A streptomycin-resistant gene, its encoded protein, and its applications

Abstract

This invention discloses a streptomycin resistance gene, its encoded protein, and its applications, belonging to the field of plant pathology technology. This invention discovers a novel streptomycin resistance gene derived from *Tomato Canker*. aph(3) This gene is located on a newly discovered plasmid pCM3 of *Tomato Canker*, encoding a protein APH(3) belonging to the aminoglycoside antibiotic phosphotransferase family. This invention verified the function of this gene through gene knockout, functional complementation, and heterologous expression experiments. Simultaneously, mass spectrometry analysis demonstrated that APH(3) catalyzes the phosphorylation of streptomycin molecules, generating streptomycin monophosphate, thereby eliminating its inhibitory activity against bacteria. Strains carrying this gene exhibit significant resistance to streptomycin. This invention relates to a streptomycin resistance gene. aph(3) It can be widely used in the study of pathogen resistance mechanisms, screening of resistant strains, and selection of molecular markers, and has important scientific research and application value.

Description

Technical Field

[0001] This invention relates to the field of plant pathology, specifically to a streptomycin resistance gene, its encoded protein, and its applications. Background Technology

[0002] Streptomycin is an aminoglycoside antibiotic that was once widely used in agriculture to control various plant bacterial diseases; it is also used in human and veterinary medicine to inhibit bacterial infections. However, streptomycin resistance in pathogens is becoming increasingly prevalent, seriously affecting control effectiveness and exacerbating the spread of resistance genes in agricultural ecosystems and environmental microorganisms. The main mechanisms of streptomycin resistance include target mutations (such as...) rpsL The role of genes and antibiotic-modifying enzymes, the latter mainly consisting of phosphotransferases (APHs).

[0003] Bacterial canker of tomato is caused by the Gram-positive bacterium Corynebacterium micranthum (Bacillus miltiorrhiza). Claviabcter michiganensis Bacterial canker (BCC) is a typical seed-borne disease caused by infection, severely impacting tomato production. Due to the lack of commercially available resistant tomato varieties, control primarily relies on preventative measures such as seed treatment, and the application of chemical pesticides in the early stages of disease development to inhibit its spread. Recent field monitoring has identified streptomycin-resistant varieties... Claviabcter michiganensis The strain has been identified, but its molecular mechanism is not fully understood. Currently, there are no systematic reports on the phosphorylation-mediated streptomycin inactivation resistance mechanism in this bacterium. Summary of the Invention

[0004] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide an anti-streptomycin gene, its encoded protein, and its applications, thereby providing a novel anti-streptomycin gene for research on pathogen resistance mechanisms, screening of resistant strains, and selection of molecular markers.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A streptomycin-resistant gene is provided. aph(3) streptomycin resistance gene aph(3) The nucleotide sequence is shown in SEQ ID NO.1.

[0006] Furthermore, the anti-streptomycin gene aph(3) Originating from Tomato Canker Bacterium Clavibacter michiganensis The plasmid pCM3 of strain TX-0702; said plasmid pCM3 includes elements associated with horizontal gene transfer.

[0007] Furthermore, elements associated with horizontal gene transfer include transposases, integrases, and conjugation transfer proteins.

[0008] This invention provides the above-mentioned streptomycin resistance gene aph(3) The encoded protein is APH(3), and the amino acid sequence of APH(3) is shown in SEQ ID NO.2.

[0009] Furthermore, D188, N193, and D208, which encode the protein APH(3), are key amino acid sites essential for catalytic activity.

[0010] This invention provides the above-mentioned streptomycin resistance gene aph(3) Applications in screening for resistant bacteria or monitoring resistance to plant pathogens.

[0011] This invention provides the above-mentioned streptomycin resistance gene aph(3) Application of the protein APH(3) encoded by phosphorylated streptomycin.

[0012] The present invention also provides a method for phosphorylating streptomycin, which uses the above-mentioned encoded protein APH(3) to phosphorylate streptomycin to generate streptomycin monophosphate in a reaction system in the presence of ATP.

