CN107258703A - Honeybee ovum for microinjection pipettes method - Google Patents
Honeybee ovum for microinjection pipettes method Download PDFInfo
- Publication number
- CN107258703A CN107258703A CN201710703765.5A CN201710703765A CN107258703A CN 107258703 A CN107258703 A CN 107258703A CN 201710703765 A CN201710703765 A CN 201710703765A CN 107258703 A CN107258703 A CN 107258703A
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- China
- Prior art keywords
- honeybee
- ovum
- bee
- honeybee ovum
- microinjection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 241000256844 Apis mellifera Species 0.000 title claims abstract description 96
- 108010000912 Egg Proteins Proteins 0.000 title claims abstract description 61
- 102000002322 Egg Proteins Human genes 0.000 title claims abstract description 61
- 210000004681 ovum Anatomy 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000000520 microinjection Methods 0.000 title claims abstract description 17
- 235000013871 bee wax Nutrition 0.000 claims abstract description 10
- 239000012166 beeswax Substances 0.000 claims abstract description 10
- 235000013601 eggs Nutrition 0.000 claims abstract description 9
- 238000009341 apiculture Methods 0.000 claims abstract description 8
- 238000009395 breeding Methods 0.000 claims abstract description 7
- 230000001488 breeding effect Effects 0.000 claims abstract description 7
- 230000004936 stimulating effect Effects 0.000 claims description 3
- 230000035558 fertility Effects 0.000 claims description 2
- 230000012447 hatching Effects 0.000 abstract description 6
- 238000010362 genome editing Methods 0.000 abstract description 4
- 238000011534 incubation Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 2
- 238000011160 research Methods 0.000 description 8
- 238000012546 transfer Methods 0.000 description 5
- 108091033409 CRISPR Proteins 0.000 description 4
- 241000238631 Hexapoda Species 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000035611 feeding Effects 0.000 description 3
- 206010052428 Wound Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000256837 Apidae Species 0.000 description 1
- 241000406668 Loxodonta cyclotis Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000001136 chorion Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000012907 honey Nutrition 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000004224 protection Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
Classifications
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Housing For Livestock And Birds (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Method is pipetted the present invention relates to a kind of honeybee ovum for microinjection, limit king's spawning is carried out first with the biological characteristics of queen to lay eggs, then uprightly sticked the characteristics of lair bottom using honeybee ovum and beeswax viscosity, the beeswax of honeybee ovum bottom is shoveled out with Moving needle for bee-keeping, is bonded on septum bar.The present invention is shifted to being largely derived from honeybee ovum in 1 hour, obtains incubation rate up to 94% effect, and solving the low problem of honeybee egg hatching rate in 1 hour, there is provided a kind of method for efficiently pipetting honeybee ovum.The honeybee ovum in a large amount of 1 hour can be shifted with fanout free region, is that application providing way of the honeybee genome editing technique in honeybee breeding field is supported.
Description
Technical field
Method is pipetted the present invention relates to a kind of honeybee ovum, and in particular to a kind of honeybee ovum side of pipetting for microinjection
Method, belongs to honeybee breeding technical field.
