CN114403158B

CN114403158B - B-lysine-resistant bacillus bacteriocin analogue and application thereof in inhibition of pseudomonas syringae tomato pathogenic varieties

Info

Publication number: CN114403158B
Application number: CN202111524823.0A
Authority: CN
Inventors: 马香; 刘柱; 唐燕琼; 陈银华; 胡新文
Original assignee: Hainan University
Current assignee: Hainan University
Priority date: 2021-12-14
Filing date: 2021-12-14
Publication date: 2022-07-05
Anticipated expiration: 2041-12-14
Also published as: CN114403158A

Abstract

The application discloses an application of a B-lysine-resistant bacillus bacteriocin analogue in preventing and treating tomato bacterial spot disease, and discloses an application of the B-lysine-resistant bacillus bacteriocin analogue in inhibiting Pseudomonas syringae tomato pathogenic varieties causing the tomato bacterial spot disease. The application also discloses a preparation method of the boron-resistant lysine-resistant bacillus bacteriocin analogue, which is characterized in that escherichia coli is used as a chassis host cell, and the bacteriocin analogue gene cluster is heterologously expressed to obtain the boron-resistant lysine-resistant bacillus bacteriocin analogue. The B-lysine-resistant bacillus bacteriocin analogue has the advantages of simple preparation method, high yield and obvious inhibition effect on Pseudomonas syringae tomato pathogenic varieties causing tomato bacterial spot diseases; and in the application of preventing and treating the tomato bacterial spot disease, the tomato bacterial spot disease is pollution-free, does not generate drug resistance on tomatoes, and can completely replace chemical prevention and treatment of the tomato bacterial spot disease.

Description

B-lysine-resistant bacillus bacteriocin analogue and application thereof in inhibition of pseudomonas syringae tomato pathogenic varieties

Technical Field

The invention relates to the technical field of microbial synthesis, in particular to a boron-resistant lysine-resistant bacillus bacteriocin analogue and application thereof in inhibiting pseudomonas syringae tomato pathogenic varieties.

Background

With the increasing of drug-resistant strains, a green and safe antibiotic substitute is urgently needed to be found at present, and microorganisms can generate various natural secondary metabolites with biological activity, so that the microbial preparation has a wide application prospect in the fields of medical treatment, agriculture, food and the like, and is expected to be one of main sources for generating new drugs. However, the problems that the biosynthesis gene cluster of some secondary metabolites is in a silent state, the products are not easy to prepare, the yield is low and the like exist at present.

The transportation of synthetic organisms will help to solve the above problems. Synthetic biology is based on the study of system biology, introducing engineering principles and methods to construct artificial biological devices and systems with predictable behavior de novo, including computational models and modular DNA, to establish metabolic pathways and to construct biological circuits to control cellular behavior through standard design and interconnections between modular components. Therefore, the design and development of the microbial underpan cells based on the system biology and the synthetic biology can realize the construction and the modification of an excellent cell factory. At present, scientists have developed a heterologous expression synthetic biology platform, and escherichia coli is considered to be a high-efficiency heterologous host due to a complete genetic tool kit, and the purposes of discovering new compounds, clarifying a product synthesis path and optimizing product yield are achieved by combining heterologous expression path reconstruction and genetic engineering.

Pseudomonas syringae tomato pathogenic strain DC3000 (Pst DC3000) is a plant pathogenic bacterium of Pseudomonas syringae, and can cause diseases of plants such as tomato, pepper, Arabidopsis thaliana and the like. Bacterial leaf spot disease in tomato caused by PstDC3000 can infect the leaves, petioles, stems, flowers and fruits of tomato, causing severe loss of tomato yield. At present, the prevention and treatment method for the diseases mainly takes chemical prevention and treatment as a main part, has quick response, low cost and easy operation, but seriously threatens the ecological environment and human health by using a large amount of chemical agents for a long time. Therefore, the search for high-efficiency, low-toxicity, pollution-free and drug-resistance-free biological control measures has become a primary task.

Bacteriocins are a class of proteins or polypeptides with bacteriostatic activity produced by bacteria during metabolism by the ribosome synthesis mechanism. Bacteriocin has the advantages of small molecular weight, easy degradation by protease without residue, no toxic or side effect on human body and the like, and can be used for food preservation, human disease control, biological control and the like at present. The synthesis of bacteriocins generally requires multiple module interactions.

