CN100415883C - A method for extracting DNA from living bees without harm - Google Patents

Abstract

The method for extracting living honeybee DNA without harm disclosed by the present invention is to extract DNA from molt tissue left at the bottom of the hive when queen bee, worker bee or drone just emerges, the operation process is simple, the living bee is not harmed, and the cost is low. The method for extracting the DNA of live honeybees without damage of the present invention has important application value in bee breeding. By extracting the DNA of honeybee molting, PCR detects the genotype of its economic traits gene locus, predicts the production performance of queen bee or drone, and can improve The accuracy of good queen bee selection and drone mating greatly improves the efficiency of bee breeding.

Description

A method for extracting DNA from living bees without harm

technical field

The invention relates to a method for extracting living honeybee DNA without damage.

Background technique

Bee breeding is to continuously expand, accumulate and strengthen a certain beneficial variation in the bee population due to natural mutation or gene recombination through selection and reproduction according to the characteristics of bee inheritance and variation, and to make this beneficial variation move towards the same variation A breeding method for the development of nature. Therefore, it is the utilization of the phenomenon of spontaneous mutation or gene recombination existing in nature. Among the existing bee varieties, a bee colony with a beneficial variation caused by spontaneous mutation or genetic recombination is selected, and it is specially bred into a system. Through continuous selection, mating is strictly controlled, and the same breeding goal is achieved. selection and breeding. In this way, after several generations, the excellent traits controlled by a beneficial mutant gene or recombined gene combination may be stably inherited in the offspring bee colony. Through the expansion of breeding, the frequency of beneficial alleles that determine a certain excellent trait is increased, and the characteristics of individual excellent individuals are rapidly diffused into common characteristics of the group, and even the excellent traits scattered among individual individuals are quickly concentrated and transformed into common characteristics of the group. specialty. So as to achieve the purpose of breeding new varieties or new strains.

It can be seen that in the breeding process, the accuracy of selection of good traits is the key to the success of breeding. Honey bees are completely social insects, and bee colonies are the basic unit of bee biology. The production performance and economic traits of bees must be reflected through the production performance of bee colonies. The production performance of bee colonies is affected by time, honey powder source Conditions, climate and other external environmental conditions are not stable enough, and it takes a lot of labor and material resources to complete. With the advancement of science and technology and the rapid development of molecular biology techniques, molecular assisted selection breeding technology using molecular markers related to economic traits has been widely used in crop and domestic animal breeding.

In the process of molecular assisted breeding, the DNA extraction of queen bees and drones is an essential link. Unlike other domestic beekeeping animals or crops, a small part of the tissue is enough to extract DNA for detection. Cutting any tissue of the queen bee or the drone will cause serious injury or even death to it, and the damage to the queen bee or the drone will affect its performance inspection and species value. Therefore, finding a method that can obtain the DNA of queen bees or drones without harming bees has important application value in bee breeding.

At present, there are mainly the following methods to extract the DNA of living bees:

1) DNA extraction method of bee chest muscles Capture queen bees or drones, kill them, dissect the chest to obtain muscles, place them in a 1.5ml centrifuge tube, and cut them into pieces with ophthalmic surgical scissors. Add 560μl of DNA extraction solution, then add 4ul of RNase A (10mg/ml), and place it in an incubator at 37°C for 2h for digestion. Take it out, add proteinase K (10mg/μl), mix thoroughly, seal the cap of the centrifuge tube with a parafilm, and digest at 55°C for 12-24h. Add an equal volume of phenol, slowly invert the centrifuge tube for 10 minutes to fully mix the two phases of the solution, centrifuge at 12000 rpm for 12 minutes, and transfer the supernatant to another clean centrifuge tube. Add an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1), mix well, centrifuge, and take the supernatant. An equal volume of chloroform/isoamyl alcohol (24:1) was added, and the above extraction step was repeated. Add 800 μl of absolute ethanol and 45 μl of 3M NaAC to the finally obtained supernatant. Centrifuge at 12000rpm for 10min, pour off the liquid carefully. Wash 2 times with 75% ethanol. The DNA was dried naturally, dissolved in an appropriate amount of TE, and stored at -20°C to obtain the DNA of adult bees.

2) DNA extraction method of bee tissue Capture queen bee or drone, use ophthalmic scissors to cut out tissues and organs such as feet, wings or antennae, put them in a 1.5ml centrifuge tube, and use ophthalmic scissors to cut them into pieces. Add 560 μl of DNA extraction solution, then add 4ul of RNase A (10mg/ml), and place in an incubator for digestion at 37°C for 2h. Take it out, add proteinase K (10mg/μl), mix thoroughly, seal the cap of the centrifuge tube with a parafilm, and digest at 55°C for 12-24h. Add an equal volume of phenol, slowly invert the centrifuge tube for 10 minutes to fully mix the two phases of the solution, centrifuge at 12000 rpm for 12 minutes, and transfer the supernatant to another clean centrifuge tube. Add an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1), mix well, centrifuge, and take the supernatant. An equal volume of chloroform/isoamyl alcohol (24:1) was added, and the above extraction step was repeated. Add 800 μl of absolute ethanol and 45 μl of 3M NaAC to the finally obtained supernatant. Centrifuge at 12000rpm for 10min, pour off the liquid carefully. Wash 2 times with 75% ethanol. The DNA was dried naturally, dissolved in an appropriate amount of TE, and stored at -20°C to obtain the DNA of adult bees.

