CN101703035A - Method for preparing silkworm eggs for transgene of eggs of bivoltine silkworm varieties - Google Patents
Method for preparing silkworm eggs for transgene of eggs of bivoltine silkworm varieties Download PDFInfo
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Abstract
The invention relates to the field of insect bioengineering, in particular to a method for transgene of eggs of bivoltine silkworm varieties. In the method, early activated eggs are obtained by utilizing combination of protection temperature and light system in a parent generation silkworm egg hatching period, and artificial hatching treatment on the silkworm eggs before offspring fertilization; and then the early activated eggs are subjected to micro-injection and screening so as to obtain transgenic silkworm eggs. The method can solve the problems that the artificial hatching treatment after the diapaused eggs of the bivoltine silkworm varieties are subjected to transgenic injection causes damage to the silkworm eggs and the silkworm eggs cannot be hatched, also can solve the problem of low hatching rate due to transgenic injection after the early hydrochloric acid hatching treatment on the diapaused eggs of the bivoltine silkworm varieties, and also solves the problems that embryo robustness is poor and larva survival rate is low for offspring non-diapause eggs of the bivoltine silkworm varieties obtained by the hydrochloric acid hatching treatment on the diapaused eggs of the bivoltine silkworm varieties and low-temperature and dark hatching conditions for the parent generation silkworm eggs.
Description
Technical field
The present invention relates to the insect bioengineering field, in particular, relate to a kind of ovum transgenic method of silkworm bivoltine race.
Background technology
Silkworm also claims silkworm, formal name used at school Bombyx mori Linnaeus, and oviparity is Lepidoptera pattern insect, and it is voltinism that silkworm has an important physical phenomenon, and so-called voltinism just is meant the generation number that takes place in a year.Silkworm has that univoltine, bivoltine, polyvoltine do not have diapause, polyvoltine has breed differences such as diapause.A generation takes place in univoltine silkworm kind in 1 year, the larval growth phase is long, the silk cocoon of tying big, but physique a little less than; Polyvoltine silkworm kind took place in 1 year more than 3 generations, and the larval growth phase is short, the silk cocoon of tying little, silk matter proterties is poor, but physique is stronger; In 2 generations, took place in the inherent natural environment in 1 year in bivoltine race, and its larval growth phase, the size of cocooing are between a change and polyvoltine silkworm kind, and physique and silk matter proterties also are well suited for silkworm and already produce required balance requirement.Therefore, silkworm already produces generally uses two to change the diapause gamogenetic egg as seed, and has formed production traits bivoltine commercial variety colony good, wide in variety.Utilize the duovoltine kind as transgenic acceptor, not only material is abundant, and can overcome a polyvoltine or a shortcoming of changing on the variety production proterties.
At present, silkworm egg phase transgenosis micro-injection method, only early stage in embryonic development, 2h-8h injection in the back 25 ℃ of protection temperature of promptly laying eggs, genetically modified success rate is just than higher, but, if two change the diapause silkworm seed in the early stage transgenosis microinjection of embryonic development, handle the silkworm seed hatching of just can hastening the hatching of silkworms owing to also need to carry out subsequently artificial incubation such as hydrochloric acid stimulation, activation excitor substances such as hydrochloric acid can enter silkworm seed by injection orifice, and also because transgenosis injection back silkworm seed is very weak, being unable to undergo strong activation stimulates simultaneously, cause death, can't realize the transgenosis injection operation; Do not stimulate if do not carry out instant hatching activation such as hydrochloric acid after injection, by gentle activation methods such as low temperature, because required time is very long, silkworm seed also can weakness or dehydration, rotten dead in the protection process, can't realize the transgenosis injection operation equally.
Therefore, set up a kind of silkworm egg processing method, before transgeneic procedure, the efficient activation problem that solves silkworm bivoltine race diapause gamogenetic egg, make silkworm bivoltine commercial variety ovum after microinjection, not need hydrochloric acid to stimulate and just can directly hatch, will the establishment of the practical cultivated silkworm breed variety of transgenosis be played an important role.
