JP3023790B1 - Gene Any-RF, dormancy controlling substance and method for producing the same - Google Patents

Abstract

A gene having a dormancy controlling activity, a dormancy controlling substance, and a method for producing the same are provided. SOLUTION: It has an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, Asp-Ile-Leu-Arg-Gly, has an amidated C-terminal, has a molecular weight of 570.959, and has a dormancy controlling activity. Any-RF, a gene encoding a protein. The dormancy controlling substance having such a gene is a peptide which is isolated and purified by adding an acid methanol solution to the silkworm, grinding, centrifugation, and treatment by an HPLC system, and controls dormancy of insects and the like. .

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the gene Any-RF, a dormancy controlling substance and a method for producing the same, and more particularly to a gene having a dormancy controlling activity derived from an insect having a prelarval form, a dormancy controlling substance and a method for producing the same. It is about. In the present specification, the novel gene isolated and purified in the present invention is hereinafter referred to as "gene Any-RF"
Abbreviated.

[0002]

2. Description of the Related Art The earth on which humans live can be compared to an "insect planet". Many insects, known as 1 million species, live on the earth in all kinds of environments more than expected.
Insects adapt and survive almost everywhere on the planet, including the tropics, temperate forests, coniferous forests, ice and snow lands, deserts, and even lakes and marshes. This is because insects have acquired various functional properties. There is much to learn from the insect world. The functional characteristics of insects include biological defense mechanisms, growth and development control mechanisms, extensive substance decomposition, production functions, sharp sensory functions, behavioral regulation mechanisms, brain and nervous mechanisms, mediation functions, etc., or environmental adaptability It is. Among the various functional characteristics of insects, the dormant function of insects can be said to be a function that should be particularly noted as a wonderful adaptation phenomenon. The significance of science of insect dormancy is as follows. (1) Dormancy can be regarded as an energy-saving type of life phenomenon in which organisms completely stop growing in the process of their development and survive bad environments such as high and low temperatures and food shortages. . (2) Dormancy is genetic control or deciphering environmental information before the habitat adversely affects the growth and development, and the growth is stopped in advance. This is a strategic adaptation strategy. (3) If dormancy can be controlled, it will be an important practical technique for controlling pests and promoting germination of crop seeds, and may be an applied technique in the agricultural field.

[0003] Therefore, if the mechanism of dormancy control of insects is clarified and the structure of dormancy controlling substance is determined and its function can be elucidated, it can be applied to the field of biological industry. In other words, the development of useful organisms for human life
It is important from the perspective of the 21st century bioindustry, as growth can be controlled at will and pests can be artificially dormant to stop pest activity. Basic research. In addition, elucidation of the functions of dormancy control substances will be an important research topic as applied research for the development of agricultural technologies for high-quality and new products and the development of technologies for new drugs.

[0004] For example, the silkworm, a kind of lepidopteran insects, like many insects, enters dormancy in the early autumn and after 4 to 10
Waking up from dormancy in May. As with silkworms, the silkworm apparently diapauses embryos in eggs, but the larvae are almost completely formed in the eggs and enter into diapause in this state, which is a type of prelarval diapause (also called prelarval diapause). It is believed that there is. As this type, more than 40 species, mainly lepidopteran insects such as gypsy moth, other than natural silkworm, are known and should be classified as a new dormant type. The prelarval dormancy of the silkworm, Bombyx mori, is not directly related to the central hormonal system of the insect, but is controlled by a repressive factor (RF) present in the mid-thoracic region of the prelarva. A model has been proposed that is controlled by a maturation factor (MF) present in the five abdominal segments (Suzuki et al.,
J. Insect Physiol., 36, 855-860, 1990). The mature factor has been partially purified and reported to be a peptide-like hormone (Naya et al., Int. Wild Silkmoth
& silk I, 195-200, 1994), but a suppressor (a dormancy regulator) has not yet been isolated.

[0005] In the silkworm that diapauses embryos, the dormant hormone is known as an inducing hormone, and the structure of this hormone is a peptide hormone composed of 24 amino acids, and the C-terminal is amidated (Imai). et al.,
Proc. Japan Acad., 67B, 98-101, 1991 ). However, in the embryo dormant type, other hormones related to dormancy are completely unknown, and in the pre-larva dormant type,
No dorman-related hormonal substance has been found.

[0006] Apart from insects, only three kinds of substances called hibernation-specific proteins have been discovered in chipmunk (K
(ondo et al., J. Biol. Chem., 267, 473-478, 1992).
However, these proteins have molecular weights of 27-, 25-,
It is 20-kDa, and its blood concentration decreases before and after hibernation, and is low during hibernation. No dormant regulator has been discovered in mammals. For example, in the case of embryonic dormancy of a wallaby (a kind of kangaroo), the pineal gland of the mother is thought to be involved in dormancy. Has not yet been isolated.