3) DNA extraction method for larvae or pupae Capture queen bee or drone larvae or pupae, place them in a 1.5ml centrifuge tube, cut them into pieces with ophthalmic surgical scissors, add 560μl DNA extraction solution, and then add RNase A (10mg/ml ) 4ul, placed in an incubator at 37°C for 2h. Take it out, add proteinase K (10mg/μl), mix thoroughly, seal the cap of the centrifuge tube with a parafilm, and digest at 55°C for 12-24h. Add an equal volume of phenol, slowly invert the centrifuge tube for 10 minutes to fully mix the two phases of the solution, centrifuge at 12000 rpm for 12 minutes, and transfer the supernatant to another clean centrifuge tube. Add an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1), mix well, centrifuge, and take the supernatant. An equal volume of chloroform/isoamyl alcohol (24:1) was added, and the above extraction step was repeated. Add 800 μl of absolute ethanol and 45 μl of 3M NaAC to the finally obtained supernatant. Centrifuge at 12000rpm for 10min, pour off the liquid carefully. Wash 2 times with 75% ethanol. After the DNA was dried naturally, it was dissolved in an appropriate amount of TE and stored at -20°C to obtain the DNA of bee larvae or pupae.

The above-mentioned bee DNA extraction either kills individual bees, or cuts off some tissues and organs of individual bees. Individual bees are very small. Such treatment will inevitably harm living bees, affecting their production performance and the use of queen bee drones. value.

Contents of the invention

The purpose of the present invention is to provide a method for extracting the DNA of living bees without harm. Utilizing the biological characteristics of honeybees, adult honeybees will leave the molt at the pupal stage at the bottom of the hive when they emerge. , from which DNA is extracted for genotyping purposes.

The method for extracting living honeybee DNA without harm of the present invention, the steps are as follows:

1) Put the queen bee, drone pupa spleen or worker bee pupa spleen cultivated to the pupal stage into an incubator at 35°C and a relative humidity of 65% to 75% for cultivation. When the queen bee, drone or worker bee has just emerged, catch the queen bee, For the drone or worker bee, stick a numbered mark on the back of its chest, collect the molt from the corresponding nest room, put it into a 1.5ml centrifuge tube and record the number;

2) Add 200ul of 5% Chelex 100, 10ul of 10mg/ml proteinase K and 7ul of DTT to the molts collected in the centrifuge tube, shake and mix to ensure that the molts are soaked in the Chelex solution, incubate at 55°C for 20-24 hours, take out and shake Mix well, denature in a water bath at 100°C for 8 minutes, centrifuge at 4°C and 12,000-13,000g for 3 minutes to obtain a supernatant containing living bee DNA.

The above-mentioned royal tower refers to the nest room built by worker bees to cultivate the queen bee. After laying eggs in the nest house, it is called the king tower. Peanut shells are generally built on both sides and the lower edge of the spleen. The queen bee goes through the egg stage of 3 days and the larval stage of 6 days after laying eggs in the royal platform, and then enters the pupal stage, and the queen bee pupal stage is 7 days.

The so-called nest room is built by worker bees with wax as a room hole for cultivating offspring or storing honey powder. Worker honeycomb, bigger claim drone room; Drone pupa spleen refers to the drone chamber that the common side is connected into one piece, contains the drone pupa, and the worker honeycomb spleen refers to the worker honeycomb that the public side is connected into one piece, and contains the worker bee pupa.

Compared with the prior art, the present invention has the beneficial effects that:

1. Adopting the method of the present invention is to extract DNA from the molting tissue discarded by honeybees in the development process, thereby avoiding killing or cutting honeybee tissue to extract DNA and causing damage to living bees.

2. Adopt the method of the present invention to extract the DNA of queen bee or drone to detect its genotype, which has important application value in bee breeding, because the queen bee is the only mother of all members of a normal bee colony, the damage to the queen bee will directly affect its production Performance inspection and species value.

3. The operation process of extracting honeybee DNA by the method of the present invention is simple and low in cost, and is suitable for extracting honeybee DNA samples in large quantities.

4. The DNA extracted by the inventive method can detect the genotypes of virgin queens and mating drones, thereby predicting the production performance of the queen bees and drones, improving the accuracy of excellent queen bee selection and drone mating, and greatly improving the breeding efficiency of honeybees.

