CN107988189B - Application of enzyme FadD1 in pseudomonas aeruginosa in degradation of DSF family signal molecules - Google Patents
Application of enzyme FadD1 in pseudomonas aeruginosa in degradation of DSF family signal molecules Download PDFInfo
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- CN107988189B CN107988189B CN201711231930.8A CN201711231930A CN107988189B CN 107988189 B CN107988189 B CN 107988189B CN 201711231930 A CN201711231930 A CN 201711231930A CN 107988189 B CN107988189 B CN 107988189B
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- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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Abstract
The invention discloses application of an enzyme FadD1 in pseudomonas aeruginosa in degradation of DSF family signal molecules. The invention obtains an enzyme FadD1 which can degrade DSF family signal molecules generated by thalli in vitro from pseudomonas aeruginosa, and the amino acid sequence of the enzyme FadD1 is shown as SEQ ID NO: 2, respectively. The enzyme FadD1 can degrade DSF family signal molecules outside the thallus, including signal molecules DSF of xanthomonas campestris and quorum sensing signals BDSF in Burkholderia cepacia, so that the enzyme FadD1 can be used for preparing preparations for degrading the DSF family signal molecules in vitro, and can be applied to preventing and treating plant or animal diseases caused by pathogenic bacteria taking DSF or BDSF as quorum sensing signals, especially plant black rot caused by xanthomonas campestris and diseases caused by Burkholderia cepacia.
Description
Technical Field
The invention relates to the technical field of enzyme application, in particular to application of an enzyme FadD1 in pseudomonas aeruginosa in degradation of DSF family signal molecules in vitro.
Background
Quorum Sensing (QS) is an important communication between microorganisms that aids bacteria in sensing Quorum and thus regulating gene expression. Xanthomonas campestris (X.campestrispv. campestris, Xcc) is a gram-negative phytopathogen that infects all crucifers causing black rot. In Xanthomonas campestris, the QS system, mediated by a DSF family (differentiated signal factor family) signal molecule, plays a key role in resisting plant infection by the immune defense system and system of the plant. Burkholderia cepacia (Burkholderia cepacia) is a conditional pathogen, can cause respiratory tract infection, pneumonia, urinary tract infection and the like of human and animals, and is harmful to the health and life of human. Burkholderia cepacia can produce a signal molecule BDSF with a structure similar to DSF, and regulates the biological function and signal conduction path of thallus. In recent years, researches show that Burkholderia and Pseudomonas aeruginosa (P. aeruginosa) coexist in lung cells of pneumonia patients, and the two bacteria have a certain competitive relationship and participate in the pathogenesis of pneumonia.
The research shows that the 'quorum quenching' is the most promising treatment strategy, and the quorum quenching is to prevent the signal molecules of pathogenic bacteria from effectively accumulating, and the expression of pathogenic genes of the pathogenic bacteria cannot be activated when the concentration of the signal molecules is reduced, so that the intercellular communication is destroyed, and the quorum sensing system is destroyed.
It is reported that the rpfB gene positioned at the upstream of the DSF synthetic gene rpfF in xanthomonas campestris is identified as fatty acyl-CoA ligase coding gene and participates in the degradation of DSF family signal molecules to regulate QS system expression. However, the enzyme RpfB in Xanthomonas campestris can only degrade DSF or BDSF in vivo, and the effect of preventing and treating related diseases of germs is not good enough, even no effect at all.
Disclosure of Invention
The first object of the present invention is to overcome the disadvantages of the prior art by providing an enzyme FadD1 for the in vitro degradation of DSF family signal molecules.
It is a second object of the invention to provide a gene encoding the enzyme FadD 1.
The third purpose of the invention is to provide the application of the enzyme FadD1 in the degradation of DSF family signal molecules in vitro.
The fourth purpose of the invention is to provide the application of the enzyme FadD1 in preparing the medicine for preventing and treating the plant black rot caused by Xanthomonas campestris or the disease caused by Burkholderia cepacia.
The fifth object of the present invention is to provide a medicament for controlling plant diseases caused by xanthomonas campestris or diseases caused by burkholderia cepacia.
The sixth purpose of the invention is to provide a medicine for preventing and treating pneumonia or plant black rot.