The invention discloses a boron-resistant lysine bacillus, which is a potential antagonistic biocontrol bacterium with an inhibiting effect on various pathogenic bacteria and pathogenic fungi.

Disclosure of Invention

Therefore, the invention aims to provide a boron-resistant lysine-resistant bacillus bacteriocin analogue and application thereof in inhibiting pseudomonas syringae tomato pathogenic varieties, and the technical scheme of the invention is realized by the following steps:

an application of B-lysine-resistant bacillus bacteriocin analogue in preventing and treating tomato bacterial spot disease. The boron-resistant lysine bacillus (Lysinibacillus boronitolorans) is preserved in the China Center for Type Culture Collection (CCTCC) in 2019, 10 and 8 months, is called CCTCC for short, addresses China-Wuhan university, and has the preservation number of CCTCC NO. M2019773.

The further technical proposal is that the boron-resistant lysine bacillus bacteriocin analogue is applied to inhibiting pseudomonas syringae tomato pathogenic varieties causing tomato bacterial spot disease.

Still a further embodiment is the use of said B.borotolerant lysine bacteriocin analogs in the inhibition of Pseudomonas syringae tomato pathogenic variants that cause bacterial blight of tomato.

According to a further technical scheme, the B.borotolerant bacteriocin analogue is applied to inhibiting the growth of the tomato pathogenic variety of Pseudomonas syringae causing bacterial spot disease of tomatoes.

The further technical proposal is that the Pseudomonas syringae tomato pathogenic variety is Pseudomonas syringae tomato pathogenic variety DC3000 Pseudomonas syringae pathovar tomato, Pst DC 3000.

The bacteriocin analogue gene cluster is a DNA fragment which is 8932bp in length and is obtained by carrying out PCR amplification by using a boron-resistant lysine bacillus whole genome as a template and Bac (10) -F and Bac (10) -R as primers.

Preferably, the sequences of the Bac (10) -F primers are as follows:

TAAGGATGATTTCTGGAATTCGGCTAAGTTTTTATATGAGTAATGTCAC, as shown in SEQ ID NO. 1.

The sequences of Bac (10) -R as primers are as follows:

GTTCCACAGGGTAGCGGATCCGCCTTCTGTTACCACTTCATCAAG, as shown in SEQ ID NO. 2.

Preferably, the sequence of the 8932bp DNA fragment is shown in SEQ ID NO. 3.

According to the preparation method of the boron-resistant lysine bacillus bacteriocin analogue, escherichia coli is used as a chassis host cell, and the boron-resistant lysine bacillus bacteriocin analogue is obtained by heterologously expressing the bacteriocin analogue gene cluster.

The further technical scheme is that the method comprises the following steps:

(1) extracting the genomic DNA of the B-resistant lysine bacillus and carrying out PCR amplification on a bacteriocin analogue gene cluster to obtain a DNA fragment;

(2) extracting plasmids, performing double digestion and recovering gel to obtain a vector;

(3) the DNA fragment is connected with a vector in a seamless cloning manner to obtain a cloned product;

(4) mixing the cloned product with escherichia coli competent cells, and then carrying out electric shock transformation to obtain thalli containing bacteriocin analogue gene cluster recombinant plasmids;

(5) the thalli is cultured and fermented to obtain fermentation liquor, the fermentation liquor is purified,

obtaining a purified product containing the B lysine-resistant Bacillus bacteriocin analog.

The further technical scheme is that the plasmid is p15A or pET.

The further technical scheme is that the host cell comprises E.coli BL 21.

Fermentation as described in the applicationThe liquid is a liquid containing bacteriocin analogs generated by fermenting the thallus, for example, the fermentation liquid of E.coli BL21 containing recombinant plasmids is 250mL of bacterial liquid (initial OD0.02OD)₆₀₀The resulting fermentation product was incubated at 37 ℃ for about 28h at 150r/min per 1000 mL/volume of medium. After fermentation, centrifuging at 9000r/min for 30min to remove precipitate, collecting fermentation supernatant, and filtering with 0.22 μm filter membrane to obtain sterile fermentation filtrate.