Background technology
Honeybee is a kind of important economic insects, and its product and its product are played increasingly in healthcare industry and medical field
Important effect.Honeybee is the important pollinator of plant again simultaneously, and its pollinate value and Ecology Action are even more inestimable.And make
For important model organism, other patterns are but lagged far behind in terms of biology of bee basic research and gene functional research
It is biological.Its reason is analyzed it can be found that it is to cause its gene work(to lack effective gene editing instrument in honeybee field
The main cause for lagging behind other model insects for a long time can be studied.Although having had Many researchers to explore is adapted to honeybee genome
The method of edit operation, for example：Semen transformation, microinjection, electroporation, transposon vector method, Crispr/Cas9 skills
Art, the method for these gene editings can contribute to us to further investigate the complicated social action of honeybee and caste differentiation phenomenon,
It will also accelerate the process of merit molecular breeding simultaneously.But these methods are built upon and obtained on intact honeybee ovum.It is special
It is not to obtain the honeybee ovum in 1 hour and 1 hour.On the one hand there is the honeybee ovum of thousands of honeybee protections unlike other productions are in leaf
The insect ovum on lower or pool side has stronger adaptability to rugged environment, once leaving bee colony for a long time, honeybee ovum is easy to extremely
Die or develop and be abnormal.On the other hand, the chorion of honeybee ovum is relatively thin and flexible, and it is difficult operation to stick in lair bottom.Even if energy
Shifted by taking ovum instrument to take out honeybee ovum, it is also difficult to which guarantee has higher incubation rate.These are observed under Stereo microscope not
The honeybee ovum of hatching, it can be seen that honeybee oophyte table has some Mechanical wounds, these wounds are that naked eyes be can't see.So a large amount of
Take the honeybee ovum in 1 hour to be shifted and ensure that more than 90% incubation rate turns into the master for hindering scientific research personnel to carry out honeybee ovum research
Want problem.If this problem can not break through, even efficient easily Crispr/Cas9 technologies can not also carry out in honeybee should
With, and want to accelerate the screening of honeybee merit molecule by honeybee genome editing technique and cultivate disease-resistant high yield new varieties
Hope is also broken in this by dream.Even hinder scientific research personnel further to study the complicated social action of honeybee with the special biology of honeybee to show
The basic research of elephant.
The content of the invention
Method is pipetted it is an object of the invention to provide a kind of honeybee ovum for microinjection.For solving largely to pipette 1
Honeybee ovum in hour, and incubation rate is not less than 90%, is that the honeybee ovum research in honeybee breeding work lays the foundation.
To realize that the object of the invention is adopted the following technical scheme that：
The present invention pipettes method for the honeybee ovum of microinjection, it is characterised in that removing step is as follows：
（1）On new honeycomb queen bee is limited with pin type prisoner king cage 24 hours；
（2）By step（1）Pin type prisoner king's cage be transferred to the other positions of the new honeycomb, limit 1 hour of queen to lay eggs；It is described
Other positions, i.e., the optional position in addition to original position；
（3）Cut off step（2）Breeding area under pin type prisoner king's cage in honeycomb, carries honeybee under being shoveled under Stereo microscope with Moving needle for bee-keeping
The beeswax of ovum, is transferred out of lair；
（4）Septum separately is taken, thickness is pressed into and is 1mm thin slice, and is cut into strip, is sticked on culture dish；
（5）By step（3）In carry honeybee ovum Moving needle for bee-keeping, be transferred to step（4）Stick on culture dish on the edge of septum bar, will
Beeswax with honeybee ovum is sticked on septum bar, as the honeybee ovum for microinjection.
Wherein, step（1）The queen bee, from starting after spawning no more than six months, the strong queen bee of fecundity；
Step（1）The new honeycomb, the new honeycomb just built from bee colony；
Step（1）Described limitation queen bee 24 hours, stimulating feeding is carried out while limitation queen bee to bee colony.
The honeycomb, pin type prisoner king cage, septum, Moving needle for bee-keeping, culture dish are bought by market.
In order to explore influence of the different disposal condition to honeybee egg hatching rate, our honeybee ovum to transfer and injection sterilized water
Honeybee ovum carries out hatching number statistical, and statistical result is as shown in table 1.
Influence of the different disposal condition of table 1 to honeybee egg hatching rate
The condition of culture of each group honeybee egg hatching is identical in table 1.The wherein control group honeycomb that cuts after king's spawning that is limited is direct
Incubator culture is put into, to honeybee ovum without any operation, its average percentage hatch rate is 98.2%.Transfer group is that honeybee ovum is transferred out of into lair
And stick on the septum bar of culture dish, its average percentage hatch rate is 94.3%.Injection group is that the honeybee ovum chosen after transfer is injected,
The average percentage hatch rate for injecting sterilized water is 64.8%.In addition, the honeybee ovum for microinjection of the present invention pipettes method, it is average
It is per minute to pipette 4 or so, honeybee ovum, more than 100, honeybee ovum can be harmlessly pipetted in 30 minutes.
