Honeybee mitochondria extraction method
By combining bee tissue-specific buffer and differential centrifugation with density gradient centrifugation, the problems of purity and activity in bee mitochondrial extraction were solved, achieving efficient bee mitochondrial extraction and laying the foundation for subsequent research.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-16
- Publication Date
- 2026-04-10
AI Technical Summary
Existing methods are insufficient for effectively extracting bee mitochondria, making it difficult to guarantee their purity and activity, which seriously affects subsequent research.
Mitochondria were extracted from bees using a combination of bee tissue-specific buffer and differential centrifugation with density gradient centrifugation. The extraction process was optimized by using a buffer containing 3-morpholinopropanesulfonic acid, sucrose, protease inhibitors, and antioxidants, combined with trypsin treatment and multiple centrifugation steps.
It significantly improved the activity and purity of bee mitochondria, providing high-quality mitochondrial samples for subsequent research.
Smart Images
Figure CN121825960A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to entomology, and more particularly to a method for extracting mitochondria from bees. Background Technology
[0002] Mitochondria are key organelles within cells, serving not only as the central site of energy metabolism, providing ATP for cellular life activities through the tricarboxylic acid cycle and oxidative phosphorylation, but also as deeply involved in crucial processes such as redox balance, calcium homeostasis regulation, and apoptosis. Their independent genetic material, mitochondrial DNA, follows maternal inheritance and has a high mutation rate, making it an important molecular marker for studying biological evolution, population genetics, and developmental mechanisms.
[0003] In social insects like bees, mitochondria play a particularly prominent functional role. Their intensive flight activities, complex social behaviors, and energy supply at each developmental stage all depend on the efficient energy production of mitochondria. Furthermore, mitochondria also play a crucial role in defense and adaptive regulation when coping with environmental stresses such as temperature fluctuations, pathogen infection, and pesticide exposure. Therefore, studying bee mitochondria is of great value for understanding their ecological adaptability, population dynamics, and colony health management, and also provides a theoretical basis for optimizing agricultural pollination services and promoting the sustainable development of beekeeping.
[0004] However, current research on bee mitochondria still faces significant methodological limitations. Although mitochondrial research is relatively advanced in the overall life sciences field, mitochondrial extraction techniques specifically tailored to the characteristics of bee tissues are still lacking. Existing methods largely draw on mammalian systems, failing to fully consider the unique anatomical structure and physiological characteristics of bee tissues. This results in mitochondria being easily damaged during extraction, making it difficult to guarantee their purity and activity, which severely restricts subsequent functional and mechanistic studies. Summary of the Invention
[0005] Purpose of the invention: The purpose of this invention is to provide a method for extracting mitochondria from bees.
[0006] Technical solution: The method for extracting mitochondria from bees according to the present invention includes the following steps: (1) After adding bee tissue-specific buffer, bee tissue was homogenized to obtain a homogenate; (2) Add trypsin to the homogenate, shake, and let stand on ice to obtain the lysis buffer; (3) The lysate was centrifuged by differential centrifugation, and the precipitate was resuspended in mitochondrial extraction buffer to obtain crude extract; (4) Using 15-35% (w / v) sucrose solution as a gradient medium, the crude extract was centrifuged by density gradient method, and the solution between 25-35% (w / v) sucrose solution was collected. The precipitate obtained after centrifugation is the bee mitochondria. The honeybee tissue-specific buffer solution is a solution containing 3-morpholinopropanesulfonic acid, sucrose, protease inhibitors, antioxidants, and metal ion chelating agents.
[0007] Preferably, the bee tissue-specific buffer solution is a solution containing 18-22 mM 3-morpholinopropanesulfonic acid, 280-320 mM sucrose, 0.8-1 mM benzomidine, 8-12 mM glutathione, and 0.8-1.2 mM ethylene glycol diaminoethyl ether tetraacetic acid, with water as the solvent.
[0008] Preferably, the pH of the bee tissue-specific buffer solution is 7.2 to 7.6.
[0009] Preferably, the bee tissue mentioned in step 1 is any one of the bee's head, thorax, or abdomen, or the whole bee.
[0010] Preferably, in step 1, the ratio of bee tissue to bee tissue-specific buffer is 1 mL of bee tissue-specific buffer added for every 4 to 6 bees.
