CN102217571A - Safe preservation method of non-diapause multivoltine silkworm variety in winter - Google Patents
Safe preservation method of non-diapause multivoltine silkworm variety in winter Download PDFInfo
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Abstract
The invention provides a safe preservation method of a non-diapause multivoltine silkworm variety in winter. The preservation method comprises the steps of: taking a micro syringe as an injection tool and the diluted fluid of an artificially synthetic insect diapause hormone as injecta, and injecting the synthetic insect diapause hormone in the pupa stage so that the non-diapause multivoltine silkworm variety lays extragenetic diapause eggs which can be preserved in winter. The preservation method of the invention overcomes the problem that the traditional non-diapause multivoltine silkworm variety is needed to be fed uninterruptedly throughout the year so as to realize succeeding preservation, in particular the difficulty caused for the preservation of the non-diapause multivoltine silkworm variety due to no production of folium mori in the winter each year. Therefore, not only is the variety preservation cost reduced, but also risks for the preservation of the non-diapause multivoltine silkworm variety in winter due to different adaptability of different silkworm variety to artificial feed are avoided.
Description
Technical field
The present invention relates to the management of bird, fish, insect, specifically be meant a kind of employing manual injection diapause hormone method, make silkworm not have duration of diapause polyvoltine kind and can give birth to the diapause ovum, thereby reach safety store method in winter efficient, that silkworm that safety is preserved does not have duration of diapause polyvoltine kind.
Technical background
Silkworm is the diapause of ovum attitude, under field conditions (factors), is divided into and one changes, two changes and the polyvoltine kinds according to difference takes place in 1 year from generation to generation.Polyvoltine silkworm kind is divided into again duration of diapause polyvoltine kind and no duration of diapause polyvoltine kind.Have duration of diapause polyvoltine kind from generation to generation the diapause of part ovum to be arranged all at each, and it is fast not have duration of diapause polyvoltine silkworm breed development, life cycle is short, and each all produces no diapause ovum from generation to generation, need raise for 7～8 generations the whole year incessantly and could preserve by subculture.Owing to the geography and climate reason, can only breed silkworms 4～5 times with mulberry leaf every year in most of area, the whole nation, do not have mulberry leaf campaigns in March November to next year then, can only utilize the silkworm artificial feed to raise subculture and preserve, bring very big difficulty for the preservation of no duration of diapause polyvoltine silkworm kind.And utilize artificial feed sericulture specification requirement height, cost height, and different silkworm races is raised the no duration of diapause polyvoltine silkworm kind of preservation with artificial feed and is had great risk artificial feed adaptability difference.Therefore, be necessary under the condition that does not influence voltinism, principal character characteristic and the economic characters of preserving kind, preservation technology to no duration of diapause polyvoltine silkworm kind winter is innovated, and can access safety, preservation efficiently winter to guarantee no duration of diapause polyvoltine kind; And can reduce by 2-3 time sericulture number of times, save great amount of manpower and material resources and financial resources, improve greatly and preserve work efficiency.
Summary of the invention
Technical problem to be solved by this invention is to preserve winter at no duration of diapause polyvoltine silkworm kind the problem of existence, a kind of Insect Diapause hormone (diapause hormone that adopts the injection synthetic in the silkworm chrysalis phase is provided, DH), make no duration of diapause polyvoltine silkworm kind give birth to the diapause ovum of nongenetic, the silkworm that can access safety, efficient preservation does not in the winter time have the safety store method in winter of duration of diapause polyvoltine kind.
