Application of amino peptidase sapm in regulating xanthomonas campestris pv. campestris virulence and motility

Abstract

The application discloses application of amino peptidase SapM in regulating virulence and motility of Xanthomonas campestris, and belongs to the technical field of biotechnology.The sapM gene function is researched by constructing a sapM gene deletion strain of Xanthomonas campestris.The research finds that knocking out the sapM gene (Delta sapM) significantly enhances the pathogenicity, extracellular polysaccharide (EPS) synthesis, amylase activity and motility of Xcc, while sapM overexpression inhibits the above-mentioned phenotypes.The flagellum morphology of the sapM mutant strain is further observed by transmission electron microscopy, and it is found that the flagellum length of the sapM mutant strain is significantly different from that of the wild type.It can be seen that the amino peptidase SapM is involved in negative regulation of virulence and motility of Xanthomonas campestris, and the application has great application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to the application of aminopeptidase SapM in regulating the virulence and motility of Xanthomonas campestris. Background Technology

[0002] Xanthomonas aeruginosa ( Xanthomonas campestris pv. campestris abbreviation Xcc Bacterium tumefaciens, also known as cruciferous black rot, widely infects cruciferous plants including Chinese cabbage, kale, radish, broccoli, cauliflower, arugula, and Arabidopsis, causing black rot in cruciferous vegetables worldwide. In recent years, with the general increase in the continuous cropping index of vegetables in my country, the occurrence of bacterial black rot in cruciferous vegetables has shown an increasing trend, posing a significant threat to cruciferous vegetable production in my country and causing huge economic losses. Conventional black rot control relies on crop rotation, disease-resistant varieties, and pesticides, but chemical control has the problem of residual pollution, and the pathogen rapidly develops resistance through genetic variation, exacerbating the difficulties in breeding disease-resistant varieties and environmental adaptability. Therefore, in-depth research is urgently needed. Xcc The study aims to investigate the physiological characteristics and pathogenic mechanisms of diseases, in order to provide a solid scientific theoretical support for building an efficient and eco-friendly integrated disease management strategy.

[0003] The M20 / M25 / M40 and M28 family of aminopeptidases are a class of conserved metallohydrolases, approximately 470 aa in size, and are found in the citrus canker pathogen (…). X. citri pv. citri ), enzyme-producing lysozyme ( Lysobacter enzymogenes ) and Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia It is widely present in bacteria such as ) and in Xcc A 92 aa SapM protein was discovered, labeled as an M20 / M25 / M40 family aminopeptidase. Further biological functions and applications of the SapM protein remain to be discovered. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the prior art and to provide the application of aminopeptidase SapM in regulating the virulence and motility of Xanthomonas campestris.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides the application of aminopeptidase SapM in regulating the virulence and motility of Xanthomonas campestris, wherein the amino acid sequence of aminopeptidase SapM is shown in SEQ ID NO.1; and the nucleotide sequence of the gene encoding aminopeptidase SapM is shown in SEQ ID NO.2.

[0007] Further, the knockout strain is obtained by gene knockout, and the virulence and motility of X. campestris are improved.

[0008] Further, the overexpression strain is obtained by gene overexpression, and the virulence and motility of X. campestris are reduced.

[0009] Further, the knockout strain is obtained by gene knockout, and the virulence and motility of X. campestris are improved. sapM The upstream and downstream fragments of the gene are obtained by primer amplification. sapM The upstream and downstream fragments of the gene are inserted into the enzyme-digested vector to construct a pK18-ΔsapM homologous recombination vector, which is transformed into the wild type strain to obtain.

[0010] Further, the suicide vector is pK18mobsacB.

[0011] Further, the overexpression strain is obtained by gene overexpression, and the virulence and motility of X. campestris are reduced. sapM The gene is taken as a target gene for amplification, and the amplified fragment is inserted into a vector by double enzyme digestion to construct an overexpression vector, which is transformed into the wild type strain to obtain.

[0012] Further, the gene overexpression vector is pBBR1-sapM.

[0013] In a second aspect, the application further provides a method for preventing and treating black rot of cruciferous crops, wherein an overexpression vector containing a SapM encoding gene is transformed into a Brassica oleracea Jingfeng No.1 plant to perform overexpression.

