Salmonella YeaZ gene deleted strain as well as construction method and application thereof

Abstract

The invention discloses a salmonella YeaZ gene deleted strain as well as a construction method and application thereof, and belongs to the technical field of gene engineering. The YeaZ gene deletion strain is constructed by utilizing a lambda-Red homologous recombination technology, and compared with a WT strain, the constructed salmonella YeaZ gene deletion strain can enter a VBNC state more easily and cannot revive in the VBNC state, and pathogenic bacteria in food can be detected by utilizing the characteristics of special biological phenotype and difficulty in reviving of the salmonella YeaZ gene deletion strain.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a Salmonella YeaZ gene deletion strain and a construction method and application thereof. Background Art

[0002] Under adverse environmental conditions, Salmonella enteritidis will lose its culturability and enter the VBNC state to cope with the adverse environmental impact, but still retain its virulence and pathogenicity. When the environmental conditions are suitable, Salmonella enteritidis in the VBNC state can be revived and restored to culturability. Conventional detection methods are difficult to detect pathogenic bacteria in the VBNC state, which is a huge hidden danger in food safety.

[0003] The YeaZ gene is widely present in Gram-positive and Gram-negative bacteria. This gene is a conserved multifunctional gene of Salmonella Enteritidis. The YeaZ gene is a homologous gene of the bacterial recovery promoting factor of Micrococcus luteus, which can restore the culturable activity of Salmonella in the viable but non-culturable state (VBNC) and can be used to detect VBNC pathogenic bacteria in food.

[0004] Chinese patent application CN117866999A discloses a method for reducing the formation of VBNC state of Salmonella, which provides a theoretical basis for the prevention and control of Salmonella in actual production by inhibiting the functional expression of ompA gene. It is of great significance to construct Enteritidis Salmonella with YeaZ gene deletion for food safety detection. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a Salmonella YeaZ gene-deficient strain and a construction method and application thereof.

[0006] The present invention adopts the following technical solutions.

[0007] A Salmonella YeaZ gene deletion strain, in which the YeaZ gene of Salmonella is knocked out, was deposited in the China Center for Type Culture Collection on January 15, 2025, with the preservation address in Wuhan, China, the preservation number is CCTCCNO: M2025159, and the classification name is Salmonella entericaSal3076ΔYeaZ::cat Salmonella entericaSal3076ΔYeaZ::cat.

[0008] A method for constructing a Salmonella YeaZ gene-deficient strain comprises the following steps: S1: Design primers H1-P1, H2-P2, H3 and H4, wherein the primers H1-P1 and H2-P2 contain 60 bp of YeaZ upstream and downstream homologous sequences, as well as the primer sequences on both sides of the chloramphenicol resistance gene (cat) of the plasmid pKD3 and the reverse transcriptase recognition site FRT; S2: Using plasmid pKD3 as template and primers H1-P1 and H2-P2, PCR amplified the chloramphenicol linear DNA fragment containing the FRT site; S3: Preparation of recombinant Salmonella Enteritidis containing pKD46 plasmid; S4: Inducible expression of recombinant Salmonella Enteritidis Red recombinase and electroporation of a 60 bp homologous targeting fragment containing the YeaZ gene; Inducing the expression of Red recombinase in the recombinant Salmonella Enteritidis, and then inserting a DNA fragment containing the homologous arm of the YeaZ gene into the recombinant Salmonella Enteritidis to obtain homologous recombinant Salmonella Enteritidis; S5: Eliminate plasmid pKD46 in homologous recombination Salmonella Enteritidis to obtain a Salmonella YeaZ gene deletion strain.

[0009] Furthermore, the H1-P1 and H2-P2 are knockout primers; H1 in the H1-P1 is the nucleotide sequence shown in SEQ ID NO.1, and P1 is the nucleotide sequence shown in SEQ ID NO.2; H1 in the H2-P2 is the nucleotide sequence shown in SEQ ID NO.3, and P2 is the nucleotide sequence shown in SEQ ID NO.4.

[0010] Furthermore, H3 and H4 are identification genes for identifying whether the YeaZ gene is recombined, and H3 is the nucleotide sequence shown in SEQ ID NO.5; and H4 is the nucleotide sequence shown in SEQ ID NO.6.