In order to achieve the purpose, the invention is realized by the following technical scheme:
an enzyme FadD1 for degrading DSF family signal molecules in vitro, wherein the amino acid sequence of the enzyme FadD1 is shown as SEQ ID NO: 2, respectively.
A gene encoding the enzyme FadD1 according to claim 1, having the nucleotide sequence shown in SEQ ID NO: 1 is shown.
Application of the enzyme FadD1 in degradation of DSF family signal molecules in vitro.
Application of the enzyme FadD1 in preparing medicine for preventing and treating plant black rot caused by xanthomonas campestris or diseases caused by Burkholderia cepacia.
Preferably, the enzyme FadD1 is the enzyme FadD1 in Pseudomonas aeruginosa (PAO 1).
Preferably, the DSF family signal molecule is the signal molecule DSF of xanthomonas campestris or the quorum sensing signal molecule BDSF in burkholderia cepacia.
A medicine for preventing and treating the plant diseases caused by xanthomonas campestris or the diseases caused by Burkholderia cepacia contains the enzyme FadD 1.
A medicine for preventing and treating pneumonia or plant black rot contains the enzyme FadD 1.
Preferably, the plant black rot is caused by Xanthomonas campestris.
Preferably, the pneumonia is caused by burkholderia cepacia.
Compared with the prior art, the invention has the following beneficial effects:
the enzyme FadD1 in Pseudomonas aeruginosa can degrade DSF family signal molecules outside the thallus, so that the enzyme FadD1 can be used for degrading signal molecules DSF and BDSF, and the preparation of the enzyme FadD1 can be used for preventing and treating plant black rot caused by xanthomonas campestris and diseases caused by Burkholderia cepacia.
Drawings
FIG. 1 is an alignment of the amino acid sequence of FadD1 in Pseudomonas aeruginosa with the amino acid sequence of RpfB in Xanthomonas.
FIG. 2 shows the expression and purification of the enzyme FadD 1; 3. lane 4 is unpurified protein; 3. lane 4 shows the purified target protein.
FIG. 3 shows the results of in vitro degradation of signal molecules BDSF and DSF by the enzyme FadD1, a is the results of detection of L C-MS after different periods of in vitro reaction of FadD1 and BDSF, and b is the results of detection of L C-MS after different periods of in vitro reaction of FadD1 and DSF.
FIG. 4 is a graph showing the effect of overexpression of fadD1 on motility, biofilm and protease activity of strain H111; a is the movement capacity of different strains; b different strains produce biofilm yield; c the magnitude of protease activity of different strains.
FIG. 5 is a graph showing the effect of overexpression of fadD1 on motility, biofilm and exopolysaccharide production after strain 8004; a is the size of the motor ability of different strains; b is the condition of biofilm produced by different strains; c is the yield of exopolysaccharide of different strains.
FIG. 6 is a graph showing the effect of overexpression of the gene fadD1 on onion infestation after strain H111.
FIG. 7 is a graph showing the effect of overexpression of the gene fadD1 on Chinese cabbage infection after strain 8004.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 obtaining of fadD1 Gene sequence
B L AST alignment is carried out on the rpfB amino acid sequence of the known Xanthomonas in the genome database of the Pseudomonas aeruginosa PAO1, a fadD1 sequence with 55 percent of homology with rpfB is obtained, and the alignment result of the amino acid sequences of the fadD1 and the rpfB is shown in a figure 1.
The genome of pseudomonas aeruginosa PAO1 is used as a template, primers are designed for PCR amplification, and the amplification primers are as follows:
fadD1-F：5’-ATGATCGAAAACTTCTGGAAGGAC；
fadD1-R：5’-TTACTTCTGGCCCGCTTTCTT，
the fadD1 gene of pseudomonas aeruginosa and the sequence thereof are obtained through cloning analysis, and are shown as SEQ ID NO: 1, the total length is 1689bp, the GC% content is 63.41%, the size of the coded protein is 61.65kDa, and the amino acid sequence is shown as SEQ ID NO: 2, respectively.