The safety store method in winter that silkworm of the present invention does not have duration of diapause polyvoltine kind is injection tool with the micro syringe, dilution with the Insect Diapause hormone of synthetic is a parenteral solution, employing is at the Insect Diapause hormone of silkworm chrysalis phase injection synthetic, make no duration of diapause polyvoltine silkworm kind give birth to the diapause ovum of nongenetic, obtain in the winter time preserving; The concrete implementation step and the controlled condition of store method are as follows:
1, selects injection period: do not have duration of diapause polyvoltine kind silkworm and carry out mulberry leaf and raise and carry out to the pupa time of last silkworm rearing season;
2, determining of elapsed time in pupa time: it is under 25.5 ℃ the condition in environmental temperature that silkworm does not have duration of diapause polyvoltine kind, and pupa time is through about 9 days, in biting through about 11 days; Treat matured silkworm weave silk finish form cocoon after, cut and open the silk cocoon of not pupating as yet, be tiled in the insulating box, temperature is controlled at 25 ℃-26 ℃, and relative moisture 70%-75% observes and puts down in writing the actual of every silkworm and pupates the time, used in order to injection, male silkworm chrysalis is normally protected, and confessionization moth mating usefulness is hour to calculate female pupa at the back institute elapsed time of pupating;
3, preparation parenteral solution: the Insect Diapause hormone of employing sterile water dilution synthetic before injection (diapause hormone, DH), being mixed with active constituent content is the parenteral solution of 0.5 μ g/ μ l; The agent of Insect Diapause prohormone needs to preserve under-70 ℃ condition, and is after former dose slowly will being thawed during prepared and diluted liquid, now with the current again;
4, injection: the female silkworm chrysalis in effective injection phase that will select, with the alcohol swabs that through volumn concentration concentration is 75% soaking in alcohol, with whole silkworm chrysalis body wiping sterilization, while disinfection experiment apparatus and both hands, select for use the micro syringe of 10 μ l to inject then, the injection term of validity for after pupate through 18h to 120h, individual injection effective dose is between 0.5 μ g/ pupa to 2 μ g/ pupa, extract the diapause hormone dilution with micro syringe, syringe needle aim at the silkworm chrysalis belly the 4th with the 5th uromere between parallel thrusting in the silkworm chrysalis body, thrust the about 1cm of the degree of depth, slowly extract syringe needle after quantitatively injecting parenteral solution, and then the injection wound of silkworm chrysalis body is used through volumn concentration concentration is that the alcohol swabs of 75% soaking in alcohol carries out disinfection;
5, the silkworm chrysalis behind the injection diapause hormone liquid and the protection of silkworm seed: the silkworm chrysalis that will inject behind the diapause hormone liquid is shared in sterile ventilative container; protection is to sprouting wings under the relative moisture condition of 25 ℃-26 ℃ temperature and 70-75%; with normal male moth mating; the nongenetic diapause silkworm seed of giving birth to is protected according to a conventional method.
Insect Diapause hormone (the diapause hormone of synthetic, DH), it is a kind of polypeptide hormone, form by 24 amino acid residues, the C terminal amideization, amino acid sequence is H-Thr-Asp-Met-Lys-Asp-Glu-Ser-Asp-Arg-Gly-Ala-His-Ser-Gl u-Arg-Gly-Ala-Leu-Cys-Phe-Gly-Pro-Arg-LeuNH2, and molecular weight is 2645.The sterile water of dilution synthetic Insect Diapause hormone adopts the bottled distilled water of triangle, and sterilizes sterilization controlled condition with autoclaving: pressure: 103.4KPa, and temperature: 121 ℃, the time: 20 minutes.The injection Disinfectant absorbent cotton is for being the alcohol swabs of 75% soaking in alcohol through volumn concentration concentration.Syringe is the micro syringe of 10 μ l.
The injection best period for after pupating through 24h to 86h.According to experimental result, 0.5 μ g/ pupa is the minimum of the individual effective dose of injection diapause hormone, the diapause ovum rate of 0.5 μ g/ pupa dosage and 1 μ g/ pupa dosage, average out to 57.61% and 69.52% respectively; The diapause rate of 2 μ g/ pupa dosage can reach more than 94% substantially.To the investigation of the diapause of being laid eggs and diapause ovum offspring's proterties, the diapause hormone injection can not produce any influence to no duration of diapause polyvoltine kind offspring's voltinism, is still keeping the characteristic of its intrinsic no duration of diapause polyvoltine behind the injection diapause hormone.
