CN113106111A - N-acyl homoserine lactone acyltransferase encoding gene aigC and application thereof - Google Patents

Abstract

The invention belongs to the technical field of molecular biological control, and discovers that N-acyl homoserine lactone acyltransferase coding genes are cloned by molecular biological technology on the basis that Pseudomonas nitroreducens HS-18 can efficiently degrade AHLs signal molecules with the chain length of C4-C14 acyl. The invention discovers in the previous research that the coding gene of the N-acyl homoserine lactone acyltransferase can be cloned by a molecular biology technology on the basis that the Pseudomonas nitroreducens HS-18 can efficiently degrade AHLs signal molecules with the chain length of C4-C14 acyl. The gene has broad-spectrum and high-efficiency quenching activity on AHLs with different side chain lengths and different side chain substituent bands. The N-homoserine lactone quenching gene expressed in pathogenic bacteria depending on AHLs can obviously weaken the motility, biofilm formation and the production of virulence factors such as extracellular enzyme of the pathogenic bacteria, and obviously weaken the pathogenicity of the pathogenic bacteria to host plants.

Description

N-acyl homoserine lactone acyltransferase encoding gene aigC and application thereof

Technical Field

The invention relates to the technical field of molecular biological control, and more particularly relates to an N-acyl homoserine lactone acyltransferase encoding gene aigC and application thereof.

Background

The use of pesticides and antibiotics is currently the most common method for controlling pathogenic bacteria, however, the long-term abuse of pesticides and antibiotics has posed a threat to environmental safety and health of human and livestock, and even caused the drug resistance of microorganisms. Therefore, a new and environmentally friendly antimicrobial approach is urgently needed.

Quorum sensing is an intercellular communication mechanism, cells synthesize and secrete a small molecule compound signal, the number of populations is determined by sensing the concentration of signal molecules diffusing to the outside of the cells, and when the concentration of the signal molecules reaches a certain threshold, the cells activate the expression of downstream target genes through a series of signal transduction. Bacteria regulate their own various physiological and biochemical functions through quorum sensing signal molecules, such as: plasmid transfer, drug resistance, motility, bioluminescence, biofilm formation, drug resistance, production of extracellular enzymes, etc., to adjust self-adaptability to the environment and viability. In particular, many gram-negative pathogens utilize quorum sensing systems to regulate the production and virulence of their own virulence factors in order to obtain a greater infectivity of the host.

N-acyl-homoserine lactones (AHLs) are the most widely existing quorum sensing signal molecules in gram-negative pathogenic bacteria and are responsible for regulating and controlling the generation and pathogenicity of virulence factors in various gram-negative pathogenic bacteria, such as: motility, formation of biofilm, and production of extracellular enzymes, extracellular polysaccharides, toxins, and the like. The first AHLs signal molecule was found in the bioluminescence study of the marine bacteria Vibrio fischeri (Vibrio fischeri) and Vibrio harveyi (Vibrio harveyi). And more AHLs are identified later, the molecular structures of the AHLs are conservative and mainly comprise an N-acyl homoserine lactone ring and acyl chains with 4-18 different carbon atoms and different substituent modifications at the No. 3 carbon position.

Quorum quenching is a way to block or destroy quorum sensing systems, and can be achieved mainly in three ways: inhibiting quorum sensing signal synthetase activity, inhibiting quorum sensing signal receptor protein activity, and modifying or enzymatically hydrolyzing quorum sensing signal molecules with a quenching enzyme. The discovery and application of group quenching genes or quenching enzymes has become one of the research hotspots for quenching of the current group. The first AHLs quencher enzyme identified was AHL lactonase AiiA found in bacillus thuringiensis, which is capable of destroying the lactone bond of the lactone ring in AHLs molecules, thereby inactivating AHL signal molecules. The expression of AiiA in pathogenic bacteria can obviously weaken the yield of pathogenic bacteria virulence factors and the virulence to host plants, and the expression of AiiA in plants can effectively improve the disease resistance of host plants to pathogenic bacteria. After the identification of AHL lactonase, AHL acyltransferase capable of cleaving amide bond between AHL lactone ring and acyl chain and AHL oxidoreductase acting on side chain hydrogen atom were also successively found.

Researches show that the expression of most of the identified AHLs quenching genes in pathogenic bacteria can weaken the toxicity of the pathogenic bacteria, the expression of the AHLs quenching genes in host plants can improve the resistance of the host to the pathogenic bacteria, and AHLs quenching enzymes, which are coded products of the AHLs quenching genes or the AHLs quenching genes, are widely and effectively applied to the aspects of biological prevention and control of agriculture and aquaculture industry and a biofilm reactor for sewage treatment. Therefore, the excavation, identification and application of the AHLs quenching genes lay a foundation for enriching AHLs colony quenching preparation resources, and have great practical application value for biological control of colony quenching ways.

Disclosure of Invention

The present invention has been made to overcome the above problems occurring in the prior art, and the present invention provides a gene encoding N-acyl homoserine lactone acyltransferase.

It is a second object of the present invention to provide a recombinant vector containing a gene encoding N-acylhomoserine lactone acylase.

The third object of the present invention is to provide a recombinant bacterium containing the recombinant vector.