Though present existing technology has to a certain degree solved the problems referred to above, there is defective separately, for example:
(1) publication number is that 101195834 (application number: Chinese invention patent Publication Specification 200710303904.1) discloses a kind of early pickling transgene method of cultivated silkworm diapause kind, give birth to back 2.25h-2.5h at the diapause silkworm seed, use 46 ℃, the hydrochloric acid of proportion 1.075, dipping 90s～210s activates silkworm seed.The egg-incubation rate has only about 5% behind this technology transgeneic procedure, and transformation efficiency is less than 1% of the processing ovum, and hatching effect product interspecific difference is very big, and the kind that has is hatched hardly.
(2) publication number is that 101195833 (application number: Chinese invention patent Publication Specification 200710303903.7) discloses a kind of low temperature transgenic method of cultivated silkworm diapause kind; environmental protection in 15 ℃ of parental generation ovum incubation period uses, relative moisture 75% impels the filial generation silkworm seed of output to become non-diapause gamogenetic egg.This technology is to the instability that influences of diapause, the egg-incubation rate can reach about 50% behind the part kind transgeneic procedure, but it is very big that hatching effect differs between kind, most of Japanese bivoltine race incubation rate is very low, and because the process of hastening the hatching of silkworms reaches 35d-40d, the newly-hatched silkworm of hatching has a delicate constitution.
(3) publication number is 101503704 (application number: Chinese invention patent Publication Specification 200910103397.6) discloses a kind of preparation method of transgenic silkworm of cultivated silkworm diapause kind, uses 15 ℃, relative moisture 75%, dark environmental protection in the parental generation ovum incubation period; 23 ℃ of hatching back 1-3 instar larvae uses, relative moisture 85%, dark environment are raised; and the absolute illumination condition protection in pupa time; impel the filial generation silkworm seed of output to become non-diapause gamogenetic egg. the same problem that exists the process length of hastening the hatching of silkworms, hatching newly-hatched silkworm to have a delicate constitution of this technology; and raise operation in the dark certain difficulty arranged; adult eclosion under this technical protection is irregular, has increased difficulty to transgeneic procedure.
Therefore, invent more simple and effective silkworm bivoltine commercial variety transgenosis silkworm seed hatching control, to improving transgene efficiency, set up the silkworm transgenic technology system of stability and high efficiency, the industrialization that realizes silkworm transgenosis achievement has important practical sense, therefore needs the preparation method of a kind of silkworm eggs for transgene of eggs of bivoltine silkworm varieties of invention.
Summary of the invention
The object of the invention provides a kind of preparation method of silkworm eggs for transgene of eggs of bivoltine silkworm varieties.
For achieving the above object, the technical solution used in the present invention is: a kind of preparation method of silkworm eggs for transgene of eggs of bivoltine silkworm varieties specifically may further comprise the steps:
(1) the parental generation ovum of silkworm bivoltine race, whole 20 ℃ of the hatching process uses of hastening the hatching of silkworms, relative moisture 75%-85%, intensity of illumination are lower than the dark condition protection of 0.1 lux (lux); Mulberry leaf or artificial feed raising play silkworm in 20 ℃-22 ℃ of hatching back larva uses, relative moisture 80%-90%, the natural daylight system condition to 3 ages; Play silkworm 3 ages and begin, use in 25 ℃-26 ℃, 75%-80% relative moisture, natural daylight system condition mulberry leaf or artificial feed to raise to cocooing; Protection obtains to produce female moth of weak diapause silkworm seed to sprouting wings in 24 ℃-26 ℃, 70%-80% relative moisture, the long light system condition of LD=18h: 6h (18h illumination every day 6h dark) then;
Wherein, LD represents Light/Dark, LD=18h: 6h represents to connect after the 18h continuous illumination every day 6h and continues dark;
(2) collect the female moth after the emergence in the step (1), after 23 ℃-26 ℃, intensity of illumination are lower than 10lux decreased light condition and male moth mating 3h-6h, mating male and female moth separately, female moth is thrown in and is producing with the paper of laying eggs, and the dark surrounds that 25 ℃-26 ℃, intensity of illumination are lower than 0.1lux is laid eggs; Collect the weak diapause gamogenetic egg that is produced in the 10min-15min and handle colony as artificial incubation, 0.5h-1.5h finishes hydrochloric acid artificial incubation processing in back 25 ℃ of-26 ℃ of environmental temperatures of laying eggs;
(3) collect artificial incubation processing colony silkworm seed in the step (2), handle, simultaneously silkworm seed is come off from the paper of laying eggs, obtain the silkworm seed of activate through hydrochloric acid activation.
