Method and application for reducing resistance genes in soil environment by protozoan amoeba

Abstract

The present invention relates to a method and application for reducing resistance genes in the soil environment by using the protozoan amoeba, belonging to the field of biotechnology. The present invention provides an application for reducing resistance genes in the soil environment by using the protozoan amoeba, wherein the protozoan amoeba reduces resistance genes in the soil environment by preying on bacteria. The protozoan amoeba is Dictyostelium discoideum QS9. The present invention provides a method for reducing resistance genes in the soil environment by using the protozoan amoeba, comprising the following steps: incubating the protozoan amoeba, collecting amoeba spores to obtain an amoeba spore solution, and adding the amoeba spore solution to the soil environment for resistance gene reduction treatment; the present invention can simultaneously reduce antibiotic resistance genes and metal resistance genes, has a wide range of pollutants to be reduced, can be used for different soil types, and is an environmentally friendly bioremediation technology with broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method and application for reducing resistance genes in the soil environment by protozoan amoeba. Background Art

[0002] Antibiotics, as effective antibacterial drugs, are widely used in clinical practice and the livestock industry. However, the extensive use and abuse of antibiotics have led to the selective enrichment of genes encoding antibiotic resistance in soil microorganisms. After pathogenic bacteria acquire antibiotic resistance genes through gene mutation or horizontal gene transfer, it results in the failure of antibiotic treatment, prolonged treatment time, and increased mortality rate, posing a severe challenge to human and animal health. On the other hand, the feed widely used in the breeding industry also has serious heavy metal over - standard problems. Under long - term selection pressure, some soil microorganisms have evolved antibiotic resistance and produced antibiotic resistance genes (ARGs) and heavy metal resistance genes (MRGs).

[0003] Research shows that resistance genes have the characteristics of long - term residue in the environment and long - distance migration and spread. Moreover, even if the environmental selection pressure is eliminated, these genes still exist. In addition to vertical transfer through bacterial proliferation and inheritance, heavy metal stress in the environment may promote the spread of antibiotic resistance genes through co - selection of antibiotic resistance genes (ARGs) and heavy metal resistance genes (MRGs). In addition to the simultaneous presence of ARGs and MRGs, another problem caused by resistance genes is that they can co - transfer multiple existing resistance genes by collecting and recombining mobile genetic elements (MGEs), thus greatly increasing their spread in different habitats and ultimately spreading to humans through the food chain, leading to the failure of antibiotic treatment. In addition, the synergistic resistance effect among bacteria makes the horizontal transfer of originally variable resistance genes more complex, generating "super bacteria", which seriously threatens human health. Therefore, it is extremely urgent and necessary to develop an economic, environmentally friendly and efficient resistance gene reduction technology.

[0004] For the resistance genes in the soil environment, the current elimination methods mainly include physical adsorption, oxidation method or microbial treatment. Due to the complex soil environment, the abundance and type of resistance genes in the soil, as well as soil physical and chemical properties and soil microbial communities, have become the entry points for reducing resistance genes. However, although agronomic regulation measures such as changing the type of fertilizer, irrigation water type, and pesticide use can affect the generation and occurrence of resistance genes in the soil, they can only control the content of resistance genes in the environment to a certain extent and cannot fundamentally remove the resistance genes in the soil environment.

[0005] At present, the use of microorganisms to eliminate resistance genes has become the mainstream method. The adsorption and degradation ability of microorganisms to antibiotics or heavy metals can effectively remove pollutants in the environment, and can also be applied to aerobic composting to improve the degradation effect of residual pollutants and resistance genes. However, due to the diversity or specificity of microbial species, it means that the mechanism for a single microorganism to reduce resistance genes is not comprehensive. In addition, the growth rate of microorganisms is slow, and it takes a long time to reach the required concentration, which will affect the efficiency of pollution treatment. Bacteriophages can also be used to eliminate ARGs. By screening, purifying and enriching cultures to obtain bacteriophages specific to ARG host bacteria, adding bacteriophage agents to the polluted environment can achieve the purpose of directional infection and lysis of host bacteria, thereby reducing ARGs in the environment. However, bacteria containing resistance genes in the soil environment also have multiple mechanisms to resist bacteriophage infection, increasing the technical difficulty. Although broad-host bacteriophages can achieve the effect of using a combination of bacteriophages, in actual soil experiments, these broad-host bacteriophages will cause a certain degree of reduction in the diversity of soil bacterial communities for their own survival, which has certain defects. Summary of the Invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method and application for reducing resistance genes in the soil environment with good biosafety and reduction effect by using the protozoan amoeba.

