Method for labeling pseudomonas fluorescens by green fluorescent protein (GFP) and application of pseudomonas fluorescens in tracking colonization of biocontrol bacteria on plants

Abstract

The invention discloses a method for labeling pseudomonas fluorescens by green fluorescent protein (GFP) and application of the method in tracking colonization of biocontrol bacteria on plants, and relates to the technical field of microorganisms, the technical key points are as follows: step S1, constructing a green fluorescent protein (GFP) gene expression vector; step S2, preparing a pseudomonas fluorescens competent state; step S3, constructing a pseudomonas fluorescens HT1-EGFP (Enhanced Green Fluorescent Protein) strain; and step S4, application of the GFP-labeled pseudomonas fluorescens in tracking colonization of biocontrol bacteria on plants. The GFP labeling technology provided by the invention has higher sensitivity and accuracy, can realize real-time tracking of the biocontrol bacteria in the plant body, and does not need to damage the plant tissue. In addition, through an electric shock transformation technology, the efficiency of transferring the exogenous gene into the strain is improved, and the method is more efficient than a traditional chemical induction or thermal shock method.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a method for marking fluorescent pseudomonas with green fluorescent protein (GFP) and an application thereof in tracking colonization of biocontrol bacteria on plants. Background Art

[0002] As an environmentally friendly disease control strategy, biological control technology has gradually received widespread attention and application. Biocontrol bacteria are an important part of biological control. They inhibit the infection and spread of pathogens through various mechanisms such as competing for nutrients and space, producing antimicrobial substances, and inducing plant resistance. Among the many biocontrol bacteria, Pseudomonas fluorescens has become a hot spot for research and application due to its diverse disease resistance mechanisms and wide adaptability.

[0003] Existing research mainly focuses on the screening of biocontrol bacteria, their disease resistance mechanisms, and their effects on plant growth. However, there are relatively few studies on the colonization process of biocontrol bacteria inside and outside plants and their interaction mechanisms with plants and pathogens. Traditional research methods usually rely on destructive methods, such as tissue sections and microscopic observations. Although these methods can provide certain quantitative and qualitative data, they are cumbersome to operate and difficult to achieve real-time and dynamic observations. In addition, the behavior of biocontrol bacteria in the natural environment is complex and changeable, and existing laboratory studies often cannot fully simulate the actual field environment, thus limiting the practical application value of the research results. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems and provide a method for marking fluorescent Pseudomonas fluorescens with green fluorescent protein (GFP) and its application in tracking the colonization of biocontrol bacteria on plants.

[0005] In order to achieve the above object, the technical scheme of the present invention is as follows: A method for marking fluorescent Pseudomonas with green fluorescent protein GFP, comprising the following steps:

[0006] Step S1, constructing a green fluorescent protein GFP gene expression vector: using BamH I and Kpn I to double-digest, respectively, the linearized PBBR1MCS2-Tac vector and the target EGFP gene fragment were cut from the eukaryotic expression vector PBBR1MCS2-Tac and the EGFP vector pEGFP-1, the amplified product and the vector after enzyme digestion were purified using a gel recovery kit, and then connected using the homologous recombinase Vazyme, and the extracted plasmid was obtained as the target vector;

[0007] Step S2, preparation of competent cells of Pseudomonas fluorescens: picking a single colony of Pseudomonas fluorescens HT1 and inoculating it into a liquid LB medium, culturing it overnight, taking the bacterial liquid of Pseudomonas fluorescens HT1 cultured overnight and inoculating it into a liquid LB medium, collecting the cells and washing them twice with sterile water precooled in an ice bath to obtain competent cells of Pseudomonas fluorescens HT1;

[0008] Step S3, constructing the fluorescent Pseudomonas HT1-EGFP strain: taking the PBBR1MCS2-Tac-EGFP plasmid and adding it into the fluorescent Pseudomonas HT1 competent cells, mixing it in an ice bath, transferring the mixture into a pre-cooled electroporation cup for electroporation, adding LB liquid culture medium into the electroporation cup immediately after the electroporation, and spreading it on an LB plate containing kanamycin for static culture;

[0009] Step S4, identifying GFP-labeled Pseudomonas fluorescens strains: using Pseudomonas fluorescens HT1 and Pseudomonas fluorescens HT1-EGFP strains as materials, amplifying the EGFP fragment by PCR amplification method, using Pseudomonas fluorescens HT1 and Pseudomonas fluorescens HT1-EGFP strains as templates, respectively, amplifying the EGFP fragment using primers pEGFP-N-3 / pEGFP-C-5, and detecting the fragment size by agarose gel electrophoresis.

