SRNA ssrS influencing Listeria monocytogenes quorum sensing system and application

Abstract

The invention belongs to the technical field of molecular biology and bioengineering, and particularly relates to sRNA ssrS for influencing a Listeria monocytogenes quorum sensing system and application of the sRNA ssrS. The invention provides a construction method of two listeria monocytogenes expression plasmids of promoter fusion eGFP related to quorum sensing. The listeria monocytogenes expression plasmids can be directly transformed and applied to research of the regulation effect of sRNA on a listeria monocytogenes quorum sensing system. The sRNA ssrS disclosed by the invention can be used as an important target spot for controlling the formation of a listeria monocytogenes biofilm in the future.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and bioengineering, and specifically relates to exploring an sRNA that affects the quorum sensing system of Listeria monocytogenes and its application. Background Art

[0002] Listeria monocytogenes (L.monocytogenes) is widely distributed in soil, water, food and the environment, among which meat products, dairy products and aquatic products have the highest degree of contamination. Listeria monocytogenes (L.monocytogenes) has a strong survival ability and can grow at 0-45℃, and can even survive repeated freezing and thawing at sub-zero temperatures. It has a strong resistance to adversity and can tolerate adverse conditions such as acid and alkali, low temperature, dryness, and hypertonicity. Listeria monocytogenes is one of the pathogenic microorganisms that cannot be detected in the national standard GB29921-2013 "Limits of Pathogenic Bacteria in Food". Listeria monocytogenes has a strong ability to infect humans. Once infected, it will cause a 90% hospitalization rate and a mortality rate of more than 20%, and the neonatal mortality rate reaches more than 50%. Listeria monocytogenes enters the host intestine through infected food, and under the action of internalization (InlA and InlB), it recognizes the receptor cells on the small intestine, and then is wrapped by the cell membrane to form a phagocytic vesicle. The phagocytic vesicle combines with the lysosome to rapidly acidify the internal environment of the phagocytic vesicle and promote the degradation of the bacteria. More than 90% of the bacteria will be acidified and degraded by the enzymes in the phagocytic vesicle. The surviving Listeria monocytogenes lyses the phagosome membrane under the action of hemolysin (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC) to escape the phagocytic vesicle. The escaped Listeria monocytogenes absorbs nutrients from the host cells for proliferation. Actin polymerization protein (ActA) aggregates the actin of the host cells to form a comet tail, thereby moving. This provides conditions for the spread of Listeria monocytogenes between cells. Under this action, Listeria monocytogenes passes from one host cell to another, forming a secondary phagocytic vesicle with a double-layer membrane structure. Under the action of LLO and broad-spectrum phospholipase (PC-PLC), the phagocytic vesicle membrane loses stability, allowing Listeria monocytogenes to quickly enter the cytoplasm for growth and reproduction. It is precisely because of the characteristics of intracellular parasitism and intercellular transmission of Listeria monocytogenes that it can be used as a vaccine vector in clinical trials after attenuation. It is not clear whether there are other regulatory factors related to virulence in Listeria monocytogenes, and the pathogenic mechanism needs to be further explored to achieve effective prevention and control of Listeria monocytogenes and its precise application.

[0003] As a type of regulatory element, sRNA precisely regulates Listeria monocytogenes-related genes at the post-transcriptional level, and has a wide range of regulatory effects on physiological activities such as virulence, stress resistance, and biofilm. The complex regulatory network of sRNA plays an important role in different stages of biofilm development, guiding free planktonic cells to switch to biofilm mode. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention clarifies that a sRNA ssrS can be used as a biofilm regulation target, affecting the formation of Listeria monocytogenes biofilm by interfering with the quorum sensing system. After the sRNA ssrS gene is deleted, the sensitivity of Listeria monocytogenes to disinfectants can be enhanced, and the sRNA can be used as a new target to deal with the threat of Listeria monocytogenes.

[0005] The object of the present invention is achieved by the following means:

[0006] In a first aspect, the present invention protects a Listeria monocytogenes strain sRNA ssrS, the nucleotide sequence of the sRNA ssrS being shown as SEQ ID NO.1.

[0007] In a second aspect, the present invention protects the use of the sRNA ssrS described above in reducing the promoter activity of key genes for quorum sensing of Listeria monocytogenes.

[0008] In a third aspect, the present invention protects the use of the sRNA ssrS described above in the preparation of a product for reducing the promoter activity of a key gene for quorum sensing of Listeria monocytogenes.

