CN107027718A - The method that Diapause transgenic bombyx mori is made by instant corona - Google Patents
The method that Diapause transgenic bombyx mori is made by instant corona Download PDFInfo
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- CN107027718A CN107027718A CN201710239491.9A CN201710239491A CN107027718A CN 107027718 A CN107027718 A CN 107027718A CN 201710239491 A CN201710239491 A CN 201710239491A CN 107027718 A CN107027718 A CN 107027718A
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- 241000255789 Bombyx mori Species 0.000 title claims abstract description 282
- 230000005058 diapause Effects 0.000 title claims abstract description 86
- 238000000034 method Methods 0.000 title claims abstract description 53
- 230000009261 transgenic effect Effects 0.000 title claims abstract description 45
- 235000013601 eggs Nutrition 0.000 claims abstract description 41
- 230000012447 hatching Effects 0.000 claims abstract description 41
- 238000011534 incubation Methods 0.000 claims abstract description 21
- 230000005684 electric field Effects 0.000 claims abstract description 10
- 238000012216 screening Methods 0.000 claims abstract description 8
- 238000003851 corona treatment Methods 0.000 claims abstract description 7
- 230000000638 stimulation Effects 0.000 claims abstract description 7
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 30
- 108090000623 proteins and genes Proteins 0.000 claims description 19
- 238000002347 injection Methods 0.000 claims description 18
- 239000007924 injection Substances 0.000 claims description 18
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- 229910052751 metal Inorganic materials 0.000 claims description 18
- 239000011521 glass Substances 0.000 claims description 17
- 102000002322 Egg Proteins Human genes 0.000 claims description 12
- 108010000912 Egg Proteins Proteins 0.000 claims description 12
- 210000004681 ovum Anatomy 0.000 claims description 12
- 102000011782 Keratins Human genes 0.000 claims description 10
- 108010076876 Keratins Proteins 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
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- 239000012153 distilled water Substances 0.000 claims description 5
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- 231100000252 nontoxic Toxicity 0.000 claims description 5
- 230000003000 nontoxic effect Effects 0.000 claims description 5
- 230000017448 oviposition Effects 0.000 claims description 5
- 230000019612 pigmentation Effects 0.000 claims description 5
- 238000009955 starching Methods 0.000 claims description 5
- 210000001136 chorion Anatomy 0.000 claims description 4
- 239000013612 plasmid Substances 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 2
- 101710200191 Feather keratin Proteins 0.000 claims 1
- 241001494479 Pecora Species 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 9
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- 241001354243 Corona Species 0.000 description 29
- 238000005554 pickling Methods 0.000 description 10
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
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- 241000256844 Apis mellifera Species 0.000 description 1
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
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- 229910052760 oxygen Inorganic materials 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
- A01K67/04—Silkworms
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
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- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of method that Diapause transgenic bombyx mori is made by instant corona, the Diapause silkworm egg after being injected using high voltage electric field to transgenosis, which carries out instant corona stimulation, makes its termination of diapause, is directly entered the incubation period.After present invention processing, subculture is raised through fluoroscopic examination screening positive individuals, cultivated silkworm diapause breed variety can be efficiently solved and carry out that transgeneic procedure is loaded down with trivial details, the low problem of efficiency.Research shows, sided corona treatment Hatching Rate of Silkworm Eggs of the present invention is up to 99%, and middle system is good as Japanese kind effect, and on the growing of later stage silkworm, the economic characters such as cocoon without influence.In a word, simple to operate, the cost ineffective rate of the present invention is high, safety non-pollution and suitable for various cultivated silkworm breed varieties, and strong technical support is provided for the preparation of transgenic bombyx mori.
Description
Technical field
Pass through instant corona the invention belongs to transgenic bombyx mori technical field, more particularly to one kind and make Diapause transgenosis
The method of silkworm.
Background technology
Silkworm is important economic insects and lepidopterous insects model organism.Silkworm has important being of physiological phenomenon
Property, i.e., the generation number occurred in 1 year.Univoltine Silkworm varieties only undergo a generation in 1 year, and the ovum then given birth to enters one and overflow
Long duration of diapause, until Second Year, termination of diapause germinates under suitable temperature and humidity conditions；Bivoltine silkworm kind 1 year
Occurred for two generations in inherent natural environment, immersion hydrochloric acid method manually can control reproductive order of generation by termination of diapause；Polyvoltinism cultivated silkworm breed variety, one
Occur more than three generations in year, non-Diapause polyvoltine kind does not have duration of diapause.Bivoltine silkworm kind larval growth phase, silk cocoon size are situated between
Between Yu Yihua and polyvoltine Silkworm varieties, its constitution, characteristics of cocoons and cocoon filament etc. are well suited for the balance requirement needed for sericultural production, so
Sericultural production generally uses bivoltine Diapausing egg, forms excellent, the wide in variety bivoltine race colony of the production traits.
