Vibrio alginolyticus phoR gene stable silencing strain and application thereof

Abstract

The invention provides a vibrio alginolyticus phoR gene silencing strain, the strain is Vibrio alginolyticus phoR-RNAi, the strain is preserved in China Center for Type Culture Collection on May 22, 2023, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: M2023799. According to the invention, a vibrio alginolyticus phoR gene silencing strain is constructed by combining a transcriptomics analysis technology with a directed and efficient gene silencing technology of RNA interference, and the virulence effect of the strain is detected. The constructed vibrio alginolyticus phoR gene silencing strain not only can be used for researching the pathogenesis of vibrio alginolyticus, but also can be used for developing vaccines for preventing or treating vibrio alginolyticus diseases, so that a new target spot is found for preventing and treating the vibrio alginolyticus diseases.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a Vibrio alginolyticus phoR gene stably silenced strain and its application. Background Art

[0002] Vibrio alginolyticus is a common Gram-negative bacterium, facultatively anaerobic, and widely survives and reproduces in aquatic ecological environments such as coastal areas and streams around the world. Vibrio alginolyticus is a thermophilic marine vibrio that can easily cause a large number of deaths of seafood during high temperatures in summer, resulting in huge economic losses. Vibrio alginolyticus has a wide range of hosts and can infect marine organisms such as Acanthopagrus schlegelii, Epinephelus coioides, Acanthopagrus latus, Litopenaeus vannamei, Ruditapes philippinarum, and Pocillopora verrucosa, mainly manifested as emaciation, skin ulceration, congestion and swelling, and even death. For different hosts, the pathogenic characteristics of Vibrio alginolyticus show specific differences. For example, Vibrio alginolyticus can cause a high mortality rate in juvenile whiteleg shrimp, accompanied by obvious symptoms of hepatopancreas necrosis; when infecting groupers, the fish body secretes too much mucus, the fins rot, the liver is congested, and the spleen is enlarged; in Pseudosciaena crocea, it can cause septicemia, bleeding, and skin surface ulcers. At present, the drugs used to prevent and control Vibrio alginolyticus are mainly antibiotics. The improper use of antibiotics is likely to cause harm such as bacterial drug resistance and drug residues. Therefore, there is an urgent need to develop biological drugs or vaccines for preventing and controlling Vibrio alginolyticus diseases.

[0003] Phosphorus is one of the essential elements for bacteria to carry out life activities. It is not only an important component of biomolecules such as nucleic acids and phospholipids, but also can store energy in the form of high-energy phosphate groups, participate in enzymatic reactions, and transmit information in signal pathways. In Gram-negative bacteria, the bacterial sensing of phosphate levels is regulated by the two-component regulator PhoB and PhoR, among which PhoR is a membrane-bound histidine kinase. In Vibrio parahaemolyticus, phoR is related to microbial metabolism and carbon metabolism, and regulates flagellar assembly and chemotactic pathways; in Salmonella typhimurium, the phoR silent body can increase the antibody response of immunoglobulins; in Pseudomonas aeruginosa, phoR can activate the expression of various genes involved in cytotoxicity by regulating the quorum sensing system; in Edwardsiella tarda, phoR has a synergistic effect with both the type III secretion system and the type VI secretion system. phoR is closely related to the virulence mechanism and self-defense mechanism of pathogenic bacteria. However, so far, the role of phoR in Vibrio alginolyticus has not been reported. Summary of the Invention

[0004] An object of the present invention is to overcome the deficiencies of the prior art, and provides a strain of Vibrio alginolyticus phoR gene stably silenced strain and its application, solving the problems in the above background technology.

[0005] One of the technical solutions adopted by the present invention to solve its technical problems is: providing a strain of Vibrio alginolyticus phoR gene stably silenced strain, which is Vibrio alginolyticus phoR-RNAi, and was deposited in the China Center for Type Culture Collection on May 22, 2023. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2023799.