In the technique scheme, the female moth that produces weak diapause silkworm seed described in the step (1) also can obtain by the following method:
The parental generation ovum of silkworm bivoltine race, use in 22 ℃-25 ℃, 75%-85% relative moisture, natural daylight system condition and protect to 19 phases of embryonic development stage the early stage of hastening the hatching of silkworms, also i.e. penta 2 phase embryos; Use 20 ℃, relative moisture 75%-85%, intensity of illumination then instead and be lower than the dark condition protection of 0.1lux; Hatching back larva is used in 20 ℃-22 ℃, relative moisture 80%-90%, natural daylight system condition mulberry leaf or artificial feed to raise to play silkworm to 3 ages and plays silkworm 3 ages and begin, and uses in 25 ℃-26 ℃, 75%-80% relative moisture, natural daylight system condition mulberry leaf or artificial feed raising to cocooing; Protection obtains to produce female moth of weak diapause silkworm seed to sprouting wings in 24 ℃-26 ℃, 70%-80% relative moisture, the long light system condition of LD=18h: 6h then.
In the technique scheme, male moth is selected from the step (2): universal method obtains the common male moth that sprouts wings simultaneously with the male moth of kind or step (1) method and female moth.
In the technique scheme, the condition that hydrochloric acid activation described in the step (3) is handled is selected from following two kinds a kind of:
A, W/V are 15% hydrochloric acid, are heated to 40 ℃, dipping silkworm seed 5min-8min;
B, W/V are 14% hydrochloric acid, are heated to 45 ℃, dipping silkworm seed 5min-8min.
The silkworm seed of the silkworm bivoltine race of the change diapause that technique scheme is obtained carries out the conventional microinjection row filter of going forward side by side, and obtains the transgenosis silkworm seed.
In the technique scheme, the described conventional microinjection row filter step of going forward side by side comprises following concrete steps:
(1) silkworm seed of hydrochloric acid artificial incubation processing after washing, is collected silkworm seed and is put in order at environment aseptic and that free nucleic acid pollutes, and the recombinant plasmid of 4000ng-5000ng is gone in every then ovum microinjection;
(2) injection back silkworm seed is coated with injection orifice with the non-toxic material envelope, then in gnotobasis, with 25 ℃-26 ℃, 75%-80% relative moisture, natural daylight system condition protection silkworm seed to hatching;
(3) hatching back larva is raised at 24 ℃-27 ℃, 75%-80% relative moisture, natural daylight system condition with mulberry leaf or artificial feed, and silkworm chrysalis is protected to adult eclosion at 24 ℃-26 ℃, 75%-80% relative moisture, natural daylight system condition;
(4) sprout wings back silkworm moth selfing or backcross after, institute lays eggs and handles through artificial incubation, hastens the hatching of silkworms at 22 ℃-26 ℃, 75%-80% relative moisture, the long light system condition of LD=18h: 6h then; In embryonic development to the 22-25 phase, oneself of promptly own 2-in 3 stages, filtered out fluorescently-labeled silkworm seed, as transgenosis G1 for individuality.
The present invention can solve the problem that can not hatch that injury caused of duovoltine kind diapause gamogenetic egg transgenosis injection back artificial incubation processing to silkworm seed; Can solve the early stage hydrochloric acid hatching of duovoltine kind diapause gamogenetic egg and handle the very low problem of back transgenosis injection incubation rate; Can effectively overcome the low and inferior shortcoming of silk of the silk amount of using the non-diapause ovum of polyvoltine transgenic line, the efficient ovum transgenic method that is fit to the practical silkworm kind of bivoltine high yield and high quality can be provided.
