Application of aminopeptidase SapM in regulation and control of virulence and motility of xanthomonas campestris
By constructing the aminopeptidase SapM gene deletion and overexpression strain, the virulence and motility of Xanthomonas wild rapeseed were regulated, and the regulation problems in the existing technology were solved, and the ecologically friendly disease management effect was achieved.
Patent Information
- Application Number
- CN202510607714.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-13
- Publication Date
- 2025-08-12
- Estimated Expiration
- 2045-05-13
AI Technical Summary
The prior art is difficult to effectively regulate the virility and motility of Xanthomonas wild rapeseed, and chemical control has problems of residual pollution and drug resistance, which affects the production of cruciferous vegetables.
By constructing the aminopeptidase SapM gene deletion strain and overexpression strain, the virulence and motility of Xenomonas wild rapeseed were regulated, and the suicide vectors pK18mobsacB and pBBR1-sapM vectors were used for gene knockout and overexpression, and ΔsapM and Xc1/sapM strains were constructed.
It significantly enhances or inhibits the pathogenicity, extracellular polysaccharide synthesis, amylase activity and motility of Xanthomonas wild rapeseed. The flagella length changes significantly, providing an ecologically friendly disease management strategy.
Smart Images
Figure CN120464608A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to application of aminopeptidase SapM in regulating the virulence and motility of Xanthomonas campestris. Background Art
[0002] Xanthomonas campestris pv. campestris (Xcc), also known as the crucifer black rot pathogen, widely infects cruciferous vegetables including cabbage, kale, radish, broccoli, cauliflower, rocket, and Arabidopsis, causing crucifer black rot worldwide. In recent years, with the widespread increase in the continuous cropping index of vegetables in my country, the incidence of bacterial black rot of cruciferous vegetables has shown an increasing trend, posing a major threat to cruciferous vegetable production in my country and causing huge economic losses. Conventional black rot prevention and control relies on crop rotation, disease-resistant varieties, and pesticides, but chemical control has residual contamination issues, and the pathogen rapidly develops resistance through genetic variation, exacerbating the difficulties in breeding disease-resistant varieties and environmental adaptability. Therefore, it is urgent to deeply explore the physiological characteristics and pathogenic mechanisms of Xcc in order to provide solid scientific theoretical support for the construction of efficient and eco-friendly integrated disease management strategies.
[0003] M20 / M25 / M40 and M28 family aminopeptidases are a conserved class of metallohydrolases, approximately 470 aminoaa in size, and are widely found in bacteria such as X. citri pv. citri, Lysobacter enzymogenes, and Stenotrophomonas maltophilia. A 92 aminoaa-sized SapM protein, annotated as an M20 / M25 / M40 family aminopeptidase, was discovered in Xcc. Further biological functions and applications of SapM proteins 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 shortcomings of the above-mentioned prior art and provide the application of aminopeptidase SapM in regulating the virulence and motility of Xanthomonas campestris.
[0005] To achieve the above object, the present invention adopts the following technical solutions:
[0006] In the first aspect, the present invention provides the use of aminopeptidase SapM in regulating the virulence and motility of Xanthomonas campestris. The amino acid sequence of the aminopeptidase SapM is shown in SEQ ID NO.1; the nucleotide sequence of the aminopeptidase SapM encoding gene is shown in SEQ ID NO.2.
[0007] Furthermore, knockout strains were obtained through gene knockout to improve the virulence and motility of Xanthomonas campestris.
[0008] Furthermore, an overexpression strain was obtained through gene overexpression to reduce the virulence and motility of Xanthomonas campestris.
[0009] Furthermore, the gene knockout strain uses a suicide vector as a vector, and primers are used to amplify the upstream and downstream fragments of the sapM gene. Through double enzyme digestion, the upstream and downstream fragments of the sapM gene are inserted into the enzyme-digested vector to construct a pK18-ΔsapM homologous recombination vector, which is then transferred into a wild-type strain for cultivation.
[0010] Furthermore, the suicide vector is pK18mobsacB.
[0011] Furthermore, the gene overexpression strain is obtained by amplifying the sapM gene as the target gene, inserting the amplified fragment into a vector through double enzyme digestion to construct an overexpression vector, and then transferring it into a wild-type strain for cultivation.
