CN1904045A - Method of harmless extracting living body bee DNA - Google Patents
Method of harmless extracting living body bee DNA Download PDFInfo
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- CN1904045A CN1904045A CN 200610052873 CN200610052873A CN1904045A CN 1904045 A CN1904045 A CN 1904045A CN 200610052873 CN200610052873 CN 200610052873 CN 200610052873 A CN200610052873 A CN 200610052873A CN 1904045 A CN1904045 A CN 1904045A
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Abstract
The present invention discloses a method for noninjuriously extracting living body honey bee DNA. It is characterized by that the DNA can be extracted from desquamate tissue of queen bee and spado or drone which is remained in the bottom portion of cell of bees. Said method is simple in operation process and does not injure living body honey bee. The invented noninjurious extraction method of living body honey bee DNA has important application value for breeding honey bee.
Description
Technical field
The present invention relates to the method for harmless extracting living body bee DNA.
Background technology
The breeding of honeybee is according to the characteristic of honeybee heredity and variation, by selecting and breeding, certain the useful variation that constantly enlarges, accumulates and strengthen in the bee colony producing because of spontaneous mutation or gene recombination, and this useful variation is developed and a kind of breeding method of carrying out towards the direction of same variation character.Therefore, it is to the spontaneous mutation of occurring in nature existence or the utilization of gene recombination.In the existing bee species, the bee colony of a certain useful variation that produces because of spontaneous mutation or gene recombination is selected, it is bred into a system specially, by Continuous Selection, strict hand breeding towards same breeding objective, is selected and is bred.Like this, through after some generations, the good character that the assortment of genes of a certain useful mutator gene or reorganization is controlled just might obtain genetic stability in the offspring bee colony.Breed by expansion, make the useful allelic increase frequency of a certain good character of decision, make the characteristics of indivedual excellent individual be diffused as the total characteristics of colony rapidly, even the good character that is dispersed in each individuality is concentrated rapidly and changed into the total characteristics of colony.Thereby reach the purpose of breeding new variety or new lines.
As seen, in breeding process, whether to the good character selection is the breeding key of success accurately.Honeybee is a social insect completely, bee colony is the fundamental unit of biology of bee, production performance of honeybee and economic characters performance must be investigated by the production performance of bee colony and just can reflect, the production performance of bee colony is investigated and influenced by multiple external environmental conditions such as time, sweet powder source condition, weather and stable inadequately, and will spend a large amount of labours, material resources and just can finish.Along with the progress of science and technology and the fast development of Protocols in Molecular Biology, utilize the molecule marker relevant to carry out molecule assisted selection technology and in farm crop and domestic animal breeding, be used widely with economic characters.
In molecule assist-breeding process, the DNA extraction of queen bee and drone the link that is absolutely necessary, the individuality of queen bee and drone is not as other family bee-keeping animal or farm crop, getting sub-fraction organizes and just is enough to extract DNA for detection, any organize all that extracts queen bee or drone will cause serious injury even dead it, will influence its performance to the injury of queen bee or drone and investigate and plant with value.Therefore, seeking method that a kind of DNA that can obtain queen bee or drone do not injure honeybee again has important use and is worth in honeybee breeding.
At present, the method for extraction living body bee DNA mainly contains following several:
1) the DNA extraction method of honeybee chest muscle is caught queen bee or drone, with its execution, dissects chest and obtains muscle, places the 1.5ml centrifuge tube, cuts with ophthalmologic operation it is shredded.Add 560 μ l DNA extracts, add RNA enzyme A (10mg/ml) 4ul again, place 37 ℃ of digestion of thermostat container 2h.Take out, add Proteinase K (10mg/ μ l), abundant mixing, film is good with the centrifuge tube cap seal with sealing, 55 ℃ of digestion 12～24h.Add isopyknic phenol, slowly put upside down the abundant mixing of two-phase that centrifuge tube 10min makes solution,, supernatant liquor is moved to another clean centrifuge tube with the centrifugal 12min of 12000rpm.Add isopyknic phenol/chloroform/primary isoamyl alcohol (25: 24: 1), fully mixing is centrifugal, gets supernatant liquor.Add equal-volume chloroform/primary isoamyl alcohol (24: 1), repeat above-mentioned extraction steps.In the supernatant liquor of last acquisition, add dehydrated alcohol 800 μ l and 3M NaAC45 μ l.With the centrifugal 10min of 12000rpm, carefully remove liquid.With 75% washing with alcohol 2 times.Dissolve among an amount of TE-20 ℃ of preservations, the DNA of the honeybee that obtains to grow up after DNA dried naturally.
