CN114807196B - Fluorescent marking method for tracking soil-borne plant pathogenic bacteria drug-resistant gene - Google Patents
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Abstract
The application discloses a fluorescent marking method for tracking soil-borne plant pathogenic bacteria drug-resistant genes, which combines a fluorescent marking technology, a lactose operon technology and a polymerase chain reaction technology to obtain the gene in the genome of the plant pathogenic bacteriaglmsThe regulatory gene is inserted after the genelacI) And a red fluorescent gene; will contain LacI repressor binding sitelacZThe multiple resistance transmissible plasmid of the promoter region and the green fluorescent gene is transferred to the plant pathogenic bacteria, and the plant pathogenic bacteria only express red fluorescence. The method for evaluating the cross-species transmission ARGs of the plant pathogenic bacteria is characterized in that the anti-plasmid in the donor strain is transferred to the receptor strain, the inhibition of plasmid fluorescence expression is relieved, and the plasmid fluorescence expression can be separated and recovered by a fluorescence microscope and a high-flux flow cytometry. The method can track and distinguish donor and acceptor strains, is favorable for evaluating the interaction mechanism of soil-borne plant pathogenic bacteria and ARGs, and provides a theoretical basis for the formulation of soil-borne disease resistance and control strategies.
Description
Technical Field
The application belongs to the technical field of microorganisms, relates to a fluorescent marking method for tracking soil-borne plant pathogenic bacteria drug-resistance genes, and in particular relates to soil-borne plant pathogenic bacteria Solanaceae Lawsonia and a red and green fluorescent gene expression regulation system.
Background
Plant soil-borne diseases caused by pathogenic bacteria of soil-borne plants are severely threatening the health of plants and sustainable economic development of agriculture (Zhu Yongguan, peng Jingjing, in Wer, shen Jirong, zhang Fusuo, 2021. Soil microbiome and soil health. Chinese science: life sciences 51, 1-11.). The discovery of antibiotics has made a tremendous contribution to human and animal-plant therapy, but as the abuse of antibiotics has led to the enrichment and spread of a large number of antibiotic resistance genes (Antibiotic Resistance Genes, ARGs) in the environment (Van Boeckel TP, pires J, silvester R, et al 2019.Global trends in antimicrobial resistance in animals in low-and MIddle-com counts.science 356: eaaw1944.). The superposition and interaction of the pathogenic bacteria of the soil plants and ARGs form the soil biological combined pollution, so that the pollution is more serious and the prevention and control are more difficult. ARGs can be transferred between different bacterial individuals and species by horizontal and vertical gene transfer, and once transferred into the food chain, would pose a serious threat to human health safety (Li B, qia Y, song Y, et al, disjunction horizontal and vertical gene transfer of antibiotic resistance plasmid in bacterial community using microfluidics, environmental International [ J ].2019,131 (C)). Studies have shown that by adding human conditional pathogenic bacteria Pseudomonas putida (Pseudomonas putida) carrying multiple resistance plasmids to soil, the carried resistance plasmids can be transferred to 15 bacteria of the genus (Xiao-Ting F, hu L, qing-Lin C, et al Fate of Antibiotic Resistant Pseudomonas putida and Broad Host Range Plasmid in Natural Soil microcosm. Front in microbiology [ J ].2019,10.). Currently, there is no study on how the risk of spread of ARGs is investigated only in e.coli and pseudomonas putida, and it is limited to the lack of suitable high-throughput tracking technology.
Disclosure of Invention
Aiming at the defects of the prior art, the first aim of the application is to provide a fluorescent marking method for tracking soil-borne plant pathogenic bacteria transmission drug-resistant genes; it is a second object of the present application to provide a method for assessing the transmission of ARGs across species of plant pathogenic bacteria.
The specific technical scheme of the application is as follows:
a fluorescent labeling method for tracking soil-borne plant pathogenic bacteria transmission drug-resistant genes; inserting sequence fragments of a fusion regulatory gene (lacI) and a first fluorescent gene into a genome of a plant pathogenic bacterium by using a fixed-point insertion type transposon Tn7 through a fluorescent labeling technology and a lactose operon technology; transferring the anti-property grain containing the operator lacZ promoter and the second fluorescent gene into the plant pathogenic bacteria to obtain the fluorescent marked plant pathogenic bacteria.
The first fluorescent gene and the second fluorescent gene are present in the plant pathogenic bacteria at this time by polymerase chain reaction technology and fluorescence microscopy technology, but the second fluorescent gene expression is inhibited, and the strain only has the first fluorescence.