Because the technique scheme utilization, the present invention compared with prior art has following advantage:
Since the protection temperature that the present invention utilizes silkworm bivoltine race parental generation ovum to hasten the hatching of silkworms hatching period make up with light system; and the artificial incubation of filial generation silkworm seed prefecundation handled; acquisition is applicable to the early stage high-efficiency artificial activation ovum of silkworm seed transgenosis injection, makes the silkworm seed of silkworm bivoltine commercial variety no longer need just can directly efficiently hatch through the overactivation stimulation after carrying out microinjection.
2. this method is fit to the transgeneic procedure of at present all silkworm bivoltine commercial variety ovum, hastens the hatching of silkworms that hatching process is short, incubation rate is high after the injection of transgenosis silkworm seed, and hatching back newly-hatched silkworm physique is strong.
Embodiment
Below in conjunction with embodiment the present invention is further described:
Embodiment one
Use spring system diapause gamogenetic egg that the silkworm bivoltine race makes greatly as the parental generation silkworm seed, use the early stage of hastening the hatching of silkworms in 22.5 ℃, 75%-85% relative moisture, natural daylight system condition and protect to the embryonic development stage 19 phases; Using dark condition that 20 ℃, relative moisture 75%-85%, intensity of illumination are lower than 0.1lux then instead hastens the hatching of silkworms and protects to egg-incubation; Hatching back larva uses mulberry leaf to raise in 20 ℃-22 ℃, relative moisture 80%-90%, natural daylight system condition; Play silkworm 3 ages and begin,, use mulberry leaf to raise to cocooing at 25 ℃-26 ℃, 75%-80% relative moisture, natural daylight system condition; Then in 24 ℃-26 ℃, 75%-80% relative moisture, the long light system condition of LD=18h: 6h protection to sprouting wings; Female moth after collect sprouting wings, 24.5 ℃, following decreased light condition of 10lux and male moth mating 5h, male moth is used with what batch obtain and makes kind hero moth greatly; Mating male and female moth separately, female moth is thrown in and is producing with the paper of laying eggs, and 25 ℃, the dark surrounds below the 0.1lux are laid eggs; Collect the interior lay eggs of 15min and handle colony as artificial incubation; Use W/V is 15% chemical pure hydrochloric acid, is heated to 40 ℃, and dipping silkworm seed 5.5min impels the silkworm seed activation when ovum 1h in age, simultaneously silkworm seed is come off from the paper of laying eggs; Silkworm seed after hydrochloric acid artificial incubation is handled, after washing, collecting silkworm seed puts in order at environment aseptic and that free nucleic acid pollutes, then at ovum 2h-6h in age, the piggyBac carrier and the helper plasmid (W/W=1: 1), inject 350 of silkworm seeds altogether of red fluorescent protein gene (ERFP) mark of 4000ng-5000ng gone in every ovum microinjection; Injection back silkworm seed is coated with injection orifice with nontoxic mucilage sealing, then in gnotobasis, extremely hatches with 25 ℃-25.5 ℃, 80%-85% relative moisture, natural daylight system condition protection silkworm seed, hatches 205 altogether, incubation rate 58.6%; Hatching back larva is raised at 24 ℃-27 ℃, 75%-80% relative moisture, natural daylight system condition with mulberry leaf, and silkworm chrysalis is protected to adult eclosion at 24 ℃-26 ℃, 75%-80% relative moisture, natural daylight system condition; Sprout wings after the silkworm moth selfing of back, the 98 moth G1 that produce of institute, hasten the hatching of silkworms at 25 ℃, 75%-80% relative moisture, the long light system condition of LD=18h: 6h through the artificial incubation processing then for silkworm seed; In embryonic development to the 22-25 phase, oneself of promptly own 2-in 3 stages, filtered out fluorescently-labeled silkworm seed, for individuality, conversion ratio is 31.5%. as transgenosis G1
