Culture method of insect symbiotic bacteria

Abstract

The invention belongs to the technical field of microorganism culture, and discloses an insect symbiotic bacterium culture method which comprises the following steps: S1, preparing an insect cell feeding layer; s2, insect eggs are collected; step S3, disinfection of insect eggs; s4, crushing insect eggs and performing inoculated culture on symbiotic bacteria; and S5, separation and amplification culture of the insect symbiotic bacteria. The insect cell feeding layer is used for culturing the insect symbiotic bacteria, and cell factors or other unknown components secreted by cells in the feeding layer are used for promoting growth and reproduction of the symbiotic bacteria which are difficult to directly culture.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial culture, and particularly relates to a method for culturing insect symbiotic bacteria. Background Art

[0002] Insect symbiotic bacteria are in a mutualistic symbiosis with insects. Many symbiotic bacteria that have a close symbiotic relationship with host insects are difficult to be directly cultured with microbial culture media. These symbiotic bacteria rely on specific nutrients or growth signals in the host body, and conventional culture media cannot meet their demanding growth requirements, resulting in failure of in vitro culture. This limits the in-depth study of the functions and application potential of these symbiotic bacteria.

[0003] By preparing an insect cell feeder layer and utilizing cytokines and other components released by the insect cell feeder layer, it may be possible to simulate the microenvironment in the insect body, thereby promoting the growth and reproduction of insect symbiotic bacteria. This method may be used to culture some difficult-to-culture insect symbiotic bacteria. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for culturing insect symbiotic bacteria using an insect cell feeder layer to solve the problem that some insect symbiotic bacteria are difficult to be cultured on conventional culture media in the prior art.

[0005] To achieve the above purpose, the present invention provides a method for culturing insect symbiotic bacteria, including the following steps: Step S1, preparation of the insect cell feeder layer.

[0006] Insect cells (such as Sf9 cells) are seeded into a cell culture dish and cultured in an incubator at 27°C until the cell confluence reaches 70%. Then, they are treated with mitomycin C at a final concentration of 100 μg / mL for 2 hours. After treatment, the medium is replaced with fresh insect cell medium. The insect cells treated in this way form a monolayer, are metabolically active but stop dividing, and constitute the feeder layer.

[0007] Step S2, collection of insect eggs.

[0008] The mated female adults are placed on their suitable host plants (such as rice) for egg laying. After 2 - 5 days, the plant tissues are dissected, and the insect eggs are collected therefrom.

[0009] Step S3, disinfection of insect eggs.

[0010] The insect eggs collected in step S2 are first disinfected with 0.25% sodium hypochlorite solution for 5 minutes, and then disinfected with 75% alcohol for 5 minutes. After disinfection, they are finally rinsed 3 times with 1×PBS (phosphate buffered saline) to remove the residual disinfectant.

[0011] Step S4, crushing of insect eggs and inoculation and culture of symbiotic bacteria.

[0012] Under sterile conditions, the eggs obtained by disinfecting in step S3 are minced with sterile ophthalmic scissors or crushed with a sterile small grinding rod. An appropriate amount of cell culture medium (such as fresh insect cell culture medium) is added, and the symbiotic bacteria in the eggs are released by pipetting or shaking. Then, the culture medium suspension containing the free symbiotic bacteria is transferred into the cell culture dish containing the insect cell feeder layer prepared in step S1 for co-culture.

[0013] Step S5: Isolation and large-scale culture of insect symbiotic bacteria.

[0014] After the symbiotic bacteria in the co-culture system of step S4 grow and reproduce, the culture is aspirated or the growth is picked and streaked on a suitable bacterial or fungal culture plate (such as a YPD medium plate). After the plate is incubated at a suitable temperature, well-shaped single colonies are picked and inoculated into the corresponding liquid medium for shaking culture to obtain a pure culture.

[0015] A method for culturing insect symbiotic bacteria of the present invention has the following remarkable advantages compared with the prior art: The present invention uses an insect cell feeder layer to culture insect symbiotic bacteria, and can utilize cytokines or other unknown components secreted by the feeder layer cells to promote the culture of symbiotic bacteria. By simulating the in vivo environment of insects, it provides an effective in vitro culture method for symbiotic bacteria that rely on host cells and are difficult to grow on conventional culture media, which is helpful for subsequent research and utilization of these symbiotic bacteria resources. Brief Description of the Drawings

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained according to these drawings, where: Figure 1 It is a comparison diagram of the culture states of symbiotic bacteria under the culture conditions with and without the Sf9 cell feeder layer in an embodiment of a method for culturing insect symbiotic bacteria of the present invention; Note: In the figure, A and B are the initial state and the state after 48 hours of culturing the symbiotic fungus on the Sf9 cell feeder layer respectively; C and D in the figure are the initial state and the state after 48 hours of culturing the symbiotic fungus without the Sf9 cell feeder layer respectively; the symbiotic fungus is circled by a circle in the figure; the scale bar is 100 μm.

[0017] Figure 2 It is a photo of the colonies of the symbiotic fungus isolated and purified on the YPD plate in an embodiment of a method for culturing insect symbiotic bacteria of the present invention. Detailed Implementation Modes

[0018] The following combines specific embodiments to detail the implementation details of a method for culturing insect symbiotic bacteria of the present invention, so that those of ordinary skill in the art can implement the present invention.

[0019] Example 1: Cultivation of Symbiotic Fungi of Nilaparvata lugens Step S1: Preparation of an insect cell feeder layer using Sf9 cells Sf9 cells are passaged in Sf-900 TM II SFM cell culture medium in a cell incubator at 27°C. When a feeder layer needs to be prepared, the cells are inoculated into a cell culture dish, the inoculation density is adjusted, and they are cultured in an incubator at 27°C until the cell confluence reaches 70%. Then, mitomycin C is added to the culture medium to a final concentration of 100 μg / mL and treated for 2 hours. After the treatment, the culture medium containing mitomycin C is aspirated, the cell layer is rinsed 3 times with 1×PBS, and finally replaced with fresh Sf-900 TM II SFM cell culture medium.