As a preferable technical scheme of the application, the plant pathogenic bacteria is Lawsonia solanaceae (Ralstonia solanacearum), and is called ralstonia solanacearum for short.
The inventor researches and discovers that the bacterial wilt is the most widely distributed and serious soil plant pathogenic bacteria, has high genome plasticity and strong natural transformation capability, and can promote the spread of ARGs. Sequence fragments of fusion regulatory gene (lacI) and red fluorescent gene were inserted into the bacterial wilt genome by the fluorescent labeling technique and lactose operon technique using the site-directed insertion transposon Tn 7. Transferring the anti-property grain containing the operator lacZ promoter and the green fluorescent gene into the bacterial wilt, and carrying out polymerase chain reaction technology and fluorescence microscopy technology on the bacterial wilt, wherein the bacterial wilt has the red green fluorescent gene, but the green fluorescent gene expression is inhibited, and the bacterial strain only has red fluorescence. Through a combination transfer experiment, the donor bacterial wilt can transfer the resistant plasmid containing the green fluorescence gene to an acceptor strain, and the inhibition of the green fluorescence expression on the resistant plasmid in the acceptor strain is relieved, so that the bacterial wilt has green fluorescence. Donor and recipient strains can be successfully obtained by high throughput cell sorting techniques. As a preferred embodiment of the present application, the first fluorescent gene is red fluorescent gene mCherry.
As a preferred embodiment of the present application, the second fluorescent gene is a green fluorescent gene gfp.
Specifically, a fluorescent labeling method for tracking soil-borne plant pathogenic bacteria transmission drug-resistant genes comprises the following specific steps:
step 1, constructing a fluorescence labeling plasmid of a plant pathogenic bacteria genome: fusing the regulatory gene lacI and the first fluorescent gene; the fusion fragment is recovered and purified and connected to a KS+ plasmid vector, and then the required fragment is connected to a Tn7 transposon vector plasmid through enzyme digestion and enzyme ligation to obtain a genome fluorescence labeling plasmid;
step 2, construction of a movable fluorescent labeling plasmid for plant pathogenic bacteria: amplifying and fusing the promoter of the operator lacZ and the second fluorescent gene, wherein the promoter of the operator lacZ comprises a repressor binding site encoded by the lacI gene; the fusion fragment is recycled and purified and connected to a KS+ plasmid vector, and then the required fragment is connected to an RP4 plasmid vector through enzyme digestion and enzyme ligation to construct a movable fluorescence labeling plasmid;
step 3, double-color fluorescent labeling of plant pathogenic bacteria: the genome fluorescence labeling plasmid which is successfully constructed in the step 1 is utilized, a target fragment is inserted into attTn7 locus at the downstream of the glmS gene of the QL-Rs1115 bacteria at fixed point through a helper plasmid containing transposase pTNS2, and a BG culture medium containing 25 mug/mL gentamycin is utilized to screen positive strains with fusion gene lacI-first fluorescence; transferring the plasmid of the step 2 into the positive strain by natural transformation, and screening Rs 1115-lacI-first fluorescent strain and second fluorescent strain by BG culture medium containing 100 mug/mL kanamycin, 25 mug/mL tetracycline hydrochloride, 100 mug/mL ampicillin and 50 mug/mL chloramphenicol; the strain has suppressed expression of the second fluorescent gene and only the first fluorescent gene as observed by fluorescence microscopy.
As a preferable technical scheme of the application, the BG medium is: 20-30 g of agar, 5g of glucose, 1g of casein amino acid, 10g of peptone, 1g of yeast powder and 1000mL of deionized water, adjusting pH to 7.2-7.4, and sterilizing at 120 ℃ for 20min.
The application also protects a cross-species transmission ARGs evaluation method of plant pathogenic bacteria, the plant pathogenic bacteria marked by fluorescence are used as donor plant pathogenic bacteria, the donor pathogenic bacteria transfer the resistance plasmid containing the second fluorescent gene to the receptor strain through a combination transfer experiment, the inhibition of the second fluorescent expression on the resistance plasmid in the receptor strain is relieved, and the second fluorescent is provided, so that the trace of the soil-borne plant pathogenic bacteria transmission drug-resistant gene is realized.
Wherein the pathogenic bacteria are Ralstonia solanacearum (Ralstonia solanacearum) which is called ralstonia solanacearum for short.