The ammonium sulfate precipitation method as referred to herein refers to a technique for precipitating and separating proteins using ammonium sulfate solutions of different concentrations. Ammonium sulfate precipitation can be used to concentrate and partially purify proteins from bulk crude preparations. High concentrations of salt ions compete with proteins for water molecules in protein solutions, thereby disrupting hydrated films on the protein surface, reducing its solubility, and allowing it to precipitate from solution. The solubility of each protein is different and thus different proteins can be precipitated using different concentrations of salt solution. This method is called salting out. Salt concentration is usually expressed in terms of saturation. Ammonium sulfate is most widely used because of its high solubility, low temperature coefficient and low tendency to denature proteins.

Compared with the prior art, the invention has the beneficial effects that:

(1) the B-lysine-resistant bacillus bacteriocin analogue has the advantages of simple preparation method, high yield and obvious inhibition effect on Pseudomonas syringae tomato pathogenic variants causing tomato bacterial spot diseases; and in the application of preventing and treating the tomato bacterial spot disease, the tomato bacterial spot disease is pollution-free, does not generate drug resistance on tomatoes, and can completely replace chemical prevention and treatment of the tomato bacterial spot disease.

(2) The boron-resistant lysine bacillus bacteriocin analogs described herein are a class of proteins or polypeptides with bacteriostatic activity produced by bacteria through the ribosome synthesis mechanism during the metabolic process. The bacteriocin has the advantages of small molecular weight, easy degradation by protease without residue, no toxic or side effect on human body and the like, and can be used for biological control, food preservation, human disease control and the like.

(3) The method uses seamless cloning to construct the vector, particularly preferentially selects a target fragment when the target fragment is large, and has high success rate and simple and convenient operation.

Drawings

FIG. 1 construction of recombinant plasmid vector. FIG. 1A is a PCR amplification gel electrophoresis of the bacteriocin analog biosynthesis gene cluster; FIG. 1B is the PCR gel electrophoresis of the gene cluster heterologous expression recombinant plasmid colony.

FIG. 2 is a graph showing the inhibition of Pseudomonas syringae tomato pathogenic mutant DC3000 by bacteriocin analogs concentrated by ammonium sulfate precipitation method using a filter paper method. Wherein, FIG. 2A is a control group, which is Pseudomonas syringae tomato var infestans DC3000 treated with fermentation broth of E.coli BL 2128 h containing empty plasmid; FIG. 2B is an experimental group of Pseudomonas syringae tomato pathogenic variety DC3000 treated with E.Coli BL 2128 h fermentation broth of recombinant plasmid containing gene cluster.

Detailed Description

For clear and complete description of the technical solutions in the present invention, the inventor has described the embodiments with reference to the drawings, but the following embodiments describe only some embodiments of the present invention, not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 predictive analysis of the bacteriocin analog Gene Cluster

1. Uploading the B-lysine-resistant bacillus genome file to an anti SMASH or BAGEL website, and predicting and analyzing the secondary metabolite biosynthesis gene cluster of the B-lysine-resistant bacillus genome file.

2. And (4) carrying out downstream analysis. After the gene cluster prediction is completed, downstream analysis is carried out, including structural domain analysis and annotation and core chemical structure prediction.

Cluster comparison analysis and smcAG (second approach Clusters of organisations Groups of Groups) analysis.

EXAMPLE 2 construction of Gene Cluster analog expression vector

1. And (3) extracting the genomic DNA and plasmid of the B-lysine-resistant bacillus.

5mL of overnight-cultured B.borascens-resistant bacillus liquid was taken, genomic DNA was extracted according to the instructions of the Vazyme Biotech bacterial genome extraction kit, and finally 2ul of the bacterial genome was taken. Likewise, 5mL of overnight-cultured E.coli DH 5. alpha. cells were harvested, and 2ul of plasmids were extracted according to the Vazyme Biotech plasmid Mini kit.

2. PCR amplification of B-lysine-resistant Bacillus bacteriocin analogue gene cluster.

And (3) performing PCR amplification on a target gene cluster by taking the extracted boron-resistant lysine bacillus whole genome as a template and Bac (10) -F and Bac (10) -R as primers respectively, and performing gel electrophoresis detection. The resulting DNA fragment (see FIG. 1A) with a clear band and an expected size (8932bp) indicates successful amplification of the gene cluster from the B.borotolerant genome. The PCR reaction system and amplification procedure are shown in tables 1 and 2:

TABLE 1 PCR amplification System

TABLE 2 PCR amplification procedure

And 3, purifying and recovering the PCR product. Product purification recovery was performed according to Vazyme Biotech product purification kit instructions, and finally 2 μ Ι _ was removed.