Embodiment two:
The spring system diapause silkworm seed that uses silkworm bivoltine race bright moon is as the parental generation silkworm seed, and whole 20 ℃ of the hatching process uses of hastening the hatching of silkworms, relative moisture 75%-85%, intensity of illumination are lower than the dark condition of 0.1lux; Hatching back larva uses mulberry leaf to raise in 20 ℃-21 ℃, relative moisture 85%-90%, natural daylight system condition; Play silkworm 3 ages and begin, mulberry leaf are raised to cocooing in 25.5 ℃-26 ℃, 75%-80% relative moisture, natural daylight system condition; Then in 25 ℃-26 ℃, 75%-80% relative moisture, the long light system condition of LD=18h: 6h protection to sprouting wings; Female moth after collect sprouting wings, 25 ℃-26 ℃, following decreased light condition of 10lux and male moth mating 4h, male moth is used common bright moon hero moth; Mating male and female moth separately, female moth is thrown in and is producing with the paper of laying eggs, and lays eggs in 25 ℃, the following dark surrounds of 0.1lux; Collect the interior lay eggs of 10min and handle colony as artificial incubation; Use W/V is 15% chemical pure hydrochloric acid, is heated to 40 ℃, and dipping silkworm seed 6.5min impels the silkworm seed activation when ovum 1.25h in age, simultaneously silkworm seed is come off from the paper of laying eggs; The silkworm seed that hydrochloric acid artificial incubation is handled, after washing, collecting silkworm seed puts in order at environment aseptic and that free nucleic acid pollutes, then at ovum 2h-6.5h in age, the piggyBac carrier and the helper plasmid (W/W=1: 1), inject 290 of silkworm seeds altogether of red fluorescent protein gene (ERFP) mark of 4000ng-5000ng gone in every ovum microinjection; Injection back silkworm seed is coated with injection orifice with nontoxic mucilage sealing, then in gnotobasis, extremely hatches with 25 ℃-25.5 ℃, 80%-85% relative moisture, natural daylight system condition protection silkworm seed, hatches 207 altogether, incubation rate 71.4%; Hatching back larva is raised at 26 ℃-27 ℃, 85%-90% relative moisture, natural daylight system condition with artificial feed, and silkworm chrysalis is protected to adult eclosion at 24 ℃-26 ℃, 75%-80% relative moisture, natural daylight system condition; Sprout wings after the silkworm moth selfing of back, the 89 moth G1 of laying eggs of institute handle through artificial incubation for silkworm seed, hasten the hatching of silkworms at 22 ℃-26 ℃, 75%-80% relative moisture, the long light system condition of LD=18h: 6h then; In embryonic development to the 22-25 phase, oneself of promptly own 2-in 3 stages, filtered out fluorescently-labeled silkworm seed, for individuality, conversion ratio is 27.8% as transgenosis G1.
Embodiment three:
Use the new spring system diapause gamogenetic egg of silkworm bivoltine race 75 as the parental generation silkworm seed, hasten the hatching of silkworms in earlier stage to use in 24.5 ℃-25 ℃, 75%-80% relative moisture, natural daylight system condition and protect to 19 phases of embryonic development stage; Use 20 ℃, relative moisture 80%-85%, intensity of illumination then instead and be lower than the dark condition protection of 0.1lux to hatching; Hatching back larva uses mulberry leaf to raise to play silkworm 3 ages to begin in 21 ℃-22 ℃, relative moisture 80%, natural daylight system condition, at 25 ℃-26 ℃, 75%-80% relative moisture, natural daylight system condition, uses the mulberry leaf raising to cocooing; Then in 25 ℃-26 ℃, 75%-80% relative moisture, the long light system condition of LD=18: 6h protection to sprouting