The fourth purpose of the invention is to provide the application of the recombinant vector.

The fifth purpose of the invention is to provide the application of the recombinant bacterium.

The purpose of the invention is realized by the following technical scheme:

an N-acyl homoserine lactone acyltransferase encoding gene aigC, the nucleotide sequence of which is shown as SEQ ID NO: 1 is shown.

The invention discovers in the previous research that the coding gene of the N-acyl homoserine lactone acyltransferase can be cloned by a molecular biology technology on the basis that the Pseudomonas nitroreducens HS-18 can efficiently degrade AHLs signal molecules with the chain length of C4-C14 acyl.

It is understood that the above-mentioned recombinant vector containing the gene encoding said N-acylhomoserine lactone acylase is also within the scope of the present invention; recombinant bacteria containing the recombinant vector are also within the scope of the present invention.

As a preferred embodiment, the recombinant vector is obtained by inserting the gene encoding N-acyl homoserine lactone acyltransferase into a broad host vector pBBR1 for heterologous expression of the gene encoding N-acyl homoserine lactone acyltransferase.

As another preferred embodiment, the recombinant vector can also be a recombinant vector for prokaryotic expression of the N-acyl homoserine lactone acyltransferase protein, which is obtained by inserting the gene encoding the N-acyl homoserine lactone acyltransferase into a prokaryotic protein expression vector pET32 a.

As a preferred embodiment, the recombinant bacterium is preferably obtained by introducing the recombinant vector expressing the gene encoding N-acyl homoserine lactone acylase in the broad host vector pBBR1 into Escherichia coli DH5 alpha.

In another preferred embodiment, the recombinant bacterium is obtained by introducing the recombinant vector expressing the gene encoding N-acylhomoserine lactone acyltransferase in the broad-host vector pBBR1 into pathogenic bacteria which cause AHL-dependent diseases.

As another preferred embodiment, the recombinant bacterium is obtained by introducing the recombinant vector for expressing N-acylhomoserine lactone acyltransferase in a prokaryotic protein expression vector pET32a into Escherichia coli BL21(DE 3).

The invention also provides N-acyl homoserine lactone acyltransferase which is named AigC, and the amino acid sequence of the N-acyl homoserine lactone acyltransferase is shown as SEQ ID NO: 2, respectively.

The invention also provides a preparation method of the N-acyl homoserine lactone acyltransferase, which is characterized in that a recombinant bacterium for expressing the N-acyl homoserine lactone acyltransferase in a protein prokaryotic expression vector pET32a is fermented and cultured, and a strain of the fermented and cultured recombinant bacterium is crushed and separated and purified to obtain the N-acyl homoserine lactone acyltransferase protein with the His label.

The invention also provides application of the recombinant bacterium or the N-acyl homoserine lactone acyltransferase in degradation of AHLs signal molecules.

Preferably, the AHLs signal molecule comprises C4-HSL, C6-SHL, 3-O-C6-HSL, C8-HSL, 3-OH-C8-HSL, C10-HSL, 3-OH-C10-HSL, C12-HSL, 3-O-C12-HSL, 3-OH-C12-HSL, 3-OH-C14-HSL; the N-acyl homoserine lactone acyltransferase has a broad spectrum of quenching activity for different AHLs signal molecules, and the reactivity of different substrates and N-acyl homoserine lactone acyltransferases may be different.

The invention also provides an application of the coding gene of the N-acyl homoserine lactone acyltransferase, the recombinant vector, the recombinant bacterium or the N-acyl homoserine lactone acyltransferase of claim 4 in preventing and treating pathogenic bacteria which depend on AHLs.

Preferably, the AHLs-dependent pathogenic bacteria include Pectinophytrium carotovorum subspecies, Burkholderia cepacia, Pseudomonas aeruginosa, Laurella solanacearum, Agrobacterium tumefaciens, Rhizoctonia solani, Erwinia europaea, and bacterial wilt of maize.

As a preferred embodiment, when the gene encoding N-acyl homoserine lactone acyltransferase is used, the gene encoding N-acyl homoserine lactone acyltransferase is expressed in AHLs mediated pathogenic bacteria, and recombinant pathogenic bacteria successfully expressing the N-acyl homoserine lactone acyltransferase are screened.

As another preferred embodiment, when the recombinant vector is used, the recombinant vector is introduced into AHLs-mediated pathogenic bacteria, and recombinant pathogenic bacteria that successfully express N-acylhomoserine lactone acyltransferase are selected.

As still another preferred embodiment, when the recombinant bacterium is used, a recombinant pathogen is selected which successfully expresses N-acylhomoserine lactone acyltransferase.

The screening of the recombinant pathogenic bacteria successfully expressing the N-acyl homoserine lactone acyltransferase obviously weakens the yield and pathogenicity of virulence factors of the pathogenic bacteria.

Preferably, the pathogenic bacteria are C8-HSL-dependent Burkholderia cepacia H111, C4-HSL-dependent Pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1 and 3-OH-C12-HSL-dependent Pseudomonas aeruginosa.

