A DSF inducible promoter, recombinant reporter bacteria and applications thereof

Abstract

A DSF-inducible promoter, a recombinant reporter bacterium and applications thereof, wherein the nucleotide sequence of the DSF-inducible promoter is shown in SEQ ID NO: 1. The recombinant reporter bacterium is formed by introducing a reporter gene and the DSF-inducible promoter into a non-DSF-producing strain in the form of a recombinant vector. The application of the recombinant reporter bacterium is to use the recombinant reporter bacterium to detect whether the strain to be tested produces DSF or whether the sample to be tested contains DSF. The DSF-inducible promoter of the present invention responds to DSF accurately and sensitively. The recombinant reporter bacterium of the present invention is easy to culture and is efficient in detecting DSF. The application of the recombinant reporter bacterium of the present invention in detecting DSF is efficient, rapid, convenient, accurate and sensitive.

Description

Technical Field

[0001] The present invention relates to a promoter, a recombinant reporter bacterium and applications thereof, and in particular to a DSF inducible promoter, a recombinant reporter bacterium and applications thereof. Background Art

[0002] Quorum sensing (QS) is a communication system for microorganisms to communicate with each other. It regulates the group behavior of microorganisms by synthesizing and releasing certain specific small molecules. Among them, Diffusible Signal Factor (DSF), as an important type of intercellular communication molecule, plays a vital role in the pathogenicity and regulation of host infection, microbial biofilm formation, and pathogen resistance in many animal and plant pathogens. Therefore, providing efficient and accurate DSF detection means and methods is expected to play an important role in the prevention and control of animal and plant diseases, clinical drug resistance research, and the treatment of membrane fouling in membrane bioreactors.

[0003] At present, the commonly used method for detecting whether bacteria produce DSF or whether DSF exists in the environment is chemical detection based on gas phase or liquid phase combined with mass spectrometry. However, such methods are time-consuming and labor-intensive and require high instrumentation and equipment.

[0004] CN111595955A discloses a method for synchronously detecting the quorum sensing signal molecules DSF and BDSF of Oligotrophomonas rhizophila. The method uses ultra-high performance liquid chromatography-triple quadrupole mass spectrometry tandem time-of-flight mass spectrometry technology to perform qualitative and quantitative detection of DSF. However, the operation of this technology is relatively complicated and has high requirements on equipment.

[0005] Therefore, it is urgent to find a DSF-inducible promoter that responds accurately and sensitively to DSF, an easy-to-cultivate and efficient recombinant reporter bacteria, and the application of efficient, rapid, convenient, accurate and sensitive recombinant reporter bacteria in detecting DSF. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the above-mentioned defects in the prior art and provide a DSF-inducible promoter that responds accurately and sensitively to DSF.

[0007] The technical problem further to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide a recombinant reporter bacteria that is easy to culture and has high efficiency in DSF detection.

[0008] A further technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide an efficient, rapid, convenient, accurate and sensitive application of recombinant reporter bacteria in detecting DSF.

[0009] The technical solution adopted by the present invention to solve the technical problem is as follows: a DSF inducible promoter, whose nucleotide sequence is shown in SEQ ID NO:1.

[0010] The technical solution adopted by the present invention to further solve the technical problem is as follows: a recombinant reporter bacterium is formed by introducing the reporter gene and the DSF inducible promoter into a DSF-free strain in the form of a recombinant vector. The method of introducing the recombinant vector is electroporation.

[0011] The principle of the recombinant reporter bacteria for detecting DSF of the present invention is as follows: since the non-DSF-producing strain cannot produce DSF, the recombinant reporter bacteria itself will not express the reporter gene; but when the strain to be tested produces DSF or DSF exists in the environment, the DSF-induced promoter regulated by DSF on the recombinant vector will start the expression of the reporter gene, and the expressed reporter gene protein will react with the chromogenic substrate to produce a blue substance, thereby forming a blue circle visible to the naked eye. The relative concentration of DSF production can be reflected by measuring the area of ​​the blue circle.

[0012] Preferably, the reporter gene is a gene encoding a detectable protein or enzyme. The expression product of the reporter gene can be easily identified or observed.

[0013] Preferably, the reporter gene includes a β-glucuronidase encoding gene or a β-galactosidase encoding gene, etc. After reacting with a color-developing substrate, the reporter gene can produce a stable blue substance visible to the naked eye, which is convenient for observation.

[0014] Preferably, the β-glucosidase encoding gene is gusA The gene has a nucleotide sequence as shown in SEQ ID NO:2.

[0015] Preferably, the recombinant vector is pBBR-DSF inducible promoter + reporter gene. The pBBR vector is a high-copy wide-host expression vector.