[0007] To achieve the above purpose, the technical solutions adopted by the present invention are as follows:

[0008] In the first aspect, the present invention provides an application of amoeba in reducing resistance genes in the soil environment, and the amoeba is Dictyostelium discoideum.

[0009] As a preferred embodiment of the application of the present invention, the protozoan amoeba is Dictyostelium discoideum QS9.

[0010] In terrestrial and aquatic ecosystems, bacteria are the main food source for amoebae. Amoebae have evolved a series of mechanisms to track, prey on, engulf, kill, and digest bacteria. First, amoebae can detect and chase bacteria at a distance through chemotaxis. Subsequently, they engulf bacteria using phagocytosis (swallowing solid particles in the surrounding environment, such as bacteria, into the cell interior in the form of vesicles). The phagosomes produced by phagocytosis fuse with lysosomes containing various digestive enzymes to form phagolysosomes, and the bacteria are digested into nutrients and transported into the cytoplasm. Dictyostelium discoideum lives in soil rich in organic matter. When it is moist, the spores inoculated on the fruiting bodies release haploid cells (myxamoebae), which exhibit the shape and lifestyle of amoebae. Therefore, as a very efficient bacterial predator, amoebae can significantly regulate and alter the microbial community. In the soil environment, the microbial community structure is complex and diverse, and most bacteria carry drug resistance genes. Therefore, while protozoan amoebae prey on bacteria, they also have an impact on the drug resistance genes in the soil environment.

[0011] As a preferred embodiment of the application described in the present invention, the resistance genes include metal resistance genes and antibiotic resistance genes.

[0012] As a preferred embodiment of the application described in the present invention, the metal resistance genes include copA, czcA, arsB, and arsC.

[0013] As a preferred embodiment of the application described in the present invention, the antibiotic resistance genes include tetA, tetM, tetC, and sul1.

[0014] As a preferred embodiment of the application described in the present invention, the application of amoebae in reducing resistance genes in the soil environment includes the following steps: adding a solution of amoeba spores to the soil environment for resistance gene reduction treatment.

[0015] As a preferred embodiment of the application described in the present invention, when the solution of amoeba spores is added to the soil environment, it is added at a ratio of 1×10 4 ~1×10 7 amoeba spores per gram of soil.

[0016] As a preferred embodiment of the application described in the present invention, the protozoan amoeba Dictyostelium discoideum QS9 reduces resistance genes in the soil environment in the form of amoeba spores; the exposure concentration of the amoeba spores in the soil is 1×10 4 ~1×10 7 spores / g of soil.

[0017] Preferably, the exposure concentration of the amoeba spores in the soil is 1×10 7spores / g soil. When the exposure concentration of amoeba spores in the soil is 1×10 7 spores / g soil, in different soil environments, the reduction effects on metal resistance genes copA, czcA, arsB and arsC, or antibiotic resistance genes tetA, tetC and sul1 are the best, and in different soil environments, the relative decrease in the abundance of total resistance genes is 71.9% - 78.11%.

[0018] As a preferred embodiment of the application described in the present invention, the treatment time of the amoeba spores in the soil is 7 - 10 days. Preferably, the treatment time of the amoeba spore solution is 7 days.

[0019] As a preferred embodiment of the application described in the present invention, the soil environments include mine soil, farm soil, and farmland vegetable plot soil.