Since Japanese Scientists in 2000 first successful incubation transgenic bombyx mori, transgenic bombyx mori is successfully reported gradually
Increase.Most Transgenic studies are all to utilize polyvoltinism cultivated silkworm breed variety, but polyvoltinism cultivated silkworm breed variety economic characters are poor, utilize
Cultivated silkworm diapause kind, which carries out transgenic research, can overcome many defect of the polyvoltinism cultivated silkworm breed variety in transgenic research.Family
The transgenic research of silkworm Diapause kind has particularly important meaning.Cultivated silkworm diapause breed variety just enters diapause after spawning
Phase, Diapausing egg utilizes the instant pickling method of routine for about 16-20 hours or so typically after spawning or passes through long-term low temperature cold
Tibetan method termination of diapause starts hatching.But, the current maximally effective silkworm transgenosis microinjection time is that 3-6 is small after spawning
When, injection aperture can be left on ovum after transgeneic procedure, if hydrochloric acid can be caused using conventional instant pickling method after injection
Penetrate into silkworm seed, make it dead, the transgenic research of cultivated silkworm diapause breed variety can not be realized at all；The aperture that transgenosis injection leaves
Also prevent silkworm seed from placing for a long time, so prolonged cold cold preserving method termination of diapause can not be used.Therefore, cultivated silkworm diapause breed variety
The diapause of ovum, very big limitation is brought to transgeneic procedure.
At present, carrying out transgene method using Diapause cultivated silkworm breed variety mainly has following several, and they respectively deposit defect：
(1) Southwestern University Zhao love spring etc. explores carries out the method for pickling in silkworm seed after fertilization and successfully obtains phase immediately
Transgenic method (early pickling transgene method of cultivated silkworm diapause breed variety, the patent No. 200710303904.1, publication date answered
On 06 11st, 2008).However, because the method needs incubation rate after pickling, microinjection relatively low, and the effect of early pickling
It is larger in interracial diversity ratio.
(2) Southwestern University Zhao love spring etc. also changes the change of cultivated silkworm diapause breed variety silkworm seed of future generation using special incubation condition
Property, and conventional silkworm transgenic technology is combined, effectively produce transgenic silkworm (the low temperature incubation transgenosis side of cultivated silkworm diapause kind
Method, the patent No. 200710303903.7, publication date 2008 year 06 month 11 days).Although the method incubation rate and polyvoltinism after injection
Silkworm seed is equally matched, but the effect of low temperature incubation is very big in interracial difference, to univoltine and Japanese duovoltine product
Plant and do not apply to substantially.
(3) Southwestern University Ma Sanyuan etc. makes parental generation using parental generation local environment condition and the relation of filial generation silkworm seed Diapause
Cocoon protection is hastened the hatching of silkworms, raised and planted to silkworm under the special environment condition through strictly controlling, and uses appropriate mating pattern
To obtain the filial generation silkworm seed of non-diapause, carry out transgenosis injection, obtain transgenic silkworm (transgenic method of cultivated silkworm diapause breed variety,
The patent No. 200910103397.6, publication date 2009 year 08 month 12 days).The method solves Japanese duovoltine product to a certain extent
The problem of transgenosis injection can not be carried out by planting, but Japanese kind still termination of diapause rate is not high, and parental generation silkworm need to be in constant temperature, perseverance
Cocoon protection is hastened the hatching of silkworms, raises and planted under the harsh environment such as wet, absolute dark, and experimental condition requires that high, cost is big；Incubation temperatures are only
There are 15 DEG C, it is necessary to which a wheat harvesting period hastens the hatching of silkworms the time；Sericulture temperature is not also high, and silkworm is long the time required to growing, and substantially prolongs
The cycle of Transgenic studies；This sericulture, seed production way and produce reality differ greatly, and the robustness to offspring has a significant impact,
Larva survival rate is low.
(4) University Of Suzhou Xu generation is waited clearly is combined using the hasten the hatching of silkworms protection temperature in hatching period of parental generation silkworm seed with light system, and
Artificial incubation processing to filial generation prefecundation silkworm seed, obtains early activation ovum, then carries out conventional microinjection and is screened,
Obtain method (a kind of preparation method of silkworm eggs for transgene of eggs of bivoltine silkworm varieties, the patent No. of transgenosis silkworm seed
200910212990.4, publication date 2010 year 05 month 12 days).This method raises low temperature incubation (20 DEG C), full age illumination and early
Phase pickling is combined, and incubation rate is improved to a certain extent, but still there are problems that aforesaid operations complexity, test period.
(5) the Diapause silkworm seed after Southwestern University Gong Jing etc. handles transgenosis is placed in closed environment and carried out at oxygenation
Reason, with termination of diapause (a kind of method of blocking transgenic bombyx mori ovum diapause, number of patent application 201610569284.5, publication date
On December 07th, 2016).The method is oxygenated (10-50h) for a long time in closed equipment, and requirement of experiment is high；And it is preceding
It has also been found that oxygen or ozonolysis are unstable except the effect of silkworm egg Diapause in the research paper of people.