Example 2 FadD1 degradation signals BDSF and DSF in vitro
1. Expression and purification of FadD1
Constructing a recombinant plasmid fadD1-pET-21a (+), transforming the recombinant plasmid fadD1-pET-21a (+) into Ecoli.B L21 (DE3), inducing expression by IPTG, analyzing the expression product by SDS-PAGE, wherein lanes 1 and 2 in figure 2 are supernatants obtained by breaking thalli, the size of the target protein FadD1 is 61.65kDa, and the C end of the target protein is connected with (His)6The tag was purified by NiSepharose 6 Fast Flow Beads using affinity chromatography.
2. FadD1 in vitro degradation signal molecules BDSF and DSF
The reaction system for degrading the BDSF by the FadD1 is that 5 mu M FadD1, 200 mu M BDSF, 30 mu L TME Buffer and 0.01MPBS Buffer are supplemented to 200 mu L, the mixture is placed at 37 ℃ and reacts for 6h, 12h, 18h and 24h respectively, then the mixture is taken out and boiled for 5min to terminate the reaction, reaction products are detected by L C-MS, three times of treatment are repeated for each time, the contrast is that an equivalent amount of inactivated enzyme solution is added, and the BDSF is changed into DSF in the reaction system for degrading the DSF by the FadD1, and the rest is the same as above.
3. Results
L C-MS measured the signal intensity (EI) of BDSF and DSF samples, and calculated the ratio of BDSF signal intensity after degradation to the control, as shown in figure 3. after 6h reaction, the amount of BDSF and DSF was reduced, and after 24h reaction, the BDSF and DSF were almost completely degraded, and the results showed that FadD1 had the function of degrading the DSF family signals.
Example 3 determination of the phenotypic Effect of FadD1 on the Regulation of the Burkholderia cepacia BDSF System
1. Construction of Burkholderia cepacia Strain overexpressing the Gene fadD1
An overexpression plasmid fadD1-pBBRI-5 is constructed, and the plasmid is transformed into wild Burkholderia cepacia H111 (hereinafter referred to as H111) to construct a Burkholderia cepacia strain (hereinafter referred to as H111(fadD1)) overexpressing a gene fadD 1.
2. Activation of Burkholderia cepacia
A wild strain H111 of Burkholderia cepacia, a mutant strain of Burkholderia cepacia, namely a gene Bcam0581 related to BDSF biosynthesis in the H111 strain (hereinafter referred to as mutant 0581) and a strain H111(fadD1) of an over-expressed gene fadD1 are activated on a L B plate and are placed in an incubator at 37 ℃ for overnight culture.
3. Effect of FadD1 on bacterial Strain biofilms
Inoculating activated Burkholderia cepacia strain H111, mutant 0581 and H111(fadD1) into L B liquid medium (tryptone 10 g/L, yeast extract 5 g/L10 g/L) for overnight culture, and measuring OD of bacterial liquid600Value, using MM medium (K)2HPO410.5g/L，KH2PO44.5g/L，(NH4)2PO42g/L，MgSO4.7H2O0.2g/L，FeSO40.005g/L，CaCl20.01g/L，MnCl20.002 g/L, mannitol 2 g/L, glycerol 2 g/L) diluted the bacterial liquid to OD600150. mu. L each was added to a 96-well plate at 0.01, 6 replicates of each treatment were added, and 5. mu.M of the BDSF mutant and overexpression were set as a reference, incubated at 37 ℃ in a shaker at 450rpm, the cells were shaken for 12h, the cell suspension was discarded, 200. mu. L0.1.1% crystal violet was added, treated at room temperature for 20min, and treated with ddH2O cleaning 96-well plate for 3 times, oven drying, adding 200 μ L95% ethanol, and determining OD570Data were processed using GraphPadPrism 6 software.
4. Effect of FadD1 on Strain motility
Inoculating activated Burkholderia cepacia strains H111, 0581 and H111(fadD1) into L B liquid culture medium, and culturing at 37 deg.C and 200rpm overnight to obtain bacterial liquid OD600The value is 1.0, 2 mu L fresh bacterial liquid is addedTo the center of a plate into which 15M L of a motile medium (tryptone 8 g/L, glucose 5 g/L, agar 3 g/L) had been poured, and a medium to which 5. mu.M BDSF had been added was set as a reference, and the set-up was repeated 5 times, the distance traveled by the test strain on the plate was measured after the plate was placed in a 30 ℃ incubator for 18 hours, and experimental data was recorded.