The present invention remarkable advantage be：
1st, during transfer honeybee ovum, honeybee ovum is contacted without any utensil, honeybee ovum is not by any injury in theory.In experiment
The average percentage hatch rate of honeybee ovum can reach 94%.
2nd, due to using new honeycomb, so being very easy to shovel out the beeswax of honeybee ovum afterbody.100 honeybees can be shifted within 30 minutes
Ovum.
3rd, using the viscosity of beeswax, honeybee ovum is secured well.It is easy to carry out microinjection to honeybee ovum and is related to honeybee ovum
Other scientific researches.
4th, it is a large amount of quickly to obtain honeybee ovum in 1 hour using queen to lay eggs habit.
Embodiment
With reference to embodiment, the present invention is further elaborated.
Embodiment 1, a kind of honeybee ovum for microinjection pipette method, comprise the following steps：
（1）Queen bee is limited with pin type prisoner king cage 24 hours, on new honeycomb while carrying out stimulating feeding, i.e., enough feedings to bee colony
Honey；
（2）By step（1）Pin type prisoner king's cage be transferred to the other positions of the honeycomb, limit 1 hour of queen to lay eggs；
（3）Cut off step（2）Breeding area under pin type prisoner king's cage in honeycomb, carries honeybee under being shoveled under Stereo microscope with Moving needle for bee-keeping
The beeswax of ovum, is transferred out of lair；Note avoiding honeybee ovum from contacting lair wall during transfer；
（4）Septum separately is taken, the thin slice that thickness is 1mm is pressed into, wide 0.5cm is cut into, long 6cm septums bar utilizes the viscous of new septum bar
Property, sticked on culture dish；
（5）By step（3）In carry honeybee ovum Moving needle for bee-keeping, be transferred to step（4）Stick on culture dish on the edge of septum bar, will
Beeswax with honeybee ovum is sticked on septum bar, as the honeybee ovum for microinjection.
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, should all belong to the covering scope of the present invention.
Claims (4)
1. a kind of honeybee ovum for microinjection pipettes method, it is characterised in that removing step is as follows：
（1）On new honeycomb queen bee is limited with pin type prisoner king cage 24 hours；
（2）By step（1）Pin type prisoner king's cage be transferred to the other positions of the new honeycomb, limit 1 hour of queen to lay eggs；It is described
Other positions, i.e., except the outside upper optional position of original position；
（3）Cut off step（2）Breeding area under pin type prisoner king's cage in honeycomb, carries honeybee under being shoveled under Stereo microscope with Moving needle for bee-keeping
The beeswax of ovum, is transferred out of lair；
（4）Septum separately is taken, thickness is pressed into and is 1mm thin slice, and is cut into strip, is sticked on culture dish；
（5）By step（3）In carry honeybee ovum Moving needle for bee-keeping, be transferred to step（4）Stick on culture dish on the edge of septum bar, will
Beeswax with honeybee ovum is sticked on septum bar；As it is used for the honeybee ovum of microinjection.
2. a kind of honeybee ovum for microinjection according to claim 1 pipettes method, it is characterised in that step（1）Institute
New honeycomb is stated, the new honeycomb just built from bee colony.
3. a kind of honeybee ovum for microinjection according to claim 1 pipettes method, it is characterised in that step（1）Institute
Queen bee is stated, from starting after spawning no more than six months, the strong new queen bee of fecundity.