[0010] Furthermore, the system in step S1 is: 100 μL (30 μL of purified PCR product or vector, 3 μL of restriction endonuclease, 10 μL of 10× restriction endonuclease buffer, 57 μL of ddH2O).

[0011] Furthermore, the connection system in step S1 is: 20 μL (20-200 ng of insert fragment amplification product, 50-200 ng of linearized cloning vector, 2 μL of recombinase II (Exnase II), 4 μL of 5× cloning expression II buffer (CE II buffer), and ultrapure water (ddH2O) is added to 20 μL); the connection conditions are 37°C, 30 min; 16°C, 2 min.

[0012] Furthermore, the electric shock conditions in step S3 are electric field strength of 1200 V / mm, resistance of 200 Ω, and capacitance of 25 μF.

[0013] Furthermore, the Pseudomonas fluorescens HT1 deposit number is CCTCC NO: M20231604.

[0014] A green fluorescent protein GFP-labeled fluorescent Pseudomonas is used to track the colonization of biocontrol bacteria on plants.

[0015] Compared with the prior art, this solution has the following beneficial effects:

[0016] 1. Real-time, non-destructive observation: By inserting the green fluorescent protein (GFP) gene into the genome of Pseudomonas fluorescens and constructing a GFP-labeled strain, the colonization process of biocontrol bacteria can be observed in real time under living conditions. This method avoids the shortcomings of traditional destructive methods and will not interfere with the normal physiological state of biocontrol bacteria and plants.

[0017] 2. Dynamic tracking and analysis: Using GFP-labeled strains, the behavior of biocontrol bacteria inside and outside plants can be dynamically tracked in natural environments to obtain continuous dynamic data. This is of great significance for studying the colonization rules of biocontrol bacteria and their interaction mechanisms with plants and pathogens.

[0018] 3. Simple and efficient operation: Compared with complex observation equipment such as electron microscopes, fluorescence microscopes are easier to operate and relatively inexpensive. The application of GFP labeling technology allows researchers to conveniently conduct real-time observation and data collection of biocontrol bacteria under conventional laboratory conditions.

[0019] 4. Improve the application value of research: By applying GFP-labeled strains in actual field environments, the behavior of biocontrol bacteria in natural environments can be more realistically simulated, improving the reliability and application value of research results. This has important guiding significance for the actual promotion and application of biological control technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of PBBR1MCS2-Tac-EGFP restriction site and gene fragment in the embodiment of the present invention;

[0021] Figure 2 It is the morphology of the fluorescent Pseudomonas EGFP-labeled strain HT1-EGFP in the embodiment of the present invention, wherein Figure A is the green fluorescence of the fluorescent Pseudomonas EGFP-labeled strain HT1-EGFP under mercury lamp irradiation, and Figure B is the morphology of the fluorescent Pseudomonas EGFP-labeled strain HT1-EGFP under normal light;

[0022] Figure 3 It is the gel electrophoresis diagram of the EGFP fragment in the embodiment of the present invention (M: DL 2000 DNA Marker; 1-2: PCR product using HT1 strain as template; 3-4: PCR product using HT1-EGFP strain as template);

[0023] Figure 4It is the inhibition of the hyphae growth of Pseudomonas fluorescens HT1 and HT1-EGFP strains on the mycelial growth of Coptis chinensis Sclerotium rolfsii LC1 in the embodiment of the present invention (LC1: the pathogenic bacterium LC1 of Coptis chinensis Sclerotium rolfsii is cultured alone; LC1+HT1: the pathogenic bacterium LC1 of Coptis chinensis Sclerotium rolfsii and Pseudomonas fluorescens HT1 are cultured in confrontation; LC1+HT1-EGFP: the pathogenic bacterium LC1 of Coptis chinensis Sclerotium rolfsii and Pseudomonas fluorescens HT1-EGFP are cultured in confrontation), wherein Figure A is the colony diameter of Coptis chinensis Sclerotium rolfsii LC1 after 3 days of culture, and Figure B is the inhibition rate of Pseudomonas fluorescens HT1 and HT1-EGFP strains on the hyphae growth of LC1;