When the safety in winter of carrying out silkworm and not having a duration of diapause polyvoltine kind is preserved, be chosen in silkworm do not have duration of diapause polyvoltine kind carry out mulberry leaf raise to the pupa time of last silkworm rearing season be injection period.In the injection phase, it is under 25.5 ℃ the condition in environmental temperature that silkworm does not have duration of diapause polyvoltine kind, and pupa time is through about 9 days, in biting through about 11 days; Treat matured silkworm weave silk finish form cocoon after, cut and open the silk cocoon of not pupating as yet, be tiled in the insulating box, temperature is controlled at 25 ℃-26 ℃, relative moisture 70%-75%; Observe and put down in writing the actual of every silkworm and pupate the time, used in order to injection, male silkworm chrysalis is normally protected, and the mating of confessionization moth is used; Hour to calculate female pupa at the back institute elapsed time of pupating.Before injection, and the Insect Diapause hormone of employing sterile water dilution synthetic (diapause hormone, DH), being mixed with active constituent content is the parenteral solution of 0.5 μ g/ μ l; The agent of Insect Diapause prohormone needs to preserve under-70 ℃ condition, and is after former dose slowly will being thawed during prepared and diluted liquid, now with the current again.After preparing parenteral solution, with the female silkworm chrysalis of having selected in effective injection phase, with the alcohol swabs that through volume percent content concentration is 75% soaking in alcohol, with whole silkworm chrysalis body wiping sterilization, while disinfection experiment apparatus and both hands, select for use the micro syringe of 10 μ l to inject then, the injection term of validity for after pupate through 18h to 120h, individual injection effective dose is between 0.5 μ g/ pupa to 2 μ g/ pupa, extract the diapause hormone dilution with micro syringe, syringe needle aim at the silkworm chrysalis belly the 4th with the 5th uromere between parallel thrusting in the silkworm chrysalis body, thrust the about 1cm of the degree of depth, slowly extract syringe needle after quantitatively injecting parenteral solution, and then the injection wound of silkworm chrysalis body is used through volume percent content concentration is that the alcohol swabs of 75% soaking in alcohol carries out disinfection.After injection diapause hormone liquid finishes; the silkworm chrysalis of injecting behind the diapause hormone liquid is shared in sterile ventilative container; temperature be 25 ℃-26 ℃ and relative moisture be under the condition of 70-75% protection to sprouting wings; with normal male moth mating; the nongenetic diapause silkworm seed of giving birth to is protected according to a conventional method.Finish the safety preservation work in winter that silkworm does not have duration of diapause polyvoltine kind.
Advantage of the present invention just is to adopt the method for manual injection's diapause hormone, makes no duration of diapause polyvoltine silkworm kind can access efficient, safe preservation in the winter time.Having overcome traditional no duration of diapause polyvoltine silkworm kind needs the whole year and raises incessantly just can to preserve by subculture, does not have particularly annual winter mulberry leaf to produce the very big difficulty that the preservation to no duration of diapause polyvoltine silkworm kind brings.Not only reduce the kind retain costs, and avoided different silkworm races the adaptive difference of artificial feed to be given no duration of diapause polyvoltine silkworm kind guarantor in winter kind of a risk of bringing.It is the characteristic that determines in pupal cell ovary maturity process that the present invention utilizes the diapause of silkworm seed, give female silkworm chrysalis injection Insect Diapause hormone (diapausehormone, DH), obtain the diapause ovum of nongenetic, help not having the subculture preservation of duration of diapause polyvoltine silkworm kind.Simultaneously, under the condition of the voltinism that does not influence kind and feature and economic characters, can either reduce the preservation risk that silkworm does not have duration of diapause polyvoltine kind, but also reduce by 2-3 time sericulture number of times, improve work efficiency significantly, saved human and material resources and financial resources.Therefore the inventive method is a kind of method of preserving no duration of diapause polyvoltine silkworm kind safe and effective winter, has broad prospect for its application.