4. a kind of honeybee ovum for microinjection according to claim 1 pipettes method, it is characterised in that step（1）Institute
The limitation queen bee stated 24 hours, stimulating feeding is carried out while limitation queen bee to bee colony.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710703765.5A CN107258703B (en) | 2017-08-16 | 2017-08-16 | Bee egg removal method for microinjection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710703765.5A CN107258703B (en) | 2017-08-16 | 2017-08-16 | Bee egg removal method for microinjection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107258703A true CN107258703A (en) | 2017-10-20 |
| CN107258703B CN107258703B (en) | 2019-12-13 |
Family
ID=60077358
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710703765.5A Active CN107258703B (en) | 2017-08-16 | 2017-08-16 | Bee egg removal method for microinjection |
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| Country | Link |
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| CN (1) | CN107258703B (en) |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1069619A (en) * | 1991-08-22 | 1993-03-10 | 陈信洁 | Anti-beemite is prevented the foulbrood beehive base |
| WO1994002600A1 (en) * | 1992-07-20 | 1994-02-03 | University Of Florida | Microinjection methods to transform arthropods with exogenous dna |
| CN2536031Y (en) * | 2002-01-15 | 2003-02-19 | 郑英花 | Honeycomb frame |
| CN102499123A (en) * | 2011-11-23 | 2012-06-20 | 江西农业大学 | Bee simulation larvae-shifting-free royal jelly production method |
| CN202958456U (en) * | 2012-12-20 | 2013-06-05 | 张�诚 | Middle queen excluder of one-eighth queen bee mating hive |
| CN203262059U (en) * | 2013-05-09 | 2013-11-06 | 北京市农林科学院 | Mini-type insect raising cage for experiments on living plants |
| CN103461222A (en) * | 2013-09-02 | 2013-12-25 | 常熟市荣兴化纺有限责任公司 | Egg fetching plug strip and comb matching structure |
| CN104304095A (en) * | 2014-10-22 | 2015-01-28 | 申磊 | Chinese bee bottle type hive honey flat box production method and device and frame for production |
| CN204860633U (en) * | 2015-08-14 | 2015-12-16 | 中国农业科学院蜜蜂研究所 | Drone ovum collection device |
| CN105360073A (en) * | 2015-10-12 | 2016-03-02 | 南宁市全健蜜蜂养殖场 | Method for selection and rearing of bee species queen |
| CN105494264A (en) * | 2015-12-14 | 2016-04-20 | 甘肃省蜂业技术推广总站 | Breeding method for good bee species for facility crop pollination |
-
2017
- 2017-08-16 CN CN201710703765.5A patent/CN107258703B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1069619A (en) * | 1991-08-22 | 1993-03-10 | 陈信洁 | Anti-beemite is prevented the foulbrood beehive base |
| WO1994002600A1 (en) * | 1992-07-20 | 1994-02-03 | University Of Florida | Microinjection methods to transform arthropods with exogenous dna |
| CN2536031Y (en) * | 2002-01-15 | 2003-02-19 | 郑英花 | Honeycomb frame |
| CN102499123A (en) * | 2011-11-23 | 2012-06-20 | 江西农业大学 | Bee simulation larvae-shifting-free royal jelly production method |
| CN202958456U (en) * | 2012-12-20 | 2013-06-05 | 张�诚 | Middle queen excluder of one-eighth queen bee mating hive |
| CN203262059U (en) * | 2013-05-09 | 2013-11-06 | 北京市农林科学院 | Mini-type insect raising cage for experiments on living plants |
| CN103461222A (en) * | 2013-09-02 | 2013-12-25 | 常熟市荣兴化纺有限责任公司 | Egg fetching plug strip and comb matching structure |
| CN104304095A (en) * | 2014-10-22 | 2015-01-28 | 申磊 | Chinese bee bottle type hive honey flat box production method and device and frame for production |
| CN204860633U (en) * | 2015-08-14 | 2015-12-16 | 中国农业科学院蜜蜂研究所 | Drone ovum collection device |
| CN105360073A (en) * | 2015-10-12 | 2016-03-02 | 南宁市全健蜜蜂养殖场 | Method for selection and rearing of bee species queen |
| CN105494264A (en) * | 2015-12-14 | 2016-04-20 | 甘肃省蜂业技术推广总站 | Breeding method for good bee species for facility crop pollination |
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| Title |
|---|
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| Publication number | Publication date |
|---|---|
| CN107258703B (en) | 2019-12-13 |
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