[0009] In a fourth aspect, the present invention protects the use of the sRNA ssrS described above in reducing the formation of Listeria monocytogenes capsule.

[0010] In a fifth aspect, the present invention protects the use of the sRNA ssrS described above in the preparation of a product for reducing the formation of Listeria monocytogenes capsule.

[0011] In a specific embodiment, the product for reducing the formation of Listeria monocytogenes capsule may be an inhibitor of inhibiting Listeria monocytogenes capsule.

[0012] In a specific embodiment, the Listeria monocytogenes is Listeria monocytogenes LMB33426. In a sixth aspect, the present invention provides a method for constructing a gene-deficient strain of Listeria monocytogenes, the method comprising the following steps:

[0013] (1) Designing primer pairs ssrS-UF, ssrS-UR and ssrS-DF, ssrS-DR, using genomic DNA of Listeria monocytogenes LMB 33426 as template, respectively amplifying the upstream gene fragment and the downstream gene fragment; overlapping extension of ssrS-UF and ssrS-DR was used to fuse the upstream and downstream fragments;

[0014] (2) Design primer pairs pKSV7-F and pKSV7-R and use pKSV7 plasmid DNA as a template to PCR amplify the linearized pKSV7 vector;

[0015] (3) using a recombinase, the fusion fragments in step (1) and step (2) are respectively connected to the linear pKSV7 linear vector to obtain a recombinant integration plasmid pKSV7-ssrS;

[0016] (4) The recombinant plasmid pKSV7-ssrS was transferred into competent Escherichia coli cells to amplify the plasmid; the plasmid was extracted using a plasmid extraction kit and transferred into competent Listeria monocytogenes cells; the homologous fragment on the recombinant plasmid pKSV7-ssrS was replaced with the genome of Listeria monocytogenes through homologous recombination, and the sRNA gene-deficient strain LM-ΔssrS was obtained through resistance screening.

[0017] In a specific embodiment, the primer sequences in step (1) and step (2) are as follows:

[0018] ssrS-UF: as shown in SEQ ID NO.2; ssrS-UR: as shown in SEQ ID NO.3;

[0019] ssrS-DF: as shown in SEQ ID NO.4; ssrS-DR: as shown in SEQ ID NO.5;

[0020] pKSV7-F: as shown in SEQ ID NO.6; pKSV7-R: as shown in SEQ ID NO.7.

[0021] In a seventh aspect, the present invention protects a recombinant plasmid fused with a key gene promoter of a quorum sensing system and eGFP, wherein the recombinant plasmid contains a key gene promoter of Listeria monocytogenes quorum sensing and a green fluorescent protein eGFP. More specifically, the promoter is a P2 promoter or a P luxS Promoter.

[0022] The recombinant plasmid is the recombinant plasmid pEL-P2 and / or the recombinant plasmid pEL-P luxS .

[0023] The recombinant plasmid constructed by the present invention is used as a fluorescent reporter plasmid, which can produce green fluorescent protein in Listeria monocytogenes and has a high fluorescence intensity under the action of a promoter.

[0024] In an eighth aspect, the present invention provides a method for constructing a recombinant plasmid of the aforementioned key gene motor of the quorum sensing system fused with eGFP, the method comprising the following steps:

[0025] (1) Design primer pairs pKSV7-F and pKSV7-R, and use pKSV7 plasmid DNA as a template to PCR amplify the linearized pKSV7 vector;

[0026] (2) Design primer pairs eGFP-F and eGFP-R, use pET28a plasmid as template, amplify by PCR, and purify the PCR product using FastPure Gel DNA Extraction Mini Kit (Vazyme) to obtain the eGFP gene fragment;

[0027] (3) Design primer pairs P2-F, P2-R and P luxS -F, P luxS -R The promoter gene fragment was obtained by PCR amplification using the genome of Listeria monocytogenes LMB 33426 as a template;

[0028] (4) The two fragments in step (2) and step (3) were fused by overlapping extension PCR, and the primers used were P2-F and eGFP-R and P luxS -F and eGFP-R, to obtain the promoter of the key gene of the quorum sensing system fused with eGFP fragment;

[0029] (5) Use a recombinase to connect the fusion fragment in step (4) and the linear vector in step (1) to obtain recombinant plasmids pEL-P2 and pEL-P luxS ;

[0030] (6) Recombinant plasmids pEL-P2 and pEL-P luxS Transfer into competent E. coli cells, amplify and extract the plasmid, and then screen.