2) the DNA extraction method of honeybee tissue is caught queen bee or drone, and the histoorgan with eye scissors clip foot, wing or feeler etc. places the 1.5ml centrifuge tube, with eye scissors it is shredded.Add 560 μ l DNA extracts, add RNA enzyme A (10mg/ml) 4ul again, place 37 ℃ of digestion of thermostat container 2h.Take out, add Proteinase K (10mg/ μ l), abundant mixing, film is good with the centrifuge tube cap seal with sealing, 55 ℃ of digestion 12～24h.Add isopyknic phenol, slowly put upside down the abundant mixing of two-phase that centrifuge tube 10min makes solution,, supernatant liquor is moved to another clean centrifuge tube with the centrifugal 12min of 12000rpm.Add isopyknic phenol/chloroform/primary isoamyl alcohol (25: 24: 1), fully mixing is centrifugal, gets supernatant liquor.Add equal-volume chloroform/primary isoamyl alcohol (24: 1), repeat above-mentioned extraction steps.In the supernatant liquor of last acquisition, add dehydrated alcohol 800 μ l and 3M NaAC45 μ l.With the centrifugal 10min of 12000rpm, carefully remove liquid.With 75% washing with alcohol 2 times.Dissolve among an amount of TE-20 ℃ of preservations, the DNA of the honeybee that obtains to grow up after DNA dried naturally.
3) the DNA extraction method of larva or pupa is caught queen bee or drone larvae or pupa, places the 1.5ml centrifuge tube, cuts with ophthalmologic operation it is shredded, and adds 560 μ l DNA extracts, adds RNA enzyme A (10mg/ml) 4ul again, places 37 ℃ of digestion of thermostat container 2h.Take out, add Proteinase K (10mg/ μ l), abundant mixing, film is good with the centrifuge tube cap seal with sealing, 55 ℃ of digestion 12～24h.Add isopyknic phenol, slowly put upside down the abundant mixing of two-phase that centrifuge tube 10min makes solution,, supernatant liquor is moved to another clean centrifuge tube with the centrifugal 12min of 12000rpm.Add isopyknic phenol/chloroform/primary isoamyl alcohol (25: 24: 1), fully mixing is centrifugal, gets supernatant liquor.Add equal-volume chloroform/primary isoamyl alcohol (24: 1), repeat above-mentioned extraction steps.In the supernatant liquor of last acquisition, add dehydrated alcohol 800 μ l and 3M NaAC 45 μ l.With the centrifugal 10min of 12000rpm, carefully remove liquid.With 75% washing with alcohol 2 times.Dissolve among an amount of TE after DNA dried naturally ,-20 ℃ of preservations obtain the DNA of bee larva or pupa.
Above-mentioned bee DNA extracts or is to kill the honeybee individuality, or is the portion of tissue organ that extracts the honeybee individuality, and the honeybee individuality is very little, and such processing all injures living body bee inevitably, and the kind that influences its production performance investigation and queen bee drone is with being worth.
Summary of the invention
The method that the purpose of this invention is to provide harmless extracting living body bee DNA, utilize the biological characteristics of honeybee, the adult honeybee of honeybee can be stayed the lair bottom to casting off a skin of pupa time when sprouting wings, by collecting casting off a skin of firm emergence honeybee comb cell bottom, therefrom extract DNA, for the usefulness of genotype detection.