[0016] Preferably, the recombinant vector pBBR-DSF inducible promoter+reporter gene is formed by fusion of the DSF inducible promoter and the reporter gene to a pBBRMCS-5 vector.

[0017] Preferably, the method for constructing the recombinant vector pBBR-DSF inducible promoter+reporter gene comprises the following steps:

[0018] 1) Using the genomic DNA of the strain encoding the DSF quorum sensing system as a template, use the primers pBBR-DSF-inducible promoter-F (e.g., pBBR-P egl-F) and DSF-inducible promoter + reporter gene-R (such as P egl +GusA-R) to PCR amplify the DSF-inducible promoter fragment;

[0019] 2) Using genomic DNA of Escherichia coli strain DH5α as template, use primers DSF inducible promoter + reporter gene-F (such as P egl +GusA-F) and pBBR-reporter-R (e.g., pBBR-GusA-R) were used to amplify the reporter gene fragment by PCR;

[0020] 3) The DSF-inducible promoter fragment obtained in step 1) and the reporter gene fragment obtained in step 2) are connected to the knockout vector double-digested with restriction endonucleases by homologous recombinase, and the connection product is transformed into Escherichia coli DH5α strain by heat shock;

[0021] 4) After the E. coli DH5α strain into which the ligation product was transferred was revived and cultured, the revived bacterial liquid was spread on LB solid medium containing gentamicin. After culture, the grown transformants were selected and verified by PCR using primers pBBR-F and pBBR-R. The transformants with the correct PCR amplification band size were selected for sequencing. After sequencing was correct, plasmid extraction was performed to obtain the recombinant vector pBBR-DSF inducible promoter + reporter gene.

[0022] Preferably, in step 3), the restriction endonuclease is Sac Ⅰ and Xba Ⅰ; The knockout vector is pBBRMCS-5 vector.

[0023] Preferably, in step 4), the temperature of the resuscitation culture is 35-39°C, and the time is 0.5-1.5 h; the content of gentamicin in the LB solid culture medium containing gentamicin is 40-60 μg / mL; the temperature of the culture after the resuscitation bacterial solution is applied to the LB solid culture medium containing gentamicin is 35-39°C, and the time is 18-30 h.

[0024] Preferably, the DSF-non-producing strain is a mutant strain lacking a DSF synthesis gene in a strain encoding a DSF quorum sensing system.

[0025] Preferably, the strain encoding the DSF quorum sensing system comprises Xanthomonas pv. citri Xanthomonas citri subsp. citri Xanthomonas campestris pv.campestris Xanthomonas campestris pv. campestris wait.

[0026] Preferably, the DSF synthetic gene comprisesrpfF wait.

[0027] Preferably, the DSF-non-producing strain is obtained by selecting a suicide knockout vector and using homologous recombination to seamlessly knock out the DSF synthesis gene in the strain encoding the DSF quorum sensing system.

[0028] The knockout method comprises the following steps:

[0029] 1) Using the genome of the strain encoding the DSF quorum sensing system as a template, use primers pK18-DSF synthetic gene-D1 (such as pK18-rpfF-D1) and pK18-DSF synthetic gene-D2 (such as pK18-rpfF-D2) to amplify the upstream fragment of the target knockout gene, and use pK18-DSF synthetic gene-D3 (such as pK18-rpfF-D3) and pK18-DSF synthetic gene-D4 (such as pK18-rpfF-D4) to amplify the downstream fragment of the target knockout gene;

[0030] 2) The upstream and downstream fragments obtained in step 1) are connected to the suicide knockout vector double-digested with restriction endonucleases by homologous recombinase, and the connection products are transformed into Escherichia coli DH5α strain by heat shock;

[0031] 3) After the E. coli DH5α strain into which the ligation product was transferred was revived and cultured, the revived bacterial solution was spread on LB solid medium containing kanamycin. After culture, the grown transformants were selected and verified by PCR with primers M13-F and M13-R. The transformants with the correct PCR amplification band size were selected for sequencing. After sequencing was correct, plasmid extraction was performed to obtain the plasmid pK18-DSF synthetic gene (such as pK18-rpfF);

[0032] 4) adding the plasmid pK18-DSF synthetic gene (such as pK18-rpfF) obtained in step 3) to the competent strain of the strain encoding the DSF quorum sensing system, and after electroporation transformation, reviving and culturing, applying the revived bacterial liquid on LB solid medium containing kanamycin, and after culturing, selecting the grown transformants to obtain a primary recombinant strain;

[0033] 5) The primary recombinant strain obtained in step 4) is cultured in a sucrose culture dish, and the obtained colonies are detected by PCR using primers pK18-DSF synthetic gene-D5 (such as pK18-rpfF-D5) and pK18-DSF synthetic gene-D6 (such as pK18-rpfF-D6), and the PCR products are sequenced. After sequencing, a strain encoding the DSF quorum sensing system of the DSF synthetic gene is knocked out, that is, a strain that does not produce DSF is obtained.