[0031] In a specific embodiment, the primer pair sequences used in the above construction method are as follows:

[0032] pKSV7-F: as shown in SEQ ID NO.6; pKSV7-R: as shown in SEQ ID NO.7;

[0033] eGFP-F: as shown in SEQ ID NO.8; eGFP-R: as shown in SEQ ID NO.9;

[0034] P2-F: as shown in SEQ ID NO.10; P2-R: as shown in SEQ ID NO.11;

[0035] P luxS -F: as shown in SEQ ID NO.12 luxS -R: as shown in SEQ ID NO.13.

[0036] In a ninth aspect, the present invention also protects the use of the recombinant plasmid described above in exploring sRNA-regulated quorum sensing systems.

[0037] In a tenth aspect, the present invention protects a method for exploring sRNA-regulated quorum sensing systems, which is achieved by introducing the recombinant plasmid described above into a wild-type and / or sRNA gene-deficient strain of Listeria monocytogenes, and then combining it with fluorescence measurement.

[0038] The present invention measures the biofilm-forming ability of the sRNA ssrS gene-deficient strain obtained above, and transfers the obtained recombinant plasmid into the wild-type Listeria monocytogenes and the sRNA ssrS gene-deficient strain, measures the fluorescence intensity in Listeria monocytogenes, measures the effect on the activity of the promoter of the key gene of quorum sensing, and evaluates the potential of sRNA ssrS as a target for controlling the biofilm formation of Listeria monocytogenes.

[0039] Beneficial Effects

[0040] The present invention describes the secondary structure of sRNA ssrS. By constructing a quorum sensing system fluorescent reporter plasmid, it is found that sRNA ssrS can affect the key genes in quorum sensing by incompletely complementary pairing with the promoter region of the key gene in quorum sensing, thereby affecting the formation and adhesion of biofilm.

[0041] Studies have shown that sRNA ssrS can be used as a target for controlling Listeria monocytogenes biofilm, thereby reducing the threat of Listeria monocytogenes and its biofilm, and laying a theoretical foundation for the development of new inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 PCR amplification and transformation verification of plasmid, promoter fragment and eGFP fragment. (Note: M is DNA Marker. Figure 1 A, Plasmid linear amplification, the theoretical size of the fragment is 7071 bp. Figure 1 B, eGFP fragment amplification, the theoretical size of the fragment is 720 bp. Figure 1 C, P2 promoter amplification, the theoretical size of the fragment is 264 bp. Figure 1 D, P2 promoter fused with eGFP amplification, the theoretical size of the fragment is 984 bp. Figure 1E, P luxS Promoter amplification, the theoretical size of the fragment is 250bp. Figure 1 F, P luxS Promoter fusion eGFP amplification, the theoretical size of the fragment is 953bp. )

[0043] Figure 2 Observation of Listeria monocytogenes under a fluorescence microscope after plasmid transformation. (The green signal indicates that the eGFP protein is successfully expressed in Listeria monocytogenes. Figure 2 A, Listeria monocytogenes control group (transformed with original plasmid). Figure 2 B, Listeria monocytogenes wild type transformed into pEL-P luxS Plasmid. Figure 2 C, Listeria monocytogenes transformed with pEL-P2 plasmid. Figure 2 D, Listeria monocytogenes LM-ΔssrS was transformed into pEL-P luxS Plasmid. Figure 2 E, Listeria monocytogenes LM-ΔssrS was transformed into pEL-P2 plasmid.

[0044] Figure 3 The fluorescence intensity was measured after the promoter of the key gene of the quorum sensing system was fused with the eGFP plasmid and transformed into the wild type of Listeria monocytogenes and LM-ΔssrS. (Control, negative control, wild type of Listeria monocytogenes and LM-ΔssrS (no plasmid transformation). LM-pEL, Listeria monocytogenes was transformed with the promoter-free pEL plasmid. pEL-Pgene, represents the plasmid constructed by fusion of different gene promoters and eGFP fragments.)

[0045] Figure 4 Prediction of ssrS secondary structure and target sites. Figure 4 A, ssrS secondary structure prediction, different colors represent different RNA structure types; green represents stem, red represents multi-branched loops, yellow represents internal loops, blue represents hairpin loops, and orange represents 5' / 3' unpaired regions. Figure 4 B, ssrS pre-P luxS Three possible interaction sites were predicted. Figure 4 C, ssrS pre-P2 predicted five possible interaction sites.