Therefore, a kind of time saving, laborsaving, technical requirements of exploration are low, and all Diapause cultivated silkworm breed varieties are can be suitably used for again turns base
It is still to realize that transgenic bombyx mori achievement is quickly applied to the active demand of sericulture there because of method.
The study hotspot that cultivated silkworm diapause is Sericulture of China circle in the nineties in last century, day are released using electrical field stimulation
The diapause that (1914) use electrification method to release silkworm seed is waited in this scholar star open country, once obtains more than 90% incubation rate, but because electrification is incubated
The constitution of change silkworm is weak and does not obtain practicality.Yang Mingguan etc. (1994) has carried out the corona artificial incubation examination of system to various silkworm eggs
Test, obtain preferable result, incubation rate is as the result of pickling method；They also found in follow-up research, sided corona treatment people
Work hatching the growing of silkworm, vital rates and cocoon yield quality are with compare no substantial differences, the feeding that corona will not be to silkworm
Support and bring harmful effect.Moreover, the method termination of diapause of corona can be used by soaking seed and refrigerate to plant.But researcher at that time
The method that invention corona releases cultivated silkworm diapause, be in order to solve environmental pollution that the pickling in silk cocoon production process brings with into
This problem, and run into production application the problems such as electric discharge is irregular and can not solve without being promoted and utilizing, subsequently
Nobody is studied again.
The content of the invention
The technical problem to be solved in the present invention is to provide one kind is simple to operate, cost ineffective rate is high, safety non-pollution
And suitable for the method that Diapause transgenic bombyx mori is made by instant corona of various cultivated silkworm breed varieties.
In order to solve the above technical problems, the present invention uses following technical scheme：
The method that Diapause transgenic bombyx mori is made by instant corona, it is stagnant after being injected using high voltage electric field to transgenosis
Fertility silkworm egg, which carries out instant corona stimulation, makes its termination of diapause, is directly entered the incubation period.
High voltage electric field comes from corona instrument, and corona instrument is mainly made up of power supply and positive and negative pole plate, and the output end of power supply is led to respectively
Cross connecting line and connect positive and negative pole plate, positive and negative pole plate is parallel to each other by insulating bar to be periodically installed on base, on negative plate
Silkworm seed metal slide glass is placed with, positive plate is provided with the pole pin of vertical silkworm seed metal slide glass.
Positive and negative electrode plate gauge lattice are 15cm × 8cm, positive and negative polar plate interval 15mm, pole pin long 10mm, needle gage 8mm.
High voltage electric field is 8.0-12KV, and instant corona stimulation time is 10s-4min.
The gene of transgenosis injection is the gene of wool keratin 3.2, the gene of wool keratin 4.3, orange-yellow fluorescin
Gene, the gene of wool keratin 5.4.
It is kind or Japanese kind that Diapause silkworm egg, which is come from cultivated silkworm diapause,.
It is that kind " 932 ", Japanese kind " 7532 " or Japanese kind are " big that Diapause silkworm egg, which is come from cultivated silkworm diapause,
Make ".
The method that Diapause transgenic bombyx mori is made above by instant corona, comprises the following steps：
<1>Routinely the method for hastening the hatching of silkworms is hastened the hatching of silkworms silkworm egg；
<2>Raise according to a conventional method；
<3>Matured silkworm is transferred in 25 DEG C of environment upper cluster, cocoond, and adopts spoiled cocoon after pupating, identification male and female are opened with knife pin, separate
Place, mated 4h separation of copulating moths after it changes moth, and female moth is invested in the silkworm egg laying eggs on paper of starching, a silkworm is collected at interval of 1h
Ovum, and the silkworm seed of collection is placed in protection in 25 DEG C of environment；
<4>After silkworm seed is rinsed together with the silkworm egg scraps of paper with running water, 2-3min is soaked in distilled water, tweezers are then used
Silkworm seed is transferred on the silkworm seed metal slide glass with 75% alcohol disinfecting, and arranged whole according to the standard of the silkworm seed outside of belly to the right
Together, the silkworm seed arranged is placed in 35-37% formaldehyde vapors and sterilizes 5min；
<5>Start to clock to collect the silkworm seed time, in 3-6h microinjection instruments after collection by 10-15nL, total concentration
400ng/ μ L transposon vector plasmid closes ovum from the sterilized silkworm seed of silkworm seed outside of belly center injection with nontoxic glue
The aperture left is injected on shell；
<6>The silkworm seed injected is placed in 35-37% formaldehyde vapors and sterilizes 5min, puts in 25 DEG C of environment and protects；
<7>Start to clock to collect the silkworm seed time, by with injecting the silkworm seed metal slide glass of silkworm seed between 20-40h
It is put on corona instrument, 8.0-12KV stimulates 10s-4min；
<8>Silkworm seed after shock treatment, is placed under conditions of 25-28 DEG C of temperature, relative humidity 80-90%, is hastened the hatching of silkworms
Until hatching, the newly-hatched silkworm for hatching and is raised, contemporary G0 silkworm moth is subjected to selfing or backcrossing, the G1 of Diapause is obtained for silkworm
Ovum, G1 scans screening after body pigmentation stage through instant sided corona treatment for silkworm seed under fluorescence microscope and obtains trans genie individual；Finally
The trans genie individual of acquisition is normally raised, subculture.
