CN102943061A - Construction and application of unmarked lysobacter enzymogenes engineering strain capable of preventing plant bacteriosis - Google Patents
Construction and application of unmarked lysobacter enzymogenes engineering strain capable of preventing plant bacteriosis Download PDFInfo
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Abstract
The invention relates to a construction strategy and a construction process of an unmarked lysobacter enzymogenes engineering strain capable of preventing plant bacteriosis, and belongs to the field of microbial genetic engineering. An excellent exogenous gene aiiA is directionally integrated onto a chromosome, and any marked genes are not brought into the unmarked lysobacter enzymogenes engineering strain. A quorum-sensing system for plant pathogenic bacteria can be efficiently damaged by the engineering strain, pathogenicity of the pathogenic bacteria on a host plant (Chinese cabbage) is remarkably reduced, and biological prevention of the plant bacteriosis is realized.
Description
(1) technical field
The invention belongs to the microbiological genetic engineering field, be specifically related to the unmarked engineering strain structure of the molten bacillus of product enzyme and application that a strain can prevent and treat phytobacterial disease.
(2) background technology
Producing the molten bacillus of enzyme (Lysobacter enzymogenes) is the type species of molten Bacillaceae (Lysobacter), belong to yellow unit cell section (Xanthomonadaceae), this bacterium bacterium colony stickiness, yellow, be rich in G+C%, coasting ability is arranged, can produce proteolytic enzyme, chitinase, β-1, the 3-dextranase, the multiple extracellular enzyme such as cellulase has good antagonistic activity to various plants pathogens such as fungi, oomycetes and nematodes.
OH1 1 strains separation is a strain biocontrol bacteria bacterial strain of domestic reported first in the capsicum rhizosphere soil.This bacterial strain all has strong antagonistic activity to various plants pathogens such as Rhizoctonia solani Kuhn (Rhizoctonia solani), cucumber corruption mould (Pythium ultimum), Phytophthora capsici (Phytophthora capsici), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), fusarium graminearum (Fusarum solani), Meloidogyne incognitas (Meloidogyne incognita).In the greenhouse pot culture test, OH11 has reached 83.6% to the capsicum epidemic disease prevention effect.Yet, up to the present, have not yet to see the report that produces the molten bacillus control of enzyme phytobacterial disease.
The QS of bacterium (Quorum sensing) system is the Information exchange mechanism that bacterium is monitored population density and regulates and control the bacterium living beings function by secreting solubility signaling molecule (autoinducer, AI).The AI majority that plant pathogenetic bacteria produces is acyl homoserine lactones (AHL), and this molecule is linked to each other with stable homoserine lactone (HSL) head by a variable acyl chain (ayslside chain) afterbody.The length of different its acyl chains of AHL molecule is 4 to 18 carbon.Pathogenic on host plant of the QS system regulation bacterium that studies confirm that in a large number plant pathogenetic bacteria destroyed the QS system of bacterium, can significantly reduce the pathogenic of germ or germ is completely lost pathogenic.The method of destroying plant pathogenetic bacteria QS system has two kinds, and a kind of is that synthase gene with AI knocks out, and makes bacterium can't produce AI, causes the QS system to destroy fully.Another is to utilize relevant foreign protein (being also referred to as degrading enzyme) to come bacterium for degrading QS signaling molecule, plays the purpose of disturbing or destroying bacterium QS, and this mechanism is called colony's cancellation (Quorum quenching, QQ).In the degrading enzyme of existing report, studying the most thorough is AiiA albumen.This albumen is located away from Bacillus sp.24 bacterial strain the earliest, the AHL class signaling molecule of the different carbon chain molecule of can degrading.
In previous work, we arrive wide host's carrier PBBR1-MCS5 with the aiiA gene clone of external source, and import the OH11 bacterial strain.OH11 bacterial strain after the conversion AHL signaling molecule that Bacteria erwinia (Pectobacteriumcarotovorum) produces of significantly degrading, and significantly reduce pathogenic on Chinese cabbage and potato of this germ, show that the external source AiiA degrading enzyme based on plasmid expression helps to realize producing the molten bacillus control of enzyme phytobacterial disease.