[0014] The nucleotide sequence of the amino peptidase SapM encoding gene is shown in SEQ ID NO. 2.

[0015] Compared with the prior art, the application has the following beneficial effects:

[0016] The application studies the function of the gene of X. campestris by constructing a gene deletion strain of X. campestris. sapM The application studies the function of the gene of X. campestris by constructing a gene deletion strain of X. campestris. sapM The research finds that knockout of the gene (Δ sapM ) significantly enhances the pathogenicity, extracellular polysaccharide (EPS) synthesis, amylase activity and motility of X. campestris, and overexpression inhibits the above-mentioned phenotypes. sapM The research finds that knockout of the gene (Δ Xcc ) significantly enhances the pathogenicity, extracellular polysaccharide (EPS) synthesis, amylase activity and motility of X. campestris, and overexpression inhibits the above-mentioned phenotypes. sapM The research finds that knockout of the gene (Δ sapM ) significantly enhances the pathogenicity, extracellular polysaccharide (EPS) synthesis, amylase activity and motility of X. campestris, and overexpression inhibits the above-mentioned phenotypes. sapM The research finds that knockout of the gene (Δ The application has great application value.

[0017] Figure 1Electrophoretogram of the mutant strain and overexpression strain in Example 1 sapM Electrophoretogram of the mutant strain and overexpression strain in Example 1 sapM Electrophoretogram of the mutant strain and overexpression strain in Example 1 sapM Electrophoretogram of the mutant strain and overexpression strain in Example 1

[0018] Figure 2 Electrophoretogram of the mutant strain and overexpression strain in Example 2 sapM Electrophoretogram of the mutant strain and overexpression strain in Example 2 sapM Electrophoretogram of the mutant strain and overexpression strain in Example 2 sapM Electrophoretogram of the mutant strain and overexpression strain in Example 2

[0019] Figure 3 Electrophoretogram of the mutant strain and overexpression strain in Example 3 ΔsapM Electrophoretogram of the mutant strain and overexpression strain in Example 3

[0020] Figure 4 Electrophoretogram of the mutant strain and overexpression strain in Example 4 sapM , ΔsapM / sapM and Xc1 / sapM Electrophoretogram of the mutant strain and overexpression strain in Example 4 DETAILED DESCRIPTION

[0021] In order to make the present application better understood, specific examples are listed as follows. Obviously, the described examples are only a part of the present application, but not all the examples. Based on the examples in the present application, other examples obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0022] The technical solutions of the present application are further described below by means of the accompanying drawings and examples.

[0023] The amino acid sequence of the aminopeptidase SapM is shown in SEQ ID NO. 1:

[0024] MLKPLGIAYEPSKGGPGPDVGPISAKGGAWAWLAQDGTDYFDLHHTADDTLDKIDPKALAQNVAAYTVFAYLAAEADGDFGSRAKSVQPPNE (SEQ ID NO. 1);

[0025] The nucleotide sequence of the aminopeptidase SapM coding gene is shown in SEQ ID NO. 2:

[0026] atgctgaagccgctgggcatcgcgtatgagcccagcaagggcggccctggcccggatgtggggccgatctctgccaagggcggtgcctgggcgtggctggcgcaggacggcaccgactacttcgatctgcaccacaccgcagacgacacgctggacaagatcgatccgaaggcgctcgcgcagaacgtggccgcctacaccgtgttcgcgtatctggccgccgaagccgatggcgatttcggcagccgcgcaaagtctgtgcagccgccgaacgagtaa (SEQ ID NO. 2);

[0027] The primer names and nucleotide sequences involved in the following examples are shown in Table 1 below:

[0028] Table 1 Primer sequences

[0029]

[0030] Note: Lowercase part is a protection base, and underlined part is a recognition sequence of restriction enzyme.

[0031] The NYG medium used in the following examples has the following formulation: 5 g / L Trypton, 3 g / L Yeast Extract, 20 g / L glycerol, pH 7.0 (using water as solvent, no agar powder is added when used in liquid medium, and 1.5% agar powder is added when used in solid medium).