[0012] Furthermore, the gene overexpression vector is pBBR1-sapM.
[0013] In a second aspect, the present invention also provides a method for preventing and controlling black rot of cruciferous crops, wherein an overexpression vector containing the SapM encoding gene is transformed into cabbage Jingfeng No. 1 plant for overexpression.
[0014] The nucleotide sequence of the aminopeptidase SapM encoding gene is shown in SEQ ID NO.2.
[0015] Compared with the prior art, the present invention has the following beneficial effects:
[0016] The present invention studies the function of the sapM gene by constructing a sapM gene-deficient strain of Xanthomonas campestris. The study found that knocking out the sapM gene (ΔsapM) significantly enhanced the pathogenicity, extracellular polysaccharide (EPS) synthesis, amylase activity and motility of Xcc, while overexpression of sapM inhibited the above phenotypes. The flagella morphology of the sapM mutant strain was further observed by transmission electron microscopy, and it was found that the flagella length of the sapM mutant strain was significantly different from that of the wild type. This shows that the aminopeptidase SapM is involved in the negative regulation of the virulence and motility of Xanthomonas campestris, and the present invention has significant application value. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 Electrophoresis patterns were constructed for the ΔsapM mutant strain and the overexpression strain in Example 1 (Figure A is the electrophoresis pattern of the ΔsapM mutant strain; Figure B is the electrophoresis pattern of the sapM gene complementation strain and the overexpression strain);
[0018] Figure 2 Figure 2 shows the pathogenicity analysis of the ΔsapM mutant strains in Example 2 (Figure A shows the infection experiment of the ΔsapM series strains on "Jingfeng No. 1" cabbage leaves; Figure B shows the statistical analysis of lesion length. Pathogenicity was calculated by measuring lesion length, with 30-50 leaves measured each time; Figure C shows the statistical analysis of CFU data of the ΔsapM series strains);
[0019] Figure 3 Figures AB and CD are analysis diagrams of ΔsapM virulence factor synthesis and motility, and phenotypic diagrams in Example 3 (wherein, Figures AB and CD are amylase assays; Figures EF and EF are motility assays; and Figure G are exopolysaccharide content assays);
[0020] Figure 4 Figure 4 shows the difference in flagellar morphology between the ΔsapM, ΔsapM / sapM, and Xc1 / sapM strains under transmission electron microscopy (Figure A shows bacterial flagellar growth under transmission electron microscopy; Figure B shows flagellar length statistics). DETAILED DESCRIPTION
[0021] To better illustrate the present invention, the following embodiments are listed. Obviously, the embodiments described are only part of the present invention, not all of the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without making any creative efforts are also within the scope of protection of the present invention.
[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.
[0023] The amino acid sequence of aminopeptidase SapM is shown in SEQ ID NO.1:
[0024] MLKPLGIAYEPSKGGPGPDVGPISAKGGAWAWLAQDGTDYFDLHHTADDTTLDKIDPKA LAQNVAAYTVFAYLAAEADGDFGSRAKSVQPPNE(SEQ ID NO.1);
[0025] The nucleotide sequence of the aminopeptidase SapM encoding gene is shown in SEQ ID NO.2:
[0026] gtgctgaagccgctgggcatcgcgtatgagcccagcaagggcggccctggcccggatgtggggccgatctctgccaagggcggtgcctgggcgtggctggcgcaggacggcaccgactacttcgatctgcaccacaccgca gacgacacgctggacaagatcgatccgaaggcgctcgcgcagaacgtggccgcctacaccgtgttcgcgtatctggccgccgaagccgatggcgatttcggcagccgcgcaaagtctgtgcagccgccgaacgagtaa(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: The lowercase part is the protection base, and the underline is the recognition sequence of the restriction endonuclease.
[0031] The NYG medium used in the following examples has a formulation of: 5 g / L Tripton, 3 g / L Lyeast Extract, 20 g / L glycerol, pH 7.0 (water is used as the solvent, no agar powder is added when used in liquid culture medium, and 1.5% agar powder is added when used in solid culture medium).