[0034] Preferably, in step 2), the restriction endonuclease isEcoR Ⅰ and Hind III; the knockout vector is pK18mobsacB.

[0035] Preferably, in steps 3) and 4), the content of kanamycin in the LB solid culture medium containing kanamycin is 40-60 μg / mL; in step 3), the temperature of the resuscitation culture is 35-39°C, and the time is 0.5-1.5 h; the temperature of the culture after the resuscitated bacterial liquid is applied to the LB solid culture medium containing kanamycin is 35-39°C, and the time is 18-30 h.

[0036] Preferably, in step 4), the preparation method of the competent strain of the strain encoding the DSF quorum sensing system is: inoculating the strain encoding the DSF quorum sensing system into LB liquid culture medium, culturing, placing on ice, centrifuging, suspending the collected bacteria, repeating the centrifugation and suspension operations, packaging, and quick freezing in liquid nitrogen to obtain the result; the culture temperature is 26-30°C, and the culture is cultured until the OD 600 The centrifugal temperature is 0-8°C, the speed is 4000-6000 rpm, and the time is 10-15 min; 8-12% of the total volume fraction is added with a pre-cooled 0-8°C sterilized glycerol solution to suspend the collected bacteria; the centrifugation and suspension operations are repeated ≥2 times; after the liquid nitrogen quick freezing, the solution is stored at -70--90°C for future use.

[0037] Preferably, in step 4), the mass volume ratio of the plasmid pK18-rpfF to the competent strain encoding the DSF quorum sensing system is 1:10-200 ng / μL; the voltage of the electroporation transformation is 1.8-2.4 kV, and the time is 5.0-5.8 ms; the temperature of the resuscitation culture is 26-30°C, and the time is 1.5-2.5 h; the temperature of the resuscitated bacterial solution after being coated on an LB solid culture medium containing kanamycin is 26-30°C, and the time is 60-84 h.

[0038] Preferably, in step 5), the concentration of the sucrose culture medium is 10-15 w / v; the culture temperature is 26-30° C., and the culture time is 72-120 h.

[0039] Preferably, the method for constructing the recombinant reporter bacteria is as follows: adding the recombinant vector to a competent strain that does not produce DSF, transforming it by electroporation, reviving it, and spreading the revived bacterial solution on an LB solid culture medium containing gentamicin. After culturing, selecting the grown transformants, performing PCR verification with primers pBBR-F and pBBR-R, and selecting the transformants with the correct PCR amplification band size as the recombinant reporter strain.

[0040] Preferably, the method for preparing the competent strain of the non-DSF-producing strain is the same as the method for preparing the competent strain of the strain encoding the DSF quorum sensing system, the only difference being that the strain encoding the DSF quorum sensing system is replaced by the non-DSF-producing strain.

[0041] Preferably, the mass volume ratio of the recombinant vector to the competent strain of the non-DSF-producing strain is 1:10-200 ng / μL; the voltage of the electric shock transformation is 1.8-2.4 kV, and the time is 5.0-5.8 ms; the temperature of the recovery culture is 26-30°C, and the time is 1.5-2.5 h; the content of gentamicin in the LB solid culture medium containing gentamicin is 40-60 μg / mL; the temperature of the culture after the recovery bacterial solution is applied to the LB solid culture medium containing gentamicin is 26-30°C, and the time is 60-84 h.

[0042] The technical solution adopted by the present invention to further solve the technical problem is as follows: an application of a recombinant reporter bacterium, wherein the recombinant reporter bacterium is used to detect whether a strain to be tested produces DSF or whether a sample to be tested contains DSF.

[0043] Preferably, the detection method comprises the following steps:

[0044] (1) inoculating the recombinant reporter bacteria into LB liquid culture medium and culturing the culture solution of the recombinant reporter bacteria; inoculating the strain to be tested into LB liquid culture medium and culturing the culture solution, centrifuging and resuspending the strain to be tested to obtain the culture solution of the strain to be tested;

[0045] (2) mixing the culture medium of the recombinant reporter bacteria obtained in step (1) with the chromogenic substrate and adding them to a sterile NYG solid culture medium to obtain a DSF detection solid culture medium;

[0046] (3) Directly inoculate the culture solution of the strain to be tested obtained in step (1) or directly add the sample to be tested to the DSF detection solid culture medium obtained in step (2), culture at a constant temperature, and observe whether a blue circle appears around the colony:

[0047] If a blue circle appears around the colony or sample, the strain to be tested produces DSF or the sample to be tested contains DSF;

[0048] If no blue circle appears around the colony or sample, the strain to be tested does not produce DSF or the sample to be tested does not contain DSF.