[0011] Furthermore, in step S3, a competent cell suspension is prepared using the target bacteria whose genes need to be knocked out, 1 to 2 μL of the pKD46 plasmid is added to 100 μL of the prepared competent cell suspension for electrotransformation, and then enzyme digestion is performed to obtain recombinant Salmonella Enteritidis containing the pKD46 plasmid.

[0012] Application of the Salmonella YeaZ gene-deficient strain prepared according to the method in food detection.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 1. The resuscitation promoting factor Rpf was first found in Micrococcus luteus and was confirmed to be able to resuscitate bacteria in the VBNC state. The isozyme YeaZ with the same function was also found in Salmonella Enteritidis, which has a certain homology with Rpf. In the present invention, in order to explore its function in Salmonella Enteritidis and its regulatory relationship with the VBNC state, the YeaZ gene was knocked out using λ-Red homologous recombination technology to obtain a Salmonella YeaZ gene-deficient strain.

[0014] 2. Knocking out the YeaZ gene using λ-Red homologous recombination technology will hinder the rod-shaped differentiation of Enteritidis Salmonella cells, making their surface smooth and reducing their toxicity. At the same time, the Salmonella YeaZ gene-deficient strain has a larger cell area and thinner cell wall than wild Enteritidis Salmonella. In addition, due to the lack of expression of the YeaZ gene, the Salmonella YeaZ gene-deficient strain enters the VBNC state earlier and more easily than wild Enteritidis Salmonella, providing a detection path for the subsequent detection of Enteritidis Salmonella in food.

[0015] 3. In the VBNC state, the YeaZ gene-deficient strain of Salmonella cannot be revived, further ensuring the safety of food preservation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the result of PCR amplification of the chloramphenicol-resistant fragment containing the YeaZ homology arm in step S2 of the present invention; (M: DNA molecular weight marker; 1: chloramphenicol-resistant fragment containing the YeaZ homology arm; 2: negative control) Figure 2 This is a diagram showing the results of plasmid PCR single enzyme digestion verification in step S3 of the present invention; Figure 3 This is a graph showing the growth of the recombinant Salmonella Enteritidis on an Amp-resistant plate in step S3 of the present invention; Figure 4 The homologous recombination process in step S4 of the present invention and the PCR verification result of Salmonella bacterial solution are shown in FIG. (a) is a schematic diagram of the homologous recombination process, and (b) is a diagram of the PCR verification result of Salmonella bacterial solution; (M: DNA molecular weight marker; 1: ΔYeaZ; 2: WT) Figure 5 The diagram shows the growth results of the Salmonella YeaZ gene-deficient strain in step S5 of the present invention when it is cultured on LB agar medium containing chloramphenicol and LB agar medium containing ampicillin; A is LB agar medium containing chloramphenicol, and B is LB agar medium containing ampicillin.

[0017] Figure 6These are scanning electron micrographs of the WT strain and the Sal3076ΔYeaZ::cat strain of Enteritidis Salmonella in the result analysis of the present invention; (a) is the WT strain, and (b) is the Sal3076ΔYeaZ::cat strain.

[0018] Figure 7 These are transmission electron micrographs of the WT strain and the Sal3076ΔYeaZ::cat strain of Enteritidis Salmonella in the result analysis of the present invention; (c) is the WT strain, and (d) is the Sal3076ΔYeaZ::cat strain.