[0013] This invention has the following beneficial effects: This invention discovers a pathogen derived from the tomato canker bacterium (… Clavibacter michiganensis Novel streptomycin resistance gene aph(3) The gene, located on a newly discovered plasmid pCM3 of *Tomato canker*, encodes APH(3), a protein belonging to the aminoglycoside antibiotic phosphotransferase family. The function of this gene was verified through gene knockout, functional complementation, and heterologous expression. Mass spectrometry analysis confirmed its catalytic product, demonstrating that APH(3) can catalyze streptomycin molecules (C...). 21 H 39 N7O 12 Phosphorylation occurs to produce streptomycin monophosphate (C...). 21 H 40 N7O 15 The streptomycin resistance gene (P) loses its inhibitory activity against bacteria, and strains carrying this gene exhibit significant resistance to streptomycin. Experiments revealed that D188, N193, and D208 of the APH(3) protein are key catalytic residues. This streptomycin resistance gene can be widely applied in research on pathogen resistance mechanisms, screening of resistant strains, and molecular marker selection, possessing significant scientific research and application value. Attached Figure Description

[0014] Figure 1 Image of pCM3 plasmid;

[0015] Figure 2 The pMarA-Chl plasmid map;

[0016] Figure 3 for aph(3) Gene mapping;

[0017] Figure 4 Among different strains of tomato canker pathogen aph(3) Expression level, MIC, and resistance phenotype;

[0018] Figure 5 The inhibition zones of streptomycin before and after APH(3) enzyme treatment against different tomato canker pathogens are shown.

[0019] Figure 6 LC-MS spectra of streptomycin and its phosphorylated products;

[0020] Figure 7 The results show the phosphorylation ability of streptomycin after mutation at the key site of APH(3); Figure A is a diagram of the APH(3) mutation site; Figure B is a diagram of the inhibition zone of the mutated tomato canker pathogen. Detailed Implementation

[0021] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0022] Example 1: aph(3) Gene identification and functional verification

[0023] anti-streptomycin gene aph(3) Originating from Tomato Canker Bacterium Clavibacter michiganensis The plasmid pCM3 of strain TX-0702 (see plasmid map) Figure 1 ). Clavibacter michiganensis The TX-0702 strain is deposited at the Seed Health Center of China Agricultural University, with the accession number TX-0702.

[0024] Bacillus transposon plasmid pMarA (Baosai Biotechnology) carries a pUC-type replicon, in C. michiganensis It cannot replicate autonomously and is a suicide plasmid. Theoretically, pMarA can only... C. michiganensis A transposition occurs, resulting in the disruption of a single gene. The original transposon fragment carries the kanamycin resistance gene; for the convenience of subsequent experiments, a gene suitable for [specific gene selection] is selected. C. michiganensis The selected chloramphenicol resistance fragment replaced kanamycin. The pMarA plasmid backbone, excluding kanamycin, was amplified by PCR. cmx The resistant fragment was ligated into a circular shape using a seamless cloning method to form a recombinant vector. The modified transposon plasmid was named pMarA-Chl (plasmid map shown). Figure 2 ).

[0025] pMarA-Chl plasmid was introduced via electroporation. Clavibacter michiganensis TX-0702 competent cells were used. Transformed cells were further screened on LBA plates containing 10 μg / mL chloramphenicol and 50 μg / mL streptomycin, yielding 30 chloramphenicol-resistant but streptomycin-sensitive mutants (i.e., resistance-loss mutants). Genomic DNA was extracted from the streptomycin-sensitive mutants and digested with restriction endonucleases XhoI, SalI, and NgoMIV to obtain... aph(3) The Suk sequence of the gene. This sequence contains an 810 bp open reading frame encoding a class of phosphotransferases belonging to the APH(3'') family (see Suk sequence). Figure 3 ).

