Induced separation method of bursaphelenchus xylophilus effect factors
By inducing the secretion effector factors of pine nematodes prepared by the corresponding substances of pine trees and performing isolation treatment, the problem of difficult to screen and isolate and isolated in the prior art is solved, providing support for the study of pathogenic mechanisms, and the method is simple to operate.
Patent Information
- Application Number
- CN202411967621.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-30
- Publication Date
- 2025-04-18
AI Technical Summary
The prior art is difficult to effectively screen and isolate the effectors secreted by pathogenic organisms of pine nematode disease, resulting in limitations on the study of pathogenic mechanisms.
By preparing inducers containing the corresponding substances of pine trees, pine nematode secretion effector factors are induced, and pine nematode secretion proteins are obtained through a series of separation steps (such as centrifugation, filtration, ultrafiltration, and freeze-drying).
The pine nematode secretes a large number of effector factors in the host body, providing a basic guarantee for the study of the pathogenic mechanism of pine nematode disease, and the method is simple to operate and easy to operate.
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Figure CN119841886A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of pine wood nematode disease, and in particular to an induction and separation method for pine wood nematode effector factors. Background Art
[0002] Pine Wilt Disease is known as the "cancer" of pine trees and is a huge threat to pine forests worldwide. Its main pathogenic agent is the pine wood nematode (Bursaphelenchus xylophilus), an invasive plant parasitic nematode originating from North America. It was first discovered in Japan in the early 20th century and later introduced to China, South Korea, Portugal and Spain. Since it was first discovered in Nanjing, my country in the 1980s, pine wood nematode disease has caused great disasters to forest ecosystems in many parts of my country. According to incomplete assessments, the economic losses caused by pine wood nematode disease to my country each year amount to tens of billions of yuan.
[0003] Research on the pathogenic mechanism of pine wood nematode disease is a prerequisite for its effective prevention and control. Through the study of various pathogenic organisms, it was found that the main way of causing disease is by secreting a variety of proteins or other small molecules into the host. Such species that can have a functional effect on the host are collectively referred to as effector factors. The screening, identification and functional verification of effector factors are important methods and prerequisites for the study of the pathogenic mechanism of pathogens, and the current screening methods are mostly through transcriptome sequencing to analyze the expression of secreted proteins. This method only reflects possible effector factors through the transcription of genes, but it is unknown whether the screened genes are actually translated, expressed and secreted into the host. Although current mass spectrometry technology can identify secreted proteins, it is difficult to separate secretions from pathogens from the host.
[0004] In view of this, the present invention is proposed. Summary of the invention
[0005] The purpose of the present invention is to provide an induction and separation method for pine wood nematode effector factors. The inducer in the induction and separation method of the present invention contains corresponding substances in pine trees. Inducing pine wood nematodes with the inducer can cause the pine wood nematodes to secrete effector factors, providing a basic guarantee for the study of the pathogenic mechanism of pine wood nematode disease.
[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:
[0007] The present invention provides an induction and separation method for pine wood nematode effector factors, and the induction and separation method for pine wood nematode effector factors comprises the following steps:
[0008] (a) Preparation of inducer:
[0009] The Masson pine branches are washed and cut into strips, and then immersed in ultrapure water and filtered to obtain a filtrate, and then the filtrate is ultrafiltered and sterilized to obtain an inducer;
[0010] (b) Induction of pine wood nematodes:
[0011] The pine wood nematodes are washed with a sucrose solution, ultrapure water and an inducer in sequence, and the pine wood nematodes are then placed in the inducer for incubation to obtain a pine wood nematode solution;
[0012] (c) Separation of pine wood nematode secretions:
[0013] The pine wood nematode solution is centrifuged, the supernatant is taken, and the supernatant is filtered and concentrated by ultrafiltration in sequence to collect the concentrated solution, and the concentrated solution is freeze-dried to obtain the pine wood nematode secretory protein.
[0014] Preferably, in step (a), the mass volume ratio of the Masson pine branches to ultrapure water is 1:(4-6); the soaking time is 20-28 hours, and the temperature is 2-6°C.