[0011] Preferably, in step 1, 8 to 12 grinding beads of 2 to 4 mm size are added to assist in homogenization.
[0012] Preferably, the final mass-volume concentration of the trypsin in step 2 is 0.001~0.003%.
[0013] Preferably, step 3 includes: (31) The lysis buffer was centrifuged at 2-8℃ and 900-1100 ×g for 4-6 min to collect the supernatant. The supernatant was then centrifuged at 2-8℃ and 11000-13000 ×g for 8-12 min to collect the precipitate. The precipitate was resuspended in bee tissue-specific buffer. (32) Repeat step 31 to obtain crude extract.
[0014] Preferably, step 4 includes: (41) Using 15%, 25%, and 35% (w / v) sucrose solutions as gradient media, add 0.2 to 0.3 times the volume of the gradient media crude extract, centrifuge at 2 to 8°C and 120,000 to 140,000 × g for 1 to 1.5 h, and collect the solution between the 25% and 35% sucrose solutions; (42) The solution obtained in step 41 is centrifuged at 2~8℃ and 12000~15000 ×g for 8-12 min, and the resulting precipitate is the bee mitochondria.
[0015] More preferably, the solvent for the sucrose solution in step 41 is a bee tissue-specific buffer solution.
[0016] More preferably, the volume ratio of the gradient medium to the crude extract in step 41 is 3~4:1.
[0017] Preferably, when the bee tissue is the bee abdomen or the whole bee, in step 2, polysorbate-20 with a final volume concentration of 0.05~0.1% is added to the homogenate.
[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The method for extracting mitochondria from bees is based on an optimized formula of bee tissue-specific buffer, which can effectively improve the activity and concentration of the extracted mitochondria compared with commercially available kits; 2. The mitochondria obtained by this extraction method have high integrity, which lays the foundation for subsequent downstream experimental research on bee mitochondria. Attached Figure Description
[0019] Figure 1 This is a schematic diagram of density gradient centrifugation after extracting mitochondria from the head of a bee based on a bee tissue-specific buffer with an optimized formulation. Figure 2 A statistical graph showing the results of ATP content determination in the extracted mitochondria; Figure 3 Transmission electron microscopy images of bee mitochondria extracted using a bee tissue-specific buffer with an optimized formulation; Figure 4 The image shows the Western blot results of bee mitochondria extracted using a bee tissue-specific buffer with an optimized formulation. Figure 5 The image shows the results of Mito-Tracker Deep Red FM staining of bee mitochondria extracted using a bee tissue-specific buffer with an optimized formulation. Detailed Implementation
[0020] The technical solution of the present invention will be further described below.
[0021] Example 1: Mitochondrial extraction from bees based on an optimized formulation of a bee tissue-specific buffer The bees used are Italian bees, provided by Yangzhou University.
[0022] The optimized honeybee tissue-specific buffer solution is an aqueous solution of 20 mM 3-morpholinopropanesulfonic acid (MOPS), 300 mM sucrose, 1 mM benzalkonium chloride, 10 mM glutathione, and 1 mM ethylene glycol diaminoethyl ether tetraacetic acid (EGTA), pH=7.4.
[0023] (1) Place the bee tissue (head, thorax, abdomen of 5 bees or 5 whole bees, with the honey sac, stinger and intestines removed from the abdomen) into a grinding tube, add 1 mL of bee tissue-specific buffer and 10 3 mm zirconia beads, and then homogenize using a Sakezi×48LR cryogenic high-throughput tissue homogenizer. The homogenization parameters are: working speed 70 Hz, working temperature 4℃, working time 60 s, interval 5 s, cycle 3 times to obtain a homogenate. (2) Add 20 μL of 0.1% (m / v) trypsin solution to the homogenate, shake for 30 s and let stand on ice for 15 min to obtain the lysis buffer. When the bee tissue used to extract mitochondria is the abdomen and the whole bee, add 1 μL of polysorbate-20 (Tween-20). (3) The lysis buffer was centrifuged at 4℃ and 1000 ×g for 5 min and the supernatant was collected. The supernatant was centrifuged at 12000 ×g for 10 min and the precipitate was resuspended in 1 mL of bee tissue-specific buffer. After resuspending, the lysis buffer was centrifuged at 4℃ and 1000 ×g for 5 min and the supernatant was collected. The supernatant was centrifuged at 12000 ×g for 10 min and the precipitate was resuspended in 1 mL of bee tissue-specific buffer pre-cooled to 4℃ to obtain the crude extract. (4) Slowly add 2 mL of 35% (w / v) sucrose-bee tissue-specific buffer solution, 3 mL of 25% (w / v) sucrose-bee tissue-specific buffer solution, and 2 mL of 15% (w / v) sucrose-bee tissue-specific buffer solution along the wall of the centrifuge tube as a density gradient medium. Slowly add 1 mL of crude extract and centrifuge at 4℃ and 130,000 ×g for 1 h. Collect the suspension between the 25% and 35% sucrose-bee tissue-specific buffer solutions. Centrifuge this solution at 4℃ and 12,000 ×g for 10 min and collect the precipitate, which is the bee mitochondria. After density gradient centrifugation in the bee head tissue extraction, as shown... Figure 1 As shown; (5) The mitochondria of the bees were resuspended in a bee tissue-specific buffer pre-cooled to 4°C to obtain purified suspensions of the bee head, bee thorax, bee abdomen and whole bee mitochondria for subsequent experiments.