[0032] Example 1 Construction of SapM gene mutant strains, complementation strains and overexpression strain mutants of Xanthomonas campestris
[0033] 1. Construction of ΔsapM mutant strain
[0034] In this study, the suicide vector pK18mobsacB was used to construct mutant strains. Using the genome of Xanthomonas campestris pv. campestris strain Xc1 as a template, following the methods reported in the literature (He et al.. Genome scale analysis of diffusible signal factor regulon in Xanthomonas campestris pv. campestris: identification of novel cell-cell communication-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 using pK18-sapMP1 / pK18-sapMP2, and the downstream fragment 2 of the sapM gene was amplified using pK18-sapMP3 / pK18-sapMP4.
[0035] The amplified upstream fragment 1 and downstream fragment 2 were double-digested with EcoRI-KpnI and KpnI-HindIII, respectively. The pK18mobsacB vector was double-digested with EcoR and HindIII, and the digested fragments and pK18mobsacB vector were recovered. T4 ligase was used to ligate upstream fragment 1, downstream fragment 2, and pK18mobsacB (vector: fragment 1: fragment 2 = 1:1:1 or 1:5:5). The upstream and downstream fragments of the sapM gene were ligated into the pK18mobsacB plasmid. Transformants were plated on LB resistance plates containing 50 μg / mL Kan and cultured to construct the pK18-ΔsapM homologous recombination vector.
[0036] Obtaining a recombinant strain:
[0037] The correctly sequenced pK18-ΔsapM recombinant plasmid was heat-shocked into S17-1 and then introduced into Xanthomonas campestris Xc1 via biparental conjugation. Primary recombinant strains were screened using the NYG dual-resistance primer containing rifampicin and kanamycin. Recombinants were verified by PCR using primers pK18-sapM P1 (EcoRI) and pK18-sapM P4 (HindIII). Strains with two PCR product bands and correct band size sequencing were considered primary recombinant strains.
[0038] Obtaining secondary recombinant strains:
[0039] Since the suicide vector pK18mobsacB carries the sacB gene, which is a commonly used negative selection marker in Gram-negative bacteria, strains carrying this gene cannot grow on a medium containing sucrose. Therefore, according to this principle, a single colony of the recombinant strain was picked and added to 5 mL of fresh NYG liquid medium containing 50 μg / mL Rif, cultured overnight at 28°C with shaking, diluted with sterile water, and spread on a NYG+Rifampicin monoclonal antibody plate containing 15% sucrose. After inverted culture at 28°C for 3-4 days, a single colony was picked and streaked on NYG+Rif+Kan and NYG+Rif plates, respectively. Colonies that grew on the NYG+Rif plate but could not grow on the NYG+Rif+Kan plate were selected for PCR verification using sapM P5 and sapM P6. The strain with the correct PCR product band was selected for sequencing verification. The strain with the correct amplified fragment and loss of kanamycin resistance was the ΔsapM gene knockout strain ( Figure 1 A).
[0040] 2. Construction of ΔsapM / sapM and Xc1 / sapM strains
[0041] To further clarify whether the phenotypic changes of the Xc1ΔsapM mutant strain were caused by the loss of the sapM gene, we complemented and overexpressed the gene. The complete sequence and promoter of the target gene were amplified using the pBBR1-sapM F (EcoRI) and pBBR1-sapM R (HindIII) primers.
[0042] The DNA fragment was inserted into the pBBR1MCS5 vector using the EcoRI and HindIII restriction sites, and the transformants were plated on LB resistance plates containing gentamicin to construct the pBBR1-sapM homologous recombination vector. The correctly sequenced recombinant plasmids were transformed into the ΔsapM mutant and Xc1 wild-type strains by electroporation, and cultured and screened on NYG+Gm+Rif double-resistance plates. After extensive PCR screening, the sapM complementation strain (ΔsapM / sapM) and overexpression strain (Xc1 / sapM) ( Figure 1 B).
[0043] Example 2 Pathogenicity Detection
[0044] Based on the characteristics of Xcc infecting leaf wounds of cruciferous plants, the leaf clipping method was used to infect Brassica oleracea cv. Jingfeng No.1 seedlings of uniform age and similar size were selected. The seedlings inoculated with Xc1 were used as the wild type, and the pathogenicity of the seedlings inoculated with ΔsapM, ΔsapM / sapM, and Xc1 / sapM strains was tested.