As a preferable technical scheme of the application, the evaluation method comprises the following specific steps: regulating the concentration of donor plant pathogenic bacteria and acceptor strain bacterial liquid to 1×10 respectively 8 Mixing CFU/mL, centrifuging to remove supernatant, adding 5mL sterile deionized water, mixing, filtering with 0.45 μm sterile filter membrane, transferring the filter membrane to receptor culture medium, and culturing at 30deg.C for 1d; and (3) washing thalli on the filter membrane with 2mL of sterile water, centrifuging, collecting 200 mu L of bacterial liquid, coating 100 mu L of bacterial liquid into a receptor culture medium containing 100 mu g/mL kanamycin, 25 mu g/mL tetracycline hydrochloride, 100 mu g/mL ampicillin and 50 mu g/mL chloramphenicol, culturing for 2d at 30 ℃, and observing fluorescence by a fluorescence microscope.
Advantageous effects
The application realizes double-color labeling of plant pathogenic bacterial wilt by means of transformation and combination, is favorable for distinguishing donor strains (red fluorescence) and acceptor strains (green fluorescence), and can track the process of spreading ARGs by the plant bacterial wilt. Meanwhile, the bacterial wilt is spread ARGs to the same species, the same genus and the cross species.
Drawings
FIG. 1 is the construction of the plasmid pUC 18-lacI-mCherry-Gm;
FIG. 2 shows construction of lacZ-GFP-RP4 plasmid;
FIG. 3 shows the QL-Rs1115-GFP strain of the bacterial wilt homospecies spreading ARGs, wherein, FIG. A shows the QL-Rs1115-GFP strain under the fluorescent microscope illumination field, and FIG. B shows the QL-Rs1115-GFP strain observed under the fluorescent microscope green filter;
FIG. 4 is a drawing of an NJQL-A6-GFP strain from which bacterial wilt spread ARGs, wherein FIG. A is an NJQL-A6-GFP strain under a fluorescence microscope illumination field, and FIG. B is an NJQL-A6-GFP strain observed under a fluorescence microscope green filter;
FIG. 5 shows a NJX-GFP strain from which bacterial wilt spread ARGs across genera, wherein FIG. A shows a NJX-GFP strain under a fluorescent microscope field, and FIG. B shows a NJX-GFP strain observed under a fluorescent microscope green filter.
Detailed Description
The technical scheme of the application is further described below with reference to the specific embodiments. The reagents or instrumentation used are not manufacturer specific and are considered to be commercially available conventional products.
Strains:
lawsonia solanaceae (Ralstonia solanacearum) QL-Rs1115 (Zhong Wei, xingming Yang, shixue Yin, et al efficiency of Bacillus-fortified organic fertiliser in controlling bacterial wilt of tomato in the field [ J ]. Applied Soil Ecology,2011,48 (2))
Ralstonia pisiformis (Ralstonia pickettii) NJQL-A6, 9/26 of 2012 deposited at China general microbiological culture Collection center, accession number address: the collection number of the strain of the Datunlu in the Yang-ward area of Beijing is CGMCC No.6628.
Bacillus subtilis (Bacillus subtilis) NJX501, 12 th year 2015, 4 th day deposited in China general microbiological culture Collection center, deposit unit address: the collection number of the strain in the Datunlu of the Yang-ward area of Beijing is CGMCC No.11823.
Culture medium:
liquid medium B: 1g of casein amino acid, 10g of peptone, 1g of yeast powder and 1000mL of deionized water, adjusting pH to 7.2-7.4, and sterilizing at 120 ℃ for 20min.
The BG solid culture medium is a liquid culture medium B, and 2-3% (w/w) of agar and 5g/L of glucose are added.
TSB broth: 15g of peptone, 5g of soybean peptone, 5g of sodium chloride and 1000mL of deionized water, and the mixture was autoclaved at 20 ℃ for 20min.
The TSA solid culture medium is TSB liquid culture medium added with 2-3% (w/w) agar.
EXAMPLE 1 fluorescent labeling of bacterial wilt-spread drug-resistance Gene
(1) Construction of a bacterial wilt genome fluorescent marker plasmid: the regulatory gene (lacI) and the red fluorescent gene mCherry were fused by polymerase chain reaction technology.Fusion fragment lacI-mCherry was recovered and purified to be linked to KS + On the plasmid vector, the desired fragment was ligated to pUC18-mini-Tn7T-Gm plasmid vector by double cleavage with HindII and BamHI, and the plasmid pUC 18-lacI-Mchery-Gm plasmid was constructed.