[0019] Figure 8 This is a cell area bar graph of the WT strain and the Salmonella YeaZ gene deletion strain in the result analysis of the present invention; Fig. 9 This is a bar graph of cell wall thickness of the WT strain and the Salmonella YeaZ gene deletion strain in the result analysis of the present invention; Fig.10 This is a monitoring graph of the culturable number of the WT strain and the Salmonella YeaZ gene deletion strain after induction of the low temperature and low pH stress factor in Example 4 of the present invention; Fig.11 The results of cell membrane integrity detection (i.e., bacterial viability detection) of the WT strain and the Salmonella YeaZ gene deletion strain after induction of low temperature and low pH stress factors in Example 4 of the present invention; (A: WT; B: ΔYeaZ; C: live bacteria control; D: heat-killed dead bacteria control) Fig.12 This is a bar graph comparing the number of viable bacteria of the WT strain and the Salmonella YeaZ gene deletion strain after induction in Example 2 of the present invention; Fig.13 The results of catalase recovery of WT strain in VBNC state and Salmonella YeaZ gene deletion strain in Example 5 of the present invention are shown in Figure 5; (1: VBNC WT inoculated in LB medium or LB agar plate; 2: VBNC Salmonella YeaZ gene deletion strain inoculated in LB medium or LB agar plate; 3: VBNC WT inoculated in LB medium or LB agar plate containing Rpf; 4: VBNC Salmonella YeaZ gene deletion strain inoculated in LB medium or LB agar plate containing Rpf) Fig.14 The expression of relA, spoT, relE and higB genes of the YeaZ gene-deficient strain and the WT strain in normal state (logarithmic phase) and VBNC state in Example 6 of the present invention; Fig.15 These are the results of measuring the proliferation activity of the WT strain and the YeaZ gene-deficient strain in Example 7 of the present invention. DETAILED DESCRIPTION

[0020] The technical scheme of the present invention is clearly and completely described below in conjunction with the embodiments. The described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. The following description of at least one exemplary embodiment is only illustrative and does not serve as any limitation to the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0021] Example 1: Construction of a Salmonella YeaZ gene deletion strain using λ-Red homologous recombination technology, comprising the following steps: S1: Primer design; 1. Design primers H1-P1 and H2-P2, whose sequences are shown in Table 1: According to the upstream and downstream sequences of the YeaZ gene of Salmonella Enteritidis published in the GenBank database, a pair of primers H1-P1 and H2-P2 were designed; H1-P1 and H2-P2 respectively contained 60 bp of YeaZ upstream and downstream homologous sequences, and also contained the primer sequences and invertase recognition site FRT on both sides of the chloramphenicol resistance gene (cat) of the plasmid pKD3; pKD3 plasmid was used as a template to amplify linear DNA, and the DNA fragment size was 1100 bp.

[0022] 2. Design identification primers H3 and H4 for identifying whether the gene is recombined, and their sequences are shown in Table 1; The size of the YeaZ gene in wild-type Salmonella Enteritidis is 696 bp, and its size after homologous recombination is 1100 bp.

[0023] S2: Using plasmid pKD3 as template, primers H1-P1 and H2-P2 were used to PCR amplify the linear DNA fragment of chloramphenicol containing the FRT site; the amplified product was identified by agarose gel electrophoresis. After successful amplification, restriction endonuclease DpnⅠ was used for digestion at 37℃ for 30min to eliminate template false positives. The digested product was recovered and purified using a DNA purification kit. The agarose gel electrophoresis results are shown in Figure 1 shown.

[0024] PCR system preparation: 2xMix 25 μL, upstream primer H1-P1 2 μL, downstream primer H2-P2 2 μL, pKD3 plasmid 4 μL, ddH 2 The reaction mixture was 17 μL of O. Reaction program: 95°C for 5 min; 30 cycles (94°C for 45 s, 55°C for 45 s, 72°C for 1 min), 72°C for 10 min.