[0024] Figure 5 The colonization of Pseudomonas fluorescens HT1 in the roots of Arabidopsis thaliana in the embodiment of the present invention (Control: Arabidopsis thaliana treated with sterile 1×PBS; HT1: Arabidopsis thaliana treated with HT1 bacterial resuspension; HT1-EGFP: Arabidopsis thaliana treated with HT1-EGFP bacterial resuspension);

[0025] Figure 6 The colonization of the sprayed fluorescent Pseudomonas HT1 in the roots of Arabidopsis thaliana in the embodiment of the present invention (Control: Arabidopsis thaliana treated with sterile 1×PBS; HT1-EGFP: Arabidopsis thaliana treated with HT1-EGFP bacterial heavy suspension);

[0026] Figure 7 1 is the effect of Pseudomonas fluorescens HT1 on the growth of Arabidopsis thaliana in the embodiment of the present invention, wherein Figure A is a photo of the whole plant, Figure B is a photo of the root system, Figure C is the plant height, Figure D is the root length, and Figure E is the fresh weight;

[0027] Figure 8 1 is the effect of Pseudomonas fluorescens HT1 on the resistance of Arabidopsis thaliana to white rot in the examples of the present invention, wherein Figure A is a photo of the experimental leaves, and Figure B is the area of ​​the lesions. DETAILED DESCRIPTION

[0028] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0029] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0030] Example:

[0031] like Figure 1-8 As shown, a method for marking fluorescent Pseudomonas with green fluorescent protein GFP comprises the following steps:

[0032] Step 1: Construct green fluorescent protein (GFP) gene expression vector:

[0033] The linearized PBBR1MCS2-Tac vector and the target EGFP gene fragment were cut from the eukaryotic expression vector PBBR1MCS2-Tac and the EGFP vector pEGFP-1 with BamH I and Kpn I. The system is: 100 μL (30 μL of purified PCR product or vector, 3 μL of restriction endonuclease, 10 μL of 10× restriction endonuclease buffer (buffer), 57 μL of ultrapure water (ddH2O)). The amplified product and vector after enzyme digestion were purified by gel recovery kit, and then connected by homologous recombinase (Vazyme). The connection system is: 20 μL (20-200 ng of inserted fragment amplification product, 50-200 ng of linearized cloning vector, 2 μL of ExnaseⅡ, 4 μL of 5×CEⅡbuffer, ddH2O is supplemented to 20 μL). Connection conditions: (37℃, 30 min; 16℃, 2 min). The connection product was directly transformed into Escherichia coli. After a single colony grows, pick a single colony and culture it in the corresponding culture medium at 37℃ on a shaker for about 8 hours, then perform PCR verification on the bacterial solution, and further verify the correct sample sequencing. After the sequencing is correct, store the correct sample bacterial solution at -80℃ for future use, and extract the plasmid, which is the obtained target vector, and store it at -20℃ for future use. Figure 1 Shown is a schematic diagram of the successfully constructed EGFP expression vector PBBR1MCS2-Tac-EGFP plasmid.

[0034] Step 2: Preparation of competent cells of Pseudomonas fluorescens

[0035] A single colony of Pseudomonas fluorescens HT1 was picked and inoculated into liquid LB medium, and cultured at 25°C overnight. The deposit number of Pseudomonas fluorescens HT1 is CCTCC NO: M20231604, and the depository is China Center for Type Culture Collection. The depository address is Wuhan University, Wuhan, China. The classification of the biological material sample is named PseudomonasfluorescensHT1-GFP, and the deposit date is September 4, 2023. The overnight cultured Pseudomonas fluorescens HT1 bacterial solution was inoculated into a fresh 10mL liquid LB medium at a ratio of 1:10, and cultured at 25°C until A600 was 0.3-0.4. Take the bacterial solution in the logarithmic phase, ice bath for 30 minutes, centrifuge at 4°C and 4000r / min for 5 minutes, collect the bacteria and wash twice with sterile water pre-cooled in an ice bath to obtain the competent state of Pseudomonas fluorescens HT1.