[0020] Step S2: Collection of Nilaparvata lugens eggs The female adults of paired Nilaparvata lugens ( Nilaparvata lugens ) are placed on rice seedlings to lay eggs. After 2 - 5 days of egg laying, the rice leaf sheaths are dissected to collect the Nilaparvata lugens eggs therein.

[0021] Step S3: Disinfection of Nilaparvata lugens eggs The collected Nilaparvata lugens eggs are first disinfected with 0.25% sodium hypochlorite solution for 5 minutes, then with 75% alcohol for 5 minutes, and finally rinsed 3 times with 1×PBS.

[0022] Step S4: Crushing of Nilaparvata lugens eggs and inoculation and culture of symbiotic fungi The disinfected eggs are cut into pieces with sterile ophthalmic scissors, and an appropriate amount of fresh Sf-900 TM II SFM cell culture medium is added to free the symbiotic fungi in the eggs. The suspension containing symbiotic fungi is pipetted into the cell culture dish containing the Sf9 cell feeder layer prepared in Step S1, and penicillin with a final concentration of 100 U / mL and streptomycin with a final concentration of 100 μg / mL can be selectively added to inhibit bacterial contamination, and it is cultured in an incubator at 27°C. The control group is inoculated into a culture dish without a feeder layer. After culturing for 48 hours and observing (see Figure 1 ), it can be seen that the fungi in the feeder layer group grow well, while the growth of fungi in the group without a feeder layer is inhibited.

[0023] Step S5: Isolation and large-scale culture of insect symbiotic fungi The symbiotic fungal samples grown and propagated in the feeder layer culture system in step S4 are streaked and isolated on a YPD solid medium plate. The plate is placed in an incubator at 27°C for 3 to 5 days until single colonies grow (see Figure 2 ). Single colonies are picked and inoculated into YPD liquid medium, and cultured with shaking in a shaker at 27°C. ITS sequence PCR and sequencing identification are performed on the obtained pure culture, and the results show that the symbiotic fungus is Moesziomyces (Ustilago maydis).

[0024] The present invention uses an insect cell feeder layer to culture insect symbiotic bacteria, and can promote the culture of symbiotic bacteria by using cytokines or other unknown components secreted by the feeder layer cells. By simulating the in vivo environment of insects, it provides an effective in vitro culture method for symbiotic bacteria that rely on host cells and are difficult to grow on conventional media, which is helpful for subsequent research and utilization of these symbiotic bacterial resources.

[0025] Although the specific embodiments of the present invention are described above, those skilled in the art should understand that these specific embodiments are only examples. Without departing from the principle and essence of the present invention, those skilled in the art can make various omissions, substitutions and changes to the details of the above methods and systems. For example, combining the above method steps so as to perform substantially the same function in a substantially the same way to achieve substantially the same result falls within the scope of the present invention. Therefore, the scope of the present invention is only defined by the appended claims.

Claims

1. A method for culturing an insect symbiotic bacterium, characterized in that, It includes the following steps: Step S1, preparation of insect cell feeder layer: Inoculate insect cells into a cell culture dish and culture them in an incubator at 27°C until the cell confluence reaches 70%. Then, treat them with mitomycin C at a final concentration of 100 μg / mL for 2 hours, and then replace with fresh insect cell medium to obtain the insect cell feeder layer; Step S2, collection of insect eggs: Transfer paired female adults onto their host plants for egg laying. After 2 - 5 days, dissect the plant tissues to collect the insect eggs; Step S3, disinfection of insect eggs: Disinfect the insect eggs collected in Step S2 with 0.25% sodium hypochlorite solution for 5 minutes, then disinfect with 75% alcohol for 5 minutes, and finally rinse 3 times with 1×PBS; Step S4, crushing of insect eggs and inoculation and culture of symbiotic bacteria: Cut or crush the eggs disinfected in Step S3 with an ophthalmic scissors or a small grinding rod, add an appropriate amount of cell medium to free the symbiotic bacteria in the eggs, and use a pipette to transfer the freed symbiotic bacteria into the cell culture dish containing the insect cell feeder layer prepared in Step S1 for culture; Step S5, isolation and large-scale culture of insect symbiotic bacteria: Streak culture the symbiotic bacteria grown and propagated in Step S4 on a bacterial or fungal culture plate, and then pick single colonies for shaking culture.

2. The culture method of an insect symbiotic bacterium according to claim 1, wherein The insect cells described in Step S1 are Sf9 cells.

3. The culture method of an insect symbiotic bacterium according to claim 1 or 2, characterized in that, The cell culture medium described in step S1 and step S4 is Sf-900 TM II SFM cell culture medium.

4. The culturing method of an insect symbiotic bacterium according to claim 1, wherein, The insect described in step S2 is Nilaparvata lugens ( Nilaparvata lugens ).

5. The culturing method of an insect symbiotic bacterium according to claim 1, characterized in that, The host plant described in Step S2 is rice.

6. The culturing method of an insect symbiotic bacterium according to claim 1, characterized in that, The crushing method described in Step S4 is to cut with an ophthalmic scissors or crush with a small grinding rod.

7. A method for culturing an insect symbiotic bacterium according to claim 1, characterized in that The fungal culture plate described in Step S5 is a YPD medium plate.

8. The culturing method of an insect symbiotic bacterium according to claim 1, characterized in that The symbiotic bacteria isolated in step S5 are fungi belonging to the genus Ustilago ( Moesziomyces ).

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