[0025] Table 1 Primer sequences for homologous recombination

[0026] S3: Preparation of recombinant Salmonella Enteritidis containing pKD46 plasmid; 1. Bacterial liquid culture. The target bacteria (Salmonella Enteritidis ATCC13076 strain) whose genes need to be knocked out is cultured at 37°C overnight to obtain Salmonella Enteritidis liquid; 2. Preparation of competent cells. The next day, take 1 mL of Enteritidis Salmonella liquid and place it in 100 mL of LB liquid culture medium, and culture it at 37°C and 200 r / min for 4 hours; then immediately place it in an ice water bath for 30 minutes; then centrifuge it at 4000 r / min for 10 minutes at 4°C, discard the supernatant, wash it twice with pre-cooled deionized water, and then wash it once with 10% pre-cooled glycerol, add 500 μL of glycerol to resuspend the cells, and obtain a competent cell suspension; divide the competent cell suspension into 5 1.5 mL centrifuge tubes, 100 μL in each tube, and store it at -80°C for later use; 3. Electroporation and enzyme digestion identification. Add 1-2μL of pKD46 plasmid to 100μL of the prepared competent cell suspension, mix well and add to the pre-cooled electroporation cup. This process is performed on ice. The electroporation conditions are set to 1800V voltage, 25μF capacitance, and 200Ω resistance. After electroporation, add 900μL of liquid LB culture medium and culture at 30℃ and 200r / min for 1h. Take 100μL of the competent cell suspension after culture and evenly spread it on a 50μg / mL ampicillin-resistant LB solid plate. After overnight culture at 30℃, the grown colonies (such as Figure 3 The plasmid was extracted and identified by enzyme digestion. The identification results are shown in Figure 2 shown.

[0027] S4: Inducible expression of Red recombinase and electroporation of homologous targeting fragments; 1. The recombinant Salmonella Enteritidis Sa-pKD46 containing the pKD46 plasmid correctly identified in S3 was cultured overnight at 30°C, and then 30 μL was taken and placed in 3 mL of LB liquid medium with Amp resistance; after culturing in a shaker at 30°C and 200 r / min for 1 hour, L-arabinose was added, and the final concentration of L-arabinose was 100 mmol / L. The culture was continued for 2 hours, and after induction to OD600 of 0.6-0.8, ice bath was performed for 30 minutes; then centrifuged at 4000 r / min for 10 minutes at 4°C, the supernatant was discarded after centrifugation, and the cells were washed twice with pre-cooled deionized water, and then washed once with pre-cooled 10% glycerol, and 500 μL of glycerol was added to resuspend the cells to obtain a recombinant competent cell suspension; the recombinant competent cell suspension was divided into 5 1.5 mL centrifuge tubes, 100 μL in each tube, one tube was taken for standby use, and the rest was stored at -80°C; 2. Take 5 μL of the targeting fragment containing the 60 bp homology arm of the YeaZ gene and place it in the recombinant competent cells prepared in step S3. After mixing, add it to a cold electroporation cup for electroporation. Set the electroporation conditions to 1800 V voltage, 25 μF capacitance, and 200 Ω capacitance. This process is performed on ice. After electroporation, add 900 μL of liquid LB culture medium and mix well. Then, culture at 30°C and 200 r / min for 1 hour. Then, evenly spread the cultured bacterial liquid of the recombinant competent cells on a 50 μg / mL chloramphenicol-resistant LB solid plate. After overnight culture at 30°C, the grown colonies are verified by bacterial liquid PCR using identification primers H3 and H4. The results are as follows: Figure 4 As shown; 3. Take 5 mL of each of the Enteritidis Salmonella and boil for 10 minutes. Centrifuge at 10000 g for 1 minute and take the supernatant as the genomic DNA to be tested. Prepare a 25 μL system (2×Taq Mix is ​​12.5 μL, upstream primer is 1.0 μL, downstream primer is 1.0 μL, ddH 2 O is 8.0 μL, bacterial solution is 2.5 μL).

[0028] S5: Eliminate plasmid pKD46 in homologous recombination of Salmonella Enteritidis to obtain a Salmonella YeaZ gene deletion strain; pKD46 plasmid is a temperature-sensitive plasmid and will automatically disappear in an environment above 37°C; place the correctly identified Salmonella Enteritidis Sal3076ΔYeaZ::cat and culture it in a shaker at 42°C and 180r / min for 15h, pick out the grown single colonies and culture them overnight, spread them evenly on LB solid culture media containing chloramphenicol and ampicillin, respectively, culture them at 30°C, and culture them on chloramphenicol plates (such as Figure 5 A) can grow on ampicillin plates (such as Figure 5 If the strain cannot grow on the plate (B), it proves that the pKD46 plasmid has been eliminated and the gene deletion strain Salmonella Enteritidis Sal3076ΔYeaZ::cat (hereinafter referred to as "ΔYeaZ") has been successfully constructed.