[0020] In the present invention, Dictyostelium discoideum QS9 is selected. It is a social amoeba. Under normal conditions, they feed on bacteria. In the case of food depletion, thousands of amoebas will gather together and migrate. When they migrate to a new location, they will finally form fruiting bodies and spores at the top of them. Therefore, in the soil environment, amoeba spores can adaptively prey on bacteria carrying resistance genes and effectively reduce the resistance genes.

[0021] In a second aspect, the present invention provides a biological agent for reducing resistance genes in a soil environment, characterized in that the biological agent contains amoeba spores, and the concentration of the amoeba spores is 2×10 4 ~2×10 7 spores / mL.

[0022] As a preferred embodiment of the method described in the present invention, the preparation method of the biological agent includes the following steps: incubating amoeba, collecting its spores in a neutral buffer solution KK2 containing potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and completing the preparation of the biological agent for reducing resistance genes in the soil environment.

[0023] In the buffer solution, each liter of the buffer contains 2.25 g of KH2PO4 and 0.67 g of K2HPO4, and the pH value of the solution is 7.0

[0024] As a preferred embodiment of the method described in the present invention, the incubation of the protozoan amoeba includes the following steps:

[0025] S1. Inoculate the Dictyostelium discoideum QS9 strain on an SM / 5 agar plate, and collect amoeba spores after the first light culture;

[0026] S2. Mix the amoeba spores collected in step S1 with the Klebsiella pneumoniae suspension, and then spread the mixed bacterial suspension on an SM / 5 agar plate by the plate coating method for the second light culture to complete incubation.

[0027] As a preferred embodiment of the method of the present invention, the light culture period is 5 - 7 days, and the light culture temperature is 20 - 23°C. Preferably, the light culture period is 5 days, and the light culture temperature is 21°C.

[0028] As a preferred embodiment of the method of the present invention, in step S2, the mixing ratio of amoeba spores to the Klebsiella pneumoniae suspension is amoeba spores: Klebsiella pneumoniae suspension = 1×10 5 spores: 100 μL.

[0029] As a preferred embodiment of the method of the present invention, the absorbance of the Klebsiella pneumoniae suspension is OD 600 = 1.5.

[0030] As a preferred embodiment of the method of the present invention, collect amoeba spores in KK2 buffer solution to obtain an amoeba spore solution, and the concentration of the amoeba spore solution is 2×10 4 ~ 2×10 7 spores / mL.

[0031] In the third aspect, the present invention provides a biological agent for reducing resistance genes in the soil environment, and the biological agent contains amoeba spores.

[0032] The concentration of the amoeba spores is 2×10 4 ~ 2×10 7 spores / mL; the amoeba is Dictyostelium discoideum QS9 strain.

[0033] In the fourth aspect, the present invention provides a method for reducing resistance genes in the soil environment by using the protozoan amoeba, including preparing and incubating the protozoan amoeba and collecting its spores, and adding the solution containing amoeba spores to the soil environment for resistance gene reduction treatment.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] 1. The present invention provides an application for reducing resistance genes in the soil environment by using the protozoan amoeba. By Dictyostelium discoideum QS9 preying on bacteria, the soil environment is repaired. Protozoan amoebas are ubiquitous in the environment, with low acquisition cost and simple and easy-to-operate technical processes. At the same time, amoebas have the ability to track, phagocytose, and digest bacteria, and have higher treatment efficiency and more stable effects compared with other microbial treatment technologies.

[0036] 2. The present invention provides a method for reducing resistance genes in the soil environment by using protozoan amoeba, which can simultaneously reduce antibiotic resistance genes (ARGs) and metal resistance genes (MRGs). The range of pollutants reduced is wide. Using this method under different soil types can reduce the total relative abundance of resistance genes by 60% - 70%. It is an environmentally friendly bioremediation technology with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a diagram of the growth of protozoan amoeba in the soil and the diagram of protozoan amoeba preying on bacteria observed under a laser confocal microscope;

[0038] Figure 2 It is a diagram of the change in the total relative abundance of resistance genes in farmland - vegetable field soil after treatment with amoeba;