5. Effect of FadD1 on protease Activity of strains
Inoculating activated Burkholderia cepacia strains H111, 0581 and H111(fadD1) into NYG liquid culture medium (tryptone 5 g/L, yeast extract powder 3 g/L, glycerol 20 g/L) at 37 deg.C and 200rpm overnight to OD600The value is 3.5-4.0, the mixture is taken out and centrifuged at 13000rpm for 10min, the supernatant is collected and filtered through a filter membrane with the aperture of 0.2 mu m for sterilization, 100 mu L of the supernatant is sucked and added with 5mg/m L azocasein with the same volume, the mixture is incubated at 30 ℃ for 60min and then added with 400 mu L TCA Buffer to terminate the reaction, the mixture is stood at room temperature for 2min and then centrifuged at 13000rpm for 1min, the supernatant is transferred to a new EP tube, 700 mu L525 mM NaOH is added and mixed evenly, and then OD is added with OD L mM NaOH442Absorbance was measured. Calculate OD442/OD600The value is the protease activity.
6. Results
As shown in fig. 4, compared with the wild strain H111, the mutant 0581 has significantly reduced motility, biofilm formation and protease activity, while the overexpression gene fadD1 strain H111(fadD1) has less motility, biofilm formation and protease activity than the wild strain, which indicates that fadD1 can affect the BDSF system-regulated phenotype in burkholderia H111 by degrading signal BDSF. While exogenous addition of 5. mu.M BDSF to the mutant and H111(fadD1) restored the phenotype.
Example 4 determination of the phenotypic Effect of FadD1 on the modulation of the DSF System by Xanthomonas campestris 8004
1. Construction of Xanthomonas strain overexpressing gene fadD1
The overexpression plasmid fadD1-pBBRI-5 was transformed into wild type Xanthomonas, and a Xanthomonas strain overexpressing the gene fadD1 (hereinafter, abbreviated as 8004(fadD1)) was constructed.
2. Xanthomonas activation
Xanthomonas wild type 8004 (hereinafter abbreviated as 8004), Xanthomonas RpfF mutant strain 8004dF, i.e., a gene RpfF knockout related to DSF synthesis in Xanthomonas (hereinafter abbreviated as mutant 8004dF), and strain 8004 (fade 1) were activated on L B plates and cultured in a 28 ℃ incubator for 48 hours.
3. Effect of FadD1 on the motility of Xanthomonas
Activated xanthomonas 8004, mutants 8004dF, and 8004(fadD1) were picked up and inoculated into L B liquid medium, and cultured at 28 ℃ and 200rpm to bacterial liquid OD600The value was 1.0. mu. L fresh bacterial solution was added to the center of a plate into which 15M L motility medium (tryptone 8 g/L, glucose 5 g/L, agar 3 g/L) had been poured, and medium to which 5. mu.M DSF had been added was set as a reference, 3 replicates each were set, and the distance traveled by the test strain on the plate was measured after the plate was placed in a 28 ℃ incubator for 48 hours, and the experimental data was recorded.
4. Effect of FadD1 on Xanthomonas biofilms
Activated Xanthomonas 8004, mutants 8004dF, and 8004(fadD1) were picked and inoculated into YEB broth (tryptone 10 g/L, yeast extract 5 g/L5 g/L, sucrose 5 g/L4·7H2O0.5g/L) was cultured at 28 ℃ and 200rpm until the OD of the bacterial liquid600The value was 1.0, and 10. mu. L of the bacterial suspension was observed under a microscope.
5. Effect of FadD1 on exopolysaccharide production by Xanthomonas
Activated Xanthomonas 8004, mutants 8004dF, and 8004(fadD1) were picked and inoculated into YEB broth, and cultured at 28 ℃ and 200rpm to bacterial liquid OD600The value was 3.0, 3 replicates per strain, and 5. mu.M DSF addition was set as a reference. Centrifuging at 12000rpm for 20min, pouring the supernatant into a weighed centrifuge tube, adding 2.5 times of anhydrous ethanol, standing at 4 ℃ for 30min, centrifuging at 10000rpm for 10min, discarding the supernatant, drying the precipitate in a 60 ℃ oven to obtain extracellular polysaccharide, weighing, and recording data.