[0006] Another technical solution adopted by the present invention to solve its technical problems is: providing the application of the Vibrio alginolyticus phoR gene stably silenced strain in the preparation of vaccines for preventing and / or treating Vibrio alginolyticus disease.

[0007] The Vibrio alginolyticus phoR gene stably silenced strain provided by the present invention is constructed by the following technical means:

[0008] S1. Through transcriptomics analysis technology, the target of screening pathogenic functional genes is locked on the phoR gene, and the phoR gene is verified by qRT-PCR;

[0009] S2. Design shRNA according to the target gene sequence and ligate it to the plasmid pACYC184 to form the recombinant plasmid pACYC184 - shRNA. The recombinant plasmid pACYC184 - shRNA is transformed into DH5α competent cells by heat shock, and the successful ligation of shRNA is verified by sequencing.

[0010] S3. After the successful ligation of shRNA, the recombinant plasmid pACYC184 - shRNA is introduced into Escherichia coli SM10 competent cells.

[0011] S4. Escherichia coli SM10 containing the recombinant plasmid pACYC184 - shRNA is introduced into wild - type Vibrio alginolyticus by conjugation and verified by gyrB sequencing.

[0012] S5. Use qRT - PCR to detect the relative expression level of this gene in the phoR stable silencing strain. Among them, shRNA at three sites is designed for each gene, and the stable silencing strain with the best silencing efficiency is named "phoR - RNAi", that is, Vibrio alginolyticus phoR - RNAi of the present invention.

[0013] S6. Verify the difference in virulence between the phoR gene stable silencing strain and wild - type Vibrio alginolyticus through adhesion experiments, biofilm formation experiments, motility experiments, and capillary chemotaxis experiments. The present invention also reveals through transcriptome sequencing of the phoR gene stable silencing strain that the phoR gene may affect the virulence of Vibrio alginolyticus by regulating related genes in bacterial secretion.

[0014] The present invention has the following beneficial effects:

[0015] 1. The adhesion ability, biofilm formation ability, motility, and chemotaxis of the stable silencing strain of the Vibrio alginolyticus phoR gene of the present invention are significantly lower than those of wild - type Vibrio alginolyticus, and it can be used for the development of vaccines for the prevention or treatment of Vibrio alginolyticus disease.

[0016] 2. The results of transcriptome sequencing analysis show that the stable silencing of the phoR gene causes significant changes in the expression levels of related virulence genes in Vibrio alginolyticus. The phoR gene may affect the virulence of Vibrio alginolyticus by regulating related genes in bacterial secretion. This indicates that the stable silencing strain of the Vibrio alginolyticus phoR gene can be used to study the pathogenic mechanism of Vibrio alginolyticus and find new targets for the prevention and treatment of Vibrio alginolyticus disease in aquatic animals. Description of the Drawings

[0017] Figure 1 It is the stable silencing efficiency diagram of the Vibrio alginolyticus phoR gene.

[0018] Data are presented as mean ± SD; three independent biological replicates were performed for each group, *** represents P < 0.001.

[0019] Figure 2 It is the growth curve of wild-type Vibrio alginolyticus and the strain with stable silencing of the phoR gene.

[0020] Figure 3 It is the result graph of the adhesion ability of wild-type Vibrio alginolyticus and phoR-RNAi strains.

[0021] Among them, A is the detection result of the adhesion ability of the wild strain of Vibrio alginolyticus;

[0022] B is the detection result of the adhesion ability of the phoR-RNAi strain;

[0023] C is the statistical histogram of adhesion ability data, and P < 0.001 is marked as ***.

[0024] Figure 4 It is the result graph of the phosphate stress experiment of wild-type Vibrio alginolyticus and the strain with stable silencing of the phoR gene.

[0025] Figure 5 It is the result graph of the artificial infection experiment of the strain with stable silencing of Vibrio alginolyticus.