The problem of existing is prepared for existing transgenic bombyx mori, inventor establishes a kind of by the making diapause of instant corona
Property transgenic bombyx mori method, the Diapause silkworm egg after being injected using high voltage electric field to transgenosis carries out instant corona and stimulates to make
Its termination of diapause, is directly entered the incubation period.After present invention processing, subculture, energy are raised through fluoroscopic examination screening positive individuals
Efficiently solve cultivated silkworm diapause breed variety and carry out that transgeneic procedure is loaded down with trivial details, the low problem of efficiency.Research shows, at corona of the present invention
Reason Hatching Rate of Silkworm Eggs is up to 99%, and with middle system is good Japanese kind effect as, and to the growing of later stage silkworm, cocooing etc. passes through
Character of helping is without influence.In a word, simple to operate, the cost ineffective rate of the present invention is high, safety non-pollution and suitable for various families
Silkworm varieties, strong technical support is provided for the preparation of transgenic bombyx mori.
Phenotypic mutation and physical mutation silkworm the genetic resources overwhelming majority preserved at present is diapause kind, and the present invention is to grind
Study carefully and established solid technical foundation using these valuable genetic resources.Moreover, transgenic technology drosophila, she
Mosquito, honeybee etc. more than 20 is planted in insect and set up, but most of reports are the non-Diapause kinds of polyvoltinism, and the present invention also will be it
The foundation of the lepidopterous insects transgenic technology of his ovum Diapause provides useful reference and reference.
Brief description of the drawings
Fig. 1 is that inventor implements the present invention and the structural representation of homemade GZ-01 types corona instrument.
In figure：1 metal shell, 2 power switches, 3 voltage regulators, 4 voltage displays, 5 power lines, 6 positive plates, 7 is negative
Pole plate, 8 positive leads, 9 negative leads, 10 insulating bars, 11 pole pins, 12 are stained with the silkworm seed metal slide glass of silkworm seed, 13 bases.
Embodiment
Below in conjunction with example in detail, how the present invention is implemented.
Wherein, Fig. 1 shows that inventor implements the present invention and homemade GZ-01 types corona instrument, and corona instrument is main by electricity
Source and positive and negative pole plate composition, the output end of power supply connect positive and negative pole plate by connecting line respectively, and positive and negative pole plate passes through insulating bar
It is parallel to each other and is periodically installed on base, silkworm seed metal slide glass is placed with negative plate, positive plate is provided with vertical silkworm seed gold
Belong to the pole pin of slide glass.Metal shell specification is 20 × 12cm；Voltage display can show 0-15KV；Power line connects family expenses 220V friendships
Stream electricity.Positive and negative electrode plate gauge lattice are 15cm × 8cm, positive and negative polar plate interval 15mm, pole pin long 10mm, needle gage 8mm.The corona instrument can
0-15KV high voltage electric field is produced, instant corona stimulation time is 10s-4min.
Following example uses homemade GZ-01 types corona instrument.