6. Results
As shown in fig. 5, compared with wild strain 8004, mutant 8004dF has a significant decrease in both motility and exopolysaccharide production, a sheet precipitate of biofilm structure can be seen in mutant bacterial liquid, and both motility and exopolysaccharide production of overexpression gene fadD1 strain 8004(fadD1) are less than wild type, which indicates that fadD1 can affect DSF system-regulated phenotype in xanthomonas 8004 through degradation signal DSF. Exogenous addition of 5. mu.M DSF to the mutant and 8004(fadD1) restored the phenotype.
Example 5 effect of FadD1 on burkholderia cepacia infection of onions:
1. burkholderia cepacia preparation
The Burkholderia cepacia strain activated in example 3, wild type H111, mutant 0581 and H111(fadD1) were selected, inoculated into L B culture medium, and cultured with shaking at 37 ℃ and 200rpm until OD of the culture medium600The value was 1.0.
2. Effect of FadD1 on Burkholderia cepacia infection of onions
Cutting fresh Bulbus Allii Cepae from the middle under aseptic condition, covering round filter paper with diameter of 1cm on the center of section of Bulbus Allii Cepae, respectively sucking 5 μ L fresh bacterial liquid with pipette gun, adding 5 μ L ddH2O as a reference, onion was observed for infestation after 48h in a 37 ℃ incubator and photographed, with three replicates per treatment.
3. Results
As shown in figure 6, compared with wild type H111, mutant Bcam0581 has obviously weakened pathogenicity on onion, and H111(fadD1) infects onion with smaller lesion area than wild type H111, which shows that FadD1 can regulate the pathogenicity of Burkholderia H111 by degrading signal molecule BDSF.
Example 6 Effect of FadD1 on Xanthomonas infection in cabbage
1. Preparation of Xanthomonas:
the activated xanthomonas strain wild type 8004, mutant 8004dF, and 8004 (fade 1) of example 4 were picked up and inoculated into L B liquid medium, and shake-cultured at 37 ℃ and 200rpm to bacterial liquid OD600The value was 1.0.
2. Influence of FadD1 on infection of Xanthomonas on Chinese cabbage
Placing fresh folium Brassicae Capitatae in a culture dish, injecting 2 μ L fresh bacterial liquid into folium Brassicae Capitatae, and injecting 2 μ L ddH2O as a reference, the cabbage was observed for infestation and photographed 7 days after being placed in an incubator at 28 ℃, and each treatment was repeated three times.
3. Results
As shown in fig. 7, compared with xanthomonas wild type 8004, mutant 8004dF is less pathogenic to cabbages, and the lesion area of the cabbages infected with 8004(fadD1) is smaller than that of wild type 8004, indicating that fadD1 can regulate the pathogenic ability of xanthomonas 8004 by degrading signal molecule DSF.