[0026] Figure 6 It is the experimental graph of the bacterial load of the strain with stable silencing of Vibrio alginolyticus.

[0027] Figure 7 It is the graph of the biofilm formation ability of the strain with stable silencing of Vibrio alginolyticus.

[0028] P < 0.001 is marked as ***.

[0029] Figure 8 It is the result graph of the motility of wild-type Vibrio alginolyticus and the strain with stable silencing of the phoR gene.

[0030] Among them, A is the colony formed by the wild strain of Vibrio alginolyticus on the semi-solid medium;

[0031] B is the colony formed by the phoR-RNAi strain on the semi-solid medium;

[0032] C is the histogram of colony diameter, and P < 0.05 is marked as *.

[0033] Figure 9 It is the result graph of the chemotaxis ability experiment of the wild strain and the phoR-RNAi strain.

[0034] P < 0.001 is marked as ***.

[0035] Figure 10It is a sample correlation result diagram of the Vibrio alginolyticus phoR gene stable silencing strain.

[0036] Figure 11 It is a differential gene statistical result diagram of the Vibrio alginolyticus phoR gene stable silencing strain.

[0037] Figure 12 It is a differential gene GO enrichment diagram of the Vibrio alginolyticus phoR gene stable silencing strain.

[0038] BP represents biological process;

[0039] CC represents cellular component;

[0040] MF represents molecular function.

[0041] Figure 13 It is a differential gene KEGG enrichment diagram of the Vibrio alginolyticus phoR gene stable silencing strain.

[0042] Figure 14 It is a schematic diagram of the PhoB-PhoR regulator participating in the phosphate regulation mechanism of Vibrio alginolyticus.

[0043] Figure 15 It is a diagram of the expression of differential genes of the phoR-RNAi strain.

[0044] Among them, A is the expression level of up-regulated genes; B is the expression level of down-regulated genes. Specific implementation manners

[0045] To better understand the present invention, the present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings. However, those skilled in the art understand that the following embodiments do not limit the protection scope of the present invention, and any changes and variations made on the basis of the present invention are within the protection scope of the present invention.

[0046] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.

[0047] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0048] The wild strain of Vibrio alginolyticus (ND-01) was isolated and identified by this laboratory, stored in physiological saline containing 40% glycerol, and stored at a constant temperature of -80°C in a refrigerator.

[0049] The competent cells of Escherichia coli DH5α were purchased from Beijing TransGen Biotech Co., Ltd. and used for preparing recombinant plasmids, and stored at -80°C. Escherichia coli SM10 was purchased from Tiangen Biotech (Beijing) Co., Ltd. and used for introducing the recombinant plasmid into the wild strain of Vibrio alginolyticus to prepare the gene silencing strain, and stored at a constant temperature of -80°C in a refrigerator.

[0050] The experimental Epinephelus coioides were purchased from the Jimei District vegetable market in Xiamen and used for the Vibrio alginolyticus adhesion experiment and the artificial infection experiment.

[0051] The PCR primers were synthesized by Xiamen Promega Biotechnology Co., Ltd., and the qRT-PCR primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0052] Example 1 Construction of a stable Vibrio alginolyticus-silenced strain

[0053] 1. Extraction of pACYC184 plasmid

[0054] The plasmid selected in this experiment was pACYC184, and the activated strain was Escherichia coli containing the plasmid. The Beijing TransGen Biotech plasmid extraction kit was used.

[0055] 2. Double digestion and purification recovery of pACYC184 plasmid

[0056] The pACYC184 plasmid was digested with the restriction endonucleases BamH I and Sph I. The reaction system was as follows:

[0057] Plasmid double digestion system

[0058] Reagent Volume (μL) 10*K Buffer 2 BamH I 1 Sph I 1 Plasmid 2 <![CDATA[ddH2O]]> 19 All 25

[0059] After preparing the double digestion system in a 200 μL centrifuge tube, it was centrifuged for 3 s with a vortex mixer and placed in a water bath at 37 °C for 3 h. After the reaction, the digestion result was judged by agarose gel electrophoresis. The successfully digested sample showed two clear bands under the gel electrophoresis imaging system, located at 150 bp and 4200 bp respectively. Subsequently, the band at 4200 bp was recovered using the Beijing TransGen Biotech purification kit.

