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

Abstract

The present application discloses the application of a borolysine-resistant bacillus bacteriocin analog in the prevention and treatment of tomato bacterial spot, and discloses that the borolysine-resistant bacillus bacteriocin analog can inhibit the cause of tomato bacterial spot Application of the disease of Pseudomonas syringae in tomato pathogenic varieties. The present application also discloses a method for preparing the borolysine-resistant Bacillus bacteriocin analogs. The boron-resistant lysine is obtained by heterologously expressing the bacteriocin analog gene cluster by using Escherichia coli as a chassis host cell. Acidobacillus bacteriocin analogs. The borolysine-resistant Bacillus bacteriocin analog described in the present application has a simple preparation method and high yield, and has obvious inhibitory effect on tomato pathogenic varieties of Pseudomonas syringae that cause bacterial spot disease of tomato; In the control and application of bacterial spot disease, it is pollution-free and does not produce resistance to tomato, and can completely replace the chemical control of tomato bacterial spot disease.

Description

A borolysine-resistant Bacillus bacteriocin analog and its application in inhibiting Pseudomonas syringae pathogenic varieties of tomato

technical field

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

Background technique

With the increasing number of drug-resistant strains, there is an urgent need to find green and safe alternatives to antibiotics, and microorganisms can produce a variety of biologically active natural secondary metabolites, which will have broad applications in medical, agricultural and food fields. The prospect is expected to be one of the main sources of new drugs. However, at present, some biosynthetic gene clusters of secondary metabolites are in a silent state, the products are not easy to prepare and the yield is low.

The emergence of synthetic biology will help solve the above problems. Synthetic biology is based on the research of systems biology, introducing engineering principles and methods to build artificial biological devices and systems with predictable behavior from scratch, including computational models and modular DNA, through standard design and modular components. Interconnect to establish metabolic pathways and build biological circuits to control cellular behavior. Therefore, the design and development of microbial chassis cells based on systems biology and synthetic biology can realize the construction and transformation of excellent cell factories. At present, scientists have developed a synthetic biology platform for heterologous expression, and Escherichia coli has been considered as an efficient heterologous host due to its complete genetic toolbox. Through the combination of heterologous expression pathway reconstruction and genetic engineering, discovery can be achieved. New compounds, elucidation of product synthesis pathways, and the purpose of optimizing product yields.

Pseudomonas syringae pathovar tomato DC3000 (Pseudomonas syringae pathovar tomato, Pst DC3000) is a class of plant pathogenic bacteria of the genus Pseudomonas syringae, which can cause diseases in tomato, pepper and Arabidopsis. Tomato bacterial spot caused by PstDC3000 can infect tomato leaves, petioles, stems, flowers and fruits, resulting in serious loss of tomato yield. At present, chemical control methods are mainly used for the prevention and control of this disease, which is quick in effect, low in cost and easy to operate. Therefore, it has become the primary task to seek high-efficiency, low-toxicity, non-polluting and non-resistance biological control measures.

Bacteriocins are a class of proteins or polypeptides with bacteriostatic activity produced by bacteria through the ribosome synthesis mechanism during metabolism. Bacteriocin has the advantages of small molecular weight, easy to be degraded by protease without residue, and no toxic and side effects to human body. The synthesis of bacteriocin often requires multiple modular interactions.

Boron-resistant lysine-resistant Bacillus is a potential antagonistic biocontrol bacterium that has inhibitory effects on a variety of pathogenic bacteria and pathogenic fungi. Host cells, through heterologous expression of bacteriocin gene clusters, use synthetic biology to redesign systems for specific targets, ultimately producing biologically active bacteriocin antibacterial actives.

SUMMARY OF THE INVENTION

In view of this, the object of the present invention is to propose a kind of boron-resistant lysine bacillus bacteriocin analog and its application in suppressing Pseudomonas syringae tomato pathogenic varieties, the technical scheme of the present invention is realized like this:

Application of a borolysine-resistant bacillus bacteriocin analog in preventing and treating tomato bacterial spot. The Lysinibacillus boronitolerans was deposited in the China Center for Type Culture Collection on October 8, 2019, referred to as CCTCC, the address is China-Wuhan-Wuhan University, and the deposit number is CCTCC NO.M2019773.