Embodiment
Embodiment 1, and silkworm does not have the safety store method in winter of duration of diapause polyvoltine kind
Adopt silkworm of the present invention not have the safety store method in winter of duration of diapause polyvoltine kind.Store method is injection tool with the micro syringe, dilution with the Insect Diapause hormone of synthetic is a parenteral solution, employing makes no duration of diapause polyvoltine silkworm kind give birth to the diapause ovum of nongenetic at the Insect Diapause hormone of silkworm chrysalis phase injection synthetic, obtains in the winter time preserving.The concrete implementation step and the controlled condition of store method are as follows:
1, selects injection period: do not have duration of diapause polyvoltine kind silkworm and carry out mulberry leaf and raise and carry out to the pupa time of last silkworm rearing season;
2, determining of elapsed time in pupa time: it is under 25.5 ℃ the condition in environmental temperature that silkworm does not have duration of diapause polyvoltine kind, and pupa time is through about 9 days, in biting through about 11 days; Treat matured silkworm weave silk finish form cocoon after, cut and open the silk cocoon of not pupating as yet, be tiled in the insulating box, temperature is controlled at 25 ℃-26 ℃, and relative moisture 70%-75% observes and puts down in writing the actual of every silkworm and pupates the time, used in order to injection, male silkworm chrysalis is normally protected, and confessionization moth mating usefulness is hour to calculate female pupa at the back institute elapsed time of pupating;
3, preparation parenteral solution: the Insect Diapause hormone of employing sterile water dilution synthetic before injection (diapause hormone, DH), being mixed with active constituent content is the parenteral solution of 0.5 μ g/ μ l; The agent of Insect Diapause prohormone needs to preserve under-70 ℃ condition, and is after former dose slowly will being thawed during prepared and diluted liquid, now with the current again;
4, injection: the female silkworm chrysalis in effective injection phase that will select, with the alcohol swabs that through volumn concentration concentration is 75% soaking in alcohol, with whole silkworm chrysalis body wiping sterilization, while disinfection experiment apparatus and both hands, select for use the micro syringe of 10 μ l to inject then, the injection term of validity for after pupate through 18h to 120h, individual injection effective dose is between 0.5 μ g/ pupa to 2 μ g/ pupa, extract the diapause hormone dilution with micro syringe, syringe needle aim at the silkworm chrysalis belly the 4th with the 5th uromere between parallel thrusting in the silkworm chrysalis body, thrust the about 1cm of the degree of depth, slowly extract syringe needle after quantitatively injecting parenteral solution, and then the injection wound of silkworm chrysalis body is used through volume percent content concentration is that the alcohol swabs of 75% soaking in alcohol carries out disinfection;
5, the silkworm chrysalis behind the injection diapause hormone liquid and the protection of silkworm seed: the silkworm chrysalis that will inject behind the diapause hormone liquid is shared in sterile ventilative container; protection is to sprouting wings under the relative moisture condition of 25 ℃-26 ℃ temperature and 70-75%; with normal male moth mating; the nongenetic diapause silkworm seed of giving birth to is protected according to a conventional method.
To adopting the explanation of material
1, silkworm cultivar origin and characteristic:
Do not have totally 13 parts of duration of diapause polyvoltine kinds (seeing Table 1) from the silkworm that external introduction is preserved in recent years by Inst. of Silkworm, Chinese Academy of Agricultural Sciences, have high-temp resisting high-humidity resisting, physique strong, grow through characteristics such as too short.