[0060] 3. Preparation of recombinant plasmid

[0061] Specific shRNAs were designed through the website http: / / rnaidesigner.thermofisher.com for the selected gene nucleotide sequence and synthesized by Shanghai Boyu Biotechnology Co., Ltd. The shRNA sequences are shown in the table.

[0062] shRNA sequence list

[0063]

[0064]

[0065] After the shRNAs were synthesized, they were placed in a centrifuge at 4 °C and centrifuged at 12000 g for 5 min. In a laminar flow hood, the corresponding volume of ddH2O was added to dilute it into a 100 μM solution, and it was pipetted evenly and stored at -20 °C for later use.

[0066] The diluted shRNA is annealed to form double strands, and the system is as follows:

[0067] shRNA annealing system

[0068] Component Volume (μL) Nuclease-free water 15 Primer F 2.5 Primer R 2.5 Annealing buffer 5

[0069] The shRNA annealing program is as follows:

[0070] shRNA annealing program

[0071] Temperature Time 95℃ 2min Drop 1℃ every 90s until 16℃ About 120min 4℃ Forever

[0072] After annealing, it is stored in a -20 °C refrigerator for later use.

[0073] The double-digested plasmid vector recovered by gel extraction is ligated to the annealing product, and the system is as follows:

[0074] Ligation system of specific shRNA and plasmid vector

[0075]

[0076]

[0077] After preparing the system, it is placed at 16 °C for overnight ligation. After ligation, 9 μL of the reaction solution is taken and 1 μL of Solution Ⅲ is added to increase the ligation efficiency.

[0078] 4. Introduction of recombinant plasmid into DH5α competent cells

[0079] 5. Preparation and transformation of Escherichia coli SM10 competent cells

[0080] 6. Bacterial conjugation

[0081] 7. Detection of silencing efficiency

[0082] After obtaining the stable Vibrio alginolyticus phoR-silenced strain, qRT-PCR is used to detect the expression level of the target gene in the stable silenced strain. The Vibrio alginolyticus containing the empty plasmid is used as a control, and gyrB is selected as the internal reference gene.

[0083] Specific shRNAs are designed based on the target gene sequence, and the shRNAs are ligated to the plasmid vector to construct recombinant plasmids. The recombinant plasmids are introduced into the wild-type Vibrio alginolyticus by conjugation to prepare stable Vibrio alginolyticus silenced strains. Three shRNAs are designed for each target gene, and stable silenced strains are prepared respectively. The stable silenced strain with the highest silencing efficiency is selected for subsequent experiments (the results are as Figure 1As shown, the silencing efficiency of phoR-RNAi-206 was 88.06%, the silencing efficiency of phoR-RNAi-524 was 95.25%, and the silencing efficiency of phoR-RNAi-973 was 94.84%. The strain with the highest silencing efficiency was screened, and phoR-RNAi-524 was used for subsequent experiments. For ease of description, phoR-RNAi-524 was named phoR-RNAi.

[0084] Example 2 Determination of the growth curve of Vibrio alginolyticus stable silencing strains

[0085] As Figure 2 shown, compared with the wild strain, the phoR-RNAi strain took longer to reach the stationary phase, and the total cell mass of the stable silencing strain was slightly lower than that of the wild strain at the stationary phase. However, the overall growth trends and growth conditions of the wild strain and the stable silencing strain were consistent. Therefore, it was shown that stable silencing of phoR did not affect the growth and reproduction of the bacteria, and subsequent experiments could be carried out on the stable silencing strain.