Sequence listing
<110> southern China university of agriculture
<120> application of enzyme FadD1 in pseudomonas aeruginosa in degradation of DSF family signal molecules
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>1689
<212>DNA
<213> Pseudomonas aeruginosa (Pseudomonas aeruginosa)
<400>1
atgatcgaaa acttctggaa ggacaagtac ccagcgggta ttgctgccga gatcaatccc 60
gaccagtacc cgaacatcct gtcggtactg aaggagtcct gccaacgttt tgccaccaag 120
cccgcgttca ccaacctggg caagaccctg acctacggcg agctgtacaa gctgtccggc 180
gacttcgccg cctacctgca gcagcacacc gatctcaagc ccggcgaccg catcgccgtc 240
cagttgccca acgtcctgca atacccgatc gtggtcttcg gcgccatgcg cgccggcctc 300
atcgtggtca acaccaaccc gctgtacacc gcgcgcgaac tggaacacca gttcaacgac 360
tccggcgcca aggcggtggt ctgcctggcc aacatggccc acctggtcga gggcgtactg 420
ccgaagaccg gcgtcaagca ggtgatcgtc accgaagtcg gcgacatcct gccgccgctg 480
aagcgcttca tcgtcaactt cgtggtcaag cacatcaaga agatggtgcc ggcctacagc 540
ctgccgcaag ccaccaagct gaccgacgcg ctggccaggg gcgccggcaa gtcgttccag 600
gaagccgcgc cgcaggccga cgacgtcgcc gtgctgcaat acaccggcgg caccaccggg 660
gtcgccaagg gcgccatgct gacccaccgc aacctggtgg ccaacatgtt gcagtgcaag 720
gcgctgatgg gcgccaacct caacgagggc tgcgagatcc tcatcgcgcc gctgccgctg 780
taccacatct atgccttcac cttccattgc atggcgatga tgctcaccgg caaccacaac 840
atcctgatca ccaacccgcg cgacctgccg tcgatgctca aggacctcgg ccagtggaag 900
ttcaccggct tcgtcggcct gaacaccctg ttcgtcgccc tgtgcaacaa cgaaaccttc 960
cgcaagctgg acttctccgc gctgaagctg accctctccg gcggcatggc gctgcaactg 1020
gccaccgcgg agcgctggaa ggaagtcacc ggctgcgcca tctgcgaagg ctacggcatg 1080
accgagaccg cgccggtggt atcggtcaac ccgttccaga acatccaggt cggcaccatc 1140
ggtatcccgg tgccctcgac cctgtgcaag gtgattggcg acgacggcca ggaagtgccc 1200
ctgggcgagc gcggcgaact ctgcgtgaag ggtccgcagg tgatgaaggg ctactggcag 1260
cgccaggagg ccaccgacga gatcctcgac gccgatggtt ggctgaagac cggcgatatc 1320
gccatcatcc aggaagacgg ctacatgcgc atcgtcgacc ggaagaagga catgatcctg 1380
gtctccggct tcaatgtgta tccgaacgaa ctggaagacg tgctggcgac cctgccgggc 1440
gtcctgcagt gcgccgcgat cggcatcccc gacgagaagt ccggcgagtc gatcaaggtc 1500
ttcgtggtgg tcaagccggg ggcgaccctg accaaggaac aggtcatgca gcacatgcat 1560
gacaacctga ccggctacaa gcggccgaag gccgtggagt tccgcgacag cctgccgacc 1620
accaacgtcg gcaagatcct gcgccgcgaa ctgcgcgacg aagagctgaa gaaagcgggc 1680
cagaagtaa 1689
<210>2
<211>562
<212>PRT
<213> Pseudomonas aeruginosa (Pseudomonas aeruginosa)
<400>2
Met Ile Glu Asn Phe Trp Lys Asp Lys Tyr Pro Ala Gly Ile Ala Ala
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Glu Ile Asn Pro Asp Gln Tyr Pro Asn Ile Leu Ser Val Leu Lys Glu
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Ser Cys Gln Arg Phe Ala Thr Lys Pro Ala Phe Thr Asn Leu Gly Lys
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Thr Leu Thr Tyr Gly Glu Leu Tyr Lys Leu Ser Gly Asp Phe Ala Ala
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Tyr Leu Gln Gln His Thr Asp Leu Lys Pro Gly Asp Arg Ile Ala Val
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Gln Leu Pro Asn Val Leu Gln Tyr Pro Ile Val Val Phe Gly Ala Met
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Arg Ala Gly Leu Ile Val Val Asn Thr Asn Pro Leu Tyr Thr Ala Arg
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Glu Leu Glu His Gln Phe Asn Asp Ser Gly Ala Lys Ala Val Val Cys
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Leu Ala Asn Met Ala His Leu Val Glu Gly Val Leu Pro Lys Thr Gly
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Val Lys Gln Val Ile Val Thr Glu Val Gly Asp Ile Leu Pro Pro Leu
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Lys Arg Phe Ile Val Asn Phe Val Val Lys His Ile Lys Lys Met Val
165 170 175