The method of harmless extracting living body bee DNA of the present invention, step is as follows:
1) royal cell of cultivating pupa time, drone pupae spleen or worker pupae spleen being put into 35 ℃, the incubator of relative humidity 65%～75% cultivates, when queen bee, drone or worker bee have just sprouted wings, catch queen bee, drone or worker bee, be stained with the mark that band is numbered at its chest backboard, centrifuge tube that collecting in corresponding lair casts off a skin puts into 1.5ml and note are good numbers;
2) add Proteinase K and the 7ul DTT of 200ul 5%Chelex 100,10ul 10mg/ml toward being collected in casting off a skin in the centrifuge tube, the vibration mixing, guarantee to cast off a skin and be immersed in the Chelex solution, 55 ℃ of temperature were bathed 20～24 hours, took out the vibration mixing, and sex change is 8 minutes in 100 ℃ of water-baths, at 4 ℃, 12,000～13, under the 000g condition centrifugal 3 minutes, obtain containing the supernatant liquor of living body bee DNA.
It is to cultivate the lair that queen bee built up that above-mentioned royal cell is meant by worker bee, produces in the lair and goes up the platform of proclaiming oneself king behind the ovum, and royal cell room shape is long, big, and the room wall is thicker, the room mouth down, and appearance is coarse as Pericarppium arachidis hypogaeae, generally builds in honeycomb both sides and lower edge.Queen bee lay eggs in royal cell 3 days ovum phase of back experience and 6 days larval stage, then enter pupa time, 7 days pupa time of queen bee.
Said lair is to build hole, room for cultivating the offspring or storing sweet powder etc. by worker bee with wax, and hole, room regular hexagon is put together by three parallelogram at the bottom of the room, littler title worker cell on same honeycomb, bigger honeycomb of holding sway over a region; The drone cell that the drone pupae spleen is meant is that common edge joins together, include drone pupae, the worker cell that the worker pupae spleen is meant is that common edge joins together, include worker pupae.
The present invention compared with prior art, the useful effect that has is:
1. adopting the inventive method is to extract DNA from the tissue of casting off a skin that honeybee abandons growth course, thereby avoids killing or extract honeybee tissue extraction DNA and living body bee is damaged.
2. the DNA that adopts the inventive method to extract queen bee or drone can detect its genotype, having important use in honeybee breeding is worth, because queen bee is unique mother of normal all members of bee colony, will directly influences its production performance to the injury of queen bee and investigate and plant with being worth.
3. adopt the operating process of the inventive method extraction bee DNA simple, cost is lower, is suitable for extracting in enormous quantities the dna sample of honeybee.
4. the DNA that adopts the inventive method to extract can detect the genotype of virgin king and mating drone, thereby predicts the production performance of this queen bee and drone, improves the accuracy that good queen bee is selected and remain and drone breeds, and improves honeybee breeding efficient greatly.
Embodiment
Embodiment 1:
For confirm from same individual muscle with cast off a skin the DNA that extracts be the same, we adopt 4 pairs of micro-satellite primers respectively to the muscle of 5 workers bee, 5 queen bees and 3 drones and the DNA that extracts in casting off a skin carry out the PCR detection.
1) honeybee muscle is tested bee farm with the Zhejiang University that is collected in of casting off a skin, select for use " No. 1, Zhejiang agricultural university " Apis mellifera kind as the test material, cultivate 20 royal cells with foster king's frame, after the royal cell capping the 5th day be put in 35 ℃, the incubator of relative humidity 65%～75% and cultivate supporting king's frame; Selecting the capping drone spleen of 18 ages in days and worker bee spleen to be put in 35 ℃, the incubator of relative humidity 65%～75% simultaneously from bee colony cultivates.Observe the developmental state of queen bee, worker bee, drone every day, when queen bee, drone or worker bee have just sprouted wings, catch 5 queen bees, 3 drones and 5 workers bee respectively, the good numbering of centrifuge tube that collecting in corresponding lair casts off a skin puts into 1.5ml and note.Queen bee, worker bee, casting off a skin of drone are numbered qc1-5, wc1-5, dc1-3 respectively.The chest muscle of queen bee, worker bee, drone is obtained in dissection, puts into corresponding 1.5ml centrifuge tube, and the muscle of queen bee, worker bee, drone is numbered qm1-5, wm1-5, dm1-3 respectively.