[0086] Example 3 Bacterial adhesion experiment of Vibrio alginolyticus stable silencing strains

[0087] As Figure 3 shown (A is the wild strain, B is the stable silencing strain), compared with the wild strain, the number of bacteria of the stable silencing strain was significantly reduced under the microscope field of view. The adhesion ability of the phoR-RNAi strain decreased by 95.48%. The results showed that stable silencing of phoR significantly affected the adhesion ability of the bacteria, and the target gene might be involved in regulating the adhesion process of the bacteria.

[0088] Example 4 Phosphate stress experiment of Vibrio alginolyticus stable silencing strains

[0089] The wild strain of Vibrio alginolyticus was subjected to phosphate stress. The results were as Figure 4 shown. When the phosphate concentration in the medium was low (0.2%, 0.3%), the expression level of the phoR gene was significantly up-regulated. As the phosphate concentration increased, the expression level of the phoR gene showed a downward trend. When the phosphate concentration was 0.2%, the expression level of phoR was up-regulated by 8.00 times; when the phosphate concentration was 0.3%, the expression level of phoR was up-regulated by 2.26 times; when the phosphate concentration was 0.4%, the expression level of phoR was up-regulated by 0.29 times; when the phosphate concentration was 0.5%, the expression level of phoR was up-regulated by 0.24 times; when the phosphate concentration was 0.6%, the expression level of phoR decreased by 18.48%; when the phosphate concentration was 0.7%, the expression level of phoR decreased by 34.45%; when the phosphate concentration was 0.8%, the expression level of phoR decreased by 42.26%; when the phosphate concentration was 0.9%, the expression level of phoR decreased by 29.06%.

[0090] Artificial Infection Experiment of Vibrio alginolyticus Stable Silencing Strain

[0091] The wild strain of Vibrio alginolyticus and the phoR-RNAi strain were used to infect Epinephelus coioides respectively, and the survival rate within 14 days after infection was recorded. As Figure 5 shown, the survival rate of Epinephelus coioides in the PBS negative control group was 100%; the Epinephelus coioides in the wild strain infection group began to die on the 2nd day after infection, and the survival rate was 0% on the 8th day; the Epinephelus coioides in the phoR-RNAi strain infection group began to die on the 5th day after infection, and the cumulative mortality rate decreased by 70%.

[0092] Example 6 Bacterial Load Experiment of Vibrio alginolyticus Stable Silencing Strain

[0093] As Figure 6 shown, with the extension of the infection time, the bacterial loads of the wild strain of Vibrio alginolyticus and the phoR-RNAi strain in the liver of Epinephelus coioides showed a trend of first increasing and then decreasing, reaching the peak on the 4th day and the 3rd day respectively. The bacterial load of the phoR-RNAi strain in the liver was always lower than that of the wild strain after infection. The bacterial loads of the wild strain and the phoR-RNAi strain in the spleen of Epinephelus coioides both reached the peak on the 2nd day, and the bacterial load of the phoR-RNAi strain in the spleen was always lower than that of the wild strain during the infection process.

[0094] Example 7 Biofilm Formation Experiment of Vibrio alginolyticus Stable Silencing Strain

[0095] As Figure 7 shown, compared with the wild strain, the biofilm formation ability of the phoR-RNAi strain decreased by 83.58%. The results showed that the stable silencing of phoR significantly affected the biofilm formation ability of the bacterial cells, indicating that phoR may be involved in regulating the biofilm formation ability of Vibrio alginolyticus.

[0096] Example 8 Motility Experiment of Vibrio alginolyticus Stable Silencing Strain

[0097] As Figure 8 shown, both the wild strain and the stable silencing strain could grow smooth round colonies on the LB solid plate, but the diameter of the colonies formed by the stable silencing strain was significantly smaller than that of the wild strain. The results showed that the motility ability of the phoR-RNAi strain decreased by 12.18% compared with the wild strain. The stable silencing of phoR affected the motility ability of the bacterial cells, and the expression of the gene may be related to the motility ability of the bacterial cells.