In a preferred embodiment, the recombinant pathogenic bacterium that successfully expresses N-acylhomoserine lactone acyltransferase comprises:

(1) the yield of AHLs is reduced; and/or the presence of a gas in the gas,

(2) the sports type is reduced; and/or the presence of a gas in the gas,

(3) reduces the formation of biofilm; and/or the presence of a gas in the gas,

(4) the production amount of protease is reduced; and/or the presence of a gas in the gas,

(5) the pathogenic force is weakened.

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

the invention discovers in the previous research that the coding gene of the N-acyl homoserine lactone acyltransferase can be cloned by a molecular biology technology on the basis that the Pseudomonas nitroreducens HS-18 can efficiently degrade AHLs signal molecules with the chain length of C4-C14 acyl. The gene has broad-spectrum and high-efficiency quenching activity on AHLs with different side chain lengths and different side chain substituent bands. The N-homoserine lactone quenching gene expressed in pathogenic bacteria depending on AHLs can obviously weaken the motility, biofilm formation and the production of virulence factors such as extracellular enzyme of the pathogenic bacteria, and obviously weaken the pathogenicity of the pathogenic bacteria to host plants.

Drawings

FIG. 1 is a diagram showing the AHLs degradation effect of recombinant Escherichia coli DH5 alpha (aigC) (FIG. 1B) expressing N-acylhomoserine lactone acyltransferase encoding gene in broad host vector pBBR1 and Escherichia coli DH5 alpha (pBBR1) (FIG. 1A) containing empty vector after culturing for 36h according to the present invention;

FIG. 2 is an AigC phylogenetic tree analysis of the N-acyl homoserine lactone acyltransferase protein according to the present invention;

FIG. 3 is the expression of AigC protein; m: a protein Marker; 1: expressing the total protein expression condition in the AigC protein recombinant bacteria; 2: BL21(DE3) total protein expression containing empty vector pET32 a; 3: expressing the intracellular protein expression condition in the AigC protein recombinant bacteria; 4: intracellular protein expression in BL21(DE3) containing empty vector pET32 a;

FIG. 4 is a graph showing the effect of expression of the gene aigC encoding N-acyl homoserine lactone acyltransferase of the present invention in Burkholderia cepacia H111, a pathogenic bacterium depending on AHL, on H111 growth, self-produced AHL production and virulence factors; FIG. 4A shows a Burkholderia cepacia growth curve; FIG. 4B shows intracellular C8-HSL production; FIG. 4C shows Burkholderia cepacia motility; FIG. 4D shows the formation of Burkholderia cepacia biofilm; FIG. 4E shows Burkholderia cepacia protease production;

FIG. 5 shows the effect of expression of gene aigC encoding N-acyl homoserine lactone acyltransferase of the present invention in H111 pathogenicity of AHL dependent pathogenic bacterium Burkholderia cepacia H111;

FIG. 6 shows the effect of expression of the gene aigC encoding N-acyl homoserine lactone acyltransferase of the present invention in the pathogenic bacterium Pseudomonas aeruginosa PAO1 dependent on AHL for growth, AHL production by self-production and virulence factors of PAO 1; FIG. 6A shows a Pseudomonas aeruginosa growth curve; FIG. 6B shows P.aeruginosa intracellular C4-HSL production; FIG. 6C shows P.aeruginosa intracellular 3-O-C12-HSL production; FIG. 6D shows Pseudomonas aeruginosa motility; FIG. 6E shows Pseudomonas aeruginosa protease production;

FIG. 7 shows the influence of the expression of the gene aigC encoding N-acyl homoserine lactone acyltransferase of the present invention in the pathogenic bacterium Pseudomonas aeruginosa PAO1 dependent on AHL pathogenicity on the pathogenicity of PAO1, FIG. 7A shows the influence on the pathogenicity of lettuce, and FIG. 7B shows the influence on the pathogenicity of cabbage.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

MM inorganic salt medium: k₂HPO₄，10.5g/L；KH₂PO₄，4.5g/L；(NH₄)₂SO₄，2.0g/L；MgSO₄·7H₂O， 0.2g/L；FeSO₄，0.005g/L；CaCl₂，0.01g/L；MnCl₂0.002 g/L; 2.0g/L of glycerol; mannitol, 2.0 g/L; sterilizing at 121 ℃ for 20min at the pH of 6.8-7.2.

LB culture medium: trypton 10.0g/L, yeast extract 5.0g/L, NaCl 10.0g/L, pH 6.8-7.2, sterilizing at 121 ℃ for 15-25 min.

PBS phosphate buffer, X-gal and the reagents needed in the culture medium are all purchased from biological reagent companies such as Guangzhou Qixiang, Dingguo, etc., and the AHLs for detecting degradation activity in the invention are purchased from Shanghai Youder chemical technology Co., Ltd and Sigma-Aldrich.

The pseudomonas nitroreducens HS-18 is separated from soil samples polluted by oil for a long time near southern China agricultural university in Guangzhou city, and is preserved in the China center for type culture Collection in 2017, 5 months and 12 days, wherein the preservation number is CCTCC NO: m2017257. HS-18 was cultured at 30 ℃ using LB medium.