[0046] Figure 5 Determination of the biofilm formation ability of wild type Listeria monocytogenes and LM-ΔssrS and their adhesion ability on food surfaces. Figure 5 A, Determination of biofilm formation ability of Listeria monocytogenes. Figure 5B, Determination of the adhesion ability of wild-type Listeria monocytogenes and LM-ΔssrS on the surface of lettuce. Water means washing with sterile water, and benzalkonium bromide means treatment with benzalkonium bromide followed by washing with sterile water. DETAILED DESCRIPTION

[0047] The present invention is further described below in conjunction with the examples. The implementation methods for which specific conditions are not specified in the following examples are generally carried out according to the well-known means in the art, or according to the conditions established by the manufacturer. The strains involved in the examples are all prior art and can be easily obtained from public commercial channels by those skilled in the art.

[0048] Example 1 Construction of sRNA ssrS gene deletion strain

[0049] (1) Primer pairs ssrS-UF, ssrS-UR (as shown in SEQ ID NO. 2-3) and ssrS-DF, ssrS-DR (as shown in SEQ ID NO. 4-5) were designed, and the genomic DNA of Listeria monocytogenes LMB 33426 was used as a template to amplify the upstream gene fragment and the downstream gene fragment respectively; ssrS-UF and ssrS-DR were overlapped and extended to fuse the upstream and downstream fragments.

[0050] (2) designing primer pairs pKSV7-F (as shown in SEQ ID NO.6) and pKSV7-R (as shown in SEQ ID NO.7), and using pKSV7 plasmid DNA as a template, PCR amplifying the linearized pKSV7 vector;

[0051] (3) using a recombinase to connect the fusion fragment in step (1) and the linearized pKSV7 vector in step (2), respectively, to obtain a recombinant plasmid pKSV7-ssrS;

[0052] (4) Take 10 μL of the recombinant plasmid pKSV7-ssrS and add it to 100 μL of competent cells. Place it on ice for 30 minutes, then place it in a water bath at 42°C for 90 seconds, and then place it on ice for 5 minutes. Add 500 μL of LB medium and culture it at 37°C and 180 rpm for 1 hour. Spread the bacterial solution on LB solid medium (containing 100 μg / mL ampicillin) and pick colonies for verification. After the verified colonies are expanded, use a plasmid extraction kit to extract the plasmid. Transform the plasmid into Listeria monocytogenes competent cells by electroporation. Take 10 μL of the recombinant plasmid and add it to 100 μL of Listeria monocytogenes competent cells thawed on ice. Mix gently and transfer it to a pre-cooled electroporation cup and place it on ice for 10 minutes. Place the electroporation cup on the electroporator and perform electroporation at 2.5 kV. After the electroporation, immediately add 1 mL of BHI medium and let it stand at 30°C for 2 hours. The bacterial liquid was spread on BHI (containing 50 μg / mL chloramphenicol) solid medium, cultured at 30°C for 2 days, and positive clones were picked for verification. The successfully transformed positive colonies were cultured in BHI (containing 50 μg / mL chloramphenicol) liquid medium at 42°C, 180rpm for 12h, subcultured 5-10 times, and homologous recombination was induced under the double pressure of temperature and antibiotics. Cultured at 30°C, 180rpm for 12h, subcultured 5-10 times, and the plasmid was discarded. Colony PCR verification obtained the sRNA gene deletion strain LM-ΔssrS.

[0053] The PCR cycle conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 57°C for 30 s, extension at 72°C (1 kb / min), for 30 cycles; and final extension at 72°C for 5 min.

[0054] PCR reaction system: template 2 μL, F / R primer 2 μL each, 2×Phanta Max Master Mix (Vazyme) 25 μL, ddH2O 19 μL, total volume 50 μL. PCR products were purified using FastPure Gel DNA Extraction Mini Kit (Vazyme).

[0055] Example 2 Construction, transformation and fluorescence observation of promoter-fused eGFP plasmid

[0056] Primer pairs pKSV7-F (as shown in SEQ ID NO.6) and pKSV7-R (as shown in SEQ ID NO.7) were designed, and the plasmid was linearized by inverse PCR using pKSV7 plasmid DNA as a template. Primer pairs eGFP-F (as shown in SEQ ID NO.8) and eGFP-R (as shown in SEQ ID NO.9) were designed, and the eGFP fragment was amplified by PCR using pET28a plasmid as a template. Primer pairs P2-F, P2-R (as shown in SEQ ID NO.10-11) and P luxS -F, P luxS -R (as shown in SEQ ID NO.12-13), using Listeria monocytogenes LMB33426 as a template to amplify P2 and P luxS Promoter.