The no diapause polyvoltine silkworm kind that table 1, Inst. of Silkworm, Chinese Academy of Agricultural Sciences preserve
| The kind numbering | The kind name | The kind numbering | The kind name | The kind numbering | The kind name |
| 7018 | Nice | 7024 | SR4 | 7029 | In the tower of Nice No. two |
| 7019 | In the tower of Nice | 7025 | SR5 | 7030 | No. two, Mysore |
| 7020 | N4 | 7026 | NL | 7031 | The Chiang Mai |
| 7021 | SR1 | 7027 | Lang how | ||
| 7023 | SR3 | 7028 | Cam |
2, injection source and characteristic:
(diapause hormone DH), is a kind of polypeptide hormone to the Insect Diapause hormone of synthetic, is made of the C terminal amideization 24 amino acid residues.Amino acid sequence is H-Thr-Asp-Met-Lys-Asp-Glu-Ser-Asp-Arg-Gly-Ala-His-Ser-Gl u-Arg-Gly-Ala-Leu-Cys-Phe-Gly-Pro-Arg-LeuNH2, molecular weight is 2645 and (is public material, list of references: Imai K, Konno T, Nakazawa Y, et al.1991.Proc.JapanAcad.67 (B), 98-101.).Inject by the female silkworm chrysalis that silkworm is not had duration of diapause polyvoltine kind, can make no duration of diapause polyvoltine bombyx mori give birth to the diapause ovum.The diapause ovum that uses this method to obtain does not have inheritance, recovers the characteristic of no duration of diapause polyvoltine kind behind its offspring's termination of diapause again.
3, the dipping alcohol of sterile water, injection Disinfectant absorbent cotton and the specification requirement of micro syringe:
The sterile water of dilution synthetic Insect Diapause hormone adopts the bottled distilled water of triangle, uses autoclaving (103.4Kpa, 121 ℃, 20 minutes) sterilization then.
The injection Disinfectant absorbent cotton is for being the alcohol swabs of 75% soaking in alcohol through volume percent content concentration.
Syringe is the micro syringe of 10 μ l.
When the safety in winter of carrying out silkworm and not having a duration of diapause polyvoltine kind is preserved, be chosen in silkworm do not have duration of diapause polyvoltine kind carry out mulberry leaf raise to the pupa time of last silkworm rearing season be injection period.In the injection phase, it is under 25.5 ℃ the condition in environmental temperature that silkworm does not have duration of diapause polyvoltine kind, and pupa time is through about 9 days, in biting through about 11 days; Treat matured silkworm weave silk finish form cocoon after, cut and open the silk cocoon of not pupating as yet, be tiled in the insulating box, temperature is controlled at 25 ℃-26 ℃, relative moisture 70%-75%; Observe and put down in writing the actual of every silkworm and pupate the time, used in order to injection, male silkworm chrysalis is normally protected, and the mating of confessionization moth is used; Hour to calculate female pupa at the back institute elapsed time of pupating.Before injection, and the Insect Diapause hormone of employing sterile water dilution synthetic (diapause hormone, DH), being mixed with active constituent content is the parenteral solution of 0.5 μ g/ μ l; The agent of Insect Diapause prohormone needs to preserve under-70 ℃ condition, and is after former dose slowly will being thawed during prepared and diluted liquid, now with the current again.After preparing parenteral solution, with the female silkworm chrysalis of having selected in effective injection phase, with the alcohol swabs that through volume percent content concentration is 75% soaking in alcohol, with whole silkworm chrysalis body wiping sterilization, while disinfection experiment apparatus and both hands, select for use the micro syringe of 10 μ l to inject then, the injection term of validity for after pupate through 18h to 120h, individual injection effective dose is between 0.5 μ g/ pupa to 2 μ g/ pupa, extract the diapause hormone dilution with micro syringe, syringe needle aim at the silkworm chrysalis belly the 4th with the 5th uromere between parallel thrusting in the silkworm chrysalis body, thrust the about 1cm of the degree of depth, slowly extract syringe needle after quantitatively injecting parenteral solution, and then the injection wound of silkworm chrysalis body is used through volume percent content concentration is that the alcohol swabs of 75% soaking in alcohol carries out disinfection.After injection diapause hormone liquid finishes; the silkworm chrysalis of injecting behind the diapause hormone liquid is shared in sterile ventilative container; temperature be 25 ℃-26 ℃ and relative moisture be under the condition of 70-75% protection to sprouting wings; with normal male moth mating; the nongenetic diapause silkworm seed of giving birth to is protected according to a conventional method.Finish the safety preservation work in winter that silkworm does not have duration of diapause polyvoltine kind.