Example 1 acquisition and identification of AHLs degradation genes

According to the whole genome sequencing result of P.nitroreducens strain HS-18 in the previous work, the gene aigC which possibly codes N-acyl homoserine lactone acyltransferase is discovered by using genome annotation and bioinformatics alignment. The software MEGA 5.10 is used for comparing the amino acid sequences of AigC and the currently known N-acyl homoserine lactone acyltransferase, and the ClustalX1.8.3 and the adjacency method are adopted for carrying out phylogenetic evolution analysis and constructing an evolutionary tree. The results are shown in FIG. 2, where AigC is compared with the known AHLs acyltransferase HacB in P.aeruginosa PAO1_PAO1With a high degree of amino acid similarity (76.6%).

Primers are designed to respectively amplify and construct primers of aigC inserted broad host vector pBBR1 and protein prokaryotic expression vector pET32a recombinant vector, and the sequences of the primers are as follows:

pBBR1-aigC-F:gtcgacggtatcgataagcttGCAGAATCGCCGCATAACA

pBBR1-aigC-R:cgctctagaactagtggatccTCAGCGACGGACGGGGAT

pET32a-aigC-F:gccatggctgatatcggatccATGAAACGCACTTTGACTGTCCT

pET32a-aigC-R:ctcgagtgcggccgcaagcttTCAGCGACGGACGGGGAT

example 2 detection of AHLs quenching Activity by recombinant Strain DH5 alpha (aigC)

AHLs degradation system setting: OD was obtained by overnight culture of the successfully constructed DH5 alpha (aigC) in LB liquid medium₆₀₀1.0 ═ 1.0Adding an equal volume of a fresh LB liquid culture medium, exogenous AHLs with different carbon chain lengths and different substituents and MOPS with a final concentration of 50mM into the seed solution to prepare a degradation system (different AHLs are selected to have proper concentrations according to different intensities for the color development of a report strain), culturing the degradation system for 36 hours in a constant temperature shaking table at 37 ℃ and 200rpm, and taking DH5 alpha (pBBR1) bacterial solution and LB liquid culture medium without the bacterial solution as controls. Secondly, the culture broth was extracted with an equal volume of ethyl acetate, and the content of residual AHLs in 10. mu.l of ethyl acetate extract was determined using a reporter strain.

Quantitatively detecting short-chain AHLs (C4-C6): short-chain AHLs (C4-C6) are detected by using a reporter strain purple bacillus CV 026. First, CV026 was activated on LB solid plates and cultured overnight on an LB liquid medium in a constant temperature shaker at 28 ℃ and 200 rpm. Preparing LB solid plate in a square culture dish of 13cm by 13cm, cutting into 0.8cm wide agar strips, loading 10 mul of ethyl acetate extract of a sample to be tested on the upper end of the agar strips, and continuously dropping a row of report strain liquid drops with similar sizes below the loading position of the sample. The area to which AHLs diffuse induces violacein production by Violaceous bacillus CV026, rendering the bacteria purple. After the sample on the agar strip is dried, culturing for 16h in a constant temperature incubator at 28 ℃, and observing and counting the distance of bacteria presenting purple CV 026. The results from FIG. 1 show that DH5 α (aigC) is capable of significantly degrading short-chain AHLs for detection (C4-HSL, C6-HSL, 3-O-C6-HSL) with high efficiency.

Medium-long chain AHLs (C8-C14) are detected by using a reporter strain Agrobacterium tumefaciens NT 1. First, NT1 was activated by LB solid plate, and NT1 was cultured overnight in a 200rpm constant temperature shaker at 28 ℃ with LB liquid medium supplemented with kanamycin to a final concentration of 50. mu.g/ml. Preparing MM solid plates in a square culture dish of 13cm by 13cm, cutting the MM solid plates into alternate agar strips with the width of 0.8cm, loading 10 mu l of ethyl acetate extract liquid of a sample to be detected at the upper ends of the agar strips, and continuously dropping a row of report strain liquid drops with similar sizes below the sample loading position. The area to which AHLs diffuse induces the production of beta-galactosidase by Agrobacterium tumefaciens NT1, which decomposes X-gal to make the thallus appear blue. The distance that Agrobacterium tumefaciens NT1 produces a blue color is proportional to the amount of AHLs to be detected. After the sample on the agar strip is dried, the agar strip is cultured for 16 hours in a constant temperature incubator at 28 ℃, and the distance of NT1 showing blue color is observed and counted. The results according to FIG. 1 show that DH5 α (aigC) pairs medium-long AHLs for detection (C8-HSL, 3-OH-C8-HSL, C10-HSL, 3-OH-C10-HSL, C12-HSL, 3-O-C12-HSL, 3-OH-C12-HSL, 3-OH-C14-HSL). Therefore, aigC has high-efficiency and broad-spectrum quenching activity on AHLs with different chain lengths and different substituents of C4-C14 to be detected.

Example 3 prokaryotic expression of AigC protein

The successfully constructed recombinant strain BL21(DE3) (pET32a-aigC) was cultured overnight in LB liquid medium supplemented with final concentration of 100. mu.g/ml ampicillin, and cultured in a constant temperature shaker at 37 ℃ and 200rpm to obtain a seed solution. And then mixing the seed liquid with the weight ratio of 1: 100 to a fresh LB liquid medium containing ampicillin at a final concentration of 100. mu.g/ml, was incubated at 37 ℃ on a 200rpm constant temperature shaker to OD₆₀₀0.6 to 0.8, the cells were induced by adding IPTG (0.5 mM final concentration) and cultured overnight in a constant temperature shaker at 200rpm at 18 ℃. Overnight cultured cells were collected, disrupted, and the expression of AigC was identified by SDS-PAGE electrophoresis. From the results in FIG. 3, it was shown that the His-tagged AigC was normally prokaryotic and was approximately 106.54kDa in size.