[0007]

As described above, a gene encoding a protein having a dormancy controlling activity and a dormancy controlling substance having a useful dormancy controlling activity are derived from insects.
The hibernation-specific protein isolated from the chipmunk, which has not yet been isolated, has a high molecular weight, so that an antigen-antibody reaction is likely to occur.In addition, the blood concentration decreases before and after hibernation, and the concentration decreases during hibernation. Because there is a problem, and no dormancy controlling substance has been found in mammals so far, the development of a substance that controls dormancy is not only controlled by insect dormancy, but mammals that have been confirmed to have a dormancy phenomenon It is strongly desired from a rather broad perspective including the control of diapause and growth of squid.

Hitherto, insect dormancy has been classified as egg dormancy, larval dormancy, pupal dormancy, and adult dormancy. However, from a biochemical point of view, the diapause stage is complex, and the endocrine stage of prelarval diapause needs to be classified as a new stage. In other words, some do not simply belong to egg dormancy, such as the silkworm, and dormantly in the prelarval stage, while others, such as poplar bees, do not fall into the pupal dormancy and dormant in the prepupal stage, which is the previous stage. is there. Therefore, in these insect species, the existence of a new dormant control hormone or a related substance is expected.

[0009] In particular, more than 40 species of insects, mainly lepidopteran insects such as pre-larval dormant type silkworms, gypsum, gypsy moth, Usbashirocho, Okakareha and Kashiwamaimai (these pre-larval dormant type insects include Yoshiro Umeya Insect egg overwintering phenomena observed from wintering eggs of silkworms, report on silk test site,
12: 393-481 (1946) and Umeya Y., Studies on embryonic
hibernation and diapause in insects, Proc.
cad., 26, 1-9 (1950)), has not yet been isolated, and the isolation and identification of this dormant regulator is important for agriculture, forestry, There is a strong demand in the fields of medicine and the like, and development of a manufacturing technique for economically and efficiently synthesizing the same has been desired.

[0010] Natural silkworm (official name: Yamamayu, scientific name: Anther
aea yamamai Guerin-Meneville) is a country of origin, has a long history with records of breeding since the Edo era, the fact that artificial feed has been developed recently and larvae are easy to breed, and it is generally bred at the farmer stage. Because of this, there is a lot of information on breeding, and it is easy to obtain. While silkworm larvae exclusively eat mulberry leaves, natural silkworms eat leaves such as oak, oak, oak, abemaki. Whereas a sericulture farmer breeds the silkworm larvae, the wild silkworm larvae grow in their natural state. The hatching rate of the silkworm species is low, and the ratio of cocoon to silk thread (thread step) is extremely small. In addition, since it is difficult to take a cocoon thread, it has significance as a rare value. The price of 1kg of silkworm silk is said to be 200,000 or 300,000 yen,
This is why it is so rare that it is called a silk diamond. Silk fabrics in which the blending ratio of tussah silk yarn, which is a wild silkworm, is increased are preferably used because the slippage of the yarn is suppressed and the slip-out resistance of the seam can be improved. Therefore, in the future, the development of the field using large silk insects, natural silkworms, is greatly expected. Therefore, the importance of isolation and structure determination of suppressors related to the life cycle control of the silkworm, Bombyx mori, is increasing.

The reasons why the identification of dormancy controlling substances of the silkworm, Bombyx mori, has been delayed are as follows. The presence of dormant regulators was predicted in 1990 (Suzuki et al., J. Insec.
t Physiol. 36, 855-860, 1990) spent a lot of time improving the extraction method and selecting columns for purification, and failed to isolate and purify the expected active fraction. . The main reason is that the substance to be identified is a very small peptide of low molecular weight, and there were unexpected difficulties in extraction and selection of an appropriate column. Therefore, when obtaining a dormancy controlling substance from a pre-larva, establishment of a purification method excellent in yield, efficiency and economy and an easy production method are desired.

An object of the present invention is to provide a gene having a dormancy controlling activity, a dormancy controlling substance and a method for producing the same. For this purpose, a gene having a dormancy controlling activity and a dormancy controlling substance are efficiently, economically isolated, identified and purified from a prelarval body of an insect.

In order to solve the above problems, larvae of the silkworm, which is easily available and has a lot of information on breeding, are conveniently used as target insects, and the present invention will be described below mainly on the silkworm.

[0014]

Means for Solving the Problems The gene Any-RF of the present invention
Is the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, Asp-Ile-
It encodes a protein having Leu-Arg-Gly, amidated at the C-terminus, and having a molecular weight of 570.959, has dormancy controlling activity, and is derived from a pre-larva of the silkworm, Bombyx mori. The dormancy controlling substance of the present invention has an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, Asp-Ile-Leu-Arg-Gly,
The C-terminus is amidated and the molecular weight is 570.959
It is. This substance is obtained, for example, by pulverizing a pre-larval body of a lepidopteran insect such as a pre-larval body of a silkworm, such as methanol:
Water: added to an acid-methanol solution consisting of acetic acid, triturated in a mortar and centrifuged, and the resulting supernatant is isolated and purified by introduction into an HPLC system, or is obtained by known peptide synthesis. Obtained according to a known method using an apparatus.