(2) Construction of a mobile fluorescence labeling plasmid for bacterial wilt: the promoter of the operator (lacZ) comprising the binding site for the repressor protein encoded by the lacI gene and the green fluorescent gene gfp are amplified and fused by polymerase chain reaction techniques. Fusion fragment lacZ-GFP was recovered and purified and ligated to KS + On the plasmid vector, the desired fragment was ligated to the RP4 plasmid vector by double cleavage with HindII and KpnI to construct the lacZ-GFP-RP4 plasmid.
(3) And (3) carrying out double-color fluorescent labeling on the bacterial wilt: the constructed pUC18-Tn7-lacI-mCherry-Gm plasmid was used, and the objective fragment lacI-mCherry was inserted at the site of attTn7 downstream of the glmS gene of the strain QL-Rs1115 by means of the electric excitation method using a helper plasmid containing the transposase pTNS2, and the Rs 1115-lacI-Mchery strain was selected using a BG medium containing 25. Mu.g/mL gentamicin. Using natural transformation, the plasmid was transferred into Rhizoctonia solani Rs1115-lacI-mCherry, and the Rs 1115-lacI-Mchery-GFP strain was selected by BG medium containing 100. Mu.g/mL kanamycin, 25. Mu.g/mL tetracycline hydrochloride, 100. Mu.g/mL ampicillin, 50. Mu.g/mL chloramphenicol. The green fluorescent gene expression of the strain is inhibited and only red fluorescence is observed by a fluorescence microscope.
EXAMPLE 2 bacterial wilt homotopy transmitting ARGs
Materials: the strain Rs1115-lacI-mCherry-GFP and QL-Rs1115 are classified and named as Lawsonia solanaceae (Ralstonia solanacearum).
Glycerol-saved Rs1115-lacI-mCherry-GFP and QL-Rs1115 were streaked on BG solid medium containing 100. Mu.g/mL kanamycin, 25. Mu.g/mL tetracycline hydrochloride, 100. Mu.g/mL ampicillin, 50. Mu.g/mL chloramphenicol and 25. Mu.g/mL gentamicin, and no antibiotic, respectively, and cultured at 30℃for 2d; single colonies on the plates were picked and transferred to liquid medium B and incubated overnight at 30℃and 170 rpm. Collecting fresh bacterial liquid, centrifuging at 4500rpm at normal temperature for 2min, collecting bacterial cells, washing bacterial cells with sterile water for 1 time, and removing cultureCulturing medium, and adjusting concentration to 1×10 with sterile water 8 CFU/mL, 1mL of bacterial liquid is evenly mixed, the bacterial liquid is centrifuged at 4500rpm for 2min at normal temperature, the supernatant is removed, 5mL of sterile deionized water is added for vortex mixing evenly, a filter membrane with the thickness of 0.45 mu m is adopted, and the filter membrane is transferred to BG culture medium for 1d at 30 ℃. The cells on the filter were washed with 2mL of sterile water into a 2mL centrifuge tube, centrifuged at 4500rpm at room temperature for 2min, 200. Mu.L of the bacterial liquid was left, 100. Mu.L of each was coated on BG medium containing 100. Mu.g/mL kanamycin, 25. Mu.g/mL tetracycline hydrochloride, 100. Mu.g/mL ampicillin, 50. Mu.g/mL chloramphenicol, cultured at 30℃for 2d, and fluorescence was observed by a fluorescence microscope to obtain QL-Rs1115-GFP strain, see FIG. 3. The results indicate that the bacterial wilt is capable of transferring ARGs between the same species
EXAMPLE 3 transfer of bacterial wilt homozygotic ARGs
Materials: rs1115-lacI-mCherry-GFP and NJQL-A6 belong to Ralstonia solanacearum (Ralstonia solanacearum) and Ralstonia pisiformis (Ralstonia pickettii), respectively.
Rs 1115-lacI-Mchery-GFP was streaked on BG solid medium containing 100. Mu.g/mL kanamycin, 25. Mu.g/mL tetracycline hydrochloride, 100. Mu.g/mL ampicillin, 50. Mu.g/mL chloramphenicol and 25. Mu.g/mL gentamicin, and incubated at 30℃for 2d; single colonies on the plates were picked and transferred to liquid medium B and incubated overnight at 30℃and 170 rpm. Collecting fresh bacterial liquid, centrifuging at 4500rpm at room temperature for 2min, collecting bacterial cells, washing bacterial cells with sterile water for 1 time, removing culture medium, and adjusting concentration to 1×10 with sterile water 8 CFU/mL, ready for use.