Experimental result shows that 0.5 μ g/ pupa is the minimum of the effective dose of injection DH.Through 18h to 120h, its individual DH injected dose can both produce the diapause ovum that quantity does not wait between 2 μ g/ pupa to 0.5 μ g/ pupas after pupate.And producing what of quantity of diapause ovum, the size with its injected dose has direct relation again.Through in 24h～86h, the dosage of DH injection is big more after pupating, and the diapause ovum rate that then obtains is just high more.2 μ g/ pupa dosage injection zone are 86.9% except the diapause rate of 72h is minimum, and the diapause rate of all the other time periods has all reached more than 94%.0.5 the diapause ovum rate of μ g/ pupa dosage injection zone and 1 μ g/ pupa dosage injection zone, average out to 57.61% and 69.52% respectively.But through the injection zone of 107h, the result is opposite after pupating, i.e. the dosage of DH injection is big more, and the diapause ovum rate that obtains is low more.
Experiment through for many years draws, cast off a skin from silkworm and pupate to pick up counting, after pupating through 18h～120h, be injection cultivated silkworm diapause hormone (diapause hormone, DH) duration of response can make the silkworm chrysalis of no duration of diapause polyvoltine kind, gives birth to nongenetic diapause ovum at the emergence post-coitum.If to injecting through silkworm chrysalis to 18h after pupating, pupal cell is broken and cause out hyperhematosis death or bacteria infection dead or can cause silkworm chrysalis stasi, can not sprout wings and dead.To carrying out the DH injection through the female pupa that surpasses 107h after pupating, the diapause ovum rate of being laid eggs then significantly descends, and is up to 30%; Through surpassing 149h, injection DH does not then fully to no effect promptly produce the diapause ovum after pupating.The best period of determining the DH injection thus for after pupating through 24h to 86h.When the many kinds of needs are injected in enormous quantities, the determining of its term of validity, after Ying Zaisheng is placed on small straw bundles to spin cocoons the 3rd day to the 5th day.(seeing table 2 for details)
Table 2, DH inject the result of the test application form of female silkworm chrysalis
Annotate: 1. the data of survey item are repeatedly the mean of experiment.2. comprise in the non-diapause ovum and go out ovum again and change blue or green dead ovum.
The diapause sex investigation that injection is laid eggs behind the DH: will be through silkworm moth after the female silkworm chrysalis of injection DH is sprouted wings and male silkworm moth mating without injection treatment, lay eggs is protected about 10 days under 25 ℃ of-26 ℃ of conditions, and the diapause of investigating silkworm seed is whether.This moment, what only annesl did not change blue or green and hatching then was the diapause silkworm seed if change the non-diapause silkworm seed that is blue or green and hatching.
Proterties to diapause ovum offspring is investigated: the diapause silkworm seed that obtains is continued to protect about 2 months under 25 ℃ of-26 ℃ of conditions, then with 15 ℃ of protections 1-2 days, move on to 5 ℃-2.5 ℃ at last and refrigerate about 3 months down.Hasten the hatching of silkworms normally, collect ants, raise to being placed on small straw bundles to spin cocoons and cocoon to April next year, selfing is laid eggs behind the change moth, and its silkworm seed (the F1 generation of diapause ovum) is non-diapause gamogenetic egg; Then, subculture is to F2 generation according to a conventional method with the F1 of diapause ovum generation, and it also all is that non-diapause ovum is hatched that institute lays eggs.Illustrate that the DH injection can not produce any influence to no duration of diapause polyvoltine kind offspring's voltinism, is still keeping the characteristic of its intrinsic no duration of diapause polyvoltine.In offspring's process of raising the diapause ovum, investigated the relevant proterties of handling kind, compare with the check plot, do not produce evident difference (, seeing table 3 for details) 7025 to be example.