[0057] PCR cycle conditions: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 57℃ annealing for 30s, 72℃ extension (1kb / min), 30 cycles; 72℃ final extension for 5min. Reaction system: 2μL template, 2μL each of F / R primers, 25μL 2×Phanta MaxMaster Mix (Vazyme), 19μL ddH2O, total volume 50μL. PCR products were purified using FastPure Gel DNA Extraction Mini Kit (Vazyme).

[0058] Use CloneExpress II One Step Cloning Kit (Vazyme) to connect P2 and P luxS The promoter fused the eGFP fragment with the linearized vector. The ligation product was transferred into JM109 competent cells by chemical transformation. Take 10 μL of the ligation product and add it to 100 μL of competent cells, place it on ice for 30 minutes, then place it in a 42°C water bath for 90 seconds, and then place it on ice for 5 minutes. Add 500 μL LB medium and culture it at 37°C and 180 rpm for 1 hour. Spread the bacterial solution on LB solid medium (containing 100 μg / mL ampicillin) and pick colonies for verification. After verifying the correct colony expansion, use FastPure Plasmid Mini Kit (Vazyme) to extract the recombinant plasmid.

[0059] The extracted plasmid was transformed into Listeria monocytogenes competent cells according to the electroporation method. Take 10μL of the recombinant plasmid and add it to 100μL of Listeria monocytogenes competent cells thawed on ice, mix gently and transfer to a pre-cooled electroporation cup, and place on ice for 10 minutes. Place the electroporation cup on the electroporator and perform electroporation at 2.5kV. After the electroporation, immediately add 1mL of BHI medium and let it stand at 30℃ for 2h. Spread the bacterial solution on BHI (containing 50μg / mL chloramphenicol) solid medium, culture at 30℃ for 2 days, and pick positive clones for verification. Inoculate a single colony of Listeria monocytogenes into the plasmid in BHI medium and culture overnight at 37℃. Take 10μL of the bacterial solution, evenly spread it on a glass slide, cover it with a coverslip after air drying, and observe and take pictures with an upright fluorescence microscope.

[0060] The results showed that the plasmid template was successfully linearized ( Figure 1 A), eGFP fragment was successfully amplified ( Figure 1 B), P2 promoter ( Figure 1 C) and P luxS Promoter ( Figure 1 E), and then fused and ligated to successfully transform into Listeria monocytogenes ( Figure 1 D and 1F). The pEL-P2 plasmid and pEL-P luxS Listeria monocytogenes wild type ( Figure 2 B and 2C) and LM-ΔssrS ( Figure 2 D and 2E) were photographed under an upright fluorescence microscope, and green fluorescence could be clearly observed, which could track Listeria monocytogenes. The control group was Listeria monocytogenes transformed with the original plasmid, and the fluorescence phenomenon could not be observed under dark field ( Figure 2 A) The results show that the pEL-P2 plasmid and pEL-P constructed in this study luxS The plasmid was able to successfully express eGFP in Listeria monocytogenes.

[0061] Example 3 sRNA ssrS gene deletion affects the promoter activity of key quorum sensing genes

[0062] The successfully transformed pEL-P2 and pEL-P obtained in Example 2 were luxS The wild type and LM-ΔssrS of Listeria monocytogenes were inoculated into BHI medium and cultured at 37°C overnight. 1 mL of bacterial solution was taken, centrifuged at 12000 rpm for 1 min, washed with sterile PBS, centrifuged again at 12000 rpm for 1 min, and the supernatant was discarded. The cells were resuspended with sterile PBS, OD600 was adjusted to 0.9, and the fluorescence intensity was measured using a fluorescence microplate reader, Em / Ex = 535 / 465 nm.

[0063] Figure 3The results showed that there was no difference in fluorescence intensity between LM-pEL (promoterless eGFP plasmid) and the control group (transferred into pKSV7 empty vector), indicating that the empty vector and the eGFP vector without promoter could not successfully express eGFP in Listeria monocytogenes. After connecting different promoters, the fluorescence intensity was significantly enhanced. Among them, the P2 promoter, as a strong promoter, fused with the eGFP recombinant plasmid had the highest fluorescence intensity in Listeria monocytogenes. Both the P2 promoter and the luxS promoter had significantly higher activity in the wild type of Listeria monocytogenes than in LM-ΔssrS, indicating that sRNA ssrS can affect the promoter activity of key genes in the quorum sensing system of Listeria monocytogenes.