[0036] Step 3: Construction of Pseudomonas fluorescens HT1-EGFP strain

[0037] Take 5μLPBBR1MCS2-Tac-EGFP plasmid and add it to 100μL fluorescent Pseudomonas HT1 competent cells, mix well and place in an ice bath for 10 minutes. Transfer the mixture to a pre-cooled 0.1cm electric shock cup and perform electric shock under the conditions of electric field strength of 1200V / mm, resistance of 200Ω, and capacitance of 25μF. Immediately after the electric shock, add 900μL of 25℃ preheated LB liquid culture medium to the electric shock cup, incubate at 25℃ for 1h, and then spread on an LB plate containing 100μg / mL kanamycin. After the colonies grow on the plate, observe the fluorescence of the colonies under a fluorescent stereomicroscope, such as Figure 2 As shown, the green fluorescence of the plate colonies was obvious under the dark field, indicating that the fluorescent Pseudomonas EGFP-labeled strain HT1-EGFP was successfully constructed.

[0038] Step 4: Identification of GFP-labeled Pseudomonas fluorescens strains

[0039] In order to clarify whether the fluorescent Pseudomonas HT1 transformed with the EGFP plasmid is authentic, the fluorescent Pseudomonas HT1 and fluorescent Pseudomonas HT1-EGFP strains were used as materials, and the EGFP fragment was amplified by PCR amplification. The fluorescent Pseudomonas HT1 and fluorescent Pseudomonas HT1-EGFP strains were used as templates, and the EGFP fragment was amplified using primers pEGFP-N-3 / pEGFP-C-5, and the fragment size was detected by agarose gel electrophoresis. The results are shown in Figure 3 As shown, the HT1-EGFP strain transformed with the EGFP plasmid amplified a band of nearly 500 bp, while the control group Pseudomonas fluorescens HT1 did not amplify it.

[0040] Step 5:

[0041] In order to clarify whether the transfer of EGFP plasmid has an effect on the antibacterial ability of Pseudomonas fluorescens HT1, Pseudomonas fluorescens HT1 and Pseudomonas fluorescens HT1-EGFP strains were used as materials, and the inhibitory effect of Pseudomonas fluorescens HT1 and Pseudomonas fluorescens HT1-EGFP strains on Sclerotium rolfsii LC1 was determined by double-dish confrontation method. The deposit number of Sclerotium rolfsii LC1 is CCTCC NO:M 20231605, and the deposit unit is China Center for Type Culture Collection, and the deposit unit address is Wuhan University, Wuhan, China. The classification of the biological material sample is named Sclerotium rolfsii LC1, and the deposit date is September 4, 2023. One dish is added with LB solid medium and coated with Pseudomonas fluorescens HT1 and Pseudomonas fluorescens HT1-EGFP strains respectively, and the other dish is added with PDA medium and inoculated with Sclerotium rolfsii LC1. The two dishes are inverted together, sealed, and cultured at 30°C. After 3 days, the colony diameter of S. rolfsii LC1 was measured, and the normal culture of S. rolfsii LC1 was used as the control. The difference in the antibacterial ability of Pseudomonas fluorescens HT1 and Pseudomonas fluorescens HT1-EGFP strains was compared. Figure 4 As shown in the figure, the introduction of EGFP plasmid did not affect the inhibitory effect of HT1 strain on the mycelial growth of LC1 of S. rolfsii. The colony diameter of the control group was 4.82 cm after 3 days of culture. After inoculation with Pseudomonas fluorescens HT1 and HT1-EGFP strains, the colony diameters of LC1 were 2.22 cm and 2.32 cm, respectively. The inhibition rates of inoculation with Pseudomonas fluorescens HT1 and HT1-EGFP strains on the mycelial growth of LC1 of S. rolfsii were 54.01% and 51.04%, respectively, with no significant difference.

[0042] Step 6: Inoculate the GFP-labeled strain into plants and track its colonization process inside and outside the plant