[0015] Preferably, in step (a), the filter used for filtration is a 0.45 μm hydrophilic PTFE needle filter; the ultrafiltration membrane used for ultrafiltration has a pore size of 3 kDa; and the filter used for filtration and sterilization is a 0.22 μm hydrophilic PTFE needle filter.
[0016] Preferably, in step (b), the final concentration of pine wood nematodes added to the inducer is 250,000 to 350,000 heads / ml.
[0017] Preferably, in step (b), the incubation is carried out on a shaker at 22-28° C. for 20-28 h, and the shaker speed is 350-500 rpm.
[0018] Preferably, in step (c), the centrifugal speed is 8000-9000 rpm, the time is 4-6 min, and the temperature is 22-28°C.
[0019] Preferably, in step (c), the filter used for filtration is a 0.22 μm hydrophilic PTFE needle filter.
[0020] Preferably, in step (c), the pore size of the ultrafiltration membrane used for ultrafiltration concentration is 3 kDa, the rotation speed of ultrafiltration concentration is 5000-5500 rpm, and the temperature is 2-6°C.
[0021] Preferably, in step (c), ultrafiltration and concentration are performed until the concentrated solution has a volume of 1 / 50 of the original solution.
[0022] Compared with the prior art, the beneficial effects of the present invention include at least:
[0023] The inducer in the induction separation method of the present invention contains corresponding substances in the pine tree body. Inducing pine wood nematodes with it can stimulate the pine wood nematodes to secrete a large number of effector factors outside the host pine tree body, providing a basic guarantee for the study of the pathogenic mechanism of pine wood nematode disease. The induction separation method of the present invention is simple and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the specific embodiments or the description of the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.
[0025] Figure 1 The expression changes of pine wood nematode effector factors before and after induction in the experimental example of the present invention;
[0026] Figure 2 The change of protein content in the supernatant concentrate of the pine wood nematode solution before and after induction in the experimental example of the present invention;
[0027] Figure 3 The peptide search result of the supernatant concentrate of the pine wood nematode solution after induction in the experimental example of the present invention;
[0028] Figure 4 This is the molecular weight distribution of the concentrated supernatant of the induced pine wood nematode solution after enzyme digestion in the experimental example of the present invention. DETAILED DESCRIPTION
[0029] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.
[0030] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the invention belongs.
[0031] The embodiment of the present invention provides a method for inducing and isolating effector factors of pine wood nematodes, and the method for inducing and isolating effector factors of pine wood nematodes comprises the following steps:
[0032] (a) Preparation of inducer:
[0033] The Masson pine branches are washed and cut into strips, and then immersed in ultrapure water and filtered to obtain a filtrate, and then the filtrate is ultrafiltered and sterilized to obtain an inducer;
[0034] (b) Induction of pine wood nematodes:
[0035] The pine wood nematodes are washed with a sucrose solution, ultrapure water and an inducer in sequence, and the pine wood nematodes are then placed in the inducer for incubation to obtain a pine wood nematode solution;
[0036] (c) Separation of pine wood nematode secretions:
[0037] The pine wood nematode solution is centrifuged, the supernatant is taken, and the supernatant is filtered and concentrated by ultrafiltration in sequence to collect the concentrated solution, and the concentrated solution is freeze-dried to obtain the pine wood nematode secretory protein.
[0038] The inducer in the induction separation method of the present invention contains corresponding substances in the pine tree body. Inducing pine wood nematodes with it can stimulate the pine wood nematodes to secrete a large number of effector factors, providing a basic guarantee for the study of the pathogenic mechanism of pine wood nematode disease; the induction separation method of the present invention is simple and easy to operate.
[0039] In some embodiments, in step (a), the mass volume ratio of the Masson pine branches to ultrapure water can be 1:(4-6); the soaking time can be 20-28 hours, and the temperature can be 2-6°C.
[0040] In some embodiments, in step (a), the filter used for filtration is a 0.45 μm hydrophilic PTFE needle filter; the ultrafiltration membrane used for ultrafiltration has a pore size of 3 kDa; and the filter used for filtration and sterilization is a 0.22 μm hydrophilic PTFE needle filter.