[0024] Comparative Example 1: Honeybee Mitochondrial Extraction Based on Conventional Universal Buffer A standard general-purpose buffer solution is an aqueous solution of 20 mM MOPS, 250 mM sucrose, and 1 mM EGTA, with a pH of 7.4.
[0025] Mitochondria were extracted from the head, thorax, and abdomen (excluding honey sacs, stingers, and intestines) of 5 bees or from 5 whole bees according to the steps in Example 1 to obtain the corresponding mitochondrial suspensions.
[0026] Comparative Example 2: Honeybee Mitochondrial Extraction Based on Commercially Available Kits Using the Solarbio Mitochondrial Extraction Kit (catalog number SM0020), mitochondria were extracted from the head, thorax, and abdomen of 5 bees (excluding the honey sac, stinger, and intestines) or from 5 whole bees to obtain the corresponding mitochondrial suspensions.
[0027] Experimental Example 1: Determination of ATP content in extracted mitochondria The ATP content of mitochondria obtained in Example 1 (bee tissue-specific buffer), Comparative Example 1 (general buffer), and Comparative Example 2 (kit) was determined using an ATP content kit (purchased from Suzhou Greens Biotechnology Co., Ltd., catalog number G0815W).
[0028] The results are as follows Figure 2 As shown, the optimized method extracts a higher mitochondrial content than the kit and conventional general buffer methods.
[0029] Experimental Example 2: Evaluation of the integrity of mitochondria extracted in Example 1 The thoracic mitochondria obtained in Example 1 were prepared and imaged by Wuhan Saiweier Biotechnology Co., Ltd. for transmission electron microscopy.
[0030] The results are as follows Figure 3 As shown, the complete membrane structure of mitochondria and the cristae formed by the inward folding of the inner mitochondrial membrane can be clearly seen.
[0031] Experimental Example 3: Purity Evaluation of Mitochondria Extracted in Example 1 (1) 1 mL of mitochondrial suspension from bee head (head), bee thorax (thorax), bee abdomen (abdomen), or whole bee (whole) obtained in Example 1 was added to 200 μL of NP-40 lysis buffer containing 1 mM benzyl sulfonyl fluoride (PMSF), gently mixed, and lysed on ice for 10 minutes. After centrifugation at 4°C and 12000 × g for 5 min, the supernatant was collected. The obtained protein sample was then added to 0.25 times the volume of BeyoWB. TM SDS-PAGE loading buffer (catalog number P0963) was mixed and heated in a 95°C water bath for 5 min, and then cooled to room temperature to obtain denatured protein samples. (2) Take the denatured protein sample and use Beyotime Biotech's Beyotime Biotech product. TM The 80-minute electrophoresis transfer and Western spectroscopy assay kit (catalog number P0002S) was used, with electrophoresis at 180V for 25 min. After electrophoresis, the separated proteins were transferred to a PVDF membrane at 500mA for 10 min, and then analyzed using Beyotime's BeyoWB assay kit. TM Sealing solution at room temperature for 5 minutes; (3) After blocking, use UQCRC2 rabbit anti-primary antibody diluted 1:300 (provided by Nankai University), incubate at 4℃ for 15 min, rinse, and then use goat anti-rabbit secondary antibody (purchased from Beyotime, catalog number P0948) to incubate at room temperature for 5 min and rinse. (4) Use BeyoWB TM The BeyoECL kit was used for development and image acquisition.