Embodiment 1
By the use of in cultivated silkworm diapause be kind " 932 " as original material, the silkworm seed that normal live box is obtained is by production
On the routine method of hastening the hatching of silkworms that uses hasten the hatching of silkworms silkworm egg：27 DEG C of temperature, relative humidity 85%.In order that hatching is neat, after silkworm egg point is blue or green
Silkworm egg is wrapped with black cloth, silkworm egg is taken out two days later and is collected ants.The newly-hatched silkworm of harvest is raised according to a conventional method：Small silkworm rearing season (1-3 ages)
25-28 DEG C, relative humidity 75-85%；Big 23-26 DEG C of silkworm rearing season (4-5 ages), relative humidity 70-80%, ventilation；Full age adopts
With natural lighting, three times on the one feeding mulberry leaf.Or full age, raise at normal temperatures could also.Matured silkworm is transferred to 25 DEG C or so
Environment in upper cluster, cocoon, adopt spoiled cocoon after pupating, identification male and female opened with knife pin, separated, mating 4h is torn open after it changes moth
It is right, female moth is invested in the silkworm egg laying eggs on paper of starching, a silkworm seed is collected at interval of 1h, and the silkworm seed of collection is placed in 25 DEG C
Environment in protect.After silkworm seed is rinsed together with the silkworm egg scraps of paper with running water, 2-3min is soaked in distilled water, tweezer is then used
Silkworm seed is transferred to the silkworm seed metal slide glass (specification with 75% alcohol disinfecting by son：76 × 26mm, thickness is 3mm) on, and according to
The standard of the silkworm seed outside of belly to the right is by its marshalling, and the silkworm seed arranged is placed in 35-37% formaldehyde vapors and sterilizes 5min.With
The collection silkworm seed time starts to clock, and carries 10-15nL, total concentration 400ng/ μ L swivel base in 3-6h microinjection instruments after collection
Constitution grain is closed with nontoxic glue leave small is injected on chorion from the sterilized silkworm seed of silkworm seed outside of belly center injection
Hole.The silkworm seed injected is placed in 35-37% formaldehyde vapors and sterilizes 5min, puts in 25 DEG C of environment and protects.To collect silkworm seed
Time starts to clock, and self-control GZ-01 type corona instrument will be put between 20-40h with the silkworm seed metal slide glass for injecting silkworm seed
On, 10kV stimulates 10s.Silkworm seed after shock treatment, is placed under conditions of 25-28 DEG C of temperature, relative humidity 80-90%, carries out
Hasten the hatching of silkworms until hatching, raise the newly-hatched silkworm for hatching and, the silkworm moth of contemporary (G0) is subjected to selfing or backcrossing, obtain the G1 of Diapause
For silkworm seed, G1 scans screening after body pigmentation stage through instant sided corona treatment for silkworm seed under fluorescence microscope and obtains trans genie individual；
Finally the trans genie individual of acquisition is normally raised, subculture, that is, complete Diapause cultivated silkworm breed variety " 932 " transgenic silkworm system
It is standby.4 experiments are carried out altogether using the kind, have obtained the transgenic silkworm kind of three carrying different genes, the results are shown in Table 1.
The instant corona Transgenic studies of the Diapause cultivated silkworm breed variety " 932 " of table 1
Remarks：The red fluorescent protein mark that pBac { 3 × P3-DsRed } starts for carrying nerve and eyes specific promoter
The piggyBac carriers of note；The green fluorescent protein that pBac { 3 × P3-EGFP } starts for carrying nerve and eyes specific promoter
The piggyBac carriers of mark；B2, B3, V1 and B5 carry different target gene respectively, and B2 is the gene of wool keratin 3.2,
B3 is the gene of wool keratin 4.3, and V1 is orange-yellow fluorescence protein gene (mKO), and B5 is the gene of wool keratin 5.4.
Embodiment 2
By the use of the Japanese kind of cultivated silkworm diapause " 7532 " as original material, the silkworm seed that normal live box is obtained is by production
On the routine method of hastening the hatching of silkworms that uses hasten the hatching of silkworms silkworm egg：27 DEG C of temperature, relative humidity 85%.In order that hatching is neat, after silkworm egg point is blue or green
Silkworm egg is wrapped with black cloth, silkworm egg is taken out two days later and is collected ants.The newly-hatched silkworm of harvest is raised according to a conventional method：Small silkworm rearing season (1-3 ages)
25-28 DEG C, relative humidity 75-85%；Big 23-26 DEG C of silkworm rearing season (4-5 ages), relative humidity 70-80%, ventilation；Full age adopts
With natural lighting, three times on the one feeding mulberry leaf.Or full age, raise at normal temperatures could also.Matured silkworm is transferred to 25 DEG C or so
Environment in upper cluster, cocoon, adopt spoiled cocoon after pupating, identification male and female opened with knife pin, separated, mating 4h is torn open after it changes moth
It is right, female moth is invested in the silkworm egg laying eggs on paper of starching, a silkworm seed is collected at interval of 1h, and the silkworm seed of collection is placed in 25 DEG C
Environment in protect.After silkworm seed is rinsed together with the silkworm egg scraps of paper with running water, 2-3min is soaked in distilled water, tweezer is then used
Silkworm seed is transferred to the silkworm seed metal slide glass (specification with 75% alcohol disinfecting by son：76 × 26mm, thickness is 3mm) on, and according to
The standard of the silkworm seed outside of belly to the right is by its marshalling, and the silkworm seed arranged is placed in 35-37% formaldehyde vapors and sterilizes 5min.With
The collection silkworm seed time starts to clock, and carries 10-15nL, total concentration 400ng/ μ L swivel base in 3-6h microinjection instruments after collection
Constitution grain (pBac { 3 × P3-DsRed }-B5, B5 are the gene of wool keratin 5.4) and helper plasmid (pHA3PIG) are from silkworm seed abdomen
In the sterilized silkworm seed of face center injection, 513 silkworm seeds of co-injection, and close what injection on chorion left with nontoxic glue
Aperture.The silkworm seed injected is placed in 35-37% formaldehyde vapors and sterilizes 5min, puts in 25 DEG C of environment and protects.To collect silkworm
The ovum time starts to clock, and self-control GZ-01 type coronas will be put between 20-40h with the silkworm seed metal slide glass for injecting silkworm seed
On instrument, 11kV stimulates 15s.Silkworm seed after shock treatment, is placed under conditions of 25-28 DEG C of temperature, relative humidity 80-90%, enters
Row is hastened the hatching of silkworms until hatching, raises 410 newly-hatched silkworms for hatching and, and the silkworm moth of contemporary (G0) is carried out into selfing or backcrossing, is obtained stagnant
112 circle G1 of fertility are for silkworm seed, and G1 scans screening after body pigmentation stage through instant sided corona treatment for silkworm seed under fluorescence microscope and obtained
38 circle transgenic positive moth circles are obtained, conversion ratio is 33.9%；Finally the trans genie individual of acquisition is normally raised, subculture, i.e., it is complete
Into the preparation of the transgenic silkworm of Diapause cultivated silkworm breed variety " 7532 ".