Double digestion and gel recovery of the p15A plasmid: the p15A plasmid was double digested with BamHI and EcoRI restriction enzymes and reacted at 37 ℃ for 6 h. After separating the cleavage products by electrophoresis on a 1% agarose gel, the gel containing the DNA fragment of interest was rapidly excised under UV light and placed in a 1.5mL EP tube, and the subsequent purification steps were performed according to the Vazyme Biotech product purification kit instructions. The double enzyme system is shown in Table 3:

TABLE 3 restriction enzyme double digestion System

5. Seamless clonal joining. According to

The Ultra One Step Cloning Kit was used for the procedures. The attachment system is shown in table 4:

TABLE 4 seamless clonal joining System

The use amount of the fragment and the vector is calculated according to the following formula, and the single fragment homologous recombination reaction is carried out. The optimum vector usage amount was [0.02 × vector base pair ] ng (0.03pmol), and the optimum insert usage amount was [0.04 × insert base pair ] ng (0.06 pmol).

② preparation of a connecting system was carried out according to Table 4.

③ reacting at 50 ℃ for 5 min; cooled to 4 ℃ or immediately placed on ice to cool.

Note: if the number of bases of the insert is much greater than the number of bases of the vector, the vector is counted as the insert.

6. The electric shock transformed BL21 competent cells. The shock conversion program was set to Bacteria. Taking out the sterilized and precooled electric rotating cup. And (3) placing the competent cells on ice for about 1min to dissolve the competent cells, adding 1-2 mu L of the ligation product or plasmid, blowing and uniformly mixing, quickly transferring the bacterial liquid into a groove of the electric rotating cup, slightly shaking for 3-5 times, quickly wiping water drops on the outer wall of the electric rotating cup, and performing electric shock transformation. After the electric transfer is finished, 1mL of LB liquid culture medium is added, the mixture is evenly blown and beaten, and the mixture is revived at 37 ℃ and 150rpm for 1 h. Centrifuging at 6000rpm for 3min to collect thallus, discarding supernatant, adding 100 μ L LB liquid culture medium, suspending thallus, spreading on spectinomycin resistant plate, and culturing at 37 deg.C for 12-16 h.

7. And (4) screening and identifying the recombinant plasmid. A single colony on the resistant plate was picked up and placed in a 1.5mL EP tube containing 40ul of sterile water, and heated in a 100 ℃ metal bath for 10min to completely lyse the thallus. Centrifuging at 12000rpm for 1min, taking 1 μ L of supernatant as a template of colony PCR, performing PCR amplification by using F3 and R3 primers, judging the size of a PCR product through 1% agarose gel electrophoresis, and finally obtaining a verification fragment of about 581bp, wherein the verification fragment conforms to the expected fragment size, and the successful construction of the vector is indicated (see FIG. 1B). Extracting positive clones to extract plasmids, and sending the plasmids to Shanghai Productivity Limited company for sequencing identification.

The F3 primer sequence is CCTTCTGCCACTGGTCGTTTATC, and the R3 primer sequence is GGCTTTACAGAACCGACACCAAT.

Example 3 detection of antibacterial Activity

The antibacterial activity of the bacteriocin analogue concentrated by the ammonium sulfate precipitation method on pseudomonas syringae tomato pathogenic variety DC3000 (target bacteria) is detected by using a filter paper method.

The extraction method comprises the following steps: ammonium sulfate was added to the E.coli BL21 fermentation broth containing the gene cluster recombinant plasmid or the E.coli BL21 fermentation broth of an empty plasmid to achieve ammonium sulfate saturation levels of 50%, 60%, 70% and 80%, and the mixture was precipitated overnight at 4 ℃. Centrifuging at 8000rpm for 10min, collecting precipitate, dissolving 1g precipitate with PBS (pH7.0), and diluting to 10mL volume to obtain bacteriocin analog solution (to-be-detected solution I) with different ammonium sulfate saturation and fermentation broth extract solution (to-be-detected solution II) of empty plasmid E.Coli BL 21.