A further technical solution is the use of the boron lysine-resistant bacillus bacteriocin analog in inhibiting the tomato pathogenic variety of Pseudomonas syringae that causes bacterial spot disease of tomato.

A still further technical solution is the use of the boron lysine-resistant bacillus bacteriocin analog in inhibiting the tomato pathogenic variety of Pseudomonas syringae that causes bacterial spot disease of tomato.

A further technical solution is the application of the borolysine-resistant bacillus bacteriocin analogs in inhibiting the growth of Pseudomonas syringae tomato pathogenic varieties that cause bacterial spot disease of tomato.

A further technical solution is that the Pseudomonas syringae pathovar tomato is Pseudomonas syringae pathovar tomato, Pst DC3000.

The above-mentioned bacteriocin analog gene cluster is a DNA with a length of 8932 bp obtained by PCR amplification using the whole genome of Bacillus boron-resistant lysine as a template and using Bac(10)-F and Bac(10)-R as primers respectively. Fragment.

Preferably, the sequence of the Bac(10)-F primer is:

TAAGGATGATTTCTGGAATTCGGCTAAGTTTTTATATGAGTAATGTCAC as shown in SEQ ID NO.

Described Bac(10)-R is the sequence of primer:

GTTCCACAGGGTAGCGGATCCGCCTTCTGTTACCACTTCATCAAG is shown in SEQ ID NO.2.

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

The preparation method of the above-mentioned boron lysine-resistant bacillus bacteriocin analogs, takes Escherichia coli as the chassis host cell, and obtains the boron lysine-resistant bacillus bacteriocin analogs by heterologously expressing the bacteriocin analog gene cluster thing.

A further technical solution includes the following steps:

(1) Extraction of genomic DNA of Boron-resistant Bacillus and PCR amplification of bacteriocin analog gene clusters to obtain DNA fragments;

(2) Extraction of plasmid, double enzyme digestion and gel recovery to obtain a vector;

(3) the DNA fragment is seamlessly cloned and connected to the carrier to obtain a cloned product;

(4) carrying out electroshock transformation after mixing the cloned product with Escherichia coli competent cells to obtain a thalline containing a bacteriocin analogue gene cluster recombinant plasmid;

(5) described thalline obtains fermented liquid through cultivation and fermentation, and fermented liquid is purified,

A purified product containing a borolysine-resistant bacillus bacteriocin analog was obtained.

A further technical solution is that the plasmid is p15A or pET.

A further technical solution is that the host cell comprises E. coli BL21.

The fermentation liquid mentioned in this application refers to the liquid containing bacteriocin analogs produced by the bacterial cells after fermentation. For example, the E.Coli BL21 fermentation liquid containing recombinant plasmids is 250 mL of bacterial liquid (initial _OD0.02OD600 /mL)/1000mLLB The filling volume of the medium, the fermentation product obtained by culturing at a temperature of 37°C at a speed of 150r/min for about 28h. After the fermentation, centrifuge at 9000 r/min for 30 min to remove the precipitate, collect the fermentation supernatant, and then filter with a 0.22 μm membrane to obtain a sterile fermentation filtrate.

The ammonium sulfate precipitation method mentioned in this application refers to the technology of precipitating and separating proteins with different concentrations of ammonium sulfate solutions. Ammonium sulfate precipitation can be used to concentrate and partially purify proteins from bulk crude preparations. High concentrations of salt ions can compete with proteins for water molecules in protein solutions, thereby destroying the hydration membrane on the surface of proteins, reducing their solubility and causing them to precipitate out of solution. The solubility of various proteins is different, so different concentrations of salt solutions can be used to precipitate different proteins. This method is called salting out. Salt concentration is usually expressed in terms of saturation. Ammonium sulfate is the most widely used because of its high solubility, low temperature coefficient and difficulty in denaturing proteins.

Compared with the prior art, the beneficial effects of the present invention are:

(1) The boron lysine-resistant bacillus bacteriocin analog described in the present application has a simple preparation method and high yield, and has an obvious inhibitory effect on tomato pathogenic varieties of Pseudomonas syringae that cause bacterial spot disease of tomato; Moreover, in the prevention and control of tomato bacterial spot, it is pollution-free and does not produce drug resistance to tomato, and can completely replace the chemical control of tomato bacterial spot.