[0043] In order to clarify whether Pseudomonas fluorescens HT1 can colonize in plants, Pseudomonas fluorescens HT1 and Pseudomonas fluorescens HT1-EGFP strains were used as materials, Arabidopsis seeds were inoculated with Pseudomonas fluorescens HT1 and Pseudomonas fluorescens HT1-EGFP strains, and the treated seeds were cultured on 1 / 2MS sucrose-free medium. After the seedlings grew, the colonization of Pseudomonas fluorescens HT1 was observed under a laser confocal microscope. Specific method: Arabidopsis seeds were first surface disinfected in 1% NaClO solution for 1 min, 75% alcohol surface disinfected for 1 min, rinsed with sterile water 3 times, and vernalized at 4℃ for 24h for use. Pseudomonas fluorescens HT1 and Pseudomonas fluorescens HT1-EGFP strains were cultured overnight in 20mL liquid LB medium (30℃, 180rmp), centrifuged at 3000rmp for 10min, and the supernatant was removed. The bacteria were resuspended in sterile 1×PBS to OD600 of 0.8 for use. The spare seeds were divided into three 1.5 mL sterile centrifuge tubes, and 1 mL of sterile 1×PBS, HT1 resuspended bacterial solution, and HT1-EGFP resuspended bacterial solution were added respectively. After soaking for 1 min, the bacterial solution was aspirated and blown dry in the clean bench. The treated seeds were evenly spotted on a 1 / 2MS sucrose-free medium plate and cultured at 20°C. After 7 days, the colonization of Pseudomonas fluorescens HT1 was observed under a laser confocal microscope. The results are shown in Figure 2. Figure 5 As shown, strong green fluorescence was observed in the roots of Arabidopsis inoculated with the HT1-EGFP strain, while no green fluorescence was observed in Arabidopsis inoculated with the HT1 strain and in the control group. This shows that seed soaking can successfully inoculate the HT1-EGFP strain, and the HT1-EGFP strain can successfully colonize in the plant. This shows that the fluorescent Pseudomonas HT1-EGFP strain can be used to study the colonization rules of antibacterial bacteria in plants.

[0044] In addition, we also explored whether the fluorescent Pseudomonas HT1 strain could colonize plants by spraying. The fluorescent Pseudomonas HT1-EGFP strain was cultured overnight in 20 mL of liquid LB medium (30°C, 180 rpm), centrifuged at 3000 rpm for 10 min, and the supernatant was removed. The bacteria were resuspended in sterile 1× PBS to an OD of 600 The prepared PBS solution was placed in a small 50 mL spray bottle sterilized with alcohol and sprayed evenly on the leaves of 20-day-old Arabidopsis seedlings. The control group was directly sprayed with 1×PBS. After 14 days, the colonization of HT1 was observed under a laser confocal microscope. The results are shown in the figure. Figure 6 As shown, green fluorescence was observed in the roots of Arabidopsis thaliana sprayed with HT1-EGFP bacterial solution, but no green fluorescence was observed in the control group. This indicates that fluorescent Pseudomonas HT1 can colonize in plants by foliar spraying.

[0045] Step 7: Determination of the growth status and resistance to white silk in Arabidopsis after inoculation with Pseudomonas fluorescens HT1

[0046] Arabidopsis thaliana inoculated with Pseudomonas fluorescens HT1 by soaking seeds was used as the material to determine the effect of inoculation with Pseudomonas fluorescens HT1 on the growth of Arabidopsis thaliana and its resistance to white rot. Specific method: The Arabidopsis seeds were surface disinfected in 1% NaClO solution for 1 min, 75% alcohol for 1 min, rinsed with sterile water 3 times, and vernalized at 4°C for 24 h for use. The Pseudomonas fluorescens HT1 strain was cultured overnight in 20 mL of liquid LB medium (30°C, 180 rpm), centrifuged at 3000 rpm for 10 min, and the supernatant was removed. The bacteria were resuspended in sterile 1×PBS to OD 600 0.8 for standby use. Divide the spare seeds into two 1.5mL sterile centrifuge tubes, add 1mL of sterile 1×PBS and HT1 to resuspend the bacteria respectively, soak for 1min, then aspirate the bacteria solution and blow dry in the clean bench. Evenly spot the treated seeds on the 1 / 2MS desucrose culture medium plate, and after culturing at 20℃ for 10d, transplant the normally developed seedlings into plastic pots filled with nutrient soil. The transplanted Arabidopsis seedlings were cultured at 20℃, 80% relative humidity, and 16:8 light: dark conditions for 30 days. Divide each group of plants into two equal parts, carefully dig out the entire plant and root system in half with tweezers, rinse with clean water, and measure the root length, plant height, fresh weight and other indicators after drying. Figure 7 As shown in the figure, inoculation with Pseudomonas fluorescens HT1 had no significant effect on the growth of Arabidopsis. There was some promotion on the root system and fresh weight of Arabidopsis, but there was no significant difference in the data analysis. It may be because the growth cycle is short and the difference cannot be reflected. If crops with long growth cycles such as Coptis chinensis are used, there may be obvious differences.