wings; Female moth after collect sprouting wings, 25 ℃-26 ℃, 10lux decreased light condition and male moth mating 5h, 75 common new male moths of male moth use; Mating male and female moth separately, female moth is thrown in and is producing with the paper of laying eggs, and 25 ℃, the dark surrounds below the 0.1lux are laid eggs; Collect the interior lay eggs of 15min and handle colony as artificial incubation; At ovum 1.25h in age, using 45 ℃, W/V is 14% chemical pure hydrochloric acid, and dipping silkworm seed 5.0min impels the silkworm seed activation, simultaneously silkworm seed is come off from the paper of laying eggs; The silkworm seed that hydrochloric acid artificial incubation is handled, after washing, collecting silkworm seed puts in order at environment aseptic and that free nucleic acid pollutes, during ovum 2.0h-5.5h in age, the piggyBac carrier and the helper plasmid (W/W=1: 1), inject 300 of silkworm seeds altogether of red fluorescent protein gene (ERFP) mark of 4000ng-5000ng gone in every ovum microinjection; Injection back silkworm seed is coated with injection orifice with nontoxic mucilage sealing, then in gnotobasis, extremely hatches with 25 ℃-25.5 ℃, 80%-85% relative moisture, natural daylight system condition protection silkworm seed, hatches 212 altogether, incubation rate 70.7%; Hatching back larva is raised at 25 ℃-27 ℃, 75%-80% relative moisture, natural daylight system condition with mulberry leaf, and silkworm chrysalis is protected to adult eclosion at 25 ℃-26 ℃, 75%-80% relative moisture, natural daylight system condition; Sprout wings after the silkworm moth selfing of back, the 99 moth G1 that produce of institute, hasten the hatching of silkworms at 24 ℃-25 ℃, 75%-80% relative moisture, the long light system condition of LD=18h: 6h through the artificial incubation processing then for silkworm seed; In embryonic development to the 22-25 phase, oneself of promptly own 2-in 3 stages, filtered out fluorescently-labeled silkworm seed, for individuality, conversion ratio is 30.5%. as transgenosis G1
Claims (4)
1. the preparation method of a silkworm eggs for transgene of eggs of bivoltine silkworm varieties is characterized in that, specifically may further comprise the steps:
(1) the parental generation ovum of silkworm bivoltine race, whole 20 ℃ of the hatching process uses of hastening the hatching of silkworms, relative moisture 75%-85%, intensity of illumination are lower than the dark condition protection of 0.1 lux; Mulberry leaf or artificial feed raising play silkworm in 20 ℃-22 ℃ of hatching back larva uses, relative moisture 80%-90%, the natural daylight system condition to 3 ages; Play silkworm 3 ages and begin, use in 25 ℃-26 ℃, 75%-80% relative moisture, natural daylight system condition mulberry leaf or artificial feed to raise to cocooing; Protection obtains to produce female moth of weak diapause silkworm seed to sprouting wings in 24 ℃-26 ℃, the long light system condition of 70%-80% relative moisture, LD=18h: 6h then;
(2) collect the female moth after the emergence in the step (1), after 23 ℃-26 ℃, intensity of illumination are lower than 10 lux decreased light conditions and male moth mating 3h-6h, mating male and female moth separately, female moth is thrown in and is producing with the paper of laying eggs, and the dark surrounds that 25 ℃-26 ℃, intensity of illumination are lower than 0.1 lux is laid eggs; Collect the weak diapause gamogenetic egg that is produced in the 10min-15min and handle colony as artificial incubation, 0.5h-1.5h finishes hydrochloric acid artificial incubation processing in back 25 ℃ of-26 ℃ of environmental temperatures of laying eggs;
(3) collect artificial incubation processing colony silkworm seed in the step (2), handle, simultaneously silkworm seed is come off from the paper of laying eggs, obtain the activate silkworm seed through hydrochloric acid activation.