Example 4 Effect of AigC expression on AHL-mediated growth of pathogen H111 and intracellular AHL production

Detecting the growth condition of H111: culturing the successfully constructed recombinant bacterium H111(aigC) seed liquid to OD overnight₆₀₀0.5, with 1: 100 portions of LB liquid medium containing a final concentration of 50. mu.g/ml kanamycin was added, and the mixture was incubated at 30 ℃ in a constant temperature shaker at 200rpm, and OD was measured every 2 hours₆₀₀H111(pBBR1) was used as a control. Growth curve results as shown in fig. 4A, the expression of aigC in H111 did not affect the growth of H111.

Detection of intracellular AHL production in H111: seed fluid was cultured overnight to OD₆₀₀0.5, with 1: adding 100 proportion of the seed solution into LB liquid culture medium containing 50 mug/ml kanamycin, culturing for 17h at 30 ℃, 200rpm constant temperature shaking table, taking equal volume of ethyl acetate extract bacteria solution, evaporating ethyl acetate extract organic phase by rotation, adding report strain NT1, culturing for 8h, crushing thalli of a culture solution, and detecting the activity of beta-galactosidase generated by NT1 induced by AHL in supernatant after cell crushing. As can be seen in FIG. 4B, the expression of aigC in H111 significantly reduced the production of C8-HSL in H111.

Example 5 Effect of AigC expression on AHL mediated biological phenotype of pathogenic bacteria H111

Detection of motility: semi-solid media (0.8% tryptone, 0.5% glucose, 0.3% agarose) were prepared for testing H111 motility. The recombinant bacterium H111(aigC) activated on the LB plate and the control H111(pBBR1) were dipped with toothpicks in the center of the motility medium plate, and after static culture in a 30 ℃ incubator for 17 hours, the motility diameter was observed and counted. The results are shown in fig. 4C, where aigC expression significantly attenuated H111 motility.

And (3) detecting a biological membrane: the overnight-cultured seed solution was adjusted to OD₆₀₀0.5, with 1: 100 portions were added to 100. mu.l of LB liquid medium containing 50. mu.g/ml kanamycin in a 96-well plate, and cultured on a 200rpm constant temperature shaker at 30 ℃ for 9 hours for biofilm measurement, 8 replicates of each treatment. First, OD was measured₆₀₀Carefully sucking away the bacteria solution with a pipette and discarding, carefully washing each well with sterile water for 3 times, adding 150 μ l of 0.1% crystal violet, standing and dyeing at room temperature for 15min, washing each well with sterile water for 3 times, air drying, adding 300 μ l of 95% ethanol, standing for 10min, measuring absorbance at 595nm, and calculating OD₅₉₅/OD₆₀₀Biofilm formation by the strain was quantified. Results as shown in fig. 4D, expression of aigC significantly attenuated biofilm formation of H111.

And (3) detecting the protease activity: the overnight-cultured seed solution was adjusted to OD₆₀₀Mu.l of the inoculum was added to wells punched out with a punch on LB + 1% mounted mill plates, 3 replicates each, at 0.5. The plate was subjected to static culture in an incubator at 30 ℃ for 17 hours, and the size of a transparent circle generated around the well by the strain was measured to quantitatively detect the protease activity. Results as shown in fig. 4E, expression of aigC significantly attenuated protease production of H111.

Example 6 Effect of AigC expression on AHL mediated pathogenicity of pathogen H111

Culturing the recombinant strain seed liquid overnight, and resuspending the strain liquid to OD with PBS buffer solution₆₀₀1.0. Equally dividing fresh onion into four parts, peeling onion sections to serve as inoculation tissues, slightly stabbing wounds in the center of the inner sides of the onion sections by using a sterile small gun head, adding 20 mu l of bacterial suspension to the wounds, repeating the steps in each treatment setting, performing moisture-preserving culture at 30 ℃ for 3 days, and observing and counting the sizes of the scabs. The results are shown in fig. 5, where aigC expression significantly attenuated H111 pathogenicity.

Example 7 Effect of AigC expression on AHL mediated growth of the pathogen Pseudomonas aeruginosa PAO1 and intracellular AHLs production

Growth of PAO 1: the successfully constructed recombinant strain PAO1(aigC) seed liquid is cultured overnight to OD₆₀₀0.5, with 1: adding 100 proportion of seed solution into LB liquid culture medium containing final concentration of 50 μ g/ml gentamicin, culturing at 37 deg.C and 200rpm constant temperature shaking table, measuring OD once every 2h₆₀₀PAO1(pBBR1) was used as a control. Growth curve results as shown in fig. 6A, the expression of aigC in PAO1 did not affect the growth of PAO 1.