(2) The boron lysine-resistant bacillus bacteriocin analogs described in the present application are a class of proteins or polypeptides with bacteriostatic activity produced by bacteria through the ribosome synthesis mechanism during the metabolic process. Bacteriocin has the advantages of small molecular weight, easy to be degraded by protease without residue, no toxic and side effects to human body, etc. In addition to being used for biological control, it can also be used in food preservation and human disease prevention and control.

(3) The present application uses seamless cloning to construct the vector, especially when the target fragment is relatively large, which not only has a high success rate but also is easy to operate.

Description of drawings

Figure 1 Construction of recombinant plasmid vector. Figure 1A: PCR-amplified gel electrophoresis image of bacteriocin analog biosynthetic gene clusters; Figure 1B: PCR gel electrophoresis image of gene clusters heterologous expression recombinant plasmid colonies.

Fig. 2 is the use of filter paper method to detect the inhibitory effect of bacteriocin analogues concentrated by ammonium sulfate precipitation method on Pseudomonas syringae tomato pathogenic variety DC3000. Among them, Fig. 2A is the control group, which is the treatment of Pseudomonas syringae tomato pathogenic variety DC3000 with the fermentation broth of E.Coli BL21 containing an empty plasmid for 28h; Fig. 2B is the experimental group, which is the E. .Coli BL21 28h fermentation broth treatment of Pseudomonas syringae tomato pathogenic variety DC3000.

Detailed ways

In order to clearly and completely describe the technical solutions of the present invention, the inventors describe with reference to the drawings and the embodiments, but the following embodiments describe only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1 Predictive analysis of bacteriocin analog gene clusters

1. Upload the Bacillus borolysine-resistant Bacillus genome file to the antiSMASH or BAGEL website, and predict and analyze the secondary metabolite biosynthesis gene clusters it possesses.

2. Downstream analysis. After the gene cluster prediction is completed, downstream analysis is performed, including the analysis and annotation of the structural domain, and the prediction of the core chemical structure.

3.ClusterBlast gene cluster comparative analysis and smCOG (secondary metabolite Clusters of Orthologous Groups of groups) analysis.

Example 2 Construction of gene cluster analog expression vector

1. Extraction of genomic DNA and plasmids of borolysine-resistant Bacillus.

Take 5 mL of the Bacillus borolysine-resistant Bacillus bacterium cultured overnight, extract genomic DNA according to the instructions of the Vazyme Biotech bacterial genome extraction kit, and finally take 2 ul. Similarly, 5 mL of overnight cultured Escherichia coli DH5α bacterial solution was taken, and the plasmid was extracted according to the instructions of the Vazyme Biotech plasmid mini-extraction kit, and finally 2 ul was taken.

2. PCR amplification of the borolysine-resistant Bacillus bacteriocin analogue gene cluster.

Using the whole genome of Bacillus borolysine-resistant Bacillus as a template and Bac(10)-F and Bac(10)-R as primers, the target gene clusters were amplified by PCR and detected by gel electrophoresis. Finally, a DNA fragment with a clear band and the expected size (8932 bp) was obtained (see Fig. 1A ), indicating that the gene cluster was successfully amplified from the genome of Bacillus borolysine resistant. The PCR reaction system and amplification procedures are shown in Table 1 and Table 2:

Table 1 PCR amplification system

Table 2 PCR amplification program

3. The PCR product was purified and recovered. Purify and recover the product according to the instructions of the Vazyme Biotech product purification kit, and finally remove 2 μL.

4. Double digestion and gel recovery of p15A plasmid: double digestion of p15A plasmid with BamHI and EcoRI restriction enzymes, and react at 37°C for 6h. After the digestion product was separated by 1% agarose gel electrophoresis, the gel containing the target DNA fragment was quickly cut off under UV light and placed in a 1.5mL EP tube. The subsequent purification steps were performed according to the Vazyme Biotech product purification kit. Instruction manual. The double enzyme digestion system is shown in Table 3:

Table 3 Restriction endonuclease double digestion system

Ultra One Step Cloning Kit试剂盒进行操作。连接体系如表4所示：5. Seamless clone connection. according to

Ultra One Step Cloning Kit for operation. The connection system is shown in Table 4:

Table 4 Seamless cloning ligation system

①According to the following formula to calculate the amount of fragment and vector used, for single-fragment homologous recombination reaction. The optimum amount of vector used = [0.02×number of base pairs of vector]ng (0.03pmol), and the optimum amount of insert used=[0.04×number of base pairs of insert]ng (0.06pmol).