2. the preparation method of a silkworm eggs for transgene of eggs of bivoltine silkworm varieties is characterized in that, specifically may further comprise the steps:
(1) the parental generation ovum of silkworm bivoltine race, use 22 ℃-25 ℃, 75%-85% relative moisture, natural daylight system condition to protect to 19 phases of embryonic development stage the early stage of hastening the hatching of silkworms; Use 20 ℃, relative moisture 75%-85%, intensity of illumination then instead and be lower than the dark condition protection of 0.1 lux; Mulberry leaf or artificial feed raising play silkworm in 20 ℃-22 ℃ of hatching back larva uses, relative moisture 80%-90%, the natural daylight system condition to 3 ages; Play silkworm 3 ages and begin, use in 25 ℃-26 ℃, 75%-80% relative moisture, natural daylight system condition mulberry leaf or artificial feed to raise to cocooing; Protection obtains to produce female moth of weak diapause silkworm seed to sprouting wings in 24 ℃-26 ℃, 70%-80% relative moisture, the long light system condition of LD=18h: 6h then;
(2) collect the female moth after the emergence in the step (1), after 23 ℃-26 ℃, intensity of illumination are lower than 10 lux decreased light conditions and male moth mating 3h-6h, mating male and female moth separately, female moth is thrown in and is producing with the paper of laying eggs, and the dark surrounds that 25 ℃-26 ℃, intensity of illumination are lower than 0.1 lux is laid eggs; Collect the weak diapause gamogenetic egg that is produced in the 10min-15min and handle colony as artificial incubation, 0.5h-1.5h finishes hydrochloric acid artificial incubation processing in back 25 ℃ of-26 ℃ of environmental temperatures of laying eggs;
(3) collect artificial incubation processing colony silkworm seed in the step (2), handle, simultaneously silkworm seed is come off from the paper of laying eggs, obtain the activate silkworm seed through hydrochloric acid activation.
3. according to the ovum transgenic method of claim 1 or 2 described silkworm bivoltine races, it is characterized in that male moth is selected from the step (2): universal method obtains the common male moth that sprouts wings simultaneously with the male moth of kind or step (1) method and female moth.
4. according to the ovum transgenic method of claim 1 or 2 described silkworm bivoltine races, it is characterized in that the condition that hydrochloric acid activation described in the step (3) is handled is selected from following two kinds a kind of:
A, W/V are 15% hydrochloric acid, are heated to 40 ℃, dipping silkworm seed 5min-8min;
B, W/V are 14% hydrochloric acid, are heated to 45 ℃, dipping silkworm seed 5min-8min.
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| CN101195833B (en) * | 2007-12-21 | 2011-04-20 | 西南大学 | Low-temperature incubation transgene method for cultivated silkworm diapause breed variety |
| CN101195834B (en) * | 2007-12-21 | 2010-06-02 | 西南大学 | Early pickling transgene method for cultivated silkworm diapause breed variety |
| CN101361474B (en) * | 2008-09-19 | 2011-01-19 | 江苏科技大学 | Preparation method of silkworm commercial race transgene silkworm egg |
| CN101503704B (en) * | 2009-03-17 | 2011-01-05 | 西南大学 | Transgenic method for cultivated silkworm diapause variety |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102706717A (en) * | 2012-07-06 | 2012-10-03 | 重庆市蚕业管理总站 | Moth grinding sample preparation system for detecting silkworm female moth corpuscule pathogeny spore |
| CN102706717B (en) * | 2012-07-06 | 2014-08-06 | 重庆市蚕业管理总站 | Moth grinding sample preparation system for detecting silkworm female moth corpuscule pathogeny spore |
| CN107484722A (en) * | 2016-06-12 | 2017-12-19 | 苏州大学 | Silkworm variety breeding method for secreting silk fibroin fibrils containing sericin protein |
| 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 |
| CN106973868A (en) * | 2017-04-13 | 2017-07-25 | 西南大学 | Pole early stage corona releases method of cultivated silkworm diapause ovum Diapause and products thereof |
| CN106973868B (en) * | 2017-04-13 | 2020-05-26 | 西南大学 | Method for eliminating silkworm diapause egg diapause fertility by extremely early corona and product thereof |
| CN114216870A (en) * | 2021-10-29 | 2022-03-22 | 辽宁省蚕业科学研究所 | Screening method and application of tussah breeding material |
| CN114216870B (en) * | 2021-10-29 | 2023-10-03 | 辽宁省蚕业科学研究所 | Screening method of tussah breeding material and application thereof |
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| CN101703035B (en) | 2011-11-30 |
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