Detection of the content of intracellular produced AHLs in PAO 1: seed fluid was cultured overnight to OD₆₀₀0.5, with 1: adding the seed solution into LB liquid culture medium containing gentamicin with final concentration of 50 mug/ml in proportion of 100, culturing for 17h in a constant temperature shaking table at 37 ℃ and 200rpm, and taking equal volume of ethyl acetate to extract bacterial liquid. PAO1 can produce both C4-HSL and 3-O-C12-HSL AHLs.

C4-detection of HSL production: after ethyl acetate extracts were evaporated to dryness, reporter strain CV026 was added and cultured overnight. And (3) centrifuging to obtain thalli, taking lysate to resuspend the thalli and lyse cells, taking 200 mu l of lysed supernatant, and detecting the light absorption value of violacein induced by AHL in CV026 at 545nm absorption wavelength.

Detection of 3-O-C12-HSL production: after ethyl acetate extract was evaporated by rotary evaporation, a reporter strain NT1 was added, the cells were disrupted after overnight culture, and the disrupted supernatant was examined for β -galactosidase activity induced by AHL to NT 1. As can be seen in FIGS. 6B and 6C, the expression of aigC in PAO1 significantly reduced the production of C4-HSL and 3-O-C12-HSL in PAO 1.

Example 8 Effect of AigC expression on the AHL mediated biological phenotype of the pathogenic bacterium PAO1

Detection of cluster motility: semisolid culture media (1% tryptone, 0.5% NaCl, 0.35% agarose) for detecting the motility of PAO1 clusters were prepared, recombinant bacteria PAO1(aigC) activated on LB plates and control PAO1(pBBR1) were dipped with toothpicks, spotted on the center of the motility culture medium plates, and after static culture in a 37 ℃ incubator for 17 hours, the motility diameters were observed and counted. The results are shown in fig. 6D, where aigC expression significantly attenuated the motility of PAO 1.

And (3) detecting the protease activity: seed culture to OD overnight₆₀₀Mu.l of the inoculum was added to wells punched out with a punch on LB + 1% mounted mill plates, 3 replicates each, at 0.5. The plate was subjected to static culture in an incubator at 37 ℃ for 17 hours, and the size of a transparent circle generated around the well by the strain was measured to quantify the protease activity. Results as shown in fig. 6E, expression of aigC significantly impaired the protease production of PAO 1.

Example 9 Effect of the expression of aigC on AHL mediated pathogenicity of the pathogenic bacterium PAO1

Culturing the recombinant strain seed liquid overnight, and resuspending the strain liquid to OD with PBS buffer solution₆₀₀1.0. Taking a fresh lettuce stalk part of 2cm by 4cm and a fresh Chinese cabbage stalk part of 5cm by 4cm, slightly stabbing wounds at the centers of the surfaces of the lettuce stalk and the Chinese cabbage stalk by using a sterile small gun head, taking 20 mu l of bacterial suspension to be added on the wounds, repeating three times for each treatment setting, carrying out moisture-preserving culture at 30 ℃ for 3 days, and observing and counting the sizes of the scabs. The results are shown in fig. 7, where aigC expression significantly attenuated the pathogenicity of PAO 1.

Sequence listing

<110> southern China university of agriculture

<120> N-acyl homoserine lactone acyltransferase encoding gene aigC and application thereof

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gatgccttct acgagctgaa ccgcgccgat acccttgcca aggcccgcac ggcggcttcg 1260