[0098] Example 9 Chemotaxis Experiment of Vibrio alginolyticus Stable Silencing Strain

[0099] As Figure 9As shown, compared with the wild strain of Vibrio alginolyticus, the chemotactic ability of the phoR-RNAi strain decreased by 78.60%. The results indicate that the stable silencing of phoR significantly affects the chemotactic function of the bacteria, and gene expression is closely related to chemotactic ability.

[0100] Example 10 Transcriptome Sequencing of the Stable Silencing Strains of Vibrio alginolyticus

[0101] I. Data Quality Control

[0102] After generating the transcriptome data, to determine the reliability of the data, quality control analysis of the data is required. The specific data statistics are shown in the table. As can be seen from the following table, the base error rate after quality control is lower than 0.03, and the Q20 index after data quality control is between 97.5% - 99.0%, indicating that the sequencing results have high accuracy and the data is available for subsequent analysis.

[0103] Data Quality Control Statistical Table

[0104] Sample name Number of reads after quality control Number of bases after quality control (bp) Base error rate after quality control (%) Q20 after quality control (%) CK_1 31349494 4094267074 0.0235 98.73 CK_2 27671014 3636377749 0.0242 98.41 CK_3 33012696 4395655350 0.0236 98.69 phoR1 24553272 3448106836 0.0259 97.65 phoR2 26170858 3632465996 0.0257 97.71 phoR3 23469880 3249878119 0.0257 97.72

[0105] II. Sample Correlation

[0106] Biological experiments need to ensure the repeatability of experimental results and at the same time ensure that the original states of parallel samples are similar to avoid the influence of sample differences on experimental results. For transcriptome sequencing, three biological replicates are set for the same treatment, and sample correlation can reflect the degree of association between parallel samples. As Figure 10 can be seen, samples under the same conditions are clustered together, indicating strong correlation. The selection of samples does not affect the sequencing results and subsequent analysis can be carried out.

[0107] III. Statistical Analysis of Differentially Expressed Genes

[0108] After obtaining the Read Counts of genes, differential expression analysis of genes between samples is performed to identify differentially expressed genes between samples, and then the functions of the differentially expressed genes are studied. For experimental designs with biological replicates, the DESeq2 software based on the negative binomial distribution is directly used to perform statistical analysis on the raw counts, and genes with differential expression between comparison groups are obtained based on certain screening conditions. The default parameters are: p-adjust < 0.05 & |log2FC| ≥ 1. According to the transcriptome results, compared with the wild strain of Vibrio alginolyticus, a total of 2095 differentially expressed genes were detected in the phoR-RNAi strain. As Figure 11 shown, among them, there are 1009 genes with up-regulated expression and 1086 genes with down-regulated expression.

[0109] IV. GO Enrichment Analysis

[0110] GO annotations enriched the genes obtained from sequencing into three major categories: biological process, cellular component, and molecular function. There were 1,813 genes related to cellular component, 2,700 genes related to molecular function, and 1,735 genes related to biological process in this sequencing. As Figure 12 shown, in the biological process category, the top ten subcategories enriched with differential genes were transcriptional regulation, DNA templated, transmembrane transport, translation, phosphorelay signal transduction system, carbohydrate metabolic process, methylation, flagellar-dependent cell motility, cell division, chemotaxis, and signal transduction; in the cellular component category, the top ten subcategories enriched with differential genes were integral component of membrane, cytoplasm, plasma membrane, ribosome, membrane, outer membrane, integral component of plasma membrane, periplasmic space, ATP-binding cassette (ABC) transporter complex, and extracellular region; in the molecular function category, the top ten subcategories enriched with differential genes were ATP binding, metal ion binding, DNA binding, DNA-binding transcription factor activity, hydrolase activity, transmembrane transporter activity, oxidoreductase activity, magnesium ion binding, zinc ion binding, and ATPase-coupled transmembrane transporter activity.