Streaking the glycerol-preserved NJQL-A6 on a TSA solid culture medium, and culturing for 1d at 30 ℃; single colonies on the plates were picked and transferred to TSA broth and incubated overnight at 30℃and 170 rpm. Collecting fresh bacterial liquid, centrifuging at 4500rpm at room temperature for 2min, collecting bacterial cells, washing bacterial cells with sterile water for 1 time, removing culture medium, and adjusting concentration to 1×10 with sterile water 8 CFU/mL, ready for use.
1mL of Rs 1115-lacI-Mchery-GFP bacterial liquid and 1mL of NJQL-A6 bacterial liquid are taken, the supernatant is removed by centrifugation at 4500rpm for 2min at normal temperature, 5mL of sterile deionized water is added for vortex mixing, a 0.45um filter membrane is passed, the filter membrane is transferred to a TSA culture medium, and the culture is carried out for 1d at 30 ℃. The cells on the filter were washed with 2mL of sterile water into a 2mL centrifuge tube, centrifuged at 4500rpm at room temperature for 2min, 200ul of the bacterial liquid was left, 100. Mu.L of each was spread on TSA medium containing 100. Mu.g/mL kanamycin, 25. Mu.g/mL tetracycline hydrochloride, 100. Mu.g/mL ampicillin, 50. Mu.g/mL chloramphenicol, cultured at 30℃for 2d, and fluorescence was observed by a fluorescence microscope to obtain NJQL-A6-GFP strain, see FIG. 4. The results indicate that ralstonia solanacearum is able to transfer ARGs between siblings.
EXAMPLE 4 spread of ARGs by ralstonia solanacearum across the gate
Materials: rs1115-lacI-mCherry-GFP and NJX501 belong to Proteus (Proteus) and Deuteromycota (Firmics), respectively.
Rs1115-lacI-mCherry-GFP was streaked on BG solid medium containing 100. Mu.g/mL kanamycin, 25. Mu.g/mL tetracycline hydrochloride, 100. Mu.g/mL ampicillin, 50. Mu.g/mL chloramphenicol and 25. Mu.g/mL gentamicin, and incubated at 30℃for 2d; single colonies on the plates were picked and transferred to liquid medium B and incubated overnight at 30℃and 170 rpm. Collecting fresh bacterial liquid, centrifuging at 4500rpm at room temperature for 2min, collecting bacterial cells, washing bacterial cells with sterile water for 1 time, removing culture medium, and adjusting concentration to 1×10 with sterile water 8 CFU/mL, ready for use.
Glycerol-saved NJX501 was streaked onto TSA solid medium and incubated at 30℃for 1d; single colonies on the plates were picked and transferred to TSA broth and incubated overnight at 30℃and 170 rpm. Collecting fresh bacterial liquid, centrifuging at 4500rpm at room temperature for 2min, collecting bacterial cells, washing bacterial cells with sterile water for 1 time, removing culture medium, and adjusting concentration to 1×10 with sterile water 8 CFU/mL, ready for use.
1mL of Rs 1115-lacI-Mchery-GFP bacterial liquid and 1mL of NJX501 bacterial liquid are taken, the solution is centrifuged for 2min at the normal temperature of 4500rpm, the supernatant is removed, 5mL of sterile deionized water is added for vortex mixing uniformly, a 0.45um filter membrane is passed, and the filter membrane is transferred to a TSA culture medium for culturing for 1d at 30 ℃. The cells on the filter were washed with 2mL of sterile water into a 2mL centrifuge tube, centrifuged at 4500rpm at room temperature for 2min, 200ul of the bacterial liquid was left, 100 ul of each was spread on TSA medium containing 100 ul of kanamycin, 25 ul of tetracycline hydrochloride, 100 ul of ampicillin, 50 ul of chloramphenicol, cultured at 30℃for 2d, and fluorescence was observed by fluorescence microscopy to obtain NJX-GFP strain, see FIG. 5. The results indicate that ralstonia solanacearum is able to transfer ARGs across the gate.
The protection of the present application is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included in the application without departing from the spirit and scope of the inventive concept, and the scope of the application is defined by the appended claims.