[0039] Figure 3 It is a diagram of the change in the total relative abundance of resistance genes in aquaculture - chicken farm soil after treatment with amoeba;

[0040] Figure 4 It is a diagram of the change in the total relative abundance of resistance genes in mine soil after treatment with amoeba;

[0041] Figure 5 It is a diagram of the effect of amoeba treatment on the abundance of different types of resistance genes in the soil. DETAILED DESCRIPTION OF THE INVENTION

[0042] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0043] Example 1 Cultivation of Protozoan Amoeba

[0044] The amoeba used in the present invention is the Dictyostelium discoideum QS9 strain, purchased from Dicty Stock Center (http: / / dictybase.org / StockCenter / StockCenter.html).

[0045] The QS9 strain was inoculated on an SM / 5 agar plate. The formula of the SM / 5 agar plate is: 2 g of glucose, 2 g of bacteriological peptone, 2 g of yeast extract, 0.2 g of MgCl2, 1.9 g of KH2PO4, 1 g of K2HPO4 and 15 g of agar per liter of medium.

[0046] After light cultivation at 21°C for 5 days, it was picked up and collected with an inoculation loop, and counted with a hemocytometer to obtain 2×10 5Amoeba spores. After culturing Klebsiella pneumoniae (purchased from ATCC) in 5 / SM culture medium at 32 °C for two days, a Klebsiella pneumoniae suspension with an absorbance of 1.5 at a wavelength of 600 nm was prepared. Mix 2×10 5 amoeba spores with 200 μL of the Klebsiella pneumoniae suspension, and then spread the mixed bacterial suspension on an SM / 5 agar plate by the plate coating method. After culturing under light at 21 °C for 5 days, collect with an inoculation loop.

[0047] In addition, inoculate a group of amoeba QS9 on an SM / 5 agar plate and repeat the above incubation operation. When the bacterial suspension after mixing amoeba QS9 and Klebsiella pneumoniae is spread on an SM / 5 agar plate for co-culture, and incubated for 12 h, wash the amoeba and Klebsiella pneumoniae on the plate with KK2 buffer, and then image under a laser confocal microscope to observe the phenomenon of amoeba preying on bacteria, as Figure 1 shown.

[0048] Example 2 Soil microcosm experiment

[0049] Collect the amoeba spores incubated on the plate with an inoculation loop and place them in a centrifuge tube containing KK2 buffer solution, and then calculate the concentration of the amoeba spore solution by the method of blood cell counting. Dilute the amoeba spore solution to 2×10 4 spores / mL, 2×10 5 spores / mL, 2×10 6 spores / mL, 2×10 7 spores / mL respectively.

[0050] To make the soil samples more representative, the soil used in this experiment was collected from soils with different land use patterns in Guangdong Province. Take Shaoguan Dabao Mountain, Guangzhou Guilong Chicken Farm and Guangzhou Intensive Vegetable Planting Base as sampling points. Adopt the method of multi-point sampling, with a sampling depth of 0 - 20 cm. Remove the coverings on the soil, including plants, roots, litter and stones, etc., and put the soil into a sterile self-sealing bag. Divide the collected soil samples into multiple portions, quickly freeze them in liquid nitrogen (or place them in an ice box), and transport them back to the laboratory as soon as possible for subsequent experiments.

[0051] Take 3 different environmental soil samples, namely farmland - vegetable field soil, breeding - chicken farm soil, and mine soil. Weigh 2 g of soil and place it in a 6-well cell culture plate (2 g of soil / well) respectively. Set up control groups and experimental groups. The control groups do not add amoeba, and the experimental groups add amoeba. Respectively pipette 1 mL of amoeba spore solution with different dilution ratios into the soil, that is, the amoeba exposure concentrations are 1×10 4 spores / g soil, 1×10 5 spores / g soil, 1×106 spores / g soil, 1×10 7 spores / g soil.

[0052] Both the control group and the laboratory set up 3 biological replicate experiments. The control group and the experimental group were placed in the incubator at the same time, and the experimental treatment time was 7 days. After the experiment, the genomic DNA of soil microorganisms was extracted separately using a DNA extraction kit for the detection and analysis of resistance genes.