[0041] In some embodiments, in step (b), the final concentration of pine wood nematodes added to the inducer can be 250,000 to 350,000 heads / ml.
[0042] In some embodiments, in step (b), the incubation is carried out on a shaker at 22 to 28° C. for 20 to 28 hours, and the shaker speed may be 350 to 500 rpm.
[0043] In some embodiments, in step (c), the centrifugal speed may be 8000-9000 rpm, the time may be 4-6 min, and the temperature may be 22-28°C.
[0044] In one embodiment, in step (c), the filter used for filtration is a 0.22 μm hydrophilic PTFE needle filter.
[0045] In some embodiments, in step (c), the pore size of the ultrafiltration membrane used for ultrafiltration concentration is 3 kDa, the ultrafiltration concentration speed can be 5000-5500 rpm, and the temperature can be 2-6°C.
[0046] In some embodiments, in step (c), ultrafiltration and concentration are performed until the concentrated solution has a volume of 1 / 50 of the original solution.
[0047] The technical solution of the present invention is further described in detail below through specific embodiments.
[0048] Example
[0049] This embodiment is a method for inducing and isolating effect factors of pine wood nematodes, and the method for inducing and isolating effect factors of pine wood nematodes comprises the following steps:
[0050] (a) Preparation of inducer:
[0051] All pine needles and tender shoots were cut off from healthy three-year-old Masson pine branches, and the remaining woody branches were washed with sterile water, cut into wood strips slightly smaller than matchsticks in a clean environment, weighed, and soaked in 5 ml of sterilized ultrapure water per gram of wood strips, and soaked at 4°C for 24 hours; the supernatant was collected and first filtered through qualitative filter paper to remove wood strips and residues, and the filtrate was further filtered through a 0.45 μm hydrophilic PTFE needle filter to further remove residues and microorganisms; the filtrate was centrifuged through an ultrafilter with a pore size of 3 kDa to remove dissolved proteins in the pine tissue, and the solution that passed through the membrane and entered the bottom of the centrifuge tube was collected; it was re-filtered and sterilized using a 0.22 μm hydrophilic PTFE needle filter to obtain an inducer, which was quickly frozen and stored below -20°C for later use;
[0052] (b) Induction of pine wood nematodes:
[0053] Wash fresh pine wood nematodes in a sterilized sucrose solution until no bacteria or debris can be observed around the nematodes under an optical microscope; wash the washed nematode solution with sterilized ultrapure water five times to remove the sucrose; wash the washed nematode solution once with an inducer to avoid diluting the inducer; soak the solution in a cell culture dish at a concentration of 300,000 nematodes per milliliter of inducer, place it on a horizontal shaker at 25°C, incubate it at 400 rpm, and after incubation for 24 hours, a large number of effector factors will be present in the solution to obtain a pine wood nematode solution;
[0054] (c) Separation of pine wood nematode secretions:
[0055] The pine wood nematode solution was centrifuged at 8000 rpm for 5 min at 25°C, and the supernatant was taken (the supernatant contained effect factors and was kept at low temperature for subsequent experiments); the supernatant was passed through a 0.22 μm hydrophilic PTFE needle filter to remove impurities; the supernatant filtrate was centrifuged at 5000 rpm at 4°C using a 3KDa ultrafiltration tube until the upper side concentrate was 1 / 50 of the volume of the original solution, the upper side concentrate was vacuum freeze-dried and stored in a solid state at low temperature, and the concentrate was the secretory protein of the pine wood nematode.
[0056] Experimental example
[0057] 6 million pine wood nematodes cultured with Botrytis cinerea in the laboratory were evenly divided into two treatments, each with three replicates: the YD treatment group was induced according to the method of the embodiment, and the CK treatment group was synchronously incubated with sterile water instead of the inducer. After the induction treatment, the nematodes were quickly frozen and then the transcriptome was detected. The supernatant was filtered and then ultrafiltered and concentrated, and the concentrate was detected by liquid chromatography-mass spectrometry.