② Prepare the connection system according to Table 4.

③Reaction at 50℃ for 5min; drop to 4℃ or cool on ice immediately.

Note: If the base number of the insert is much larger than the base number of the vector, the vector should be counted as the insert.

6. Transform BL21 competent cells by electric shock. Set the electroshock transformation program to Bacteria. Remove the sterilized and pre-cooled rotor cup. Place the competent cells on ice for about 1 min to dissolve, add 1-2 μL of ligation product or plasmid, mix by pipetting, quickly transfer the bacterial solution to the groove of the electric rotor, shake gently for 3-5 times, and wipe quickly Water droplets on the outer wall of the electric rotor cup for electric shock conversion. After electroporation, 1 mL of LB liquid medium was added, mixed by pipetting, and recovered for 1 h at 37°C and 150 rpm. The cells were collected by centrifugation at 6000 rpm for 3 min, the supernatant was discarded, 100 μL of LB liquid medium was added, the cells were suspended, coated on a spectinomycin-resistant plate, and cultured at 37° C. for 12-16 h.

7. Screening and identification of recombinant plasmids. Pick a single colony on the resistant plate into a 1.5 mL EP tube filled with 40 ul of sterile water, and place it in a 100°C metal bath for 10 min to completely lyse the bacterial cells. Centrifuge at 12,000 rpm for 1 min, take 1 μL of the supernatant as the template for colony PCR, use F3 and R3 primers for PCR amplification, and judge the size of the PCR product by 1% agarose gel electrophoresis. Finally, a verification fragment of about 581bp is obtained, which is in line with expectations. The fragment size indicated that the vector was constructed successfully (see Figure 1B). Positive clones were extracted for plasmid extraction and sent to Shanghai Sangon Co., Ltd. for sequencing identification.

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

The detection of embodiment 3 antibacterial activity

The antibacterial activity of the bacteriocin analogues concentrated by ammonium sulfate precipitation against Pseudomonas syringae tomato pathogenic variety DC3000 (target bacteria) was detected by filter paper method.

Extraction method: Add ammonium sulfate to the fermentation broth of E.Coli BL21 containing gene cluster recombinant plasmid or to the fermentation broth of E.Coli BL21 with empty plasmid to make ammonium sulfate saturation reach 50%, 60%, 70% and 80%, Precipitate overnight at 4°C. Centrifuge at 8000 rpm for 10 min, collect the precipitate, dissolve 1 g of the precipitate with PBS (pH 7.0) and dilute to 10 mL to obtain bacteriocin analogue solutions with different ammonium sulfate saturations (solution I) and empty plasmid E.Coli BL21 The fermentation broth extract solution (testing solution II).

A single colony of the target bacteria was picked and cultured in 5 mL of LB liquid medium at 37° C. and 150 rpm for 12 h. According to the initial inoculation amount of OD ₆₀₀ = 0.02, it was transferred to 20 mL of LB liquid medium, and cultured to OD ₆₀₀ = 0.4-0.6. Take 10 ⁸ bacterial liquid into a 1.5 mL EP tube, centrifuge at 6000 rpm for 3 min, discard the supernatant, add 1 mL of fresh LB medium, and mix by pipetting.

Pipette 500 μL onto the LB solid medium and spread evenly with a sterile cotton swab. A 6 mm piece of sterile filter paper (5 layers) was placed on the surface of the medium coated with the target bacteria.

Drop the test solution I prepared in advance with different ammonium sulfate saturation on the filter paper, and the total amount is 80 μL (a small amount of drops are added several times); add the test solution II with the corresponding ammonium sulfate saturation to the filter paper as a control. The test I filter paper and the test liquid II filter paper with the same ammonium sulfate saturation are placed on the same plate, and three parallel plates are set for each ammonium sulfate saturation, for a total of 12 plates. After culturing at 30°C for 12 hours, the growth of the target bacteria was observed. The results showed that the bacteriocin analogue solution with ammonium sulfate saturation of 80% had obvious growth inhibition effect on the growth of Pseudomonas syringae tomato pathogenic variety DC3000, as shown in Figure 2 shown.