[0045] The strains in different treatment groups were cultured overnight at 220 rpm and 28 °C for 24 h, and the OD 600 =1.0. Use sterilized scissors to cut the leaf perpendicular to the midrib 1 cm away from the tip, and use a sterile cotton swab to dip the bacterial solution and gently smear the cut part of the leaf. Inoculate 30-50 leaves with each strain, observe the length of the lesions after 10 days, and use the plants inoculated with Xc1 bacterial solution as the wild type control. It was found that the average length of the lesions inoculated with Xc1 bacterial solution was 31.761 mm, while the length of the lesions caused by inoculation with ΔsapM (40.72 mm) was significantly higher than that of the Xc1 wild type. The length of the lesions caused by inoculation with ΔsapM / sapM (32.234 mm) returned to the wild type. On the contrary, the average length of the lesions caused by Xc1 / sapM was 23.199 mm, which was significantly lower than that of the wild type ( Figure 2 AB). Further analysis of the colonization of each strain in plants revealed that the number of bacterial colonies in plants with ΔsapM was significantly greater than that of wild-type Xc1. On the contrary, the ratio of Xc1 / sapM was significantly lower than that of wild-type Xc1 ( Figure 2 C) These results suggest that SapM is involved in the negative regulation of Xcc virulence.
[0046] Example 3 Bacterial virulence factors and motility determination
[0047] Virulence factors are crucial for the pathogenicity of pathogenic bacteria. To this end, the effect of sapM on the synthesis of Xcc virulence factors was studied, and the synthesis of amylase, protease and EPS in ΔsapM, ΔsapM / sapM and Xc1 / sapM was quantitatively analyzed.
[0048] 1. Extracellular enzyme detection
[0049] Sample processing: The strain to be tested was inoculated into 5 mL of NYG liquid medium and cultured in a shaking incubator at 220 rpm at 28°C for 16 h. The bacterial concentration was adjusted to OD 600 =1.0;
[0050] (a) Extracellular protease detection: 2 μL of bacterial solution was pipetted onto a NYG plate (containing 1% skim milk), left to rest for 10 min, and then incubated at 28°C for 48 h. The outer diameter (R) and inner diameter (r) of the transparent zone around the colony were observed and measured. The relative enzyme activity was calculated as (R 2 -r 2 ) / r 2 ;
[0051] (b) Extracellular amylase assay: 2 μL of bacterial culture was pipetted onto NYG plates (containing 0.1% soluble starch) and allowed to rest for 10 min. Incubated inverted at 28°C for 48 h. The plates were stained with an I2 / KI mixture (with a molar ratio of I2 to KI of 1:108) for 3 min, then destained with 70% ethanol. The size of the clear zone surrounding the colonies was measured.
[0052] (c) Extracellular cellulase assay: Use a pipette to spot 2 μL of bacterial culture onto a NYG plate (containing 0.5% sodium carboxymethyl cellulose). Allow to rest for 10 minutes and then incubate inverted at 28°C for 24 hours. Add 20 mL of 0.1% Congo red solution to the plate and stain for 30 minutes. Decolorize with 1 M NaCl for 20 minutes each, repeating twice. Observe and measure the size of the clear zone surrounding the colony.
[0053] 2. Extracellular polysaccharide detection
[0054] The strain to be tested was inoculated into 5 mL of NYG liquid medium and cultured in a shaking incubator at 30°C and 220 rpm for 16 h. The bacterial concentration was adjusted to OD 600 =1.0. Transfer 1 mL of the culture medium to 100 mL of NYG liquid medium containing 4% glucose (including 50 μg / mL rifampicin). Incubate at 30°C and 220 rpm on a shaker for 5 days. Add 4 volumes of anhydrous ethanol to precipitate the exopolysaccharide while stirring. Remove the flocculent precipitate, dry it at 42°C, and weigh it.