2) extraction of DNA respectively the muscle in the centrifuge tube, casting off a skin adds 200ul 5%Chelex 100 (Bio-Rad Laboratories, CA), 10ul 10mg/ml Proteinase K and 7ul DTT, the vibration mixing, guarantee that sample is immersed in the Chelex solution, 55 ℃ of temperature were bathed 20 hours, take out the vibration mixing, sex change is 8 minutes in 100 ℃ of water-baths, at 4 ℃, 13, under the 000g condition centrifugal 3 minutes, supernatant liquor is transferred in the centrifuge tube of another clean 1.5ml, contains the bee DNA that extracts in the supernatant liquor.
3) PCR detects and adopts 4 pairs of micro-satellite primers, primer sequence and PCR reaction conditions separately see Table 1, primer IRD 700/800 (METABION, Martinsried Germany) fluorescent mark, the muscle of queen bee, drone, worker bee and the DNA of the tissue extraction of casting off a skin are carried out pcr amplification, pipette the 4ul supernatant liquor as dna profiling, the PCR reaction system is undertaken by table 2.The PCR reaction product is the sample electrophoretic separation on LI-COR 4300 DNAAnalyzer sequenators, with SAGA software electrophoresis result is carried out data analysis, detects each individual genotype, the results are shown in Table 3.
As can be seen from the test results, from the muscle of 5 queen bees, 5 workers bee, 3 drones and the PCR result of the DNA that extracts of casting off a skin all in full accord, that is to say that the middle DNA that extracts of casting off a skin of a honeybee individuality is just the same with the DNA that extracts from this individuality muscle, the genotype of the DNA that detection is extracted from cast off a skin just can accurately be measured this individual genotype, utilizes dna molecular marker harmlessly to measure the genotypic purpose of living body bee thereby reach.This test is proof also, the DNA sample that extracts from a honeybee casts off a skin is enough to satisfy the requirement that PCR detects, as long as adopt the PCR primer relevant with economic characters, just can carry out pcr amplification to queen bee, drone or the worker bee DNA that extracts of casting off a skin, detect the genotype of this living body bee, reach production performance and the kind value of this queen bee of expection or drone.
The sequence of 4 pairs of micro-satellite primers of table 1 honeybee and PCR reaction conditions
| The site | Primer sequence | [Mg 2+] (mM) | Annealing temperature (℃) | Cycle index |
| A14 A29 A43 B124 | 5’-GTGTCGCAATCGACGTAACC-3’ 5’-GTCGATTACCGATCGTGACG-3’ 5’-AAACAGTACATTTGTGACCC-3’ 5’-CAACTTCAACTGAAATCCG-3’ 5’-CACCGAAACAAGATGCAAG-3’ 5’-CCGCTCATTAAGATATCCG-3’ 5’-GCAACAGGTCGGGTTAGAG-3’ 5’-CAGGATAGGGTAGGTAAGCAG-3’ | 2.5 2.5 2.5 2.5 | 58 54 55 55 | 40 40 40 40 |
The little satellite PCR of table 2 honeybee reaction system
| H 2O(μL) | 8.4-X |
| Buffer(μL) MgCl 2(25mM)(μL) dNTPs(2mM each)(μL) Primer1(10μM)(μL) Primer2(10μM)(μL) Taq polymerase(5U/μL)(μL) DNA Template(μL) | 1.25 1.25 1.00 0.25 0.25 0.10 X |
| Total(μL) | 12.5 |
The PCR reaction result of the table 3 honeybee muscle and the extraction DNA of casting off a skin
| The sample title | Primer | |||
| A14 | A29 | A43 | B124 | |
| wc1 wc2 wc3 wc4 wc5 wm1 wm2 wm3 wm4 wm5 qc1 qc2 qc3 qc4 qc5 qm1 qm2 qm3 qm4 qm5 dc1 dc2 dc3 dm1 dm2 dm3 | 214/214 214/214 214/224 214/214 214/224 214/214 214/214 214/224 214/214 214/224 214/224 214/230 214/224 214/224 214/230 214/224 214/230 214/224 214/224 214/230 216 214 216 216 214 216 | 148/176 148/176 148/162 148/178 150/150 148/176 148/176 148/162 148/178 150/150 148/150 148/148 148/150 150/162 148/148 148/150 148/148 148/150 150/162 148/148 156 148 156 156 148 156 | 123/139 123/123 123/139 125/125 125/125 123/139 123/123 123/139 125/125 125/125 125/139 139/139 123/123 123/123 123/139 125/139 139/139 123/123 123/123 123/139 123 123 123 123 123 123 | 212/214 212/214 214/222 212/214 214/216 212/214 212/214 214/222 212/214 214/216 214/216 214/214 214/216 214/222 214/214 214/216 214/214 214/216 214/222 214/214 216 214 214 216 214 214 |
Annotate: wc1-5 represents the worker bee sample 1-5 of casting off a skin; Wm1-5 represents worker bee chest muscle sample 1-5;
Qc1-5 represents the queen bee sample 1-5 of casting off a skin; Qm1-5 represents queen bee chest muscle sample 1-5;
Dc1-3 represents the drone sample 1-3 of casting off a skin; Dm1-3 represents drone chest muscle sample 1-3;
Wc1 and wm1 representative cast off a skin and muscle tissue from same worker bee individuality;
Qc1 and qm1 representative cast off a skin and muscle tissue from same queen bee individuality, other the rest may be inferred these samples are all from same bee colony.