As shown in Figure 2, the E.Coli BL21 28h fermentation broth containing the recombinant plasmid of the gene cluster treated Pseudomonas syringae tomato pathogenic variety DC3000 with a bacteriostatic zone, while the E.Coli BL21 fermentation broth containing the empty plasmid treated cloves Pseudomonas tomato pathogenic variety DC3000 did not show a bacteriostatic zone, indicating that the bacteriocin analogues of borolysine-resistant bacillus described in this application, after heterologous expression, can endow recombinant Escherichia coli to inhibit Pseudomonas syringae Biocontrol activity of the pathogenic bacterium tomato varietal DC3000.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the scope of the present invention. within the scope of protection.

sequence listing

<110> Hainan University

<120> A borolysine-resistant Bacillus bacteriocin analog and its application in inhibiting Pseudomonas syringae pathogenic varieties of tomato

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aattgtggat ggtgtactac atagttgagg gcatcaaagc caatatgacc aaagccaata 5640

ttttcgtggc gatccttacc tgcaccgcgc acatttttgg agtcattgat atgcaatact 5700

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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 ccttttttagg 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 (6)

1. the application of a boron-resistant lysine bacillus bacteriocin analog in the prevention and treatment of tomato bacterial spot, it is characterized in that, taking Escherichia coli as a chassis host cell, by heterologous expression bacteriocin analog gene cluster, obtain The boron lysine-resistant bacillus bacteriocin analog; The bacteriocin analog gene cluster is obtained by PCR amplification using the whole genome of Lysinibacillus boronitolerans as a template and using Bac(10)-F and Bac(10)-R as primers respectively. A DNA fragment with a length of 8932 bp; The sequence of the Bac(10)-F primer is shown in SEQ ID NO.1; The sequence of the Bac(10)-R primer is shown in SEQ ID NO.2; The boron-resistant lysine bacillus was deposited in the China Type Culture Collection CCTCC on October 8, 2019, and the deposit number is CCTCC NO.M 2019773. 2. the application of a kind of boron-lysine-resistant bacillus bacteriocin analogue according to claim 1 in preventing and treating tomato bacterial spot, it is characterized in that: the sequence of the DNA fragment of described 8932bp is as SEQ ID NO. 3 shown. 3. A borolysine-resistant bacillus bacteriocin bacteriocin analog is characterized in that: taking Escherichia coli as a chassis host cell, by heterologous expression bacteriocin analog gene cluster, the borolysine-resistant bacillus bacterium is obtained hormone analogs; The bacteriocin analog gene cluster is obtained by PCR amplification using the whole genome of Lysinibacillus boronitolerans as a template and using Bac(10)-F and Bac(10)-R as primers respectively. A DNA fragment with a length of 8932 bp; The sequence of the Bac(10)-F primer is shown in SEQ ID NO.1; The sequence of the Bac(10)-R primer is shown in SEQ ID NO.2; The sequence of the 8932bp DNA fragment is shown in SEQ ID NO.3; The boron-resistant lysine bacillus was deposited in the China Type Culture Collection CCTCC on October 8, 2019, and the deposit number is CCTCC NO.M 2019773. 4. a preparation method of boron lysine-resistant bacillus bacteriocin analogs as claimed in claim 3, is characterized in that: take Escherichia coli as chassis host cell, by heterologous expression bacteriocin analog gene cluster, obtain The boron lysine-resistant bacillus bacteriocin analog. 5. the preparation method of a kind of boron-resistant lysine bacillus bacteriocin analogue according to claim 4, is characterized in that: comprise the following steps: (1) Extraction of genomic DNA of Bacillus borolysine-resistant and PCR amplification of bacteriocin analog gene clusters to obtain DNA fragments; (2) Extraction of plasmid, double enzyme digestion and gel recovery to obtain a vector; (3) The DNA fragment is seamlessly cloned and connected to the carrier to obtain a cloned product; (4) The cloned product is mixed with Escherichia coli competent cells and electroporated to obtain a bacterial cell containing the recombinant plasmid of the bacteriocin analog gene cluster; (5) The bacterial cells are cultured and fermented to obtain a fermentation liquid, centrifuged to remove the precipitate, the fermentation supernatant is collected, and the fermentation supernatant is purified to obtain a purified product containing the borolysine-resistant bacillus bacteriocin analogues . 6. The preparation method of a borolysine-resistant bacillus bacteriocin analogue according to claim 5, wherein the plasmid is p15A or pET, and the Escherichia coli comprises E. coli BL21.

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