Claims (7)
1. A fluorescent marking method for tracking soil-borne plant pathogenic bacteria drug-resistant gene is characterized in that fusion regulating genes are fused by utilizing fixed-point insertion type transposon Tn7 through a fluorescent marking technology and a lactose operon technologylacIAnd inserting the sequence fragment of the first fluorescent gene into the genome of the plant pathogenic bacterium; will contain an operatorlacZTransferring the resistant plasmids of the promoter and the second fluorescent gene into plant pathogenic bacteria to obtain fluorescent marked plant pathogenic bacteria; wherein the plant pathogenic bacteria is Laurella of SolanaceaeRalstonia solanacearum）。
2. The method for fluorescent labeling of a pathogenic soil-borne plant resistance gene according to claim 1, wherein the first fluorescent gene is a red fluorescent genemCherry。
3. The method for fluorescent labeling for tracking a soil-borne plant pathogenic bacteria-borne drug-resistant gene according to any one of claims 1-2, wherein the second fluorescent gene is a green fluorescent genegfp。
4. The fluorescence labeling method for tracking soil-borne plant pathogenic bacteria transmission resistance genes according to claim 1, comprising the following specific steps:
step 1, constructing a fluorescence labeling plasmid of a plant pathogenic bacteria genome: will regulate geneslacIFusing with a first fluorescent gene; fusion fragment recovery purification ligation to KS + The plasmid vector is connected with a required fragment to a Tn7 transposon vector plasmid through enzyme digestion and enzyme ligation to obtain a genome fluorescence labeling plasmid;
step 2, movable fluorescent marker for plant pathogenic bacteriaAnd (3) particle construction: will manipulate the genelacZAmplifying and fusing the promoter and the second fluorescent gene of (C), wherein the operatorlacZThe promoter of (a) compriseslacIA gene encoding a repressor binding site; fusion fragment recovery purification ligation to KS + The plasmid vector is connected with the required fragment through enzyme digestion and enzyme ligation to an RP4 plasmid vector, so as to construct a movable fluorescent labeling plasmid;
step 3, double-color fluorescent labeling of plant pathogenic bacteria: the genome fluorescence labeling plasmid successfully constructed in the step 1 is utilized to insert the target fragment into the QL-Rs1115 bacteria at fixed point through the auxiliary plasmid containing the transposase pTNS2glmSAn attTn7 site at the downstream of the gene, and screening a positive strain with fusion gene lacI-first fluorescence by utilizing a BG culture medium containing 25 mug/mL gentamicin; transferring the plasmid in the step 2 into the positive strain by utilizing natural transformation, and screening the RS 1115-lacI-first fluorescent strain and the second fluorescent strain by using a BG culture medium containing 100 mug/mL kanamycin, 25 mug/mL tetracycline hydrochloride, 100 mug/mL ampicillin and 50 mug/mL chloramphenicol; the strain has suppressed expression of the second fluorescent gene and only the first fluorescent gene as observed by fluorescence microscopy.
5. The fluorescence labeling method for tracking soil-borne plant pathogenic bacteria transmission resistance genes according to claim 4, wherein the BG medium is: 20-30 g of agar, 5g of glucose, 1g of casein amino acid, 10-g g of peptone, 1g of yeast powder and 1000-mL g of deionized water, adjusting pH to 7.2-7.4, and sterilizing at 120 ℃ for 20min.
6. A plant pathogenic bacteria cross-species transmission ARGs assessment method is characterized in that the plant pathogenic bacteria marked by the fluorescence of claim 1 are taken as donor plant pathogenic bacteria, the donor pathogenic bacteria transfer the resistance plasmid containing a second fluorescent gene to an acceptor strain through a combination transfer experiment, the inhibition of the second fluorescent expression on the resistance plasmid in the acceptor strain is relieved, and the second fluorescent is provided, so that the tracking of the soil-borne plant pathogenic bacteria transmission drug-resistant gene is realized; wherein the pathogenic bacteria are Laurella of SolanaceaeRalstonia solanacearum）。
7. The method of claim 6, wherein the concentration of each of the donor plant pathogenic bacteria and the recipient strain bacterial fluid is adjusted to 1X 10 8 Mixing CFU/mL with 1mL bacteria solution, centrifuging to remove supernatant, adding 5mL sterile deionized water, vortex mixing, passing through 0.45 μm sterile filter membrane, transferring the filter membrane onto receptor culture medium, and culturing at 30deg.C for 1d; and (3) washing thalli on the filter membrane with 2mL sterile water until the thalli are in a centrifuge tube, centrifuging, reserving 200 mu l of bacterial liquid, coating 100 mu l of bacterial liquid into a receptor culture medium containing 100 mu g/mL kanamycin, 25 mu g/mL tetracycline hydrochloride, 100 mu g/mL ampicillin and 50 mu g/mL chloramphenicol, culturing at 30 ℃ for 2d, and observing fluorescence through a fluorescence microscope.
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