Insects that are dormant in the prelarval body include not only natural silkworms, but also the above-mentioned gem moths, which are the major pests of the world's forests; Other larvae of 40 or more lepidopteran insects and larvae of orthoptera insects such as grasshoppers can also be applied and used.

As described above, the dormancy controlling substance of the present invention has Asp-Il
e-Leu-Arg-Gly, which is not a compound whose C-terminus is free-oxidized, but is amidated. This is evident from the bioassay test for the synthesized product described below, since only the amidated C-terminal has an active function. The C-terminus of the active substance is amidated, even if it is an insect-derived active peptide hormone having a relatively large molecular weight of 24 to 40 amino acids or a low molecular weight of 5 as in the present invention. I know that. In addition, a composition having an amino acid sequence similar to that of the dormancy controlling substance obtained from lepidopteran insects such as the silkworm can efficiently control the dormancy of the above-mentioned various lepidopteran and orthoptera insects. This is obvious from the point of view of the circadian rhythm (biological clock) of living things. That is, Beck, S.
T., Insect photoperiodism, pp. 288, Academic Pres
s, London (1968) and Takao Kondo, Biological Clock of Plants, Chemistry and Biology, 28: 810-819 (1990), reproductive activities of animals (including kangaroo dormancy) using circadian rhythms, It has been shown to control the developmental phenomena of many organisms, such as insect dormancy and flower bud formation (including dormancy) in plants.

[0017] As described above, the amino acid sequence structure of the peptide is inhibitory factors involved in the maintenance of prior larval dormancy wild silkworm is Asp-Ile-Leu-Arg- Gly-NH 2. This peptide was also revealed by computer search (BLAST and FASTA) to be a novel dormancy-controlling peptide in the living world, and was identified as Antheraea yamamai-Repr
It was named essive Factor (Any-RF). The present peptide, in which the C-terminal of 5 amino acids is amidated, was not found in the biological world as a free mode until the present invention was completed, but the amino acid sequence of some biological proteins was Some are the same --- Asp-Ile-Leu-Agr-Gly-
An array of-can be seen. For example, according to a computer search, the yeast hypothesis 22.1 KD protein (193
166 to 170 amino acids), 142 of human leukemia inhibitory factor precursor (202 amino acids)
The sequences up to # 146 are the same. However, the function of the amino acid sequence of this portion has not been clarified at all. That is, even though the amino acid sequence is the same, no peptide having a physiological function in a free existence mode by interposing between the N-terminus and the C-terminus and amidating the C-terminus has been found.

Thus, according to the present invention, a novel peptide hormone that controls dormancy of insects can be isolated and identified with a substance that has never been known in insect endocrinology, and the mechanism of insect dormancy mechanism has been clarified. Dormant organisms have been identified not only as insects but also marine products, plants, and mammalian wallabies (companies of kangaroos). It is an important clue to elucidate the life mechanism of certain low energy metabolism. In the future, it will be a substance that promotes long-term life support, and may eventually be a lead compound for the development of a longevity promoting substance.

The novel peptide of the present invention, which is found from the silkworm, is a pentapeptide composed of five amino acids, and does not easily cause an antigen-antibody reaction even when administered from outside the body. In addition, it has a growth inhibitory activity that delays the dormancy awakening of the silkworm, which is completely destined for dormancy awakening with the conventional artificial compound for breaking dormancy (dormant awakening), and reduces the dormancy awakening rate. From the above-mentioned literature, it is obvious from the literature that this structure can be directly administered to not only different insect species but also marine animals, plants, and mammals in view of the circadian rhythm of living organisms. In insects, this dormancy regulator is not limited to silkworms, but rather a wide variety of insect species.
Moreover, each stage can act as a variety of growth regulators. Therefore, the novel peptide of the present invention is not only a dormant regulator of pre-larval insects, but also has excellent physiological functions as a wide-ranging growth regulator including other insects and mammals.

In order to promote the diapause of insects, usually, (1)
As a method that can be applied to eggs at any time during dormancy,
The shell was removed using tweezers, the pre-diapause larva was taken out, and the imidazole compound (1-benzyl-5-[(E) -2,6-dimethyl-1,5-) dissolved in acetone was placed on the ventral surface of the pre-larva. Heptadienyl] imidazole (hereinafter KK-4
Also called 2. )) Is applied at 0.1 μg / 0.5 μl; and (2) As a method with a limited stage, it is stored at 25 ° C. for about one month after laying eggs, and then stored at 25 ° C. for 2 to 3 months.
And low-temperature treatment at ℃. In order to clarify the dormancy control function, a dormancy control substance was inoculated as follows into 24 hours after the pre-larvae destined to wake to dormancy by either of the above two methods. I just need.
For example, using a glass capillary tube, 0.05 μl of the dormancy controlling substance of the present invention at various concentrations dissolved in distilled water is inoculated percutaneously or orally from the neck or the like into the prelarva.