[0049] Preferably, in step (1), the recombinant reporter bacteria are cultured at 26-30°C and a rotation speed of 160-200 rpm until OD 600 It is 1.2 to 1.8.

[0050] Preferably, in step (1), the conditions for culturing the strain to be tested are: culturing at 26-30°C and a rotation speed of 160-200 rpm until OD 600 It is 1.0~2.2.

[0051] Preferably, in step (1), the centrifugal speed is 4000-6000 rpm and the time is 4-6 min.

[0052] Preferably, in step (1), the supernatant after centrifugation is removed and an equal volume of sterile water is added for re-suspending.

[0053] Preferably, in step (2), the volume ratio of the culture solution of the recombinant reporter bacteria to the chromogenic substrate is 1:0.5-1.0.

[0054] Preferably, in step (2), the volume ratio of the recombinant reporter bacteria culture solution to the sterile NYG solid culture medium is 1:80-120.

[0055] Preferably, in step (2), the concentration of the chromogenic substrate is 15-25 mg / mL.

[0056] Preferably, in step (2), the chromogenic substrate is a solution of 5-bromo-4-chloro-3-indole-β-D-glucuronic acid cyclohexylamine salt (abbreviated as X-Gluc).

[0057] Preferably, in step (2), the temperature of the sterile NYG solid culture medium is 40-44°C, and the concentration of agar is 1.2-1.8 w / v%.

[0058] Preferably, in step (3), the amount of direct inoculation or addition is 10 to 15 μL.

[0059] Preferably, in step (3), the constant temperature culture temperature is 26-30°C and the time is 18-50 h.

[0060] The formula of the LB liquid culture medium used in the present invention is: 8-12 g / L tryptone, 4-6 g / L yeast powder, 8-12 g / L NaCl, and the pH value is adjusted to 7.0-7.4.

[0061] The LB solid culture medium used in the present invention is prepared by adding agar with a final concentration of 1.2 to 1.8 w / v% to the LB liquid culture medium.

[0062] The preparation method of the NYG solid culture medium used in the present invention is as follows: first prepare a NYG liquid culture medium with the formula of 18-22 g / L of glycerol, 4-6 g / L of peptone, and 2-4 g / L of yeast extract, adjust the pH value to 7.0-7.4, and then add agar with a final concentration of 1.2-1.8 w / v% to the NYG liquid culture medium.

[0063] The beneficial effects of the present invention are as follows:

[0064] (1) The DSF-inducible promoter of the present invention responds accurately and sensitively to DSF;

[0065] (2) The recombinant reporter bacteria of the present invention are easy to culture and highly efficient in detecting DSF. The blue circle area / colony area (i.e., the relative value of DSF production) of different tested strains has a large numerical difference, which makes it easy to judge whether the tested strain produces DSF or not;

[0066] (3) The application of the recombinant reporter bacteria of the present invention in detecting DSF provides a method for detecting DSF, which can enable researchers of bacterial QS phenomena to efficiently, quickly, conveniently, accurately and sensitively detect DSF signal molecules during the research process, thereby meeting the needs of scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 The recombinant reporter strain Δ of Example 3 of the present invention rpfF / pBBR-P egl The color development photos of +GusA on different strains i, ii, and iii after constant temperature culture for 48 hours (as shown in Figure A, where the strains i, ii, and iii are Xcc8004, Xcc, Δ rpfF ) and DSF yield bar graph (as shown in Figure B);

[0068] Figure 2 The recombinant reporter strain Δ of Example 3 of the present invention rpfF / pBBR-P egl The color development photos of different strains i, ii, and iii after being cultured at constant temperature for 48 hours (as shown in Figure A, where the strains i, ii, and iii are Δ rpfC, Xcc, Δ rpfF ) and DSF yield bar graph (as shown in Figure B);

[0069] Figure 3 This is a color development photograph of different strains i, ii, and iii after constant temperature culture for 48 hours using the reported reporter strain FE58 in Example 1 (as shown in Figure A, where the strains i, ii, and iii are Xcc8004, Xcc, Δ rpfF ) and DSF yield bar graph (as shown in Figure B);

[0070] Among them, DSF yield = blue circle area / colony area. DETAILED DESCRIPTION

[0071] The present invention will be further described below in conjunction with the embodiments and drawings.