[0053] Example 3 Extraction of Genomic DNA of Soil Microorganisms

[0054] In the present invention, the genomic DNA of soil microorganisms was extracted using a kit ( Pro Kit, QIAGEN). The steps are as follows:

[0055] (1) According to the richness of the microbial biomass in the soil, 250 - 500 mg of soil was placed in the PowerBead Pro centrifuge tube provided with the kit, 800 μL of lysis solution Solution CD1 was added, and then it was vortexed thoroughly.

[0056] (2) Then the centrifuge tube in (1) was fixed on the vortex homogenization plate and homogenized continuously at the highest speed for 10 minutes to completely release the genomic DNA of the microorganisms in the soil.

[0057] (3) The centrifuge tube in (2) was centrifuged at 15000×g for 1 minute.

[0058] (4) After centrifugation, the supernatant was transferred to a clean 2 mL centrifuge tube, and then 200 μL of SolutionCD2 solution was added and vortexed for 5 s to mix evenly.

[0059] (5) At room temperature, it was centrifuged at 15000×g for 1 minute. Then, 700 μl of the supernatant was transferred to a clean 2 ml centrifuge tube.

[0060] (6) 600 μL of Solution CD3 solution was added to the centrifuge tube and vortexed for 5 s.

[0061] (7) 650 μL of the lysis solution was transferred to the DNA filtration column and centrifuged at 15000×g for 1 minute.

[0062] (8) The filtrate was discarded, and then the filtration column was centrifuged at 15000×g for 1 minute again to ensure that there was no lysis solution residue in the filtration column.

[0063] (9) The filtration column was carefully placed in a clean 2 mL collection tube, and 500 μL of SolutionEA solution was added to the filtration column.

[0064] (10) Centrifuge at 15,000×g for 1 minute and discard the filtrate;

[0065] (11) Add 500 μL of Solution C5 to the filter column. Centrifuge at 15,000×g for 1 minute and discard the filtrate;

[0066] (12) Centrifuge the empty column at 16,000×g for 2 minutes. Carefully place the filter column into a new 1.5 ml centrifuge tube;

[0067] (13) Add 50 - 100 μL of DNA elution buffer to the center of the white filter membrane in the filter column;

[0068] (14) Centrifuge at 15,000×g for 1 minute. Discard the filter column and collect the DNA sample in the centrifuge tube. Store the DNA sample in a -80°C refrigerator for future use.

[0069] Example 4 Effect of Protozoan Amoeba on the Total Relative Abundance of Soil Resistance Genes

[0070] In the present invention, quantitative polymerase chain reaction (qPCR) was used to quantitatively detect and analyze the resistance genes in different soils in the soil microcosm experiment of Example 2, and the change trends between the total relative abundances of resistance genes in the experimental treatment group and the control group soils were compared.

[0071] iTaq TM Universal Greensupermix (BIO - RAD, USA) was used as the green fluorescent dye, and the volume of each reaction system was 20 μL, including 10 μL of iTaq TM Greensupermix, 7 μL of ddH2O, 1 μL each of the forward and reverse primers, and 1 μL of the template DNA. The quantitative detection of resistance genes was carried out on a BIO - RAD CFX instrument (BIO - RAD, USA). The reaction conditions for the whole process were as follows: pre - denaturation at 95°C for 3 min, then 40 cycles of the amplification stage with denaturation at 95°C for 10 s, followed by annealing at different temperatures for 30 s, where the annealing temperature was determined by the properties of the resistance gene primers. The melting curve detection program for the specificity of the amplification products was between 65°C and 95°C, with a 5 - s increment for every 0.5°C increase. During the qPCR quantitative detection process, 3 replicates were set for each sample for machine detection.

[0072] Relative abundance of resistance gene = (copy number of target resistance gene / copy number of 16S rDNA gene).