[0032] Example 1 Construction of a gene mutation strain of Xanthomonas campestris SapM Construction of mutant strains, complemented strains and overexpression strains of mutants

[0033] 1. Δ sapM Construction of a mutant strain

[0034] In this study, a suicide vector pK18mobsacB was used to construct the mutant strain. The genome of Xanthomonas campestris Xc1 (strain Xc1) was used as a template, and the reported method (He et al., 2011) was referred to. Xanthomonas campestris pv. campestris strain Xc1) was used as a template, and the reported method (He et al., 2011) was referred to. et al.. Genome scale analysis of diffusible signal factor regulonin Xanthomonas campestris pv. campestris: identification of novel cell-cellcommunication-dependent genes and functions. 2006b; Li et al.. RpoN1 and RpoN2 play different regulatory roles in virulence traits, flagellar biosynthesis, and basal metabolism in Xanthomonas campestris (2020), the upstream fragment 1 of the sapM gene was amplified by pK18-sapM P1 / pK18-sapM P2; and the upstream fragment 1 of the sapM gene was amplified by pK18-sapM P3 / pK18-sapM P4. sapM Downstream segment 2 of the gene.

[0035] The amplified upstream fragment 1 and downstream fragment 2 were double-digested with EcoRI-KpnI and KpnI-HindIII, respectively. The pK18mobsacB vector was then double-digested with EcoRI and HindIII, and the digested fragments and pK18mobsacB vector were recovered. The upstream fragment 1, downstream fragment 2, and pK18mobsacB were ligated using T4 ligase (vector:fragment 1:fragment 2 = 1:1:1 or 1:5:5). sapM The upstream and downstream fragments of the gene were ligated into the pK18mobsacB plasmid, and the transformants were plated on LB resistant plates containing 50 μg / mL Kan to construct the pK18-ΔsapM homologous recombination vector.

[0036] Obtaining a recombinant strain in one step:

[0037] The correctly sequenced pK18-ΔsapM recombinant plasmid was heat-shocked into S17-1, and then transferred into Xanthomonas spp. Xc1 via a biparental conjugation method. Primary recombinant strains were screened using NYG antibiotics containing rifampicin and kanamycin. The recombinants were verified by PCR using pK18-sapM P1 (EcoRI) and pK18-sapM P4 (HindIII) primers. Strains with two bands in the PCR product and correctly sequenced band sizes were considered primary recombinant strains.

[0038] Obtaining the secondary recombinant strain:

[0039] Because the suicide vector pK18mobsacB carries sacBGenes are commonly used negative selection markers in Gram-negative bacteria; strains carrying these genes cannot grow on sucrose-containing media. Therefore, based on this principle, a single colony of a recombinant strain was picked and placed in 5 mL of fresh NYG liquid medium containing 50 μg / mL Rif, incubated overnight at 28°C with shaking. After dilution with sterile water, the culture was plated onto NYG+rifamidine monoclonal antibody plates containing 15% sucrose. After incubation at 28°C upside down for 3-4 days, single colonies were picked and streaked onto NYG+Rif+Kan and NYG+Rif plates respectively. Colonies that grew on NYG+Rif plates but not on NYG+Rif+Kan plates were selected for PCR verification using SAPMP5 and SAPMP6. Strains with correct PCR product bands were selected for sequencing verification; the strains with correct amplified fragments and loss of kanamycin resistance were identified as Δ strains. sapM Gene knockout strains ( Figure 1 A).

[0040] 2. Δ sapM / sapM and Xc1 / sapM strain construction

[0041] To further clarify Xc1Δ sapM Are the phenotypic changes in mutant strains caused by... sapM The gene deletion caused this, so we performed complementation and overexpression of the gene. The complete sequence of the target gene and the promoter sequence were amplified using pBBR1-sapM F (EcoRI) and pBBR1-sapM R (HindIII) primers.

[0042] DNA fragments were inserted into the pBBR1MCS5 vector using EcoRI and HindIII restriction sites. Transformants were plated on LB filtrates containing gentamicin and cultured to construct the pBBR1-sapM homologous recombination vector. Recombinant plasmids with correct sequencing results were transformed into ΔpM via electroporation. sapM From mutant strains and Xc1 wild-type strains, selection was performed by culturing with NYG+Gm+Rif double antibody plates, followed by extensive PCR screening, ultimately yielding... sapM Complementary strains (Δ sapM / sapM ) and overexpression strain (Xc1) / sapM ) ( Figure 1 B).