[0072] Xanthomonas pv. citri used in the present invention Xanthomonas citri subsp. citri (The present invention is referred to as Xcc ), purchased from China Center for Type Culture Collection, the address of the depository unit is Wuhan University, Luojiashan, Wuhan, the deposit date is December 13, 2023, and the deposit number is CCTCC FB 2023518; the wild campestris pv. campestris used Xanthomonas campestris pv. campestris (The present invention is referred to as Xcc 8004) was purchased from the National Collection of Plant Pathogenic Bacteria, United Kingdom, with the accession number: NCPPB No.1145;

[0073] The gentamicin (Gm) and kanamycin (Kan) used were purchased from Beijing Solebow Technology Co., Ltd.; the Escherichia coli strain used was ( Escherichia coli )DH5α was purchased from Beijing Qingke Biotechnology Co., Ltd.; the endonucleases used Sac Ⅰ. Xba Ⅰ. EcoR Ⅰ. Hind III were purchased from TaKaRa, and homologous recombination enzymes were purchased from Novartis. The pBBRMCS-5 and pK18mobsacB vectors used were purchased from Invitrogen. The 5-bromo-4-chloro-3-indole-β-D-glucuronic acid cyclohexylamine salt (X-Gluc) used was purchased from Beijing Coolbo Technology Co., Ltd.

[0074] The formula of the LB liquid medium used is: tryptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, and the pH value is adjusted to 7.2; the LB solid medium used is prepared by adding agar with a final concentration of 1.5 w / v% to the LB liquid medium; the preparation method of the NYG solid medium used is: first prepare NYG liquid medium with a formula of: glycerol 20 g / L, peptone 5 g / L, yeast extract 3 g / L, adjust the pH value to 7.2, and then add agar with a final concentration of 1.5w / v% to the NYG liquid medium;

[0075] Unless otherwise specified, the raw materials or chemical and biological reagents used in the examples and comparative examples of the present invention were obtained through conventional commercial channels;

[0076] The primer synthesis and sequencing in the examples and comparative examples of the present invention were all completed by Hangzhou Youkang Biotechnology Co., Ltd.

[0077] A DSF inducible promoter example 1

[0078] The nucleotide sequence of the DSF inducible promoter is shown in SEQ ID NO:1.

[0079] A recombinant reporter bacteria example 2

[0080] The recombinant reporter bacterium Δ rpfF / pBBR-P egl +GusA is a reporter gene encoding β-glucuronidase ( gusA gene, the nucleotide sequence of which is shown in SEQ ID NO: 2) and the DSF-inducible promoter described in Example 1 (abbreviated as P egl , nucleotide sequence as shown in SEQ ID NO: 1) to the recombinant vector pBBR-P egl +GusA was introduced into the non-DSF-producing strain Δ rpfF Become.

[0081] Wherein, the recombinant vector pBBR-P egl +GusA is induced by the DSF-inducible promoter P egl With reporter gene gusA The method for constructing the pBBRMCS-5 vector comprises the following steps:

[0082] 1) Xcc The genomic DNA of the strain was used as a template and the primers pBBR-P egl -F and P egl +GusA-R (as shown in Table 1 ) was used to PCR amplify the DSF-inducible promoter fragment;

[0083] 2) Using genomic DNA of Escherichia coli strain DH5α as template, primer P egl +GusA-F and pBBR-GusA-R (as shown in Table 1) for PCR amplification gusA Reporter gene fragment;

[0084] 3) The DSF-inducible promoter fragment obtained in step 1) and the gusA Reporter gene fragment, with restriction endonuclease Sac Ⅰ and Xba Ⅰ The double-digested pBBRMCS-5 vector was ligated using homologous recombination enzymes, and the ligation product was transformed into E. coli DH5α strain by heat shock;

[0085] 4) After the E. coli DH5α strain into which the ligation product was transferred was revived and cultured at 37°C for 1 hour, the revived bacterial solution was spread on LB solid medium containing 50 μg / mL Gm and cultured at 37°C for 24 hours. The grown transformants were selected and verified by PCR with primers pBBR-F and pBBR-R (as shown in Table 1). The transformants with the correct PCR amplification band size were selected and sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing. After sequencing was correct, plasmid extraction was performed to obtain the recombinant vector pBBR-P egl +GusA.