Further, in species other than insects, the peptide of the present invention is a kind of oligopeptide consisting of 5 amino acids, and therefore, is not only a potential as a dormancy controlling substance or a growth inhibitory substance, but also for example, mammals. The effects of sleep regulating substances, such as, are also expected. In particular, in higher animals, as described above, short, low-molecular-weight peptides such as the present peptide are unlikely to become antigens, and therefore, a simple study on possible functions by administration of a synthetic peptide from outside the body can be easily performed. It has the feature of being able to. Further, the novel peptide of the present invention can be a new pharmaceutical agent having no antigen-antibody reaction and having a cell growth inhibitory effect.

The above amino acid sequence Asp-Ile-Leu-Arg-
5'-GAY-ATH-YTN-MGN-GGN-3 'as base sequence for Gly
Can be considered.

The biologically active substances first discovered in insects such as ecdysone and juvenile hormone that control molting and metamorphosis are:
Today, they are found not only in insects, but also in crustaceans (crabs and shrimps), annelids (trophies and earthworms), and plants such as Togaribamaki and Hinata Kozuchi (eds. Seiichiro Kawashima, Endocrinology, pp. .159,199
5). A dormant hormone, which is known as a hormone that induces the dormancy of silkworms, is also known as FXPR, which has been found in other insects such as Bombyx mori and Armyworm.
It is said to belong to the amide family. Judging from these facts, the pentapeptide of the present invention is similarly singly used by insects other than the silkworm, since it is particularly commonly involved in the maintenance of dormancy, a common phenomenon of many insects. The technical probability of separation and identification is very high.

Furthermore, the natural silkworm, at the pupal stage as well as the prelarval stage, is dormant around August for a short period of about one month, as is evident from a physiological phenomenon called aesthesia. Insect pupa dormancy has been described as dormancy due to lack of molting hormone (ecdysone). Therefore, the pentapeptide discovered this time has very high technical probability of controlling not only the pupal dormancy of the silkworm, but also the pupal dormancy of other insects such as the cabbage butterfly, the tussah, the American white tortoise, and the tobacco. In other words, since pentapeptide has a function of maintaining a specific physiological state of low energy metabolism (refers to the function of maintaining dormancy of pre-larvae of the silkworm), the pentapeptide is a pupa and maintains the same physiological state for a long time. It is thought that pupal dormancy works in the same way. Therefore, similar pentapeptides can be found in pupal dormant cabbage butterflies, sacsans, white-winged squirrels and tobacco. On the other hand, not only pupal dormancy but also larval stages of other insects may be found. That is, as described above, in the case of insect hormones, different insect species and different stages may cause the same structural substance to have different functions. Thus, the same pentapeptide and its related substances can be identified from many insect species as dormancy regulators or growth regulators.

The pentapeptide can be extracted from the whole individual of such many insect species in the same manner as the pre-larvae of the silkworm, according to the method shown in FIG. In the case of direct extraction from body fluid, a part of a living tissue such as a leg of a larva body is incised to allow the body fluid to flow out, and methanol: water: acetic acid in FIG.
A 90% acid / methanol solution prepared by replacing a 90: 9: 1 water portion with a body fluid is mixed in ice cooling, and then mixed.
It may be extracted according to FIG.

[0026]

DETAILED DESCRIPTION OF THE INVENTION As described above, the novel dormancy controlling substance of the present invention, which is a novel peptide having a dormancy controlling function, has Asp-Ile-Leu-Ar amino acid sequence up to 5 cycles from the N-terminus.
g-Gly, amidated at the C-terminus and having a molecular weight of 5
It is a low molecular substance of 70.959, and can be isolated and purified from, for example, the prelarva of the silkworm, or can be synthesized by a conventional method since the amino acid sequence has been elucidated.

The novel peptide is expected to exhibit functions not only at the prelarva stage but also at other stages.
That is, insects generally hatch to the larval stage after the pre-larva (the larva body is formed in the egg). The same substance may be found in many insects, including the armyworm that sleeps at this larval stage. This is because it is well known that insect hormones exert different functions with the same substance in different insects and at different stages. For example, in the case of juvenile hormones, larvae of insects of the order Lepidoptera (for example, armyworm nymphs) act to maintain dormancy and maintenance of larval traits. It can be inferred from the fact that if it is missing, it will be dormant. Therefore, the novel peptide isolated from the silkworm this time is not limited to silkworms, but may be a wide variety of insect species, and may act as various growth regulators at each stage.

As a conventionally known substance related to insect dormancy,
As noted above, only a few dormant hormones have been reported in silkworms. It is a peptide amide, and many peptide hormones in insects fall into this group,
The ends are common and are Phe-X amino acid-Pro-Arg-Leu-amide. That is, it is a kind of FXPRL amide family. However, this dormant hormone is a hormone involved in inducing dormancy in silkworms and has no function of maintaining dormancy.

In the case of silkworms, the dormant hormone secreted from the sub-esophageal ganglion of the mother's pupa stage acts on the ovaries, and thereafter determines the dormancy of the embryos in the eggs laid. That is,
There is a temporal gap between the time of the action of the dormant hormone and the time when the dormancy is performed. However, the feature of the silkworm is that the action of the novel peptide and the execution of dormancy are at the same time. However, the novel peptides of the present invention are completely different from dormant hormones of known structure and function.