[0029] Example 2: Verification of the induction of the Salmonella YeaZ gene deletion strain into the VBNC state by low temperature and high salt solution stress, comprising the following steps: (1) Preparation of bacterial suspension and stress induction: The prepared WT strain (wild-type Enteritidis Salmonella) and the Salmonella YeaZ gene-deficient strain (ΔYeaZ) were adjusted to a bacterial suspension concentration of 1×10 8CFU / mL, take 3 tubes of wild Enteritidis Salmonella suspension and Salmonella YeaZ gene deletion strain suspension, each with 10mL, and centrifuge at 4℃, 8000r / min for 5min, gently remove the supernatant; resuspend in physiological saline with a pH of 3.0 and a mass fraction of 15% NaCl, and vortex to mix evenly. Place at 2-8℃ for induction treatment. Take the bacterial suspension every 12 hours and detect the culturable number. When the culturable number is monitored to 0CFU / mL, use CTC dye to detect the total number of viable bacteria; (2) Cultivable number test: Take 100 μL of the bacterial solution to be tested from each tube, make 10-fold serial dilutions with sterile PBS buffer solution (10 mmol, pH = 7.2-7.4), and apply 100 μL of each dilution on tryptic soy agar medium. The dilutions are 10 3 times, 10 4 times, 10 5 times, 10 6 times, and cultured overnight in a 37°C biochemical incubator. The next day, the colonies were observed and counted. Fig.10 As shown; (3) Qualitative detection of viable bacterial activity: After stress induction to culturable count of 0, 0.1 mL of bacterial solution was taken to detect bacterial activity. After staining the cells with the LIVE / DEAD™ BacLight™ Bacterial Viability Detection Kit (Thermo), the bacterial activity was observed under a laser confocal microscope in random fields of view. After induction, the bacterial suspension contained bacteria in two states: dead (orange) and VBNC (green). The results are as follows: Fig.11 As shown; (4) Quantitative detection of total viable bacteria: After the stress induction to culturable count of 0, take 0.1 mL of bacterial solution to detect the total viable bacteria. The CTC Bacterial Viability Detection Kit (Tongren Chemical Research Institute) can be used to stain cells with respiratory activity, and then the total viable bacteria can be detected by flow cytometry. The results are as follows: Fig.12 As shown; (5) Calculation of VBNC bacteria: VBNC bacteria = total number of viable bacteria - number of culturable bacteria.

[0030] Embodiment 3: The logarithmic phase of Salmonella Enteritidis WT and ΔYeaZ were fixed with electron microscopy fixative and sent to Wuhan Sewell Biotechnology Co., Ltd. for scanning electron microscopy and transmission electron microscopy. The differences in morphological structure between the two bacteria were observed by scanning electron microscopy and transmission electron microscopy. 20 cells were randomly selected and the cell size and cell wall thickness were measured using ImageJ.

[0031] Example 4: Verification of the recovery of the Salmonella YeaZ gene deletion strain, comprising the following steps; The concentration of the bacterial solution of the VBNC-treated Salmonella enteritidis WT strain and ΔYeaZ strain was adjusted to a total viable count of 1×10 4 CFU / ml. Take 100μL of each bacterial suspension and add it to 3mL of LB liquid medium and 3mL of LB liquid medium containing 10μg / mL catalase, place it in a shaker and shake it at 37℃, 180r / min for 24 hours to observe the growth status; at the same time, take 100μL of each bacterial suspension and spread it on LB agar medium plate and 3mL of LB agar medium plate containing 10μg / mL catalase, and place it in a 37℃ biochemical incubator for 24 hours. Its growth status is shown in the figure. Fig.13 shown.