[0043] Example 2 Pathogenicity Detection

[0044] in accordance with Xcc The characteristics of infection through wounds on cruciferous plant leaves were studied. The leaf-cutting method was used to infect plants at the 5-leaf stage. Cabbage variety Jingfeng No. 1 (of similar age and size) was selected. Brassica oleraceacv. Jingfeng No.1) seedlings, with seedlings inoculated with Xc1 bacterial suspension as wild type, were used to control the inoculation. ΔsapM , ΔsapM / sapM and Xc1 / sapM Pathogenicity testing was performed on seedlings of the strain.

[0045] Strains from different treatment groups were cultured overnight at 220 rpm and 28°C with shaking for 24 h, and OD was measured. 600 =1.0. Using sterile scissors, cut the leaf perpendicularly to the midrib, 1 cm from the leaf tip. Gently apply the bacterial solution to the cut surface with a sterile cotton swab. Inoculate 30-50 leaves with each strain. Observe the length of lesions after 10 days, using plants inoculated with Xc1 bacterial solution as a wild-type control. The average lesion length was found to be 31.761 mm for plants inoculated with Xc1 bacterial solution, while... ΔsapM The resulting lesion length (40.72 mm) was significantly higher than that of the Xc1 wild type, after inoculation. ΔsapM / sapM The lesion length caused by Xc1 (32.234 mm) returned to that of the wild type; conversely, Xc1... / sapM The average length of the lesions caused was 23.199 mm, significantly lower than that of the wild type ( Figure 2 (AB). Further analysis of the colonization of each strain in plants revealed... ΔsapM The number of bacterial colonies in plants was significantly higher than that of wild-type Xc1, while Xc1... / sapM It is significantly lower than wild-type Xc1 ( Figure 2 C). These results indicate that SapM participates in negative regulation. Xcc Poisoning power.

[0046] Example 3 Determination of bacterial virulence factors and motility

[0047] Virulence factors are crucial for the pathogenicity of pathogenic bacteria; therefore, research has been conducted on... sapM right Xcc The influence of virulence factor synthesis was quantitatively analyzed. ΔsapM , ΔsapM / sapM and Xc1 / sapM Synthesis of amylase, protease and EPS in the middle.

[0048] 1. Extracellular enzyme detection

[0049] Sample preparation: The test strain was inoculated into 5 mL of NYG liquid medium and cultured at 28°C with shaking at 220 rpm for 16 h. The bacterial concentration was then adjusted to the OD value. 600 =1.0;

[0050] (a) Extracellular protease detection: 2 μL of bacterial solution was spotted on NYG plate (containing 1% skim milk) with a pipette, and incubated at 28°C for 48 h. The size of the transparent circle around the colony was observed and measured, and the relative enzyme activity = (R 2 -r 2 ) / r 2 ;

[0051] (b) Extracellular amylase detection: 2 μL of bacterial solution was spotted on NYG plate (containing 0.1% soluble starch) with a pipette, and incubated at 28°C for 48 h. The plate was stained with I2 / KI mixture (the molar concentration ratio of I2 and KI in the mixture was 1:108) for 3 min, and then decolorized with 70% ethanol. The size of the transparent circle around the colony was observed and measured;

[0052] (c) Extracellular cellulase detection: 2 μL of bacterial solution was spotted on NYG plate (containing 0.5% carboxymethyl cellulose sodium) with a pipette, and incubated at 28°C for 24 h. 20 mL of 0.1% Congo red solution was added to the plate and stained for 30 min, and then decolorized with 1 M NaCl for 20 min twice. The size of the transparent circle around the colony was observed and measured;

[0053] 2. Extracellular polysaccharide detection

[0054] The test strain was inoculated into 5 mL of NYG liquid medium, and incubated at 30°C with 220 rpm shaking for 16 h. The bacterial concentration was adjusted to OD 600 = 1.0. 1 mL of the bacterial culture was transferred into 100 mL of NYG liquid medium containing 4% glucose (containing 50 μg / mL rifampicin), and incubated at 30°C with 220 rpm shaking for 5 days. The extracellular polysaccharide was precipitated by adding 4 times the volume of anhydrous ethanol while stirring, and then the flocculent precipitate was removed and dried at 42°C. The weight of the dried precipitate was measured.