[0086] Wherein, the non-DSF producing strain is Xcc DSF synthesis gene is missing in the strain rpfF The mutant strain is denoted as Δ rpfF The suicide knockout vector pK18mobsacB was used to perform scarless knockout using homologous recombination. Xcc DSF synthesis gene in strains rpfF and become;

[0087] The knockout method comprises the following steps:

[0088] 1) Xcc The genome of the strain was used as a template, and primers pK18-rpfF-D1 and pK18-rpfF-D2 (as shown in Table 1) were used to amplify the upstream fragment of the target knockout gene, and primers pK18-rpfF-D3 and pK18-rpfF-D4 (as shown in Table 1) were used to amplify the downstream fragment of the target knockout gene;

[0089] 2) The upstream and downstream fragments obtained in step 1) were cleaved by restriction endonuclease EcoR Ⅰ and Hind III double-digested suicide knockout vector pK18mobsacB, connected by homologous recombination enzyme, and the ligation product was transformed into Escherichia coli DH5α strain by heat shock;

[0090] 3) After the E. coli DH5α strain into which the ligation product was transferred was revived and cultured at 37°C for 1 hour, the revived bacterial solution was spread on LB solid medium containing 50 μg / mL Kan, and cultured at 37°C for 24 hours. The grown transformants were selected and verified by PCR with primers M13-F and M13-R (as shown in Table 1). The transformants with the correct PCR amplification band size were selected and sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing. After sequencing was correct, plasmid extraction was performed to obtain plasmid pK18-rpfF;

[0091] 4) Add 2.5 ng of the plasmid pK18-rpfF obtained in step 3) to 100 μL XccThe competent strain of the strain was transformed by electroporation at 2.1 kV for 5.4 ms, and then revived and cultured at 28°C for 2 h. The revived bacterial solution was spread on LB solid medium containing 50 μg / mL Kan, and cultured at 28°C for 72 h. The grown transformants were selected to obtain the primary recombinant strain.

[0092] Said Xcc The preparation method of the competent strain of the strain is as follows: Xcc The strain was inoculated into LB liquid medium and cultured at 28°C until OD 600 The concentration of the culture medium was 0.7. After being placed on ice for 1 h, the mixture was centrifuged at 4 °C and 5000 rpm for 10 min. A 10% precooled 4 °C sterile glycerol solution was added to suspend the collected bacteria. The centrifugation and suspension operation was repeated twice at 4 °C and 5000 rpm. The bacteria finally suspended with a 10% precooled 4 °C sterile glycerol solution were divided into 1.5 mL sterile centrifuge tubes, 100 μL per tube, and quickly frozen in liquid nitrogen and stored at -80 °C for later use.

[0093] 5) The recombinant strain obtained in step 4) was placed in a 10 w / v% sucrose culture dish and cultured at 28 °C for 96 h. The resulting colonies were tested by PCR using primers pK18-rpfF-D5 and pK18-rpfF-D6 (as shown in Table 1). The PCR product of 1336 bp was sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing. After sequencing was confirmed, the DSF synthesis gene was knocked out. rpfF of Xcc strain, i.e., strain Δ that does not produce DSF rpfF .

[0094] The recombinant reporter bacterium Δ rpfF / pBBR-P egl The construction method of +GusA is as follows: 2.5 ng of recombinant vector pBBR-P egl +GusA was added to 100 μL of the non-DSF-producing strain Δ rpfF The competent strain was transformed by electroporation at 2.1 kV for 5.4 ms, then revived at 28 °C for 2 h. The revived bacterial solution was spread on LB solid medium containing 50 μg / mL Gm. After culturing at 28 °C for 72 h, the grown transformants were selected and verified by PCR with primers pBBR-F and pBBR-R. The transformants with the correct PCR amplification band size were selected as the recombinant reporter strain Δ rpfF / pBBR-P egl +GusA;

[0095] The non-DSF producing strain ΔrpfF The method for preparing the competent strain is as follows: the non-DSF-producing strain Δ rpfF Inoculate in LB liquid medium and culture at 28°C until OD 600 The alginate was placed on ice for 1 h, and then centrifuged at 4°C and 5000 rpm for 10 min. A 4°C sterile glycerol solution was added to 10% of the total volume to suspend the collected bacteria. The centrifugation and suspension operations were repeated twice at 4°C and 5000 rpm. The bacteria finally suspended with a 4°C sterile glycerol solution accounting for 10% of the total volume were divided into 1.5 mL sterile centrifuge tubes, 100 μL per tube, and quickly frozen in liquid nitrogen and stored at -80°C for later use.