[0032] Example 5: Verification of the different gene expression between the Salmonella YeaZ gene deletion strain and the WT strain (wild-type Salmonella Enteritidis), the method is as follows: (1) RNA extraction: A micro RNA extraction kit was used to isolate the total RNA of the WT strain and the Salmonella YeaZ gene deletion strain in the logarithmic phase and VBNC state. The final concentration of the RNA sample was measured using a NANODROP micro UV spectrophotometer, and the mRNA was reverse transcribed into cDNA using a reverse transcription kit. Reaction procedure: incubate at 42°C for 60 min and terminate the reaction by heating at 70°C for 5 min; (2) Real-time fluorescence quantitative PCR: Synthetic primers: Primers and primer sequences are shown in Table 2; Using the primers of the target gene to be detected synthesized in Table 2, the cDNA sample obtained by reverse transcription in step (1) of this embodiment was subjected to qPCR detection using a SYBR qPCR Master Mix kit. The 10 μL reaction system included: 5 μL of 2×ChamQUniversal SYBR qPCR Master Mix, 0.25 μL of each upstream and downstream primer, 1 μL of cDNA sample, and 3.5 μL of sterile water; Reaction conditions: pre-denaturation at 95°C for 30 s, 40 cycles (denaturation at 95°C for 10 s, annealing and extension at 50°C for 30 s), melting curve: 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s; The expression of relA, spoT, relE and higB genes in WT and ΔYeaZ strains under normal conditions (logarithmic phase) and VBNC conditions was detected by qPCR using specific primers. The expression level of the target gene was compared with the internal reference gene (16S rDNA) and then 2 -ΔΔCt The results were calculated by the method. Each experiment was repeated 3 times independently. Fig.14 shown.

[0033] Table 2 Primer sequences of target genes

[0034] Example 6: Verification of the proliferation activity of the Salmonella YeaZ gene deletion strain, the method is as follows.

[0035] Fresh bacterial suspensions of WT and ΔYeaZ were taken and counted, and the concentrations were 10 6 CFU / mL was inoculated into 200mL LB liquid culture medium. Cultured at 37℃ and 180r / min for 24h, and 1mL bacterial sample was taken every two hours to measure the OD600 value. The test results are shown in Fig.15 .

[0036] Results and Analysis: 1. The present invention successfully constructed a Salmonella YeaZ gene deletion strain.

[0037] 2. The differences in morphology and structure between the YeaZ gene-deficient strain of Salmonella and wild-type Enteritidis Salmonella were observed by scanning electron microscopy and transmission electron microscopy. Figure 6 and Figure 7 As shown, there are differences in bacterial size. The WT strain is irregularly rod-shaped and larger, while the Salmonella YeaZ gene-deficient strain is relatively smaller and regular spherical, with thicker cell walls and blocked cell wall septum division. The results show that the loss of the YeaZ gene leads to abnormal cell wall peptidoglycan cycling.

[0038] In addition, it was proved that the YeaZ gene affects the rod differentiation and cell wall thickness of Salmonella by affecting the peptidoglycan cycle of the Salmonella cell wall. 20 cells were randomly selected from the gene-deficient strain of Salmonella enteritidis and the wild-type strain and placed in the field of view. The cell area and cell wall thickness of the two strains were calculated using ImageJ. The results are shown in Figure 8 and Fig. 9 As shown, the cell area of ​​wild-type Salmonella is significantly larger than that of the gene-deficient strain. Similar to Rpf, YeaZ has peptidoglycan hydrolase activity, which can be used for cell wall decomposition and release of peptidoglycan fragments (cell wall peptides), as well as for peptidoglycan recycling and cell wall remodeling. However, the deletion of the YeaZ gene leads to the obstruction of peptidoglycan rearrangement, which is not conducive to bacterial proliferation.

[0039] 3. According to Embodiment 3 and Figure 10-12 It can be seen that the Salmonella YeaZ gene deletion strain is more likely to enter the VBNC state than the WT strain under the same level of stress conditions, which indicates that the deletion of the YeaZ gene will lead to a weakened ability of Enteritidis Salmonella to resist stress and reduced stress resistance.

[0040] 4. By Fig.13As shown in Example 4, the WT strain of Enteritidis Salmonella in the VBNC state can be revived by catalase and its culturability is restored. However, the YeaZ gene-deficient strain of Salmonella cannot be revived by catalase. This shows that YeaZ is a necessary condition for the resuscitation of Enteritidis Salmonella in the VBNC state, and the YeaZ gene-deficient strain of Salmonella cannot be revived in the VBNC state.