[0073] In different soil types, the results of the change trends of the total relative abundances of resistance genes in the control group without amoeba addition and the experimental group with amoeba exposure are as follows:

[0074] In farmland-vegetable field soil, the results of the change trends of the total relative abundances of resistance genes are as Figure 2 shown (CK represents the control group without amoeba addition in vegetable field soil; C4, C5, C6, C7 represent the amoeba exposure amounts in vegetable field soil of 1×10 4 spores / g soil, 1×10 5 spores / g soil, 1×10 6 spores / g soil, 1×10 7 spores / g soil, respectively). Among them, when the amoeba spore addition amount is 1×10 4 spores / g soil, the total relative abundance of resistance genes in vegetable field soil decreased by 44.33%. When the amoeba spore addition amount is 1×10 5 spores / g soil, the total relative abundance of resistance genes in vegetable field soil decreased by 52.58%. When the amoeba spore addition amount is 1×10 6 spores / g soil, the total relative abundance of resistance genes in vegetable field soil decreased by 69.81%. When the amoeba spore addition amount is 1×10 7 spores / g soil, the total relative abundance of resistance genes in vegetable field soil decreased by 78.11%. It shows that the amoeba QS9 in the present invention can significantly reduce the total relative abundance of resistance genes in vegetable field soil through predation.

[0075] In aquaculture-chicken farm soil, the results of the change trends of the total relative abundances of resistance genes are as Figure 3 shown (CK represents the control group without amoeba addition in chicken farm soil; J4, J5, J6, J7 represent the amoeba exposure amounts in chicken farm soil of 1×10 4 spores / g soil, 1×10 5 spores / g soil, 1×10 6 spores / g soil, 1×10 7 spores / g soil, respectively). When the amoeba spore addition amount is 1×10 4 spores / g soil, the total relative abundance of resistance genes in chicken farm soil decreased by 39.18%. When the amoeba spore addition amount is 1×10 5 spores / g soil, the total relative abundance of resistance genes in chicken farm soil decreased by 48.98%. When the amoeba spore addition amount is 1×10 6 spores / g soil, the total relative abundance of resistance genes in chicken farm soil decreased by 70.33%. When the amoeba spore addition amount is 1×10 7When the amoeba spore dosage was 1×10⁶ spores / g soil, the total relative abundance of resistance genes in the chicken farm soil decreased by 77.86%. This indicates that Amoeba QS9 in the present invention can significantly reduce the total relative abundance of resistance genes in the soil of chicken farms through predation.

[0076] In the mine soil, the results of the change trend of the total relative abundance of resistance genes are as Figure 4 shown (CK represents the control group without adding amoeba in the mine soil; K4, K5, K6, and K7 represent the amoeba exposure amounts in the mine soil are 1×10⁴ 4 spores / g soil, 1×10⁵ 5 spores / g soil, 1×10⁶ 6 spores / g soil, 1×10⁷ 7 spores / g soil, respectively). When the amoeba spore dosage was 1×10⁴ 4 spores / g soil, the total relative abundance of resistance genes in the mine soil decreased by 24.66%. When the amoeba spore dosage was 1×10⁵ 5 spores / g soil, the total relative abundance of resistance genes in the mine soil decreased by 38.03%. When the amoeba spore dosage was 1×10⁶ 6 spores / g soil, the total relative abundance of resistance genes in the mine soil decreased by 48.38%. When the amoeba spore dosage was 1×10⁷ 7 spores / g soil, the total relative abundance of resistance genes in the mine soil decreased by 71.90%. This indicates that Amoeba QS9 in the present invention can significantly reduce the total relative abundance of resistance genes in the mine soil through predation.

[0077] Generally, with the increase of amoeba exposure amount, the reduction degree of resistance genes is better. However, due to the overall level of resistance genes in the soil within a certain range (for example, the relative abundance of resistance genes in farmland soil is 1.4E-03; the relative abundance of resistance genes in breeding soil is 9.0E-02; the relative abundance of resistance genes in mine soil is 2.5E-04), and limited by the ability of amoeba spores to prey on bacteria and its growth cycle, when the amoeba exposure amount exceeds 1×10⁷ 7 spores / g soil, the reduction effect of resistance genes tends to be stable, and the reduction effect is maintained at about 1.0 log. Therefore, when the amoeba exposure amount is 1×10⁷ 7 spores / g soil, the best reduction effect of resistance genes can be obtained.