[0064] Example 4 sRNA ssrS secondary structure and binding site prediction

[0065] The RNAfold server predicted the secondary structure of sRNA ssrS based on the minimum free energy (MFE) algorithm. The IntaRNA server predicted the secondary structure of ssrS with P2 and P luxS Possible interaction sites. According to the default parameter settings, the specific parameters are as follows: the number of interactions per RNA pair is 5, overlap is allowed in the query, no single base pair, and no GU at the end of the helix.

[0066] Figure 4 A shows that the secondary structure of sRNA ssrS contains multiple stem-loop structures. These stem-loop structures are usually the sites where sRNA interacts with target mRNA and play an important role in studying the function of sRNA. Figure 4 B and 4C show the sRNA ssrS and P predicted by IntaRNA server, respectively. luxS The possible binding site between ssrS and P2. ssrS may regulate the activities of the two promoters through incomplete base pairing.

[0067] Example 5 sRNA ssrS affects the formation of biofilm by affecting quorum sensing and its application

[0068] Pick single colonies of Listeria monocytogenes wild type and ssrS gene deletion strain and place them in LB liquid culture medium, culture overnight at 37°C, 180rpm, and adjust OD600 to 0.5±0.02. Add 180μL of sterile LB liquid culture medium to a 96-well plate and inoculate the seed solution at a 1% inoculation rate. After culture at 37°C, discard the bacterial solution, wash three times with 200μL of sterile PBS, and air-dry in the air. Add 200μL of 1% crystal violet to stain for 30 minutes, discard the staining solution, wash three times with 200μL of sterile PBS, and air-dry naturally. Add 200μL of 95% ethanol and incubate at room temperature for 5 minutes, and measure OD with an enzyme reader.595 .

[0069] Fresh lettuce was purchased from Nanjing Market and cut into 4 cm × 4 cm pieces. The lettuce was washed with sterile water and exposed to ultraviolet light for 15 min on each side. The cultured wild-type Listeria monocytogenes and LM-ΔssrS bacterial suspensions were adjusted to OD 600 0.5±0.02, add 100μL of bacterial suspension on the surface of lettuce. Then incubate at 37℃ for 36h. Rinse three times with sterile water. When treated with benzalkonium bromide, soak the lettuce in 0.1% benzalkonium bromide solution for 5 minutes, then wash three times with sterile water. Homogenize the washed lettuce, dilute it continuously, and spread it on the plate. Incubate at 37℃ for 24h to count the microbial colonies.

[0070] The results showed that the biofilm of Listeria monocytogenes showed a trend of first increasing and then decreasing. The biofilm formation amount of LM-ΔssrS was significantly lower than that of wild-type Listeria monocytogenes at all stages. Figure 5 A), the results show that ssrS can affect key genes by binding to the promoter region of key genes in the quorum sensing system, thereby affecting the formation of biofilms. Wild-type Listeria monocytogenes and LM-ΔssrS were added to fresh lettuce, and after the formation of biofilms, they were washed with sterile water, or treated with the disinfectant benzalkonium bromide and then washed with sterile water. The results showed that the number of bacteria on the surface of lettuce washed with sterile water or treated with benzalkonium bromide and then washed with sterile water was significantly lower than that of wild-type Listeria monocytogenes ( Figure 5 B). The results show that sRNA ssrS has an important influence on the adhesion ability of Listeria monocytogenes on food surfaces. sRNA ssrS can be used as a new target in the future to treat Listeria monocytogenes and reduce the harm of Listeria monocytogenes and its biofilm.

[0071] The above embodiments are merely exemplary implementations used to illustrate the principles of the invention, and the invention is not limited thereto. Those skilled in the art may make various improvements and changes without departing from the essence of the invention, and these improvements and changes also fall within the protection scope of the invention.

Claims

1. sRNA ssrS of Listeria monocytogenes strain, the nucleotide sequence of the sRNA ssrS is shown in SEQ ID NO.

1.