Detailed ways

Example 1:

In order to confirm that the DNA extracted from the muscle and molting of the same individual is the same, we used 4 pairs of microsatellite primers to detect the DNA extracted from the muscle and molting of 5 worker bees, 5 queen bees and 3 drones respectively.

1) Collection of honeybee muscle and molted skin In the experimental apiary of Zhejiang University, the Italian bee variety "Zhe Nong Da No. 1" was selected as the experimental material, and 20 king bees were cultivated with the king-raising frame. The king box is placed in an incubator at 35°C and a relative humidity of 65% to 75% for cultivation; at the same time, 18-day-old sealed drone spleen and worker honeycomb are selected from the bee colony and placed in an incubator at 35°C and a relative humidity of 65% to 75%. Cultured in an incubator. Observe the development of queen bees, worker bees, and drones every day. When the queen bees, drones, or worker bees have just emerged, capture 5 queen bees, 3 drones, and 5 worker bees respectively, and collect the molt from the corresponding nest room and put them into a 1.5ml centrifuge tube and write down the serial number. The molts of queen bees, worker bees and drones are numbered qc1-5, Wc1-5 and dc1-3 respectively. The chest muscles of queen bees, worker bees, and drones were dissected and put into corresponding 1.5ml centrifuge tubes. The muscles of queen bees, worker bees, and drones were numbered qm1-5, wm1-5, and dm1-3, respectively.

2) Extraction of DNA Add 200ul of 5% Chelex 100 (Bio-Rad Laboratories, CA), 10ul of 10mg/ml proteinase K and 7ul of DTT to the muscle and molt in the centrifuge tube, shake and mix well to ensure that the sample is soaked in the Chelex solution , incubate at 55°C for 20 hours, take it out and shake and mix well, denature in 100°C water bath for 8 minutes, centrifuge at 4°C and 13,000g for 3 minutes, transfer the supernatant to another clean 1.5ml centrifuge tube, The serum contains extracted bee DNA.

3) PCR detection 4 pairs of microsatellite primers were used. The primer sequences and their respective PCR reaction conditions are shown in Table 1. The primers were fluorescently labeled with IRD 700/800 (METABION, Martinsried Germany) to extract the muscle and molting tissues of queen bees, drones, and worker bees. The DNA was amplified by PCR, and 4 ul of the supernatant was pipetted as a DNA template, and the PCR reaction system was carried out according to Table 2. The PCR reaction products were separated by electrophoresis on the LI-COR 4300 DNAAnalyzer sequencer, and the electrophoresis results were analyzed using SAGA software to detect the genotype of each individual. The results are shown in Table 3.

It can be seen from the test results that the PCR results of the DNA extracted from the muscles and molts of 5 queen bees, 5 worker bees, and 3 drones are completely consistent. The DNA extracted from the molting is exactly the same, and the genotype of the DNA extracted from the molting can be accurately determined to determine the genotype of the individual, thereby achieving the purpose of using DNA molecular markers to determine the genotype of living bees without harm. The test also proved that the DNA sample extracted from the molt of a bee is sufficient for PCR detection, as long as the PCR primers related to economic traits are used, the DNA extracted from the molt of a queen bee, drone or worker bee can be PCR amplified Detecting the genotype of the living bee to achieve the expected production performance and breeding value of the queen bee or drone.

Table 1 Sequences and PCR reaction conditions of 4 pairs of microsatellite primers in honey bees

Table 2 Honey bee microsatellite PCR reaction system

Table 3 PCR reaction results of DNA extracted from honeybee muscle and molt

Note: wc1-5 means worker bee molting samples 1-5; wm1-5 means worker bee chest muscle samples 1-5;

qc1-5 represents queen bee molting samples 1-5, qm1-5 represents queen bee chest muscle samples 1-5;

dc1-3 represents drone molting samples 1-3; dm1-3 represents drone chest muscle samples 1-3;

wc1 and wm1 represent molting and muscle tissue from the same individual worker bee;

qc1 and qm1 represent that the molting and musculature come from the same queen individual, and so on.

These samples were all from the same colony.

Claims (1)

1. The method for extracting living honeybee DNA without harm, is characterized in that the steps are as follows: 1) Put the queen bee, drone pupa spleen or worker bee pupa spleen cultivated to the pupal stage into an incubator at 35°C and a relative humidity of 65% to 75% for cultivation. When the queen bee, drone or worker bee has just emerged, catch the queen bee, For the drone or worker bee, stick a numbered mark on the back of its chest, collect the molt from the corresponding nest room, put it into a 1.5ml centrifuge tube and record the number; 2) Add 200ul of 5% Chelex 100, 10ul of 10mg/ml proteinase K and 7ul of DTT to the molt collected in the centrifuge tube, shake and mix to ensure that the molt is soaked in the Chelex solution, incubate at 55°C for 20-24 hours, take out the shaking mixture homogeneous, denatured in a 100°C water bath for 8 minutes, centrifuged at 4°C and 12,000-13,000g for 3 minutes to obtain a supernatant containing living bee DNA.