A single colony of the target bacterium was picked up and cultured in 5mL of LB liquid medium at 37 ℃ for 12 hours at 150 rpm. By OD₆₀₀Initial inoculum size of 0.02 was transferred to 20mL LB liquid medium and cultured to OD₆₀₀0.4-0.6. Get 10⁸The bacterial solution was centrifuged at 6000rpm for 3min in a 1.5mL EP tube, the supernatant was discarded, 1mL of fresh LB medium was added, and the mixture was blown up and mixed.

Pipette 500. mu.L onto LB solid medium and spread well with sterile cotton swab. A 6mm sterile filter paper sheet (5 layers) was placed on the surface of the medium coated with the target bacteria.

Dripping the solution I to be detected with different ammonium sulfate saturation degrees prepared in advance onto a filter paper sheet, wherein the total amount is 80 mu L (dripping a small amount of solution for multiple times); and dropping the solution II to be detected with the corresponding ammonium sulfate saturation onto a filter paper sheet as a control, placing the filter paper I to be detected and the filter paper II to be detected with the same ammonium sulfate saturation on the same flat plate, and arranging three parallel plates for each ammonium sulfate saturation for 12 flat plates. After the culture at 30 ℃ for 12h, the growth of the target bacteria is observed, and as a result, the growth inhibition effect of the bacteriocin analogue solution with the saturation degree of ammonium sulfate of 80% on the growth of the pseudomonas syringae tomato pathogenic variety DC3000 is obvious, as shown in figure 2.

As shown in FIG. 2, the E.coli BL 2128 h fermentation broth of the recombinant plasmid containing the gene cluster treated the Pseudomonas syringae tomato pathogenic variant DC3000 showed a zone of inhibition, while the E.coli BL21 fermentation broth treated the Pseudomonas syringae tomato pathogenic variant DC3000 containing the empty plasmid did not show a zone of inhibition, which indicates that the bacteriocin analog of B-lysine-resistant Bacillus described in this application can be used to inhibit the biocontrol activity of the Pseudomonas syringae tomato pathogenic variant DC3000 by heterologous expression of the bacteriocin analog.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> university of Hainan

<120> B-lysine-resistant bacillus bacteriocin analogue and application thereof in inhibition of pseudomonas syringae tomato pathogenic varieties

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gttccacagg gtagcggatc cgccttctgt taccacttca tcaag 45

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taggtacttc gagtgccttc atcgcttgta ctagctttgc tacattttca attaccgttt 660

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tctctacagt aaacatcctg aacacctctt tttcttttct taataatatt atacaaaaac 780

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tatcattttt attgcaataa aaaatattta atatacggaa aatataactt aatttcaaaa 960

atacctttct atttgttcgt ttttaatgaa aaatacctgc ttaaatgtgt acattttgtt 1020

gcgagcctgt cgccattctg ctcaaatatt aagaatttta ttttttctct tgaaaattag 1080

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gtgcatacaa tttcaacgca ggtccatctg ctctaccaca agaagtatta gaaaatgctc 1200

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tgcctatgaa cttcttaaca actgagcaaa aagctagcta tgtattatca ggctcatggt 1440

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gcacaggaaa tgttccatta gtggcggata tgtcaagtga catcctatca aaaccagtgg 1680

atattgaaaa attcggtatc atctatgcag gagctcaaaa aaaccttggc ccttcaggtg 1740

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tcgaaggaaa tatgaaatct atccccatac aattagcttg attagatgtt tatagcaata 2340

tttcagctta gggctttaca cattgcaaaa gtttttgaga aaaatataga tttttaagct 2400

gcctatcgga catatcgcca gtctctagct atgcaccaaa catagattat tactgtaagg 2460

gttaccttta cgaaactaga ggaagggagg tctagacaat ggaagataaa agatatatgt 2520

cacatatgaa ctggcaaggc tgtaaatgtc ctaaaaaaga ggaaaaatgc tgtcaacgta 2580

aagttgaaaa atgtcattgc caaaaagagg aaaaatgcca ttgtaaaaaa gaagaaaaat 2640

gtacaccaag aatgagcagc cactgtagag gctgtatttg tcaccaatta agaaaattag 2700

agctagctac tacactagat atcttcttat ctggaggtat tagcttctta ggtgtgactt 2760

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aaatccatat aaaatgcact aattagacca attagtacag cagtttctcc aaaaacaata 3540

gcataaatga tggtttgctt aaagcccttt gccaagcgca ttgctgctgc gacaggtaat 3600

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gcagtcacaa tcataaataa aagatgaacc caacgagctg gaagtccact agcttttgca 3720

