, Yoshii-cho, Tano-gun, Gunma (72) Inventor Nobuo Kuwahara             33-9 Tsurugayacho, Maebashi City, Gunma Prefecture F term (reference) 4B024 AA10 CA04 DA02 EA04 GA12                 4B065 AA90X AA99Y AB01 BA04                       CA60

Claims (11)

[Claims] 1. A method for introducing a polynucleotide into a silkworm having the property of producing dormant eggs, which comprises: (a) laying dormant eggs under the condition that grown adults produce non-diapause eggs. A step of culturing dormant eggs and breeding larvae of a silkworm having a characteristic, and letting grown adults produce non-diapausing eggs,
(B) introducing a polynucleotide into the non-diapausing egg produced in step (a). 2. The method according to claim 1, wherein the silkworm is a practical variety. 3. The method according to claim 2, wherein there are as many practical varieties as possible. 4. The method according to claim 2, wherein the practical variety is 200. 5. The condition in which the silkworm having the property of yielding a dormant egg in step (a) yields a non-dormant egg is 15
The method according to claim 3, which is a condition of culturing at from ℃ to 21 ℃. 6. The condition for the silkworm of the step (a) having the property of yielding dormant eggs to yield non-diapause eggs is 15
The method according to claim 4, which is a condition of culturing at a temperature of 21 ° C to 21 ° C, or a condition of rearing larvae in a clear light. 7. A method of producing a transgenic silkworm for a silkworm having a property of producing dormant eggs, which comprises introducing the polynucleotide into the silkworm by the method according to claim 1. 8. A transgenic silkworm produced by the method according to claim 7. 9. A method for obtaining non-diapausing eggs of Gunma, which comprises culturing the dormant eggs of Gunma at 15 ° C. to 21 ° C. and producing non-diapausing eggs from grown adults. 10. A method for obtaining 200 non-diapausing eggs, which comprises culturing 200 dormant eggs at 15 ° C. to 21 ° C. or rearing larvae under total light conditions, and producing non-diapausing eggs from grown adults. A method characterized by: 11. A non-diapause egg obtained by the method according to claim 9.