[0111] V. KEGG Enrichment Analysis

[0112] Through KEGG mapping analysis, all differentially expressed genes were mapped to 41 KEGG pathways in total. There were 122 differentially expressed genes enriched in the human disease branch, 841 differentially expressed genes enriched in the metabolism branch, 66 differentially expressed genes enriched in the organismal systems branch, 334 differentially expressed genes enriched in the cellular processes branch, 210 differentially expressed genes enriched in the genetic information processing branch, and 415 differentially expressed genes enriched in the environmental information processing branch. At the KEGG three-level classification level, the top twenty differentially expressed gene enrichment pathways were selected, and the gene distribution was as Figure 13 shown. Differentially expressed genes were mainly enriched in the two-component system (map02020), ABC transporters (map02010), and bacterial secretion system (map03070) in the environmental information processing branch; flagellar assembly (map02040), quorum sensing (map02024), biofilm formation - Vibrio cholerae (map05111), bacterial chemotaxis (map02030), and biofilm formation - Escherichia coli (map02026) in the cellular processes branch; ribosome (map03010) in the genetic information processing branch; and eleven pathways in the metabolism branch. Among them, the pathway with the largest number of enriched differential genes was the two-component system, followed by the ABC transporters, and then the flagellar assembly pathway.

[0113] According to the KEGG analysis, a schematic diagram of the phosphate regulation mechanism involving the PhoB-PhoR regulator in Vibrio alginolyticus was drawn (asFigure 14 ) When the environment where the bacterial cells are located is under phosphate-limiting conditions, PhoR located on the cell membrane senses the change in phosphate concentration, phosphorylates its own histidine residue, and transfers the phosphate group to PhoB and PhoP. After receiving the signal from PhoR, PhoB will transmit the signal to PhoA and PstS, and then a phosphate assimilation reaction occurs inside the bacterial cells to maintain the phosphate level in the pathogenic bacteria. RegX3 can also receive the signal of reduced phosphate concentration and transmit the signal to PhoA and PstS simultaneously, promoting the generation of the phosphate assimilation reaction. At the same time, when PhoP receives the signal from PhoR, it will transmit the signal to PhoD, which can also cause phosphate assimilation inside the bacterial cells to maintain the phosphate concentration required by the bacterial cells.

[0114] VI. Verification experiment on the expression levels of differentially expressed genes in the transcriptome

[0115] The expression levels of 20 differentially expressed genes in the phoR-RNAi strain were verified by qRT-PCR to ensure the reliability of the transcriptome sequencing data. As Figure 15 shown, 10 up-regulated genes and 10 down-regulated genes were selected. The experiment showed that the results of qRT-PCR detection were consistent with the data trend of the transcriptome sequencing, proving that the sequencing data this time was available.

[0116] In summary, the present invention constructed a Vibrio alginolyticus phoR gene-silenced strain and detected the virulence effect of this strain. The constructed Vibrio alginolyticus phoR gene-silenced strain can not only be used to study the pathogenic mechanism of Vibrio alginolyticus, but also be used to develop vaccines for preventing or treating Vibrio alginolyticus disease, so as to find new targets for the prevention and treatment of Vibrio alginolyticus disease.

[0117] Although the specific implementation manners of the present invention have been described above, those skilled in the art of this technology should understand that the specific embodiments we described are illustrative rather than used to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should all be covered by the scope protected by the claims of the present invention.

Claims

1. A Vibrio alginolyticus phoR gene stably silenced strain, characterized in that: It is Vibrio alginolyticus phoR-RNAi, which was deposited at the China Center for Type Culture Collection on May 22, 2023. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2023799.

2. Use of the Vibrio alginolyticus phoR gene stably silenced strain according to claim 1, characterized in that: The application of the strain with stable silencing of the phoR gene of Vibrio alginolyticus in the preparation of a vaccine for preventing and / or treating Vibrio alginolyticus disease.

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