Claims (1)
1. the method for harmless extracting living body bee DNA is characterized in that step is as follows:
1) royal cell of cultivating pupa time, drone pupae spleen or worker pupae spleen being put into 35 ℃, the incubator of relative humidity 65%～75% cultivates, when queen bee, drone or worker bee have just sprouted wings, catch queen bee, drone or worker bee, be stained with the mark that band is numbered at its chest backboard, centrifuge tube that collecting in corresponding lair casts off a skin puts into 1.5ml and note are good numbers;
2) add Proteinase K and the 7ul DTT of 200ul_5%Chelex 100,10ul 10mg/ml toward being collected in casting off a skin in the centrifuge tube, the vibration mixing, guarantee to cast off a skin and be immersed in the Chelex solution, 55 ℃ of temperature were bathed 20～24 hours, took out the vibration mixing, and sex change is 8 minutes in 100 ℃ of water-baths, at 4 ℃, 12,000～13, under the 000g condition centrifugal 3 minutes, obtain containing the supernatant liquor of living body bee DNA.
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| CNB2006100528732A CN100415883C (en) | 2006-08-10 | 2006-08-10 | Method of harmless extracting living body bee DNA |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101319213B (en) * | 2007-06-07 | 2010-10-20 | 上海市血液中心 | Bacteria genome DNA extraction liquid, preparation and application thereof |
| CN103184215A (en) * | 2013-04-10 | 2013-07-03 | 中国水产科学研究院长江水产研究所 | Method for extracting genomic DNA from giant salamander skin mucus |
| CN110277210A (en) * | 2019-06-24 | 2019-09-24 | 黑龙江八一农垦大学 | The extracting method of magnetic particle in a kind of honeybee |
| CN111575277A (en) * | 2020-06-04 | 2020-08-25 | 天津师范大学 | Method for extracting molting DNA (deoxyribonucleic acid) in pupal stage of single chironomidae |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5013644A (en) * | 1987-12-07 | 1991-05-07 | Wisconsin Alumni Research Foundation | Identification of africanized honey bees |
| CN1306004A (en) * | 2000-12-25 | 2001-08-01 | 武汉市康源蜂业有限公司 | Application of bee larva in preparing nucleic acid |
-
2006
- 2006-08-10 CN CNB2006100528732A patent/CN100415883C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101319213B (en) * | 2007-06-07 | 2010-10-20 | 上海市血液中心 | Bacteria genome DNA extraction liquid, preparation and application thereof |
| CN103184215A (en) * | 2013-04-10 | 2013-07-03 | 中国水产科学研究院长江水产研究所 | Method for extracting genomic DNA from giant salamander skin mucus |
| CN110277210A (en) * | 2019-06-24 | 2019-09-24 | 黑龙江八一农垦大学 | The extracting method of magnetic particle in a kind of honeybee |
| CN111575277A (en) * | 2020-06-04 | 2020-08-25 | 天津师范大学 | Method for extracting molting DNA (deoxyribonucleic acid) in pupal stage of single chironomidae |
| CN111575277B (en) * | 2020-06-04 | 2023-10-24 | 天津师范大学 | Method for extracting single molting DNA in midge period of midge family |
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