Yet, the engineering strain that contains exogenous plasmid because often contain the microbiotic encoding gene on the plasmid, causes this class engineering strain to be used in the field and has certain risk, may horizontal transfer such as resistant gene, also may spoiled soil or the microflora of plant rhizosphere form.For this situation, alternative scheme is that the external source excellent genes is incorporated on the karyomit(e) of biocontrol bacteria, constructs the engineering strain of antibiotic-free mark.The structure of unmarked engineering strain generally needs two conditions, and the one, seek or clone the strong promoter of a constitutive expression, be convenient to the expression that on karyomit(e) high strength starts foreign gene, the 2nd, seek a suitable integration target.Because when exogenous origin gene integrator behind target gene, target gene can be inserted into the sudden change inactivation, causes the afunction of target gene.Therefore, we need to find a functional gene that the molten bacillus biocontrol effect of product enzyme is had negative regulation to be used as the target gene of exogenous origin gene integrator.
In previous work, we have made up novel, an effective promoter probe, and have cloned the strong promoter PB500 of a constitutive expression from the OH11 bacterial strain.Simultaneously, we have cloned a pigment synthesis genes involved hmgA (gene accession number: EU717786) again from produce the molten bacillus of enzyme.After this transgenation, the OH11 bacterium colony can produce melanochrome at the LB flat board, but that mutant strain improves the prevention effect of the fungal diseases of plants such as Rhizoctonia solani Kuhn is nearly 20%, shows that hmgA is a suitable exogenous origin gene integrator target.
(3) summary of the invention
The present invention will be take hmgA as target gene, SacB is reverse selection markers, on the unmarked karyomit(e) that is incorporated into OH11 of fusion gene PB500-aiiA, make up unmarked, genetic stability, and have the molten bacillus engineering strain of the product enzyme OH11PA of degraded AHL class signaling molecule ability.Engineering strain OH11PA has preferably biocontrol effect to bacterial soft rot.
Beneficial effect:
The construction strategy of unmarked genetically engineered bacteria provided by the invention can be the follow-up foreign gene directional integration that other are good and provides Technical Reference to producing in the molten bacillus of enzyme.Simultaneously, multifunctional engineering strain OH11PA provided by the invention can prevent and treat fungal diseases of plants, had again the ability of control phytobacterial disease, widened the biological and ecological methods to prevent plant disease, pests, and erosion scope of producing the molten bacillus of enzyme, for solid basis has been established in the industrialization fermentation and the field application that further realize this bacterium.
(4) description of drawings
The structure schematic diagram of Fig. 1 engineering strain OH11PA
The pcr amplification of Fig. 2 PB500, aiiA and PB500-aiiA
The electrophoresis detection of Fig. 3 dna fragmentation hmgA-F, hmgA-R, PB500-aiiA and pEX18GM
The enzyme of Fig. 4 recombinant vectors pEX18PAT is cut checking
The collection of illustrative plates of Fig. 5 plasmid pEX18PAT
Resistance and the phenotype of the doubtful engineering strain OH11PA of Fig. 6
The PCR checking of the doubtful engineering strain OH11PA of Fig. 7
The inhibition phenomenon that Fig. 8 glucose produces OH11PA melanochrome
Fig. 9 bacterial strain OH11A is to the degrading activity of signaling molecule AHLA
Figure 10 bacterial strain OH11PA is to the stripped biocontrol effect of soft rot of Chinese cabbage
(5) embodiment
Below the present invention is further elaborated by implementation, but do not limit the present invention
Embodiment 1: the structure that contains the unmarked engineering strain of the molten bacillus of product enzyme of PB500-aiiA
1 materials and methods
1.1 test materials
The used coli strain DH5 α of this test purchases the precious biotech firm (TaKaRa) in Dalian, and SMlO λ pir and DH5 α λ pir bacterial strain are so kind as to give by Life Science College professor Zhu Jun of Agricultural University Of Nanjing.Producing the molten bacillus of enzyme is separated and preservation by the contriver.Carrier pEXl8GM is so kind as to give by Life Science College professor Zhu Jun of Agricultural University Of Nanjing.Plasmid pME6863 is so kind as to give by professor Molina of Swiss Federal Institute of Technology.The LB substratum is used in the cultivation of coli strain, and culture temperature is 37 ℃, and the rotating speed of liquid culture is 225 rpm/min.Produce the cultivation of the molten bacillus of enzyme and use the LB substratum, culture temperature is 30 ℃, and the rotating speed of liquid culture is 225rpm/min.Microbiotic gentamicin (Gm), Streptomycin sulphate (Sm), 5-bromo-4-chloro-3-indoles-β-D-galactoside (X-gal) are all purchased Nanjing assistant research fellow biotech firm.