[0078] Example 5: Effects of Protozoan Amoeba on the Abundance of Different Kinds of Resistance Genes in Soil

[0079] In this invention, quantitative polymerase chain reaction (qPCR) was used to analyze the changing trends of the abundances of different types of resistance genes in soil by the protozoan amoeba in Example 2, including metal resistance genes copA, czcA, arsB, arsC and antibiotic resistance genes tetA, tetM, tetC, sul1.

[0080] iTaq was used in the fluorescence quantitative PCR analysis system TM Universal Greensupermix (BIO-RAD, USA) was used as the green fluorescent dye, and the volume of each reaction system was 20 μL, which included 10 μL iTaq TM Greensupermix, 7 μL ddH2O, 1 μL each of the forward and reverse primers, and 1 μL of the template DNA.

[0081] Quantitative detection of resistance genes was carried out on a BIO-RAD CFX instrument (BIO-RAD, USA). The reaction conditions for the whole process were as follows: pre-denaturation at 95 °C for 3 min, then a 40-cycle amplification stage at 95 °C with denaturation for 10 s, followed by annealing for 30 s at different temperatures, where the annealing temperature was determined by the nature of the resistance gene primers. The melting curve detection program for the specificity of the amplification products was between 65 °C and 95 °C, with a 5-s increase for every 0.5 °C rise. During the qPCR quantitative detection process, 3 replicates were set for each sample for machine detection.

[0082] The abundance of resistance genes was taken as the logarithmic value, i.e., log(N t / N0), where N t : represents the abundance of resistance genes in the soil after amoeba exposure; N0: represents the abundance of resistance genes in the control group. The results are as Figure 5 shown.

[0083] In farmland-vegetable field soil, the effects of the protozoan amoeba on the abundances of different types of resistance genes are as Figure 5 (a) shows that amoeba QS9 can significantly reduce the relative abundances of the antibiotic resistance genes tetA, tetM and sul1 in farmland-vegetable field soil through predation. When the amoeba exposure concentration is 1×10 7 spores / g soil, the reduction effect of the resistance gene abundance is the best, with a decrease of 0.6 - 0.9 logs.

[0084] In breeding-chicken farm soil, the effects of the protozoan amoeba on the abundances of different types of resistance genes are as Figure 5As shown in 7 (b), Amoeba QS9 can significantly reduce the relative abundances of metal resistance genes copA and czcA, and antibiotic resistance genes tetA, tetC, and sul1 in the soil of chicken farms through predation. When the amoeba exposure concentration is 1×10

[0085] spores / g soil, the reduction effect of the resistance gene abundance is the best, decreasing by 0.6 - 1.0 logs. Figure 5 (c), Amoeba QS9 can significantly reduce the relative abundances of metal resistance genes arsB and arsC in mine soil through predation. When the amoeba exposure concentration is 1×10 7 spores / g soil, the reduction effect of the resistance gene abundance is the best, decreasing by 0.5 - 0.9 logs.

[0086] 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 the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of an amoeba in reducing resistance genes in the soil environment, characterized in that, The amoeba is Dictyostelium discoideum, and the strain of Dictyostelium discoideum is QS9; The resistance genes include at least one of metal resistance genes and antibiotic resistance genes; the metal resistance genes include at least one of copA, czcA, arsB, and arsC; the antibiotic resistance genes include at least one of tetA, tetM, tetC, and sul1.

2. The application according to claim 1, wherein The application includes the following steps: adding a solution of amoeba spores to the soil environment for resistance gene reduction treatment.

3. The application according to claim 2, characterized in that, When the solution of amoeba spores is added to the soil environment, 1×10 4 ~1×10 7 The solution was added in a ratio of 100% amoebae spores.

4. The application according to claim 2, characterized in that, The treatment time of the amoeba spores in the soil is 7 - 10 days.

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