[0047] The other half was used to determine the effect of inoculation with Pseudomonas fluorescens HT1 on the ability of Arabidopsis to resist white rot. Select the second round of Arabidopsis leaves of uniform size and no obvious disease in each group, clean them and place them neatly in a plastic basin covered with moist absorbent paper to keep them moist. Ten leaves were selected from each HT1 treatment group and PBS control group, and a 3mm diameter bacterial cake taken from the edge of the freshly activated white rot fungus LC1 colony was inoculated in the center of the leaf. After the inoculation is completed, use a spray bottle to spray a certain amount of sterile water to keep it moist, and seal the entire experimental pot with plastic wrap and place it in a 28°C incubator. After 48 hours, the diameter of the lesions was measured and the disease situation was recorded. The results are as follows Figure 8 As shown in the figure, the incidence and severity of Arabidopsis inoculated with HT1 were significantly lower than those in the control group. The use of HT1 significantly increased the rate of mild disease in inoculated leaves.

[0048] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for labeling Pseudomonas fluorescens with green fluorescent protein (GFP), characterized in that: The following steps are involved: Step S1, constructing a green fluorescent protein GFP gene expression vector: using BamH I and Kpn I to double-digest, respectively, the linearized PBBR1MCS2-Tac vector and the target EGFP gene fragment were cut from the eukaryotic expression vector PBBR1MCS2-Tac and the EGFP vector pEGFP-1, the amplified product and the vector after enzyme digestion were purified using a gel recovery kit, and then connected using the homologous recombinase Vazyme, and the extracted plasmid was obtained as the target vector; Step S2, preparation of competent cells of Pseudomonas fluorescens: picking a single colony of Pseudomonas fluorescens HT1 and inoculating it into a liquid LB medium, culturing it overnight, taking the bacterial liquid of Pseudomonas fluorescens HT1 cultured overnight and inoculating it into a liquid LB medium, collecting the cells and washing them twice with sterile water precooled in an ice bath to obtain competent cells of Pseudomonas fluorescens HT1; Step S3, constructing the fluorescent Pseudomonas HT1-EGFP strain: taking the PBBR1MCS2-Tac-EGFP plasmid and adding it to the fluorescent Pseudomonas HT1 competent cells, mixing it in an ice bath, transferring the mixture to a pre-cooled electroporation cup for electroporation for 15 seconds, and immediately adding LB liquid culture medium to the electroporation cup after the electroporation, and spreading it on an LB plate containing kanamycin for static culture; Step S4, identifying GFP-labeled Pseudomonas fluorescens strains: using Pseudomonas fluorescens HT1 and Pseudomonas fluorescens HT1-EGFP strains as materials, amplifying the EGFP fragment by PCR amplification method, using Pseudomonas fluorescens HT1 and Pseudomonas fluorescens HT1-EGFP strains as templates, respectively, amplifying the EGFP fragment using primers pEGFP-N-3 / pEGFP-C-5, and detecting the fragment size by agarose gel electrophoresis.

2. A method for labeling Pseudomonas fluorescens with green fluorescent protein (GFP) as claimed in claim 1, characterized in that: The system in step S1 is: 100 μL (30 μL of purified PCR product or vector, 3 μL of restriction endonuclease, 10 μL of 10× restriction endonuclease buffer, and 57 μL of ultrapure water).

3. A method for labeling Pseudomonas fluorescens with green fluorescent protein (GFP) as claimed in claim 1, characterized in that: The connection system in step S1 is: 20 μL (20-200 ng of insert fragment amplification product, 50-200 ng of linearized cloning vector, 2 μL of recombinase II, 4 μL of 5× cloning expression II buffer, and ultrapure water to 20 μL); the connection conditions are 37° C., 30 min; 16° C., 2 min.

4. A method for labeling Pseudomonas fluorescens with green fluorescent protein (GFP) as claimed in claim 1, characterized in that: The electric shock conditions in step S3 are as follows: electric field strength 1200 V / mm, resistance 200 Ω, and capacitance 25 μF.

5. The method for labeling Pseudomonas fluorescens with green fluorescent protein (GFP) as claimed in claim 1, characterized in that: The Pseudomonas fluorescens HT1 deposit number is CCTCCNO:M 20231604.

6. An application of green fluorescent protein (GFP) labeled fluorescent Pseudomonas in tracking the colonization of biocontrol bacteria on plants.

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