Table 3, F
2In generation, is the proterties comparison sheet mainly
| Project | Through the injection treatment district | Without the injection treatment district |
| Kind | SR5(7025) | SR5(7025) |
| The ovum look | Faint yellow | Faint yellow |
| The chorion look | Milky | Milky |
| Single moth number (grain) of on average laying eggs | 376 | 385 |
| Common incubation rate (%) | 94.41 | 92.47 |
| The newly-hatched silkworm body colour | Pitchy | Pitchy |
| 5 ages are through (d:h) | 8:00 | 7:12 |
| Full age is through (d:h) | 25:12 | 25:00 |
| The grown silkworm body colour | Bluish white | Bluish white |
| The grown silkworm speckle | System | System |
| The cocoon look | Golden yellow | Golden yellow |
| Cocoon shape | Long ellipse | Long ellipse |
| Cocoon weight (g) | 1.020 | 1.058 |
| Cocoon shell weight (g) | 0.140 | 0.143 |
| Cocoon layer rate (%) | 13.73 | 13.52 |
| Following generation ovum diapause situation | Not diapause | Not diapause |
Owing to adopt the method for manual injection's diapause hormone, make no duration of diapause polyvoltine kind can access efficient, safe preservation in the winter time.Having overcome traditional no duration of diapause polyvoltine silkworm kind needs the whole year and raises incessantly just can to preserve by subculture, does not have particularly annual winter mulberry leaf to produce the very big difficulty that the preservation to no duration of diapause polyvoltine silkworm kind brings.Not only reduce feeding cost, and avoided different silkworm races the adaptive difference of artificial feed to be given no duration of diapause polyvoltine silkworm kind guarantor in winter kind of a risk of bringing.
Claims (10)
1. a silkworm does not have the safety store method in winter of duration of diapause polyvoltine kind, it is characterized in that with the micro syringe being injection tool, dilution with the Insect Diapause hormone of synthetic is a parenteral solution, employing is at the Insect Diapause hormone of silkworm chrysalis phase injection synthetic, make no duration of diapause polyvoltine silkworm kind give birth to the diapause ovum of nongenetic, obtain in the winter time preserving; The concrete implementation step and the controlled condition of store method are as follows:
⑴, select injection period: do not have duration of diapause polyvoltine kind silkworm and carry out mulberry leaf and raise and carry out to the pupa time of last silkworm rearing season;
⑵ determining of, elapsed time in pupa time: it is under 25.5 ℃ the condition in environmental temperature that silkworm does not have duration of diapause polyvoltine kind, and pupa time is through 9 days, in biting through 11 days; Treat matured silkworm weave silk finish form cocoon after, cut and open the silk cocoon of not pupating as yet, be tiled in the insulating box, temperature is controlled at 25 ℃-26 ℃, and relative moisture 70%-75% observes and puts down in writing the actual of every silkworm and pupates the time, used in order to injection, male silkworm chrysalis is normally protected, and confessionization moth mating usefulness is hour to calculate female pupa at the back institute elapsed time of pupating;
⑶, preparation parenteral solution: adopt the Insect Diapause hormone of sterile water dilution synthetic before injection, being mixed with active constituent content is the parenteral solution of 0.5 μ g/ μ l; The agent of Insect Diapause prohormone needs to preserve under-70 ℃ condition, and is after former dose slowly will being thawed during prepared and diluted liquid, now with the current again;
⑷, injection: the female silkworm chrysalis in effective injection phase that will select, with the alcohol swabs that through volumn concentration concentration is 75% soaking in alcohol, with whole silkworm chrysalis body wiping sterilization, while disinfection experiment apparatus and both hands, select for use the micro syringe of 10 μ l to inject then, the injection term of validity for after pupate through 18h to 120h, individual injection effective dose is between 0.5 μ g/ pupa to 2 μ g/ pupa, extract the diapause hormone dilution with micro syringe, syringe needle aim at the silkworm chrysalis belly the 4th with the 5th uromere between parallel thrusting in the silkworm chrysalis body, thrust the about 1cm of the degree of depth, slowly extract syringe needle after quantitatively injecting parenteral solution, and then the injection wound of silkworm chrysalis body is used through volumn concentration concentration is that the alcohol swabs of 75% soaking in alcohol carries out disinfection;
⑸ the silkworm chrysalis behind, the injection diapause hormone liquid and the protection of silkworm seed: the silkworm chrysalis that will inject behind the diapause hormone liquid is shared in sterile ventilative container; protection is to sprouting wings under the relative moisture condition of 25 ℃-26 ℃ temperature and 70-75%; with normal male moth mating; the nongenetic diapause silkworm seed of giving birth to is protected according to a conventional method.