[0041] 5. By Example 5 and Fig.14 It can be seen that after the gene-deficient strain and the WT strain of Enteritidis Salmonella enter the VBNC state, the expression of the stringent response-related gene relA is upregulated, the expression of spoT is downregulated, and the expression of the TA system-related genes relE and higB is upregulated, proving that the knockout of the YeaZ gene activates the stringent response and the TA system, thereby promoting the corresponding stress of the bacteria and making the mutant strain more likely to enter the VBNC state. In the logarithmic phase and VBNC state, the YeaZ gene-deficient strain is compared with the WT strain, and relA, spoT, relE and higB are all upregulated, indicating that the loss of the YeaZ gene upregulates the stringent response, leading to the accumulation of ppGpp, and then leading to the accumulation of polyphosphates, affecting the expression of genes related to the TA system.

[0042] 6. By Example 7 and Fig.15 It can be seen that the proliferation rate of the gene-deficient strain in 0-12 hours is lower than that of the WT strain, and the growth rates of the two strains are basically the same after 14 hours. This shows that the presence of the YeaZ gene has a certain promoting effect on the growth of Salmonella Enteritidis.

[0043] In summary, the YeaZ gene-deficient strain of Salmonella constructed by the method of the present invention has a different biological phenotype compared with the WT strain; at the same time, it has low toxicity and is more likely to enter the VBNC state.

Claims

1. A Salmonella YeaZ gene-deficient strain, characterized in that: The YeaZ gene deletion strain of Salmonella enterica Sal3076ΔYeaZ::cat was constructed through the λ-Red gene knockout technology and deposited in the China Center for Type Culture Collection on January 15, 2025, with the preservation address in Wuhan, China, the preservation number is CCTCC NO: M2025159, and the classification name is Salmonella enterica Sal3076ΔYeaZ::cat.

2. A method for constructing a Salmonella YeaZ gene-deficient strain according to claim 1, characterized in that: The following steps are involved: S1: Design primers H1-P1, H2-P2, H3 and H4, wherein the primers H1-P1 and H2-P2 contain 60 bp of YeaZ upstream and downstream homologous sequences, as well as the primer sequences on both sides of the plasmid pKD3 chloramphenicol resistance gene cat and the reverse transcriptase recognition site FRT; S2: Using plasmid pKD3 as template and primers H1-P1 and H2-P2, PCR amplified the chloramphenicol linear DNA fragment containing the FRT site; S3: Preparation of recombinant Salmonella Enteritidis containing pKD46 plasmid; S4: Inducible expression of recombinant Salmonella Enteritidis Red recombinase and electroporation of a 60 bp homologous targeting fragment containing the YeaZ gene; Inducing the expression of Red recombinase in the recombinant Salmonella Enteritidis, and then inserting a DNA fragment containing the homologous arm of the YeaZ gene into the recombinant Salmonella Enteritidis to obtain homologous recombinant Salmonella Enteritidis; S5: Eliminate plasmid pKD46 in homologous recombination Salmonella Enteritidis to obtain a Salmonella YeaZ gene deletion strain.

3. The construction method according to claim 2, characterized in that: The H1-P1 and H2-P2 are knockout primers; H1 in the H1-P1 is the nucleotide sequence shown in SEQ ID NO.1, and P1 is the nucleotide sequence shown in SEQ ID NO.2; H2 in the H2-P2 is the nucleotide sequence shown in SEQ ID NO.3, and P2 is the nucleotide sequence shown in SEQ ID NO.

4.

4. The construction method according to claim 2, characterized in that: The H3 and H4 are identification genes for identifying whether the YeaZ gene is recombined, the H3 is the nucleotide sequence shown in SEQ ID NO.5; the H4 is the nucleotide sequence shown in SEQ ID NO.

6.

5. The construction method according to claim 2, characterized in that: In the step S3, a competent cell suspension is prepared using the target bacteria whose genes need to be knocked out, 1 to 2 μL of the pKD46 plasmid is added to 100 μL of the prepared competent cell suspension for electrotransformation, and then enzyme digestion is performed to obtain recombinant Salmonella Enteritidis containing the pKD46 plasmid.

6. Use of a Salmonella YeaZ gene-deficient strain constructed by the construction method according to any one of claims 2 to 5 in food detection.

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