2. Use of the sRNA ssrS according to claim 1 in any one of the following (A1)-(A4): (A1) Application in reducing the promoter activity of key quorum sensing genes of Listeria monocytogenes; (A2) Use in the preparation of a product for reducing the promoter activity of a key gene for quorum sensing of Listeria monocytogenes; (A3) Application in reducing the film formation of Listeria monocytogenes; (A4) Use in the preparation of a product for reducing the film formation of Listeria monocytogenes.

3. The use according to claim 2, characterized in that: The Listeria monocytogenes is Listeria monocytogenes LMB33426.

4. A method for constructing a gene-deficient strain of Listeria monocytogenes, the method comprising the following steps: (1) Design primer pairs ssrS-UF / R and ssrS-DF / R, and use the genomic DNA of Listeria monocytogenes LMB 33426 as a template to amplify the upstream gene fragment and the downstream gene fragment, respectively; overlap extension of ssrS-UF and ssrS-DR was used to fuse the upstream and downstream fragments; (2) Design primer pairs pKSV7-F and pKSV7-R and use pKSV7 plasmid DNA as a template to PCR amplify the linearized pKSV7 vector; (3) using a recombinase, the fusion fragments in step (1) and step (2) are respectively connected to the linear pKSV7 linear vector to obtain a recombinant integration plasmid pKSV7-ssrS; (4) The recombinant plasmid pKSV7-ssrS was transferred into competent Escherichia coli cells to amplify the plasmid; the plasmid was extracted using a plasmid extraction kit and transferred into competent Listeria monocytogenes cells; the homologous fragment on the recombinant plasmid pKSV7-ssrS was replaced with the genome of Listeria monocytogenes through homologous recombination, and the sRNA gene-deficient strain LM-ΔssrS was obtained through resistance screening.

5. The method according to claim 4, characterized in that The primer sequences in step (1) and step (2) are as follows: ssrS-UF: as shown in SEQ ID NO.2; ssrS-UR: as shown in SEQ ID NO.3; ssrS-DF: as shown in SEQ ID NO.4; ssrS-DR: as shown in SEQ ID NO.5; pKSV7-F: as shown in SEQ ID NO.6; pKSV7-R: as shown in SEQ ID NO.

7.

6. A recombinant plasmid fused with a key gene promoter of a quorum sensing system and eGFP, wherein the recombinant plasmid contains a key gene promoter of a quorum sensing system of Listeria monocytogenes and a green fluorescent protein eGFP. More specifically, the promoter is a P2 promoter or a P luxS Promoter.

7. The method for constructing a recombinant plasmid according to claim 6, characterized in that: The method comprises the following steps: (1) Design primer pairs pKSV7-F and pKSV7-R, and use pKSV7 plasmid DNA as a template to PCR amplify the linearized pKSV7 vector; (2) Design primer pairs eGFP-F and eGFP-R, and obtain the eGFP gene fragment by PCR amplification using pET28a plasmid as template; (3) Design primer pairs P2-F / R and P luxS -F / R used the genome of Listeria monocytogenes LMB 33426 as a template and obtained the promoter gene fragment by PCR amplification; (4) The two fragments in step (2) and step (3) were fused by overlapping extension PCR, and the primers used were P2-F and eGFP-R and P luxS -F and eGFP-R, to obtain the promoter of the key gene of the quorum sensing system fused with eGFP fragment; (5) Use a recombinase to connect the fusion fragment in step (4) and the linear vector in step (1) to obtain recombinant plasmids pEL-P2 and pEL-P luxS ; (6) Recombinant plasmids pEL-P2 and pEL-P luxS Transfer into E. coli JM109 competent cells, amplify and extract the plasmid, and screen.

8. The method according to claim 7, characterized in that The primer pair sequences used in the construction method are as follows: pKSV7-F: as shown in SEQ ID NO.6; pKSV7-R: as shown in SEQ ID NO.7; eGFP-F: as shown in SEQ ID NO.8; eGFP-R: as shown in SEQ ID NO.9; P2-F: as shown in SEQ ID NO.10; P2-R: as shown in SEQ ID NO.11; P luxS -F: as shown in SEQ ID NO.12 luxS -R: as shown in SEQ ID NO.

13.

9. Use of the recombinant plasmid according to claim 6 in exploring sRNA-regulated quorum sensing systems.

10. A method for exploring sRNA-regulated quorum sensing system, comprising introducing the recombinant plasmid of claim 6 into wild-type Listeria monocytogenes and / or sRNA gene-deficient strains, and combining the method with fluorescence measurement.

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