aagatccagt cgccgggcct caacgtggta tgggccaacg cccgcggcga catcggctgg 1320

tgggcatcgg cgcaattgcc ggtacgcccg gacggggtca acccgaactt cctgctcgac 1380

ggcgccagcg gccaggccga caagaccggc ttctacccct tcagcgagaa cccgcaggaa 1440

gaaaacccgg cgcgcggcta catcgtctcg gccaacttcc agccggtacc ggccaacggt 1500

cgcccggtgc cgggctacta caacctgccc gaccgcggtc agcaactgaa caagcgcctg 1560

tccgacgatt cggtgaaatg ggacctgcag aacagccagg cgctgcaact ggacaccgcc 1620

accggctatg gcccgcgctt cctcaagccg ctgctgccga tcctgcgtga agccgcggca 1680

accgacgaag agaaggcgct ggtggagagc ctggccaact ggcagggcga ccatccgctg 1740

gactccgtga ccgccacgct gttcaaccag ctcctctacc aggtggcaga cggcgccatg 1800

cgcgacgaga tgggcgacgc cttcttcgac aacctgctgt ccacccgcgt gctcgacgtg 1860

gccctgccgc gcctggccgc cgacgagggc tcgccctggt gggacaaccg caagacgccg 1920

cagaaggaaa ctcgcgcgga tatcgtcaag gccgcctgga aaggcagcct ggcgcatctg 1980

cgcaccaccc tcggccagga ctcgaaacag tggctgtggg gtaaggcgca caccctgacc 2040

cacggccacc cgctgggcca gcagaagccg ctggatcgcc tcttcaatgt cggcccattc 2100

gccgcgccgg gcggccacga ggtaccgaac aacctctccg cccgcgtcgg cccggcaccc 2160

tggcaggtgg tctacggccc gtccacccgt cgcctgatcg acttcgccga cccggcccac 2220

agcctgggca tcaacccggt gggccagagc ggcgtgccct tcgacaagca ctacgacgac 2280

caggccgaag cctacatcga aggccagtac ctgccgcagc actacgatga aaacgaggtg 2340

aaggccaaca ccaagggcat cctgcggctg atccccgtcc gtcgctga 2388

<210> 2

<211> 795

<212> PRT

<213> germ

<400> 2

Met Lys Arg Thr Leu Thr Val Leu Ala Val Val Val Val Ala Ala Ala

1 5 10 15

Ala Gly Ala Gly Trp Tyr Leu His Gly Lys Gln Pro Val Arg Asp Gly

20 25 30

Gln Leu Pro Leu Ala Gly Leu Ala Ser Glu Val Thr Val Arg Tyr Asp

35 40 45

Glu Arg Gly Val Pro His Ile Lys Ala Gly Ser Glu Glu Asp Met Tyr

50 55 60

Arg Ala Ile Gly Tyr Val His Ala Gln Asp Arg Leu Phe Gln Met Glu

65 70 75 80

Ile Leu Arg Arg Leu Ser Arg Gly Glu Leu Ala Glu Val Leu Gly Pro

85 90 95

Lys Leu Val Asp Thr Asp Arg Met Phe Arg Ser Leu Arg Ile Arg Asp

100 105 110

His Ala Ala Glu Tyr Val Lys Thr Gln Asp Lys Asn Ser Pro Ala Trp

115 120 125

Lys Ala Leu Val Ala Tyr Leu Asp Gly Val Asn Gln Phe Gln Asp Ser

130 135 140

His Pro Arg Pro Val Glu Phe Asp Ile Leu Gly Ile Pro Lys Arg Pro

145 150 155 160

Phe Thr Pro Glu Asp Thr Val Ser Val Ala Gly Tyr Met Ala Tyr Ser

165 170 175

Phe Ala Ala Ala Phe Arg Thr Glu Pro Val Leu Thr Tyr Val Arg Asp

180 185 190

Gln Leu Gly Ala Asp Tyr Leu Lys Val Phe Asp Leu Asp Trp His Pro

195 200 205

Asn Gly Val Leu Thr Pro Ser Pro Leu Ala Ala Ala Asp Trp Gln Asp

210 215 220

Met Ser Ala Ile Ala Gln Leu Ser His Ala Ala Leu Glu Lys Ala Gly

225 230 235 240

Leu Pro Gln Phe Glu Gly Ser Asn Ala Trp Ala Val Ser Gly Ser Arg

245 250 255

Thr Lys Ser Gly Lys Pro Leu Leu Ala Gly Asp Pro His Ile Arg Phe

260 265 270

Ala Val Pro Ala Val Trp Tyr Glu Met Gln Ala Ser Ala Pro Gly Phe

275 280 285

Glu Leu Tyr Gly His Tyr Gln Ala Leu Asn Pro Phe Ala Ser Leu Gly

290 295 300

His Asn Leu Gln Phe Gly Trp Ser Leu Thr Met Phe Gln Asn Asp Asp

305 310 315 320

Val Asp Leu Val Ala Glu Lys Val Asn Pro Asp Asn Pro Asn Gln Val

325 330 335

Trp Tyr His Gly Gln Trp Val Asp Leu Lys Ser Glu Glu Gln Ser Ile

340 345 350

Ala Val Lys Gly Glu Ala Pro Val Lys Ile Thr Leu Arg Ser Ser Pro

355 360 365

His Gly Pro Leu Val Asn Asp Ala Leu Gly Thr Ala Ala Gly Lys Thr

370 375 380

Pro Val Ala Met Trp Trp Ala Phe Leu Glu Thr Gln Asn Pro Ile Leu

385 390 395 400

Asp Ala Phe Tyr Glu Leu Asn Arg Ala Asp Thr Leu Ala Lys Ala Arg

405 410 415

Thr Ala Ala Ser Lys Ile Gln Ser Pro Gly Leu Asn Val Val Trp Ala

420 425 430

Asn Ala Arg Gly Asp Ile Gly Trp Trp Ala Ser Ala Gln Leu Pro Val

435 440 445

Arg Pro Asp Gly Val Asn Pro Asn Phe Leu Leu Asp Gly Ala Ser Gly

450 455 460

Gln Ala Asp Lys Thr Gly Phe Tyr Pro Phe Ser Glu Asn Pro Gln Glu

465 470 475 480

Glu Asn Pro Ala Arg Gly Tyr Ile Val Ser Ala Asn Phe Gln Pro Val

485 490 495

Pro Ala Asn Gly Arg Pro Val Pro Gly Tyr Tyr Asn Leu Pro Asp Arg

500 505 510

Gly Gln Gln Leu Asn Lys Arg Leu Ser Asp Asp Ser Val Lys Trp Asp

515 520 525

Leu Gln Asn Ser Gln Ala Leu Gln Leu Asp Thr Ala Thr Gly Tyr Gly

530 535 540

Pro Arg Phe Leu Lys Pro Leu Leu Pro Ile Leu Arg Glu Ala Ala Ala

545 550 555 560

Thr Asp Glu Glu Lys Ala Leu Val Glu Ser Leu Ala Asn Trp Gln Gly

565 570 575