[0096] Application Example 3 of a Recombinant Reporter Bacteria

[0097] The recombinant reporter bacteria Δ rpfF / pBBR-P egl +GusA is used to detect the strains i, ii, and iii to be tested (wherein the strains i, ii, and iii to be tested are Xcc8004, Xcc, Δ rpfF ) Whether to generate DSF;

[0098] The detection method comprises the following steps:

[0099] (1) The recombinant reporter strain Δ rpfF / pBBR-P egl +GusA was inoculated into LB liquid medium and cultured at 28°C and 180 rpm until OD 600 is 1.5, and the recombinant reporter strain Δ rpfF / pBBR-P egl +GusA culture solution;

[0100] The strains to be tested, ⅰ, ⅱ, and ⅲ, were inoculated into LB liquid medium and cultured at 28°C and 180 rpm until OD 600 is 2.0, centrifuge at 5000 rpm for 5 min, remove the supernatant after centrifugation, add an equal volume of sterile water to resuspend, and obtain the culture solutions of the tested strains i, ii, and iii respectively;

[0101] (2) Add 1 mL of the recombinant reporter strain Δ rpfF / pBBR-P egl+ GusA culture solution was mixed with 750 μL of 20 mg / mL color substrate X-Gluc solution and added into 100 mL of sterile NYG solid medium at 42°C and 1.5 w / v% agar concentration to obtain DSF detection solid medium;

[0102] (3) Inoculate 10 μL of the culture solution of the test strains i, ii, and iii obtained in step (1) directly onto the DSF solid medium obtained in step (2), and culture at 28 °C for 48 h to observe whether a blue circle appears around the colony:

[0103] If a blue circle appears around the colony, the strain to be tested produces DSF;

[0104] If no blue ring appears around the colony, the strain to be tested does not produce DSF.

[0105] like Figure 1 As shown, the culture broth of the test strains i, ii, and iii was in the presence of the recombinant reporter strain Δ rpfF / pBBR-P egl +GusA DSF detection solid medium, after constant temperature culture for 48 h, the test strain I can be observed Xcc 8004 and test strain II Xcc All of them produced obvious blue circles, but the strains to be tested that did not synthesize DSF ⅲ Δ rpfF No obvious blue circle is produced, indicating that the recombinant reporter bacteria of the present invention can quickly, sensitively and accurately detect whether different types of test strains produce DSF.

[0106] Application Example 4 of a Recombinant Reporter Bacteria

[0107] The difference between this embodiment and embodiment 3 is that the recombinant reporter strain Δ rpfF / pBBR-P egl +GusA is used to detect the strains i, ii, and iii to be tested (wherein the strains i, ii, and iii to be tested are Δ rpfC, Xcc, Δ rpfF ) Whether to generate DSF;

[0108] The detection method is the same as in Example 3.

[0109] The Δ rpfC(DSF high-yielding strain, Slater H, Alvarez-morales A, Barber CE, et al. A two-component system involving an HD-GYP domain protein links cell-cellsignalling to pathogenicity gene expression in Xanthomonas campestris [J].Molecular Microbiology, 2000,38(5): 986-1003.) The suicide knockout vector pK18mobsacB was used to perform scarless knockout using homologous recombination. Xcc In strains rpfC and become;

[0110] The knockout method comprises the following steps:

[0111] 1) Xcc The genome of the strain was used as a template, and primers pK18-rpfC-D1 and pK18-rpfC-D2 (as shown in Table 1) were used to amplify the upstream fragment of the target knockout gene, and primers pK18-rpfC-D3 and pK18-rpfC-D4 (as shown in Table 1) were used to amplify the downstream fragment of the target knockout gene;

[0112] 2) The same as step 2 of the knockout method in Example 2);

[0113] 3) The same as step 3 of the knockout method in Example 2) to obtain plasmid pK18-rpfC;

[0114] 4) Add 2.5 μL of the plasmid pK18-rpfC obtained in step 3) to 100 μL Xcc In the competent strain of the strain, the rest is the same as step 4 of the knockout method in Example 2);

[0115] 5) The recombinant strain obtained in step 4) was placed in a 10 w / v% sucrose culture dish and cultured at 28 °C for 96 h. The resulting colonies were tested by PCR using primers pK18-rpfC-D5 and pK18-rpfC-D6 (as shown in Table 1). The PCR product of 1323 bp was sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing. After sequencing was confirmed, Δ rpfC .

[0116] like Figure 2 As shown, the culture broth of the test strains i, ii, and iii was in the presence of the recombinant reporter strain Δ rpfF / pBBR-P egl+GusA DSF detection solid medium, after constant temperature culture for 48 h, the strain ⅰ Δ rpfC and strain II Xcc Both produce obvious blue circles, and Δ rpfC The blue circle area of ​​the strain is significantly higher than that of the wild type Xcc ; Δ rpfF The strain did not form an obvious blue circle; this indicates that the recombinant reporter strain Δ rpfF / pBBR-P egl +GusA can not only quickly, sensitively and accurately detect whether different types of test strains produce DSF, but also the blue circles produced are proportional to the DSF production.