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acaatggcag caattccgat caccacccat aaatcttgac gagatacagc agaaaccgag 3840

ccaaacaaat aactcattaa atctgtgcta aagccactgg caagtgagat aaagatggcg 3900

ctaatcccaa taccacctga cataatgatt ggaatggcaa gctcctcata gtgcttatat 3960

aaacgtctta aacgctcaat caaaatagag ccactaacag aggctagtat acctaaataa 4020

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cacggaatgg ccataccaag catctagcgc gtcctgagaa atattatcaa attcattttt 4320

atagccgtgg aagtgaatgg tttgatttaa acatgccaca tgactaatac gatttgatac 4380

cgtatcgaca tcgtgtgtga caagaatcat cgtaattcct tgttcccgat ttaatttcgc 4440

cagcatatca taaaatgact gcacgttctc gtgatcaatg ccgacagttg gctcatctaa 4500

aatgagcagt ttaggatcac tgactaaagc tcttgcaata aaaacacgtt gttgttgtcc 4560

accagataat tctccaatat tgcgattcat ataagcatcc atgccaactg cctttaaagc 4620

ttcttgaact ttgtctttgg cgtctcggcc aggtctttta aaaagcccta atttttttgt 4680

taatccactt ttcacgacct cttgcacggt tgctgggaac ccagaattaa aggcattgga 4740

tttttgtgag acatagccaa tccattcacg gtgcttaaag ttcgcgcttg tttcaccaaa 4800

tagctttatt tcaccagata aaggtttcaa caaacctaaa ataattttta ataatgttga 4860

ttttcccgaa ccatttggac cgagaagtgc taaaaaatct ccctcttcta cacgaaaaga 4920

aatgtttttt aaaacttgcg tatagtcata ttgaaatgac acattttcaa tgtcaataag 4980

tgttgttttc atgcatgatc gcttctttct aattcaaaat cattacgatt tacaaattgt 5040

aagtataaag gaaagtgcag tttttgtaaa gaaaagaaga tggatgcttt ttcgaatagg 5100

ctgatggcaa gtgtgtaaaa cgtgaaaata aaatggggta aaggaggatt gactttgagt 5160

attcaatttt tacagcaatt agcacaaagc acggataagg aaattgtcgc gattgctcgt 5220

gaagaaggtt ttagcattac gacttctgaa gtaaaaaaac ttcgacctta tttggagcaa 5280

ttctcatttt cttggctatt tttaggtatt cctaaagata ttcttgttga agtggaagct 5340

gttttaggga gaaagcgctc tcgccaactg attgccttgt ttacaaaata aaaaaagccc 5400

gaaatcagaa ccagcctgat ttcgggttag ttatattagc cctttaaagc ttcaatatgc 5460

tcaggcttaa atgtaccgtt tcgtaacata tcgatttcca ctttatacgg agctacctta 5520

gatttagcgt cactagtgag agggacaaac ggtgtctcta aaattttagg aacatccatt 5580

aattgtggat ggtgtactac atagttgagg gcatcaaagc caatatgacc aaagccaata 5640

ttttcgtggc gatccttacc tgcaccgcgc acatttttgg agtcattgat atgcaatact 5700

ttaatgcgtt ccacacctat tattttatca aattcgttta ataccccatc aaaatcttcc 5760

acaatattat agccagcatc atgtgtatga caagtatcaa agcatacaga aagtcgctcg 5820

ttatttgtta caccatcaat aattttagca agctcttcaa aggaacgtcc acattctgtc 5880

cccttccctg ccatggtttc taatgcaatt tgaacgggat agtcttgaga taaaacctca 5940

tttaaacctt caataatttt agcaatacca gcatcagcac cagctcctac atgggcacca 6000

ggatgtaaca caatttgcgt tgcctccagt gctgcagtac gttcaatttc cgattgtagg 6060

aaatctacac cgagacggaa tgtttctggt ttttctgtat tgccaatatt aataatgtaa 6120

ggtgcatgta caacaatatt ggtcatacca tgctctttca tatgcaggag tccattcatg 6180

atgtttaagt cagcaatggc tttgcgacgc gtattttgcg gtgctccagt ataaatcata 6240

aatgtattgg ctccgtatga cagtgcctct ttactagagc caagtagcat ttctttcccg 6300

ctcatcgaaa catgtgagcc aagtaacatg taaaagcccc cttatttttt acctcgtgct 6360

tttaagcgac gttcacgttt tttaattttt tccatttccc acttcatatt acgtttgtat 6420

ccaggtttta cctttttagg cttacgtacc aacgcttttg ctttcacatc aatttcattt 6480