Embodiment 3
By the use of the Japanese kind of cultivated silkworm diapause " making greatly " as original material, the silkworm seed that normal live box is obtained is by production
On the routine method of hastening the hatching of silkworms that uses hasten the hatching of silkworms silkworm egg：27 DEG C of temperature, relative humidity 85%.In order that hatching is neat, after silkworm egg point is blue or green
Silkworm egg is wrapped with black cloth, silkworm egg is taken out two days later and is collected ants.The newly-hatched silkworm of harvest is raised according to a conventional method：Small silkworm rearing season (1-3 ages)
25-28 DEG C, relative humidity 75-85%；Big 23-26 DEG C of silkworm rearing season (4-5 ages), relative humidity 70-80%, ventilation；Full age adopts
With natural lighting, three times on the one feeding mulberry leaf.Or full age, raise at normal temperatures could also.Matured silkworm is transferred to 25 DEG C or so
Environment in upper cluster, cocoon, adopt spoiled cocoon after pupating, identification male and female opened with knife pin, separated, mating 4h is torn open after it changes moth
It is right, female moth is invested in the silkworm egg laying eggs on paper of starching, a silkworm seed is collected at interval of 1h, and the silkworm seed of collection is placed in 25 DEG C
Environment in protect.After silkworm seed is rinsed together with the silkworm egg scraps of paper with running water, 2-3min is soaked in distilled water, tweezer is then used
Silkworm seed is transferred to the silkworm seed metal slide glass (specification with 75% alcohol disinfecting by son：76 × 26mm, thickness is 3mm) on, and according to
The standard of the silkworm seed outside of belly to the right is by its marshalling, and the silkworm seed arranged is placed in 35-37% formaldehyde vapors and sterilizes 5min.With
The collection silkworm seed time starts to clock, and carries 10-15nL, total concentration 400ng/ μ L swivel base in 3-6h microinjection instruments after collection
Constitution grain (pBac { 3 × P3-DsRed }-B2, B2 are the gene of wool keratin 3.2) and helper plasmid (pHA3PIG) are from silkworm seed abdomen
In the sterilized silkworm seed of face center injection, 438 silkworm seeds of co-injection, and close what injection on chorion left with nontoxic glue
Aperture.The silkworm seed injected is placed in 35-37% formaldehyde vapors and sterilizes 5min, puts in 25 DEG C of environment and protects.To collect silkworm
The ovum time starts to clock, and self-control GZ-01 type coronas will be put between 20-40h with the silkworm seed metal slide glass for injecting silkworm seed
On instrument, 10.5kV stimulates 30s.Silkworm seed after shock treatment, is placed under conditions of 25-28 DEG C of temperature, relative humidity 80-90%,
Hastened the hatching of silkworms until hatching, raise 382 newly-hatched silkworms for hatching and, the silkworm moth of contemporary (G0) is subjected to selfing or backcrossing, obtained
98 circle G1 of Diapause are for silkworm seed, and G1 scans screening after body pigmentation stage through instant sided corona treatment for silkworm seed under fluorescence microscope and obtained
35 circle transgenic positive moth circles are obtained, conversion ratio is 35.7%；Finally the trans genie individual of acquisition is normally raised, subculture, i.e., it is complete
Into the preparation of the transgenic silkworm of Diapause cultivated silkworm breed variety " making greatly ".
Practice have shown that, compared with prior art, the present invention has advantage following prominent：
<1>Corona releasing silkworm seed diapause technology is combined by the present invention with Gene Transfer Methods in Silkworms first, is dexterously broken through
The bottleneck of cultivated silkworm diapause breed variety transgenic technology, about the defect of cultivated silkworm diapause kind transgenic method before overcoming.And
Experiment also indicates that termination of diapause efficiency high, stability are strong, and effect compares favourably with conventional pickling method, the life to later stage silkworm body
Long development etc. is also without influence.
<2>The hastening the hatching of silkworms of silkworm seed in the present invention, raise and termination of diapause is simpler than the method for forefathers, easy to operate, silkworm
Raise mulberry leaf in the environment of can using ambient temperature and moisture to feed, be conventional method for breeding, without special installation and processing,
It is consistent with produce reality.Compared with existing low temperature incubation mode is raised, high degree reduces feeding cost, simplified
Raise operation.