[0032] The results are as follows Figure 4 As shown, the core subunit UQCRC2 of bee mitochondrial complex III was detected in the extracted mitochondrial suspension, indicating successful extraction. The trend of UQCRC2 content was consistent with the ATP content measurement results. The amount of mitochondria from the whole bee was the highest in the obtained mitochondrial suspension, followed by those from the thorax, and then those from the abdomen and head.
[0033] Experimental Example 4: Staining of mitochondria extracted in Example 1 with fluorescent probes The mitochondrial suspension obtained in Example 1 was stained with 200 nM Beyotime Mito-Tracker Red CMXRos (catalog number C1049B) solution preheated at 37°C for 30 min, and then observed and images were acquired using a confocal microscope.
[0034] The results are as follows Figure 5 As shown, the extracted mitochondria exhibit high activity.
Claims
1. A method for extracting mitochondria from bees, characterized in that the steps include... include: (1) After adding bee tissue-specific buffer, bee tissue was homogenized to obtain a homogenate; (2) Add trypsin to the homogenate, shake, and let stand on ice to obtain the lysis buffer; (3) The lysate was centrifuged by differential centrifugation, and the precipitate was resuspended in mitochondrial extraction buffer to obtain crude extract; (4) Using 15-35% (w / v) sucrose solution as a gradient medium, the crude extract was centrifuged by density gradient method, and the suspended matter between 25-35% (w / v) sucrose solution was collected. The precipitate obtained after centrifugation is the bee mitochondria. The honeybee tissue-specific buffer solution is a solution containing 3-morpholinopropanesulfonic acid, sucrose, protease inhibitors, antioxidants, and metal ion chelating agents.
2. The method for extracting bee mitochondria according to claim 1, characterized in that, The honeybee tissue-specific buffer solution is a solution containing 18-22 mM 3-morpholinopropanesulfonic acid, 280-320 mM sucrose, 0.8-1 mM benzomidine, 8-12 mM glutathione, and 0.8-1.2 mM ethylene glycol diaminoethyl ether tetraacetic acid, with water as the solvent.
3. The method for extracting bee mitochondria according to claim 2, characterized in that, The pH of the honeybee tissue-specific buffer solution is 7.2-7.
6.
4. The method for extracting bee mitochondria according to claim 1, characterized in that, The bee tissue mentioned in step 1 can be any one of the bee's head, thorax, or abdomen, or the entire bee.
5. The method for extracting bee mitochondria according to claim 1, characterized in that, In step 1, the ratio of bee tissue to bee tissue-specific buffer is 1 mL of bee tissue-specific buffer added for every 4 to 6 bees.
6. The method for extracting bee mitochondria according to claim 1, characterized in that, The final mass-volume concentration of trypsin in step 2 is 0.001~0.003%.
7. The method for extracting bee mitochondria according to claim 1, characterized in that, Step 3 includes: (31) The lysis buffer was centrifuged at 2~8℃ and 900~1100 ×g for 4~6 min to collect the supernatant. The supernatant was then centrifuged at 2~8℃ and 11000~13000 ×g for 8~12 min to collect the precipitate. The precipitate was resuspended in bee tissue-specific buffer. (32) Repeat step 31 to obtain crude extract.
8. The method for extracting bee mitochondria according to claim 1, characterized in that, Step 4 includes: (41) Using 15%, 25%, and 35% (w / v) sucrose solutions as gradient media, add 0.2 to 0.3 times the volume of the gradient media crude extract, centrifuge at 2 to 8°C and 120,000 to 140,000 × g for 1 to 1.5 h, and collect the solution between the 25% and 35% sucrose solutions; (42) The solution obtained in step 41 is centrifuged at 2~8℃ and 12000~15000 ×g for 8-12 min, and the resulting precipitate is the bee mitochondria.
9. The method for extracting bee mitochondria according to claim 8, characterized in that, The solvent for the sucrose solution in step 41 is a bee tissue-specific buffer.
10. The method for extracting bee mitochondria according to claim 1, characterized in that, When the bee tissue is the bee abdomen or the whole bee, in step 2, 0.05-0.1% of polysorbate-20 by volume is also added to the homogenate.