[0026] Using TX-0702 as the wild type, knockout was achieved through homologous recombination. aph(3) Gene (nucleotide sequence as shown in SEQ ID NO. 1). Compared with wild type (MIC = 128 μg / mL), the knockout mutant Δ aph(3) The streptomycin MIC was reduced to 4 μg / mL; the complete aph(3) The gene was cloned into the expression vector pHN216 (vector construction reference: Laine, MJ, Nakhei, H., Dreier, J., Lehtilä, K., Meletzus, D., Eichenlaub, R., and Metzler, MC (1996). Stable transformation of the gram-positive phytopathogenic bacterium Clavibacter michiganensis subsp. sepedonicus (with several cloning vectors. Appl. Environ. Microbiol. 62, 1500-1506.), electrical shock converts back to Δ aph(3) The streptomycin MIC in the transformant was restored to 128 μg / mL; overexpression in wild-type TX-0702 was also observed. aph(3) The MIC was increased to 256 μg / mL; further... aph(3) Introducing streptomycin-sensitive strains C. michiganensis The overexpression strain BT-0505+ was obtained from BT-0505 (original MIC = 4 μg / mL). aph(3) Its MIC increased to 32 μg / mL (see Figure 4 The above results confirm that... aph(3) Genes independently mediate streptomycin resistance.

[0027] Example 2: Verification of phosphorylation reaction

[0028] Will aph(3) Gene cloning to a gene with a 6×His tag pEASY -Blunt E1 expression vector (purchased from Beijing TransGen Biotech), transformation Escherichia coli BL21(DE3) was used to induce and purify APH(3) protein. The reaction system consisted of 40 mM Tris-HCl (pH 7.5), 40 mM KCl, 10 mM MgCl2, 4 mM ATP, and 1 mg of purified APH(3) protein. Then, 1 mg of streptomycin was added and the mixture was incubated at 37°C for 2 h. Subsequently, 5 μL of the reaction product was added to filter paper and placed on a filter paper inoculated with... C. michiganensis (1×10) 6 LBA plates (CFU / mL). Results are as follows: Figure 5 As shown, treatment with APH(3) protein completely eliminated the effect of streptomycin on sensitive strains Δ aph(3) The antibacterial activity of BT-0505.

[0029] Using the above reaction system, the products of streptomycin before and after treatment with APH(3) protein were detected by LC-MS (liquid chromatography-mass spectrometry), with the untreated group serving as a blank control. Cation mode was used. The chromatographic column was a ThermoScientific™ Hypersil GOLD™ HILIC (2.1 mm × 150 mm); mobile phase A was water, and mobile phase B was acetonitrile; gradient elution: 0-5 min, 5% A; 5-15 min, 5% A-65% A; 15-20 min, 65% A-90% A; 20-25 min, 90% A; flow rate was 0.2 mL / min; injection volume was 15 μL; ion source was ESI; nebulizer was 30 psi; high-purity nitrogen was used as the spray dry gas at a flow rate of 11 L / min; temperature was 325℃; and capillary voltage was 4000 V. Results are as follows: Figure 6 As shown, streptomycin molecule (C 21 H 39 N7O 12 The hydrogenation peak of streptomycin monophosphate (MW=581.57 Da) was 582.27 m / z; after treatment with APH(3) protein, a peak of 662.24 m / z was detected, which is consistent with the theoretical value of streptomycin monophosphate (C). 21 H 40 N7O 15 P, MW = 661.55 Da) matching, confirming that APH(3) catalyzes the transfer of monophosphate groups.

[0030] Example 3: Analysis of key site mutations

[0031] By performing multiple sequence alignment of the APH(3) protein with homologous proteins of Actinobacteria, it was deduced that D188, N193, and D208 are key catalytic sites, and at the same time, [the following was selected]. C. michiganensis The unique non-conserved site T148 was used as a negative control, and this site was mutated to alanine (Ala). The recombinant protein was expressed and purified according to the method described in Example 2, and then subjected to in vitro phosphorylation. The results showed that mutation of any one amino acid in D188, N193, and D208 to Ala resulted in complete loss of phosphorylation function, while mutation of T148 to Ala had no effect, indicating that these three sites are the catalytic core of the APH(3) protein (see Example 2). Figure 7 ).