2. silkworm according to claim 1 does not have the safety store method in winter of duration of diapause polyvoltine kind, the Insect Diapause hormone that it is characterized in that employed synthetic is a kind of polypeptide hormone, form by 24 amino acid residues, the C terminal amideization, amino acid sequence is H-Thr-Asp-Met-Lys-Asp-Glu-Ser-Asp-Arg-Gly-Ala-His-Ser-Gl u-Arg-Gly-Ala-Leu-Cys-Phe-Gly-Pro-Arg-Leu-NH2, and molecular weight is 2645.
3. silkworm according to claim 1 does not have the safety store method in winter of duration of diapause polyvoltine kind, it is characterized in that the sterile water that dilutes synthetic Insect Diapause hormone adopts the bottled distilled water of triangle, and sterilize with autoclaving, sterilization controlled condition: pressure: 103.4KPa, temperature: 121 ℃, the time: 20 minutes.
4. silkworm according to claim 1 does not have the safety store method in winter of duration of diapause polyvoltine kind, after it is characterized in that selecting in injection period to pupate through 24h to 86h.
5. silkworm according to claim 1 does not have the safety store method in winter of duration of diapause polyvoltine kind, it is characterized in that the individual injected dose of silkworm chrysalis is 0.5 μ g/ pupa.
6. silkworm according to claim 1 does not have the safety store method in winter of duration of diapause polyvoltine kind, it is characterized in that the individual injected dose of silkworm chrysalis is 2 μ g/ pupas.
7. silkworm according to claim 1 does not have the safety store method in winter of duration of diapause polyvoltine kind, it is characterized in that the individual injected dose of silkworm chrysalis is 1 μ g/ pupa.
8. silkworm according to claim 1 does not have the safety store method in winter of duration of diapause polyvoltine kind, and through 24h, the individual injected dose of silkworm chrysalis is 2 μ g/ pupas after it is characterized in that selecting in injection period to pupate.
9. silkworm according to claim 1 does not have the safety store method in winter of duration of diapause polyvoltine kind, and through 55h, the individual injected dose of silkworm chrysalis is 2 μ g/ pupas after it is characterized in that selecting in injection period to pupate.
10. silkworm according to claim 1 does not have the safety store method in winter of duration of diapause polyvoltine kind, and through 86h, the individual injected dose of silkworm chrysalis is 2 μ g/ pupas after it is characterized in that selecting in injection period to pupate.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106719467A (en) * | 2016-11-14 | 2017-05-31 | 江苏科技大学 | A kind of Winter safety store method without diapause polyvoltinism cultivated silkworm breed variety |
| CN109792973A (en) * | 2019-01-28 | 2019-05-24 | 江苏科技大学 | Silkworm polypeptide functional study method and its application |
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2011
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106719467A (en) * | 2016-11-14 | 2017-05-31 | 江苏科技大学 | A kind of Winter safety store method without diapause polyvoltinism cultivated silkworm breed variety |
| CN109792973A (en) * | 2019-01-28 | 2019-05-24 | 江苏科技大学 | Silkworm polypeptide functional study method and its application |
| CN109792973B (en) * | 2019-01-28 | 2022-01-04 | 江苏科技大学 | Silkworm polypeptide function research method and application thereof |
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