Pro Ala Tyr Ser Leu Pro Gln Ala Thr Lys Leu Thr Asp Ala Leu Ala
180 185 190
Arg Gly Ala Gly Lys Ser Phe Gln Glu Ala Ala Pro Gln Ala Asp Asp
195 200 205
Val Ala Val Leu Gln Tyr Thr Gly Gly Thr Thr Gly Val Ala Lys Gly
210 215 220
Ala Met Leu Thr His Arg Asn Leu Val Ala Asn Met Leu Gln Cys Lys
225 230 235 240
Ala Leu Met Gly Ala Asn Leu Asn Glu Gly Cys Glu Ile Leu Ile Ala
245 250 255
Pro Leu Pro Leu Tyr His Ile Tyr Ala Phe Thr Phe His Cys Met Ala
260 265 270
Met Met Leu Thr Gly Asn His Asn Ile Leu Ile Thr Asn Pro Arg Asp
275 280 285
Leu Pro Ser Met Leu Lys Asp Leu Gly Gln Trp Lys Phe Thr Gly Phe
290 295 300
Val Gly Leu Asn Thr Leu Phe Val Ala Leu Cys Asn Asn Glu Thr Phe
305 310 315 320
Arg Lys Leu Asp Phe Ser Ala Leu Lys Leu Thr Leu Ser Gly Gly Met
325 330 335
Ala Leu Gln Leu Ala Thr Ala Glu Arg Trp Lys Glu Val Thr Gly Cys
340 345 350
Ala Ile Cys Glu Gly Tyr Gly Met Thr Glu Thr Ala Pro Val Val Ser
355 360 365
Val Asn Pro Phe Gln Asn Ile Gln Val Gly Thr Ile Gly Ile Pro Val
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Pro Ser Thr Leu Cys Lys Val Ile Gly Asp Asp Gly Gln Glu Val Pro
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Leu Gly Glu Arg Gly Glu Leu Cys Val Lys Gly Pro Gln Val Met Lys
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Gly Tyr Trp Gln Arg Gln Glu Ala Thr Asp Glu Ile Leu Asp Ala Asp
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Gly Trp Leu Lys Thr Gly Asp Ile Ala Ile Ile Gln Glu Asp Gly Tyr
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Met Arg Ile Val Asp Arg Lys Lys Asp Met Ile Leu Val Ser Gly Phe
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Asn Val Tyr Pro Asn Glu Leu Glu Asp Val Leu Ala Thr Leu Pro Gly
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Val Leu Gln Cys Ala Ala Ile Gly Ile Pro Asp Glu Lys Ser Gly Glu
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Ser Ile Lys Val Phe Val Val Val Lys Pro Gly Ala Thr Leu Thr Lys
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Glu Gln Val Met Gln His Met His Asp Asn Leu Thr Gly Tyr Lys Arg
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Pro Lys Ala Val Glu Phe Arg Asp Ser Leu Pro Thr Thr Asn Val Gly
530 535 540
Lys Ile Leu Arg Arg Glu Leu Arg Asp Glu Glu Leu Lys Lys Ala Gly
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Gln Lys
Claims (2)
1. The amino acid sequence is shown as SEQ ID NO: 2 in the degradation of DSF family signal molecules in vitro;
the DSF family signal molecule is xanthomonas campestris quorum sensing signal molecule DSF or quorum sensing signal molecule BDSF in Burkholderia cepacia.
2. The amino acid sequence is shown as SEQ ID NO: 2 in the preparation of a medicament for preventing and treating plant black rot caused by Xanthomonas campestris (xanthmonas campestris pv. campestris) or plant diseases caused by Burkholderia cepacia (Burkholderia cepacia).
Priority Applications (1)
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| CN102071163A (en) * | 2010-11-29 | 2011-05-25 | 山东省科学院中日友好生物技术研究中心 | Multifunctional engineering strain for degrading chlorpyrifos and construction method thereof |
| CN106916831A (en) * | 2015-12-28 | 2017-07-04 | 广西大学 | A kind of application of the pathogenic related gene of Xanthomonas campestris |
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| CN102071163A (en) * | 2010-11-29 | 2011-05-25 | 山东省科学院中日友好生物技术研究中心 | Multifunctional engineering strain for degrading chlorpyrifos and construction method thereof |
| CN102071163B (en) * | 2010-11-29 | 2012-10-24 | 山东省科学院中日友好生物技术研究中心 | Multifunctional engineering strain for degrading chlorpyrifos and construction method thereof |
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