[0032] In this invention aph(3) The nucleotide sequence of the gene is shown below:

[0033] ATGCGCAGTCACACTCGCTCACTCGTCCAGCGACTGGAACGCGCCGCTGCCGGATGGTCGAGAGTCACCGCTGGCGACTCTGACGCGCTTGTGCTTCGCAGCGCGGACGGCGATCGGTACGCGAAGGTCGTGGATGGCAGGGGAGTCGAGGATCTGCAGGCTGAGCGAGATCGCATCGAATGGCTAGCGTCCACAAGCATTCCCGGCGCGTCCGTACTTGACTGGCACGAGGAACACGATGGCGCATTCTTGGTGACTTCCTCGGTCGCGGGGGTGCCAGCGGACGTGCTCGATGCAGCGGCTCTCACACGAGCGTGGCCGTTCATAACTGCGCAGGTGCGCGAACTGCACGCACTACCGGTTGAAGACTGCCCGTTCGAGTTCCGTGTGAACGAGAGGATGCAGGCGGCCCGCAAAGTCGTCGGGGGCGGGCGGGTCCATACCGAGTTCCTGCCCCTCGAAGTAGTGCATGTAGCGCCGAACGAGATCTTCGTGGAACTCGAGCGGCAGGCGTCAGCGCGTGAGGCGCAGGAAGCAGCCGGAATGGCGGTCTGTCACGGTGACCTGTGCTTGCCGAACATCCTGATCGACCCCGCGACGTCACGCGTGACTGGCCTGATCGACGTTGGCCGACTGGGACGAGCCGACGCGCACACCGACCTTGCACTGCTGTTCGCGACAGCGCGCGAGACGTGGGCCGATTCCAAGCGAGCAGCTGTCGCAGAAGCTGCGTTCCGGCGGACCTACGGCGGAACCATCGACGATGAACGACTAAGGTTCTACCTCTGGCTCGACCCACTCACATGGTAG (SEQ ID NO.1);

[0034] The amino acid sequence of the APH(3) protein in the present invention is as follows:

[0035] MRSHTRSLVQRLERAAAGWSRVTAGDSDALVLRSADGDRYAKVVDGRGVEDLQAERDRIEWLASTSIPGASVLDWHEEHDGAFLVTSSVAGVPADVLDAAALTRAWPFITAQVRELHALPVEDCPFEFRVNERMQA ARKVVGGGRVHTEFLPLEVVHVAPNEIFVELERQASAREAQEAAGMAVCHGDLCLPNILIDPATSRVTGLIDVGRLGRADAHTDLALLFATARETWADSKRAAVAEAAFRRTYGGTIDDERLRFYLWLDPLTW (SEQ ID NO.2).

[0036] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An anti-streptomycin gene aph(3) characterized in that, The anti-streptomycin gene aph(3) The nucleotide sequence of the anti-streptomycin gene is shown as SEQ ID NO.

1.

2. The anti-streptomycin gene of claim 1 aph(3) encoding the protein APH(3), characterized by The amino acid sequence of the APH(3) is shown as SEQ ID NO.

2.

3. The streptomycin resistance gene according to claim 2, encoding the protein APH(3), characterized in that, aph(3) The amino acid sites D188, N193 and D208 of the encoded protein APH(3) are essential for catalytic activity. ​ 4. The streptomycin resistance gene of claim 1 aph(3) Use in screening for resistant bacteria or monitoring of plant pathogen resistance.

5. The streptomycin resistance gene of claim 2 or 3 aph(3) for use in phosphorylating streptomycin to form streptomycin monophosphate.

6. A method of phosphorylating streptomycin to produce streptomycin monophosphate, characterized by, The encoded protein APH(3) of claim 2 or 3 is used to phosphorylate streptomycin to generate monophosphoryl streptomycin in the presence of ATP in a reaction system.

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