tcttgcttca cacgattttt acgtgcatga cgctctttta actctgacca ttcgccatct 6540

tttacatctt tttgcacaaa aggaatgccc attttttcta cgcgaactac tgcatcctcg 6600

tctgatggct caaataatgt aatggctgtc cctttattgc ctgcacgcgc tgttcgacca 6660

acacggtgaa taaagaattc taagtcctct ggaatctcgt agttaataac atgagaaatg 6720

ccttggatat caataccacg agcagctaag tcagttgcta caatgtattg gtagtcaagc 6780

tcacggattt gcttcatcat ttttttacga tcacgtggac ttaagtcacc atgaatttga 6840

ccacaacgga taccatgctc atttaagtag ccagcaacgt gttctgctgt tttacgtgtg 6900

ttggtaaaga taacagctaa aaagggatta atgccctcaa tgacttctaa taaacgttta 6960

ttacgcgatt ttgagcgcac tggtacaagc acaaaatcga tgccttctgc cactggtcgt 7020

ttatcattca tatggacgtg cactggcgct tccatatatt tctttaaaaa tggctgaagt 7080

ttttctggaa ttgttgcaga aaaaacatac atttcaagct tttctggcat ctgtgaagca 7140

aatccatcaa tctcttcaat gaagcccata tcaaaagcta gatccgcttc atcaacaact 7200

aagattggtg ctgtatgcac aaataacgct tgtgctgaaa caaggtcacg aatacgtcct 7260

ggtgttccaa caacaatttg tggctgtgtt tttaatttat caatggagcg ttgtttgtcc 7320

gtacccccaa taaatagttt tgcttgaatc gcagttcctt ctattaattg atttaatgca 7380

tcaaaaattt gttgtgctaa ctctcgagta ggagaagtaa taacagcctg tacttcctgt 7440

ttttctacat caatacgttg aacaatcggg attaaaaaac tatgtgtttt ccctgttcct 7500

gtatgtgcct gtccaatagc actttttcct tttaataaaa gcggaataat ttctttttga 7560

attggtgtcg gttctgtaaa gcctagattt gcaatggcgt cctgcaaaaa tggctgaaaa 7620

ttataatcag tatattttga catcattcgt tcctcctaac atctttttca ttgtaccatg 7680

attcgaactt taaggtatat ttacgttcta tttcttacta gcataaacat atttttaatg 7740

ttttattatc accgtaacaa agcccttatc aaaggtactt gaaatacgca gaatatgcaa 7800

tcaaaaaagc gctgcaacaa tgttcgaaaa cgcttgtaat atgtaatttt tatgtcaatt 7860

tatagggaac taatgattag cagatttccc acatgttatg atagagctgt aagatattat 7920

gggaggttgg tagattatga aagcagcaag atggtataaa gcaaaagaca tccgtgtaga 7980

aaacattgaa gaaccagtca tcgcacctgg aaaagtaaaa attaaagtac attggacagg 8040

gatttgtggc agtgatttac atgaatacgc agctgggcca atctttattc cagtggagca 8100

acctcactat gtaagtaaag acatcgcacc cattgtgatg ggacatgaat tttcaggaga 8160

agtagttgaa attggcgatg gtgtaacatc tgttcaaata ggagatcctg tagtagtaga 8220

accaatcctt gcatgtggcg aatgtgctgc ttgtaaaaaa ggtaaatata atatttgtaa 8280

acatttaggt ttccacggtc tttcaggcgg tggcggagga ttctccgaat atacaatggt 8340

agatgaaaaa atggttcaca aaatgccaga agggctttct tatgagcaag gagcacttgt 8400

agaaccagcg gcagtcgctt tacatgccgt gcgtcaaagt aaattaaaag ccggtgacaa 8460

agctgccgtc tttggaacag gaccaatcgg gcttcttgtt attgaagcat tacgtgcagc 8520

aggcgcagca gaaatctatg ctgtagagct ttcagcagag cgtgccgcaa aagctttaga 8580

acttggtgca acagccgtca ttaaccctaa agaggaagat gccgttgtgc gtctgcatga 8640

attaacaaat ggtggtgtag atgttgcctt tgaagtaacg ggcgttcctg tggttttaca 8700

acaagctatt gactcaacaa cttttgaagg tgaaacaatc attgtgtcga tttgggagtc 8760

tacagctgct atccaaccga acaatattgt tctatcagag cgaacagtca aaggaattat 8820

tgcctaccgt gatattttcc cagccgtgat ggagctaatg acacaaggtt acttcccagc 8880

agacaagctt gttacaaaac gaattgctct tgatgaagtg gtaacagaag gc 8932

Claims