In the present invention, in order to isolate and identify a novel peptide from the pre-larvae of the silkworm, the pre-larvae are excised from the dormant eggs within one month after laying, frozen immediately with liquid nitrogen, and then frozen. -80 ° C until used in the isolation process
To save. In the isolation process, a 10-fold volume of an acid methanol solution (for example, methanol: water: acetic acid = 90:
9: 1), grind in a mortar, and 10,000 g for 3
Centrifuge for 0 minutes to prepare the supernatant. This operation 3
Iteratively, the combined supernatant may be concentrated with a centrifugal vaporizer, and the resulting supernatant may be introduced into an HPLC system.

The reason why acetic acid is used for the acid methanol solution is as follows. This extraction method is also used when extracting peptide hormones from other insects. By adding acetic acid, it is possible to suppress protease activity of 90% or more, and to suppress peptide degradation. it can. As the acid methanol liquid, methanol: water: acetic acid =
90: 9: 1 (% by volume) is optimal, but is not particularly limited to this range.

As described above, it is best to use a dormant egg within one month after laying eggs as described above, for the following reason. It is considered that the dormancy depth is strong within about 20 days after the onset of dormancy from about 10 days after spawning, but the dormancy of insects generally becomes shallower as time elapses. In the present invention, the prelarvae 1,
The dormancy control substance, a hormone-like substance finally obtained from 500 animals, is considerably small. Activity of Table 1 and about 57
Estimating from a molecular weight of 1, the prelarva of the silkworm, 1,50
Only 21.4 μm of the insect dormancy controlling substance of the present invention from 0 animals
It is calculated that g can be isolated. However, compared to silkworms, the natural silkworm has not developed a perfect artificial feed and has a difficulty in spawning technology, so it costs as high as 5 to 20 yen per egg. Therefore, it is not efficient and economical to prepare the novel substance of the present invention using the silkworm.

In general, the important factors in the extraction of hormone-like substances are what to select as a raw material to obtain a high recovery rate and what to select as a column resin for efficient separation. However, if it can be synthesized, there is nothing better than that. However, since the amino acid sequence of the dormancy controlling substance has been determined as described above, an active substance having this sequence can be economically and efficiently synthesized by a conventional method.

If the gene of the novel peptide can be identified, it can be widely applied not only to insects but also to the field of the biological industry including many biological species, and it is possible to freely control the growth of living organisms. Therefore, an antisense recombinant of this peptide, that is, a gene that is paired with the peptide gene, is introduced, and a method of suppressing the original gene expression is used to create a non-dormant gypsy moth. Gypsum populations have declined and are expected to make a significant economic contribution to the North American forest industry. Further, by using the peptide as a medium additive for various cultured cells, long-term preservation of the cultured cells becomes possible, which leads to the development of a long-term preservative for the cultured cells, and finally, the insect cultured cells Can be the core of technology in developing long-term preservatives for

It has been reported that a high molecular weight protein from the pupa of the cabbage butterfly can suppress the growth of human cancer cells (Koyama et al, Jpn. J. Cancer Res., 87, 12).
59-1262, 1996), the antigen-antibody reaction becomes a major obstacle when it is actually used. However, the peptides of the present invention
An oligopeptide consisting of only 5 amino acids,
It has an excellent feature that it does not become an antigen. This is because the antigen-antibody reaction by vertebrate immunoglobulins does not work with small oligopeptides that enter from outside the body.

[0036]

Next, the present invention will be described in detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples. (1) Isolation of Active Fraction The dormancy controlling substance of the present invention was isolated and purified as follows according to the process shown in FIG. 1 and used in the following Examples.

The prelarvae were excised from the dormant eggs of the silkworm within one month after laying eggs, immediately frozen with liquid nitrogen, and then stored at -80 ° C until use. About 1,500 (about 6 g)
10 times the volume of acid methanol solution (methanol:
(Water: acetic acid = 90: 9: 1 (% by volume)), and the mixture was ground in a mortar and centrifuged at 10,000 g for 30 minutes to obtain a supernatant. This operation was repeated three times, and the combined supernatant was concentrated with a centrifugal vaporizer. The concentrate was heat-treated at 100 ° C. for 10 minutes and centrifuged at 10,000 g for 15 minutes. Cold acetone was added to the supernatant thus obtained so that the final concentration of the supernatant was 80%, and the mixture was centrifuged at 10,000 g for 15 minutes to obtain a precipitate. Dissolve this precipitate in water,
The solution passed through a filter (mesh size: 0.5 μm) was eluted twice by high-performance liquid chromatography using a reversed-phase column, and finally was subjected to elution processing by high-performance liquid chromatography using a mixed separation mode column to obtain an isolated and purified product. .