<3>, can be high as long as hatching of silkworm egg at most 8 days, are shortened more than 20 days than low temperature incubation (more than 30 days) in the present invention
The acquisition test material of efficiency, substantially reduces the test period.And silkworm is weak, difficulty after the egg-incubation of low temperature incubation
Raising, health be not good, and the silkworm raised in the present invention is consistent with produce reality, hatching homogeneous, easily silkworm body health, raising.
<4>Silkworm seed after being injected by transgenosis only needs to be put on self-control GZ-01 type corona instrument, 8.0-12KV high-tension electricities
Field stimulates 10s-4min, you can termination of diapause, and screening of hastening the hatching of silkworms then is carried out in the usual way, with using immersion hydrochloric acid termination of diapause
Mode compared to safe and pollution-free.
<5>The method of existing prepare transgenosis silkworm, equal centering system kind effect is good, but Japanese kind effect is not good；And this
Invention is applied to all Diapause cultivated silkworm breed varieties, and the effect of Japanese, middle system is equally good.
<6>Although it is consistent with forefathers' report to make GZ-01 types corona instrument operation principle by oneself, voltage regulation limits are wide and can connect
Continuous regulation, machine is compact, light, easy to control and operates.By experimental study, GZ-01 type corona instrument can guarantee that working region electricity
Field is uniform, preferably reaches target requirement.
Claims (8)
1. a kind of method that Diapause transgenic bombyx mori is made by instant corona, it is characterised in that using high voltage electric field to turning base
Make its termination of diapause because the Diapause silkworm egg after injection carries out instant corona stimulation, be directly entered the incubation period.
2. the method according to claim 1 that Diapause transgenic bombyx mori is made by instant corona, it is characterised in that：Institute
High voltage electric field is stated from corona instrument, corona instrument is mainly made up of power supply and positive and negative pole plate, the output end of power supply passes through connection respectively
Line connects positive and negative pole plate, and positive and negative pole plate is parallel to each other by insulating bar to be periodically installed on base, is placed with negative plate
Silkworm seed metal slide glass, positive plate is provided with the pole pin of vertical silkworm seed metal slide glass.
3. the method according to claim 2 that Diapause transgenic bombyx mori is made by instant corona, it is characterised in that：Institute
Positive and negative electrode plate gauge lattice are stated for 15cm × 8cm, positive and negative polar plate interval 15mm, pole pin long 10mm, needle gage 8mm.
4. the method according to claim 3 that Diapause transgenic bombyx mori is made by instant corona, it is characterised in that：Institute
High voltage electric field is stated for 8.0-12KV, the instant corona stimulation time is 10s-4min.
5. the method according to claim 1 that Diapause transgenic bombyx mori is made by instant corona, it is characterised in that：Institute
The gene for stating transgenosis injection is the gene of wool keratin 3.2, the gene of wool keratin 4.3, orange-yellow fluorescence protein gene, sheep
The gene of feather keratin 5.4.
6. the method according to claim 1 that Diapause transgenic bombyx mori is made by instant corona, it is characterised in that：Institute
It is kind or Japanese kind that Diapause silkworm egg, which is stated, in cultivated silkworm diapause.
7. the method according to claim 6 that Diapause transgenic bombyx mori is made by instant corona, it is characterised in that：Institute
It is kind " 932 ", Japanese kind " 7532 " or Japanese kind " making greatly " that Diapause silkworm egg, which is stated, in cultivated silkworm diapause.