[0058] 1. Effector expression profile
[0059] The transcription level of nematodes after induction treatment was measured, and the results were as follows Figure 1 As shown;
[0060] Depend on Figure 1 It can be seen that the expression level of nematode effector factor genes after induction treatment increased significantly, indicating that treating pine wood nematodes with induction solution simulates the expression pattern of pine wood nematodes in pine trees, thereby promoting the expression of pine wood nematode pathogenicity-related genes.
[0061] 2. Proteomic results
[0062] The protein concentration of the supernatant concentrate after pine wood nematode induction was determined by Bradford method. Figure 2 As shown;
[0063] Depend on Figure 2 It can be seen that:
[0064] After induction, the protein concentration in the supernatant of the nematode solution increased significantly, indicating that the inducer can stimulate pine wood nematodes to secrete a large amount of protein.
[0065] 3. Identification of protein components in the supernatant concentrate after pine wood nematode induction
[0066] The protein solution was purified and then hydrolyzed by LC-MS / MS (such as Figure 3 ) and compared with the peptide mass fingerprint of the pine nematode proteome (as shown in Figure 4 shown).
[0067] Depend on Figure 3-4 It can be seen that 19355 mass spectra were successfully matched, and 5155 peptides were identified by comparison with the database, 980 proteins from pine wood nematodes, and the protein molecular weights were mainly distributed between 10-70kDa.
[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.
Claims
1. A method for inducing and isolating effector factors of pine wood nematodes, characterized in that: The induction and separation method of pine wood nematode effector factors comprises the following steps: (a) Preparation of inducer: The Masson pine branches are washed and cut into strips, and then immersed in ultrapure water and filtered to obtain a filtrate, and then the filtrate is ultrafiltered and sterilized to obtain an inducer; (b) Induction of pine wood nematodes: The pine wood nematodes are washed with a sucrose solution, ultrapure water and an inducer in sequence, and the pine wood nematodes are then placed in the inducer for incubation to obtain a pine wood nematode solution; (c) Separation of pine wood nematode secretions: The pine wood nematode solution is centrifuged, the supernatant is taken, and the supernatant is filtered and concentrated by ultrafiltration in sequence to collect the concentrated solution, and the concentrated solution is freeze-dried to obtain the pine wood nematode secretory protein.
2. The method for inducing and isolating effector factors of pine wood nematodes according to claim 1, characterized in that: In the step (a), the mass volume ratio of the Masson pine branches to the ultrapure water is 1:(4-6); the soaking time is 20-28 hours, and the temperature is 2-6°C.
3. The method for inducing and isolating effector factors of pine wood nematodes according to claim 1, characterized in that: In the step (a), the filter used for filtration is a 0.45 μm hydrophilic PTFE needle filter; the ultrafiltration membrane used for ultrafiltration has a pore size of 3 kDa; and the filter used for filtration and sterilization is a 0.22 μm hydrophilic PTFE needle filter.
4. The method for inducing and isolating effector factors of pine wood nematodes according to claim 1, characterized in that: In the step (b), the final concentration of pine wood nematodes added to the inducer is 250,000 to 350,000 heads / ml.
5. The method for inducing and isolating effector factors of pine wood nematodes according to claim 1, characterized in that: In the step (b), the incubation is carried out on a shaker at 22 to 28° C. for 20 to 28 hours, and the shaker speed is 350 to 500 rpm.
6. The method for inducing and isolating effector factors of pine wood nematodes according to claim 1, characterized in that: In the step (c), the centrifugal speed is 8000-9000 rpm, the time is 4-6 min, and the temperature is 22-28°C.
7. The method for inducing and isolating effector factors of pine wood nematodes according to claim 1, characterized in that: In the step (c), the filter used for filtration is a 0.22 μm hydrophilic PTFE needle filter.
8. The method for inducing and isolating effector factors of pine wood nematodes according to claim 1, characterized in that: In the step (c), the pore size of the ultrafiltration membrane used for ultrafiltration concentration is 3 kDa, the ultrafiltration concentration speed is 5000-5500 rpm, and the temperature is 2-6°C.
9. The method for inducing and isolating effector factors of pine wood nematodes according to claim 1, characterized in that: In the step (c), ultrafiltration and concentration are performed until the concentrated solution has a volume of 1 / 50 of the original solution.