In the first reversed phase high performance liquid chromatography (RP-HPLC) in this isolation method, TSKgel was used as a column.
The second RP-H using ODS-80Ts (Tosoh Corporation)
The same columns were used in the PLC system. In the third HPLC system using a column having a reverse phase and an ion exchange mode, an RSpak NN-614 column (manufactured by Showa Denko KK) was used. In each system, the concentration fraction of acetonitrile (%) was changed while adding acetonitrile to a 0.1% trifluoroacetic acid (TFA) aqueous solution (vol%), and the active fraction was eluted using the concentration gradient. did. The absorbance is measured at 220 nm and the flow rate is 1 ml / min or 0.5 m
1 / min. (2) Biological Assay Generally, non-dormant type is used to measure inhibitory factor activity, and a dormancy induction rate is used. Therefore, dormant prelarvae were treated with imidazole compound KK-42 for dormancy awakening (Suzuki e
t al., Int.Society for wild Silkmoth, 79-84, 198
9; Suzuki et al., J. Insect Physiol., 855-860, 199
0) After 24 hours, the active fraction was injected, and the sleep control function was determined based on the delay in the average number of days until sleep awakening and the decrease in the sleep awakening rate. (3) Determination of N-terminal amino acid sequence The N-terminal amino acid sequence was determined using a protein sequencer. The N-terminal amino acid sequence of the isolated / purified dormancy control substance was analyzed using a peptide sequencer G1000A (Hewlett Packard). (4) Mass spectrometry MALDI-TOF MS (Matrix-Assisted Laser Desorption Ion
(Time-of-Flight mass spectrometer) (Voyager PerSept
ive Biosystems) and a sample obtained by synthesizing a peptide having the same amino acid sequence as this substance and a peptide having the same amino acid sequence by a known method in an amount of 0.5 μl to each well of the sample plate of the mass spectrometer. Inject and mix with an equal volume of matrix (a mixture of 0.1% TFA aqueous solution and acetonitrile in a ratio of 50:50 (vol%) saturated with α-cyano-4-hydroxycinnamic acid) did. After drying, the molecular mass was determined as a cationization matrix. (5) Synthetic peptide A peptide that completely matches the primary structure of the dormant regulator of the present invention isolated and purified as described above was synthesized using a peptide synthesizer (PSSM-8; manufactured by Shimadzu Corporation). . The purity of the synthetic peptide was confirmed by HPLC and MALDI-TOF MS. Asp-Ile-Leu-Agr-Gly-NH as a peptide
₂ and Asp-Ile-Leu-Agr-Gly-COOH were synthesized,
It was used in the following examples. Example 1 Isolation of Active Fraction A dormancy controlling substance was isolated from about 1,500 pre-larvae of the silkworm,
For purification, the process detailed in the section “Isolation of the active fraction” above was used. In the first reverse-phase HPLC system, the active fraction was eluted at elution times of 11 to 17 minutes (FIG. 2). Second reverse phase HPLC with injection of this active fraction
The system identified an active fraction in the first peak eluting at about 3 minutes (FIG. 3). Next, in the third HPLC system into which the active fraction was injected, activity was observed in a peak eluted at about 9.5 minutes (FIG. 4). Assuming that the isolation and purification were completed by this series of operations, the final active fraction from about 1,500 pre-diapause larvae was analyzed with a peptide sequencer (G1000A manufactured by Hewlett Packard) as described below. . Example 2 Determination of Structure of Control Substance 100 μl of the dormant control substance obtained in Example 1 was dissolved in pure water, and N was dissolved in an aqueous solution containing 25 μl of the dormant control substance.
Sequence analysis was performed up to 10 cycles from the end. As a result, the amino acid sequence of the active substance having the dormancy inhibitory activity is Asp-Ile-
It was confirmed to be Leu-Agr-Gly. C of the active substance
Terminal is amidated (-NH ₂ ) or free oxidation
(-COOH), this isolated and purified product and the synthetic peptide were subjected to MALDI-TOF MS (mass spectrometry).
Was analyzed by 571.858 in the isolated and purified product
When 572.846 and two large peaks were observed (Fig. 5), the maximum peak was observed at Asp-Ile-Leu-Agr- Gly-NH 2 at 571.959 synthetic peptides (Fig. 6), and synthetic 573.045 for Asp-Ile-Leu-Agr-Gly-COOH
A maximum peak was observed in FIG. 7 (FIG. 7).