8. the method according to claim 2 that Diapause transgenic bombyx mori is made by instant corona, it is characterised in that bag
Include following steps：
<1>Routinely the method for hastening the hatching of silkworms is hastened the hatching of silkworms silkworm egg；
<2>Raise according to a conventional method；
<3>Matured silkworm is transferred in 25 DEG C of environment upper cluster, cocoond, and adopts spoiled cocoon after pupating, opens identification male and female with knife pin, separately put
Put, mated 4h separation of copulating moths after it changes moth, and female moth is invested in the silkworm egg laying eggs on paper of starching, a silkworm seed is collected at interval of 1h,
And the silkworm seed of collection is placed in protection in 25 DEG C of environment；
<4>After silkworm seed is rinsed together with the silkworm egg scraps of paper with running water, 2-3min is soaked in distilled water, then with tweezers by silkworm
Ovum is transferred on the silkworm seed metal slide glass with 75% alcohol disinfecting, and according to the standard of the silkworm seed outside of belly to the right by its marshalling,
The silkworm seed arranged is placed in 35-37% formaldehyde vapors and sterilizes 5min；
<5>Start to clock to collect the silkworm seed time, in 3-6h microinjection instruments after collection by 10-15nL, total concentration 400ng/ μ
L transposon vector plasmid closes note on chorion from the sterilized silkworm seed of silkworm seed outside of belly center injection, and with nontoxic glue
Penetrate the aperture left；
<6>The silkworm seed injected is placed in 35-37% formaldehyde vapors and sterilizes 5min, puts in 25 DEG C of environment and protects；
<7>Start to clock to collect the silkworm seed time, will be put into the silkworm seed metal slide glass for injecting silkworm seed between 20-40h
On corona instrument, 8.0-12KV stimulates 10s-4min；
<8>Silkworm seed after shock treatment, is placed under conditions of 25-28 DEG C of temperature, relative humidity 80-90%, hastened the hatching of silkworms until
Hatching, raises the newly-hatched silkworm for hatching and, and contemporary G0 silkworm moth is carried out into selfing or backcrossing, obtains the G1 of Diapause for silkworm seed, G1
Screening is scanned under fluorescence microscope after body pigmentation stage through instant sided corona treatment for silkworm seed and obtains trans genie individual；It will finally obtain
Trans genie individual normally raise, subculture.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109566530A (en) * | 2018-12-07 | 2019-04-05 | 广东省生物资源应用研究所 | A kind of method of the red ear tortoise ovum diapause of low temperature induction |
| CN113621619A (en) * | 2021-07-15 | 2021-11-09 | 西南大学 | Essential gene Bmpnd-2 for diapause of silkworm eggs and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1362520A (en) * | 2001-01-02 | 2002-08-07 | 成都天友发展有限公司 | Constitution process of blank expression system for transferring spider's dragline protein gene into Bombyx mori |
| KR20040049909A (en) * | 2002-12-05 | 2004-06-14 | 진병래 | Expression vector for transgenesis of silkworms and transgenic silkworm sustained the fifth instar larval period |
| CN1774507A (en) * | 2003-03-13 | 2006-05-17 | 科学研究国家中心 | Nucleic acid controlling the expression of a useful polypeptide in the posterior silk glands of a lepidoptera and application thereof |
| CN101195833A (en) * | 2007-12-21 | 2008-06-11 | 西南大学 | Low-temperature incubation transgene method for cultivated silkworm diapause breed variety |
| CN101195834A (en) * | 2007-12-21 | 2008-06-11 | 西南大学 | Early pickling transgene method for cultivated silkworm diapause breed variety |
| CN101503704A (en) * | 2009-03-17 | 2009-08-12 | 西南大学 | Transgenic method for cultivated silkworm diapause variety |
| CN106191111A (en) * | 2016-07-19 | 2016-12-07 | 西南大学 | A kind of method blocking transgenic bombyx mori ovum diapause |
-
2017
- 2017-04-12 CN CN201710239491.9A patent/CN107027718A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1362520A (en) * | 2001-01-02 | 2002-08-07 | 成都天友发展有限公司 | Constitution process of blank expression system for transferring spider's dragline protein gene into Bombyx mori |
| KR20040049909A (en) * | 2002-12-05 | 2004-06-14 | 진병래 | Expression vector for transgenesis of silkworms and transgenic silkworm sustained the fifth instar larval period |
| CN1774507A (en) * | 2003-03-13 | 2006-05-17 | 科学研究国家中心 | Nucleic acid controlling the expression of a useful polypeptide in the posterior silk glands of a lepidoptera and application thereof |
| CN101195833A (en) * | 2007-12-21 | 2008-06-11 | 西南大学 | Low-temperature incubation transgene method for cultivated silkworm diapause breed variety |
| CN101195834A (en) * | 2007-12-21 | 2008-06-11 | 西南大学 | Early pickling transgene method for cultivated silkworm diapause breed variety |
| CN101503704A (en) * | 2009-03-17 | 2009-08-12 | 西南大学 | Transgenic method for cultivated silkworm diapause variety |
| CN106191111A (en) * | 2016-07-19 | 2016-12-07 | 西南大学 | A kind of method blocking transgenic bombyx mori ovum diapause |
Non-Patent Citations (3)
| Title |
|---|
| 徐晓忠等: "影响家蚕电晕人工孵化的若干因素", 《浙江大学学报(农业与生命科学版)》 * |
| 徐汉福等: "适用于实用家蚕品种转基因的蚕卵滞育解除方法", 《蚕业科学》 * |
| 杨明观 等: "桑蚕卵电晕人工孵化技术研究1.即时电晕处理的人工孵化效果", 《蚕桑通报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109566530A (en) * | 2018-12-07 | 2019-04-05 | 广东省生物资源应用研究所 | A kind of method of the red ear tortoise ovum diapause of low temperature induction |
| CN113621619A (en) * | 2021-07-15 | 2021-11-09 | 西南大学 | Essential gene Bmpnd-2 for diapause of silkworm eggs and application thereof |
| CN113621619B (en) * | 2021-07-15 | 2023-09-12 | 西南大学 | Essential gene Bmpnd-2 for diapause of silkworm eggs and application thereof |
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