[0055] 3. Motility detection

[0056] The test strain was inoculated into 5 mL of NYG liquid medium, and incubated at 28°C with 220 rpm shaking for 16 h. The OD 600 was adjusted to 1.0. 2 μL of the bacterial solution was spotted on NYG plate (containing 0.3% agarose) with a pipette, and incubated at room temperature for 2 days. The colony diameter was counted, and the wild-type strain (Xc1) was used as a control. Each test strain was repeated at least 3 times.

[0057] The experimental results showed that, sapMGene mutations lead to Xcc Significantly increases the synthesis of amylase, protease and EPS (amylase, protease, and EPS). Figure 3 AC), on the contrary, sapM When overexpressed, it significantly reduces Xcc Synthesis of amylase, protease and EPS (amylase, protease and EPS) Figure 3 AC). Additionally, through analysis... sapM right Xcc The effects of motion were found in SapM. Xcc Exercise has a negative regulatory effect ( Figure 3 D). These findings illustrate SapM Participating in negative regulation Xcc Synthesis and motility of virulence factors.

[0058] Example 4: Observation of bacterial flagella morphology using transmission electron microscopy

[0059] Xc1, Δ sapM Δ sapM / sapM and Xc1 / sapM The culture was inoculated into NYG liquid medium and cultured for 24 h. Fresh bacterial culture was then streaked onto NYG plates and incubated at 28°C for 12 h. The method was followed according to the literature (Li...). et al.. RpoN1 andRpoN2 play different regulatory roles in virulence traits, flagellarbiosynthesis, and basal metabolism in Xanthomonas campestris 2020; Yu et al.. RpoN2- and FliA-regulated fliTX is indispensible for flagellar motility and virulence in Xanthomonas oryzae pv. oryzae (2017) Newly formed colonies were dissolved in sterile water, and the bacterial solution was then dropped onto a copper grid with a carbon support film. The cells were stained with 2% uranium acetate for 30 seconds and then air-dried for 10 minutes. Finally, the morphology of the bacterial flagella was observed using a Hitachi H-7650 transmission electron microscope.

[0060] Xc1 was observed using a transmission electron microscope. ΔsapM , ΔsapM / sapM and Xc1 / sapM The integrity of the flagella was found. sapM Mutation caused Xcc Increased flagella length sapM Complementary strains ( ΔsapM / sapMthe length of flagella of the overexpression strain (Xc1 sapM ) was significantly shorter than that of the wild type ( sapM ). Figure 4 ), indicating that sapM is involved in the negative regulation of Xcc flagellum synthesis.

[0061] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.

Claims

1. Use of the aminopeptidase SapM for modulating virulence and motility of Xanthomonas campestris, characterized in that, The amino acid sequence of the amino peptidase SapM is shown as SEQ ID NO. 1; the nucleotide sequence of the amino peptidase SapM coding gene is shown as SEQ ID NO. 2; the application is to knockout the amino peptidase SapM coding gene to improve the virulence and motility of Xanthomonas campestris.

2. Use according to claim 1, characterized in that, Gene knockout strains were obtained by primer amplification using a suicide vector. sapM Upstream and downstream gene fragments are digested with two enzymes to... sapM The upstream and downstream fragments of the gene were inserted into an enzyme digestion vector to construct the pK18-ΔsapM homologous recombination vector, which was then transformed into a wild-type strain for culture.

3. Use according to claim 2, characterized in that, The suicide vector is pK18mobsacB.

4. Use of the aminopeptidase SapM for modulating virulence and motility of Xanthomonas campestris, characterized in that, The amino acid sequence of the amino peptidase SapM is shown as SEQ ID NO. 1; the nucleotide sequence of the amino peptidase SapM coding gene is shown as SEQ ID NO. 2; the application is to overexpress the amino peptidase SapM coding gene to reduce the virulence and motility of Xanthomonas campestris.

5. Use according to claim 4, characterized in that, Gene overexpression strains are sapM The target gene is amplified, and the amplified fragment is inserted into a vector through double enzyme digestion to construct an overexpression vector, which is then transformed into a wild-type strain for culture.

6. Use according to claim 5, characterized in that, The gene overexpression vector is pBBR1-sapM.

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