Thus, since it is considered that both types of amino acid sequences exist in the living body, the following examples were used in order to clarify which C-terminal the true active body has. Then, the physiological activities of the isolated and purified product and both types of synthetic peptides were examined. Example 3 Bioassay of isolated and purified product and synthetic peptide An isolated and purified product, having the same amino acid sequence, and
Two types of synthetic peptides, of which the C-terminus is amidated or free-oxidized, are injected into the dormant pre-larvae of the dormant Bombyx mori, treated with the imidazole compound KK-42 to fate dormancy awakening. The state of diapause and the average number of days until diapause were examined, and the diapause rate was evaluated. That is, KK-42 dissolved in acetone was applied to the abdomen of the larva in an amount of 0.1 μg / 0.5 μl per animal to wake the larva to 100% dormancy. After dormancy awakening treatment, 0.05μ per animal at 24 hours
One injection of 1 L of distilled water was used as a control. In the case of an isolated and purified product, 0.05 μl of distilled water is injected after adjusting the concentration so that 100 pmols of the isolated and purified product from the pre-larva is contained. Is 100 pmols / 0.
Injected 05 μl. In each case, the number of individuals that were dormant, the number of individuals that awakened to dormancy, the number of dead individuals, and the average number of days required for awakening of dormancy were examined, and the dormancy awakening rate was evaluated. Table 1 shows the obtained results. As is clear from Table 1, the average number of days to awakening of dormancy in the control group was 5.79 days, and the dormancy awakening rate was 100%. In the isolated and purified product, the average number of days to awakening of dormancy was 6.46 days, which was extended by about one day compared to the control group, and the dormancy awakening rate was reduced to 43.3%. In the synthetic peptide, the free acid type is similar to the control group in terms of both the average number of days until dormancy awakening and the dormancy awakening rate, and the average number of days until dormancy awakening and the dormancy awakening rate of the amidated type are data of isolated and purified products. Was very similar to The above results demonstrate that the C-terminus of the isolated and identified peptide is amidated, which is important as a physiological function for maintaining dormancy.

Therefore, the amino acid sequence of the inhibitory factor derived from the silkworm
Column structure is Asp-Ile-Leu-Arg-Gly-NH _TwoAnd its molecules
The mass is calculated from the peak measurement of the mass spectrum.
It is sufficient to reduce the mass 1 of the component, which is 570.959 Da.
You can see that. Example 4 Physiological Activity of Synthetic Peptide The peptide of the present invention whose structure was determined (C-terminal was amidated)
Peptide with the same structure as above
And elucidate the relationship between the activity and the concentration of the synthetic peptide.
Was analyzed. That is, the imidazole compound KK-42 (0.1
μg / 0.5 μl) to the dormant pre-larval larvae
Sleep awakening treatment and synthetic pep
How much can you prevent dormancy by injecting pide
Number of dormant individuals, number of awake dormants, number of dead
The body number was examined and the dormancy awakening rate was evaluated.

First, the imidazole compound KK-42 having a diapause awakening effect was dissolved in acetone, and this solution was applied to the ventral side of the dormant pre-larvae of the silkworm, 0.1 μg / 0.5 μl per animal. 0.05 at 24 hours after treatment with KK-42
μl of distilled water, then a second injection of distilled water at 24 hours, and a third injection of distilled water 24 hours after the second injection (Table 2 below). Inside
(Represented as Control-1, 2, 3), respectively, and the dormancy awakening rate was evaluated at each time point. In addition, a synthetic peptide having an activity to maintain dormancy of the silkworm, 100 per animal,
pmoles / 0.05 μl injection (1 like control)
, Two, three injections), the dormancy awakening rate was evaluated for each plot. Table 2 shows the obtained results. As is clear from Table 2, the numbers of the dormant larvae, dormant awake larvae, and dead larvae of the pre-larvae and the dormancy awakening rate show a similar tendency among the controls-1, 2, and 3. That is, even if the number of injections of distilled water is increased, almost all of the prelarvae are in a diapause state. This means that treatment with KK-42 is destined to awaken dormancy of the prelarvae of the silkworm, but that injection of distilled water does not interfere with the activity of KK-42.
On the other hand, in the case of the synthetic peptide, the dormancy awakening rate can be reduced from 53.3% to 30% when injected twice compared with the single injection, and further increased to 13.3% when injected three times. Can be reduced. The above specifically demonstrates that the novel peptide whose structure has been determined is a factor that maintains dormancy. Example 5 Demonstration of Dormancy Control Factor by Antiserum Injection Cys-ε in which ε-Acp and cystine are bound to the peptide of the present invention
-Acp (aminocaproic acid) -Asp-Ile-Leu-Arg-Gly-NH ₂ (CX
It was synthesized DILRG-NH _2), using the KLH as a carrier protein, peptide ~KLH create a conjugate which was injected to immunize domestic rabbits with Freund's complete adjuvant as immunogen. After confirming antibody production by ELISA, whole blood was collected after a 2-month immunization period. The serum from which the antibody was produced in this manner was used as an antiserum in the experiment. The blood collected before the injection did not produce any antibody, and was used as a preserum in the control.

The dormant pre-larval larvae were injected with 0.1 μl or 0.2 μl per animal of preserum and antiserum, respectively. After that, the number of dormant individuals,
The number of dead individuals was examined, and the dormancy awakening rate was evaluated. Table 3 shows the obtained results. As is clear from Table 3, when the control group was injected with the preserum, no pre-larvae awakening to dormancy were observed in both 0.1 μl and 0.2 μl, and all the larvae remained dormant. However, when the antiserum was injected, 10% at 0.1 μl,
With 0.2 μl, about 37% of dormancy could be awakened, and the dormancy awakening rate increased with an increase in the injection volume. These results demonstrate that the peptide of the present invention is a substance that controls the maintenance of dormancy in vivo. FIG. 8 (A)
FIG. 8B is a photograph showing the state of diapause continuation of the prelarvae of the silkworm, which had been injected with 0.2 μl of preserum, and FIG.
1 is a photograph showing a state of awakening of dormancy in a pre-larval larvae injected with antiserum. From the results shown in Table 3 and FIG. 8, it was proved that the peptide of the present invention whose structure was determined was a factor that controls the maintenance of dormancy in the pre-larva of the silkworm, Bombyx mori.