Asp His Pro Leu Asp Ser Val Thr Ala Thr Leu Phe Asn Gln Leu Leu

580 585 590

Tyr Gln Val Ala Asp Gly Ala Met Arg Asp Glu Met Gly Asp Ala Phe

595 600 605

Phe Asp Asn Leu Leu Ser Thr Arg Val Leu Asp Val Ala Leu Pro Arg

610 615 620

Leu Ala Ala Asp Glu Gly Ser Pro Trp Trp Asp Asn Arg Lys Thr Pro

625 630 635 640

Gln Lys Glu Thr Arg Ala Asp Ile Val Lys Ala Ala Trp Lys Gly Ser

645 650 655

Leu Ala His Leu Arg Thr Thr Leu Gly Gln Asp Ser Lys Gln Trp Leu

660 665 670

Trp Gly Lys Ala His Thr Leu Thr His Gly His Pro Leu Gly Gln Gln

675 680 685

Lys Pro Leu Asp Arg Leu Phe Asn Val Gly Pro Phe Ala Ala Pro Gly

690 695 700

Gly His Glu Val Pro Asn Asn Leu Ser Ala Arg Val Gly Pro Ala Pro

705 710 715 720

Trp Gln Val Val Tyr Gly Pro Ser Thr Arg Arg Leu Ile Asp Phe Ala

725 730 735

Asp Pro Ala His Ser Leu Gly Ile Asn Pro Val Gly Gln Ser Gly Val

740 745 750

Pro Phe Asp Lys His Tyr Asp Asp Gln Ala Glu Ala Tyr Ile Glu Gly

755 760 765

Gln Tyr Leu Pro Gln His Tyr Asp Glu Asn Glu Val Lys Ala Asn Thr

770 775 780

Lys Gly Ile Leu Arg Leu Ile Pro Val Arg Arg

785 790 795

<210> 3

<211> 40

<212> DNA

<213> germ

<400> 3

gtcgacggta tcgataagct tgcagaatcg ccgcataaca 40

<210> 4

<211> 39

<212> DNA

<213> germ

<400> 4

cgctctagaa ctagtggatc ctcagcgacg gacggggat 39

<210> 5

<211> 44

<212> DNA

<213> germ

<400> 5

gccatggctg atatcggatc catgaaacgc actttgactg tcct 44

<210> 6

<211> 39

<212> DNA

<213> germ

<400> 6

ctcgagtgcg gccgcaagct ttcagcgacg gacggggat 39

Claims (10)

1. An N-acyl homoserine lactone acyltransferase encoding gene aigC, which is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO: 1 is shown.

2. A recombinant vector comprising the gene encoding N-acylhomoserine lactone acylase of claim 1.

3. A recombinant bacterium comprising the recombinant vector of claim 2.

4. An N-acyl homoserine lactone acyltransferase characterized in that its amino acid sequence is as shown in SEQ ID NO: 2, respectively.

5. The recombinant bacterium of claim 3, or the use of the N-acylhomoserine lactone acyltransferase of claim 4 for degrading AHLs signal molecules.

6. The use of claim 5, wherein said AHLs signal molecules comprise C4-HSL, C6-SHL, 3-O-C6-HSL, C8-HSL, 3-OH-C8-HSL, C10-HSL, 3-OH-C10-HSL, C12-HSL, 3-O-C12-HSL, 3-OH-C12-HSL, 3-OH-C14-HSL.

7. Use of the gene encoding N-acyl homoserine lactone acyltransferase according to claim 1, or the recombinant vector according to claim 2, or the recombinant bacterium according to claim 3, or the N-acyl homoserine lactone acyltransferase according to claim 4 for controlling pathogenic bacteria which cause AHLs-dependent diseases.

8. The use of claim 7, wherein said AHLs-dependent pathogenic bacteria comprise Pectinobacterium carotovorum subspecies, Burkholderia cepacia, Pseudomonas aeruginosa, Laurella solanacearum, Agrobacterium tumefaciens, Rhizoctonia solani, Erwinia carotovora, bacterial wilt bacteria of maize.

9. The use according to claim 8, wherein when the gene encoding N-acyl homoserine lactone acyltransferase of claim 1 is used, the gene encoding N-acyl homoserine lactone acyltransferase is expressed in AHLs mediated pathogenic bacteria, and recombinant pathogenic bacteria for successful expression of N-acyl homoserine lactone acyltransferase are selected; when the recombinant vector of claim 2 is used, the recombinant vector is introduced into AHLs mediated pathogenic bacteria, and recombinant pathogenic bacteria for successfully expressing N-acyl homoserine lactone acyltransferase are screened; when the recombinant bacterium according to claim 3 is used, a recombinant pathogen is selected which successfully expresses N-acylhomoserine lactone acylase.

10. Use according to claim 9, wherein the recombinant pathogen that successfully expresses an N-acyl homoserine lactone acyltransferase:

(1) the yield of AHLs is reduced; and/or the presence of a gas in the gas,

(2) the sports type is reduced; and/or the presence of a gas in the gas,

(3) reduces the formation of biofilm; and/or the presence of a gas in the gas,

(4) the production amount of protease is reduced; and/or the presence of a gas in the gas,

(5) the pathogenic force is weakened.

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