1. The application of the boron-resistant lysine-resistant bacillus bacteriocin analog in preventing and treating bacterial spot diseases of tomatoes is characterized in that escherichia coli is used as a chassis host cell, and the boron-resistant lysine-resistant bacillus bacteriocin analog is obtained by heterologously expressing a bacteriocin analog gene cluster;

the bacteriocin analogue gene cluster is B-lysine resistant bacillus (B)Lysinibacillus boronitolerans) The whole genome is used as a template, respectivelyUsing Bac (10) -F and Bac (10) -R as primers, and carrying out PCR amplification to obtain a DNA fragment with the length of 8932 bp;

the sequence of the Bac (10) -F primer is shown as SEQ ID NO. 1;

the sequence of the Bac (10) -R primer is shown as SEQ ID NO. 2;

the boron-resistant lysine bacillus is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 and 8 months, and the preservation number is CCTCC NO. M2019773.

2. The use of a B lysine-resistant Bacillus bacteroides bacteriocin analog according to claim 1 for the control of bacterial spot disease in tomato: the sequence of the 8932bp DNA fragment is shown in SEQ ID NO. 3.

3. A lysine-resistant bacillus boronisin analog, comprising: taking escherichia coli as a chassis host cell, and obtaining the boron-resistant lysine bacillus bacteriocin analogue through heterologous expression of a bacteriocin analogue gene cluster;

the bacteriocin analogue gene cluster is B-lysine resistant bacillus (B)Lysinibacillus boronitolerans) Taking a whole genome as a template, and respectively taking Bac (10) -F and Bac (10) -R as primers, and carrying out PCR amplification to obtain a DNA fragment with the length of 8932 bp;

the sequence of the Bac (10) -F primer is shown as SEQ ID NO. 1;

the sequence of the Bac (10) -R primer is shown as SEQ ID NO. 2;

the sequence of the 8932bp DNA fragment is shown as SEQ ID NO. 3;

the B-lysine-resistant bacillus is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 and 8 months, and the preservation number is CCTCC NO. M2019773.

4. A method of preparing a boron-tolerant lysine bacillus bacteriocin analog of claim 3, wherein: and (3) taking escherichia coli as a chassis host cell, and obtaining the boron-resistant lysine bacillus bacteriocin analogue through heterologous expression of a bacteriocin analogue gene cluster.

5. The method of claim 4 for the preparation of a borotolerant lysine bacillus bacteriocin analog, wherein: the method comprises the following steps:

(3) the DNA fragment is in seamless cloning connection with a vector to obtain a cloned product;

(5) and (3) performing culture fermentation on the thalli to obtain fermentation liquor, centrifuging, removing precipitates, collecting fermentation supernatant, and purifying the fermentation supernatant to obtain a purified product containing the boron-resistant lysine bacillus bacteriocin analogue.

6. The method of claim 5, wherein the method comprises the steps of: the plasmid is p15A or pET, and the Escherichia coli comprisesE. coli BL21。