[0055] 3. Motility Detection
[0056] The strain to be tested was inoculated into 5 mL of NYG liquid medium and cultured in a shaking incubator at 220 rpm at 28°C for 16 h. The OD 600 If the amplification factor is 1.0, use a pipette to spot 2 μL of bacterial solution on a NYG plate (containing 0.3% agarose). Let it rest for 10 minutes and then incubate upright at room temperature for 2 days. Count the colony diameters, using the wild-type strain (Xc1) as a control. Repeat at least three times for each strain to be tested.
[0057] The experimental results showed that the mutation of sapM gene caused Xcc to significantly increase the synthesis of amylase, protease and EPS ( Figure 3AC), on the contrary, when sapM was overexpressed, the synthesis of Xcc amylase, protease and EPS was significantly reduced ( Figure 3 AC). In addition, by analyzing the effect of sapM on Xcc motility, it was found that SapM plays a negative regulatory role in Xcc motility ( Figure 3 D) These findings suggest that SapM is involved in negatively regulating Xcc virulence factor synthesis and motility.
[0058] Example 4 Transmission electron microscopy observation of bacterial flagella morphology
[0059] Xc1, ΔsapM, ΔsapM / sapM, and Xc1 / sapM were inoculated into NYG liquid medium and cultured for 24 hours. Fresh bacterial suspension was streaked onto NYG plates and incubated at 28°C for 12 hours. Following the methods reported in the literature (Li et al.. RpoN1 and RpoN2 play different regulatory roles in virulence traits, flagellar biosynthesis, and basal metabolism in Xanthomonas campestris. 2020; Yu et al.. RpoN2- and FliA-regulated fliTX is indispensable for flagellar motility and virulence in Xanthomonas oryzae pv. oryzae. 2017), newly formed bacterial colonies were dissolved in sterile water and the bacterial droplets were placed on copper grids coated with carbon support film. The cells were stained with 2% uranyl acetate for 30 seconds and air-dried for 10 minutes. Finally, the morphology of bacterial flagella was observed using a Hitachi H-7650 transmission electron microscope.
[0060] The integrity of the flagella of Xc1, ΔsapM, ΔsapM / sapM, and Xc1 / sapM was observed by transmission electron microscopy. The results showed that the sapM mutation caused the length of Xcc flagella to increase. The flagella length of the sapM complementation strain (ΔsapM / sapM) was the same as that of the wild type, and the flagella length of the sapM overexpression strain (Xc1 / sapM) was significantly shorter than that of the wild type ( Figure 4 ), indicating that sapM is involved in the negative regulation of Xcc flagellar synthesis.
[0061] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.
Claims
1. Application of aminopeptidase SapM in regulating the virulence and motility of Xanthomonas campestris, characterized in that: The amino acid sequence of the aminopeptidase SapM is shown in SEQ ID NO.1; the nucleotide sequence of the aminopeptidase SapM encoding gene is shown in SEQ ID NO.
2.
2. The use according to claim 1, characterized in that Knockout strains were obtained by gene knockout to improve the virulence and motility of Xanthomonas campestris.
3. The use according to claim 1, characterized in that An overexpression strain was obtained by gene overexpression, which reduced the virulence and motility of Xanthomonas campestris.
4. The use according to claim 2, characterized in that The gene knockout strain uses a suicide vector as a carrier, primers are used to amplify the upstream and downstream fragments of the sapM gene, and the upstream and downstream fragments of the sapM gene are inserted into the enzyme-cut vector through double enzyme digestion to construct a pK18-ΔsapM homologous recombination vector, which is then transferred into a wild-type strain for cultivation.
5. The use according to claim 4, characterized in that The suicide vector is pK18mobsacB.
6. The use according to claim 3, characterized in that The gene overexpression strain is obtained by amplifying the sapM gene as the target gene, inserting the amplified fragment into a vector through double enzyme digestion to construct an overexpression vector, and then transferring the vector into a wild-type strain for cultivation.
7. The use according to claim 6, characterized in that The gene overexpression vector is pBBR1-sapM.
8. A method for preventing and controlling black rot of cruciferous crops, characterized in that: The overexpression vector containing the SapM encoding gene was transformed into cabbage Jingfeng No. 1 plants for overexpression.
9. The method according to claim 8, characterized in that The nucleotide sequence of the aminopeptidase SapM encoding gene is shown in SEQ ID NO.2.