[0117]

[0118] Comparative Example 1

[0119] The difference between this comparative example and Example 3 is that the reported reporter strain FE58 (Wang LH, He Y, Gao Y, et al. A bacterial cell-cell communication signal with cross-kingdom structural analogues [J]. Molecular Microbiology, 2004, 51 (3): 903-912.) is used to detect the test strains i, ii, and iii (wherein the test strains i, ii, and iii are Xcc8004, Xcc, Δ rpfF ) Whether to generate DSF;

[0120] In step (3) of the detection method, constant temperature culture is performed for 48 h; the rest is the same as in Example 3.

[0121] like Figure 3 As shown in the figure, the culture liquid of the test strains i, ii, and iii was cultured on the DSF detection solid medium containing the reported reporter strain FE58 for 48 h at constant temperature. Xcc The 8004 strain produced a smaller blue circle. Figure 1 By comparison, the recombinant reporter strain Δ rpfF / pBBR-P egl +GusA is more sensitive and accurate in detecting DSF.

Claims

1. A recombinant reporter bacterium, characterized in that: The reporter gene and the DSF inducible promoter are introduced into a DSF-non-producing strain in the form of a recombinant vector; the nucleotide sequence of the DSF inducible promoter is shown in SEQ ID NO: 1; the DSF-non-producing strain is a mutant strain lacking the DSF synthesis gene in the strain encoding the DSF quorum sensing system; the strain encoding the DSF quorum sensing system includes Xanthomonas pv. citri Xanthomonas citri subsp. citri Xanthomonas campestris pv.campestris Xanthomonas campestris pv. campestris .

2. The recombinant reporter bacteria according to claim 1, characterized in that: The reporter gene is a gene encoding a detectable protein or enzyme; the enzyme gene includes a β-glucosidase encoding gene or a β-galactosidase encoding gene; the β-glucosidase encoding gene is gusA The gene has a nucleotide sequence as shown in SEQ ID NO: 2; the recombinant vector is pBBR-DSF inducible promoter + reporter gene; the recombinant vector pBBR-DSF inducible promoter + reporter gene is formed by fusion of DSF inducible promoter and reporter gene to pBBRMCS-5 vector; the DSF synthetic gene includes f The DSF-non-producing strain is obtained by selecting a suicide knockout vector and using homologous recombination to seamlessly knock out the DSF synthesis gene in the strain encoding the DSF quorum sensing system.

3. A use of the recombinant reporter bacteria as claimed in claim 1 or 2, characterized in that: The recombinant reporter bacteria according to claim 1 or 2 are used to detect whether the strain to be tested produces DSF or whether the sample to be tested contains DSF.

4. The use of the recombinant reporter bacteria according to claim 3, characterized in that: The detection method comprises the following steps: (1) inoculating the recombinant reporter bacteria of claim 1 or 2 into LB liquid culture medium, culturing to obtain a culture solution of the recombinant reporter bacteria; inoculating the strain to be tested into LB liquid culture medium, culturing, centrifuging, and resuspending to obtain a culture solution of the strain to be tested; (2) mixing the culture medium of the recombinant reporter bacteria obtained in step (1) with the chromogenic substrate and adding them to a sterile NYG solid culture medium to obtain a DSF detection solid culture medium; (3) Directly inoculate the culture solution of the strain to be tested obtained in step (1) or directly add the sample to be tested to the DSF detection solid culture medium obtained in step (2), culture at a constant temperature, and observe whether a blue circle appears around the colony: If a blue circle appears around the colony or sample, the strain to be tested produces DSF or the sample to be tested contains DSF; If no blue circle appears around the colony or sample, the strain to be tested does not produce DSF or the sample to be tested does not contain DSF.

5. The use of the recombinant reporter bacteria according to claim 4, characterized in that: In step (1), the recombinant reporter bacteria are cultured at 26-30°C and 160-200 rpm until OD 600 The conditions for culturing the strain to be tested are: 26-30 °C, 160-200 rpm, and culturing until OD 600 The centrifugal speed is 4000-6000 rpm and the time is 4-6 min. The supernatant after centrifugation is removed and an equal volume of sterile water is added for resuspending. In step (2), the volume ratio of the recombinant reporter bacteria culture solution to the chromogenic substrate is 1:0.5-1.

0. The volume ratio of the recombinant reporter bacteria culture solution to the sterile NYG solid culture medium is 1:80-120. The concentration of the chromogenic substrate is 15-25 mg / mL. The chromogenic substrate is 5-bromo-4-chloro-3-indole-β-D-glucuronic acid cyclohexylamine salt solution. The temperature of the sterile NYG solid culture medium is 40-44 °C and the concentration of agar is 1.2-1.8 w / v. In step (3), the amount of direct inoculation or addition is 10-15 μL. The temperature of the constant temperature culture is 26-30 °C and the time is 18-50 h.

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