[0043]

According to the dormancy controlling substance having the gene Any-RF of the present invention, even if the dormant pre-larva or the like is awakened to dormancy and is destined for development, it can prolong or stop dormancy awakening. can do. Various physiological activities and simple administration methods can be expected for the peptide which is a dormant substance.

Since the mechanism of the insect dormancy mechanism has been elucidated by the present invention, it is essential that these organisms dormant by using the dormancy controlling substance of the present invention for organisms that have been confirmed to be dormant. It is an important clue for elucidating the life mechanism of low energy metabolism. In the future, it will lead to the development of a substance that promotes long-term life support, and may eventually be a lead compound for the development of a longevity promoting substance.

Further, the peptide of the present invention is a pentapeptide, which is unlikely to cause an antigen-antibody reaction even when administered from outside a living body, and has a remarkable growth inhibitory activity. Can be administered directly to dormant organisms. In particular, even in higher animals, short, low-molecular-weight peptides such as the present peptide are unlikely to become antigens. I have. Further, the peptide of the present invention can be a new pharmaceutical agent having no antigen-antibody reaction and having a cell growth inhibitory effect.
Furthermore, the peptide of the present invention can also be a preservative for cultured cells.

[0046]

[Sequence list]

SEQUENCE LISTING <110> Kimura, Shigeru; Director General of Nationa
l Institute of Sericultural and Entomological Scie
nce Ministry of Agriculture, Forestry and Fisherri
es <120> Substance for Repressing Diapause of Insects
and Method of Obtaining the Same <130> 990 137 <160> 1 <210> 1 <211> 5 <212> PRT <213> Antheraea yamamai Guerin-Meneville <400> 1 Asp Ile Leu Agr Gly 1 5

[Brief description of the drawings]

FIG. 1 is a flow sheet showing a process for isolating and purifying a dormancy controlling substance of the present invention.

FIG. 2 is a spectrum showing an elution peak of an active fraction by a first reverse-phase HPLC system in a process for isolating a dormant control substance of the present invention.

FIG. 3 is a spectrum showing an elution peak of an active fraction by a second reversed-phase HPLC system in the process of isolating the dormant control substance of the present invention.

FIG. 4 is a spectrum showing an elution peak of an active fraction by a third HPLC system in the process of isolating a dormant control substance of the present invention.

FIG. 5 is a mass spectrometry spectrum obtained by measuring the molecular mass of a peptide of an isolated and purified product that is a dormancy controlling substance of the present invention.

FIG. 6 is a mass spectrometry spectrum obtained by measuring the molecular mass of a synthetic peptide having the same amino acid sequence as the dormancy controlling substance of the present invention and having the same C-terminus.

FIG. 7 is a mass spectrometry spectrum obtained by measuring the molecular mass of a synthetic peptide having the same amino acid sequence as the dormancy controlling substance of the present invention but having a different C-terminus.

FIG. 8 (A) is a photograph showing the state of diapause continued in the pre-serum injected larvae of the silkworm. (B) Photograph showing the state of awakening of dormancy in the pre-larvae of the silkworm, injected with antiserum.

────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yang Ping 1-92-36 Shuhoso, Morioka-shi, Iwate Prefecture 102 (56) References Insect. Physio l. , 36 (11) (1990), p. 855-860 (58) Field surveyed (Int. Cl. ⁷ , DB name) BIOSIS (DIALOG) REGISTRY (STN) WPI (DIALOG)

Claims (6)

(57) [Claims] 1. A gene encoding a protein having the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing, Asp-Ile-Leu-Arg-Gly, amidated at the C-terminal and having a molecular weight of 570.959. Any-RF. 2. The gene Any-RF according to claim 1, wherein the protein has a dormancy controlling activity. 3. The gene Any-RF according to claim 1, wherein the protein is derived from a pre-larva of a silkworm, Bombyx mori. 4. It has an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, Asp-Ile-Leu-Arg-Gly, is amidated at the C-terminal, has a molecular weight of 570.959, and has a dormancy controlling activity. A dormancy controlling substance characterized by having: 5. The dormancy controlling substance according to claim 4, wherein the dormancy controlling substance is derived from a pre-larva of a silkworm. 6. A pre-larva of an insect is crushed with an acid-methanol solution of methanol: water: acetic acid, ground, centrifuged, and the resulting supernatant is subjected to reverse-phase high-performance liquid chromatography and mixing. By introduction into separation mode high-performance liquid chromatography, it has the amino acid sequence of Asp-Ile-Leu-Arg-Gly shown in SEQ ID NO: 1 in the sequence listing, is amidated at the C-terminus, and has a molecular weight of 570.959 A method for producing a dormancy controlling substance, characterized by obtaining a dormancy controlling substance.