1.2 PB500-aiiA merges the structure of fragment
According to PB500 and aiiA sequence, design following primer:
PBA-1:5 '-AA
GGATCCTGCGAGACAACTTTTTTCCG-3, (lower stroke latitude is BamHI); PBA-2:5'-GAAATAAAGCTTCTTTACTGTCATGGATCGAATATTCCTGGTTTTTC-3 '; PBA-3:5'-GAAAAACCAGGAATATTCGATCCATGACAGTAAAGAAGCTTTATTTC-3 '; PBA-4:5'-AA
AAGCTTCTATATATATTCAGGGAACAC-3 ' (underscore is HindIII).Take the genomic dna of OH11 bacterial strain as template, utilize primer PBA-1/PBA-2, pcr amplification promotor PB500; Take plasmid pME6863 as template, utilize primer PBA-3/PBA-4, pcr amplification aiiA gene.Afterwards, the PCR product of PB500 and aiiA is mixed than 1: 1 according to mol, and take this mixed solution as template, utilize primer PBA-1/PBA-4, by overlapping PCR PB500 and aiiA are connected to become PB500-aiiA.PB500-aiiA carries out " TA " clone, the exactness of sequence verification sequence after reclaiming purifying.
1.3hmgA-F and the pcr amplification of hmgA-R
Sequence according to hmgA. design primer: EHmgA-1:5 ,-AA
GAATTCCGGCGCGTTCTCGCCGTTCG-3 ' (underscore is restriction enzyme site EcoRI); BhmgA-2:5 ,-AA
GGATCCCGGCCCCAGATCCGGCAGCC-3 ' (underscore is BamHI); A HhmgA-3:5 ' AA
AAGCTTATCGGTTCCAACGGCCTGGC-3 ' (underscore is HindIII); PhmgA-4:5'-AA
CTGCAGCCCGGGGCGAAGCCGCCGGC-3, (underscore be enzyme cut the position account for PstI).Take the genomic dna of OH11 bacterial strain as template, utilize primer EhmgA-1/BhmgA-2, the dna fragmentation of pcr amplification hmgA gene 5 ' end 420bp, called after HmgA-F; Equally, take the OH11 genomic dna as template, utilize primer HmgA-1/PhmgA-4, the dna fragmentation of pcr amplification hmgA gene 3 ' end 430bp, called after hmgA-R.
1.4 the structure of the pEX18PAT of recombinant vectors and checking
Set up the enzyme of 200 μ L and cut system.Use respectively restriction endonuclease EcoRI/BamHI, BamHI/HindIII, HindIII/PstI, EcoRI/PstI that hmgA-F, PB500-aiiA, HmgA-R and pEX18GM are carried out double digestion, reclaim purifying.Afterwards, hmgA-F behind the purifying, PB500-aiiA, hmgA-R, pEX18GM are connected in 16 ℃ under the effect of T4 dna ligase, behind the reaction 20h, transform bacillus coli DH 5 λ pir competent cell, at LA dull and stereotyped (containing 20 μ g/ml Gm) screening transformant.The screening of positive transformant at first uses the Auele Specific Primer (PBAT3/PBAT4) of aiiA to carry out pcr amplification, afterwards, cultivate the PCR positive colony, and extraction plasmid, carry out the double digestion checking with EcoRI/PstI, can observation hmgA-F, PB500-aiiA, hmgA-R be connected on the pEX18GM carrier simultaneously.At last, the pEX18GM that carries 3 external source Insert Fragments (hmgA-F, PB500-aiiA, hmgA-R) is named as pEX18PAT.
1.5 the structure of unmarked engineering strain OH11PA and checking
The building process of unmarked engineering strain OH11PA is seen Fig. 1.The pEXl8PAT electric shock is transformed SM10 λ pir competent cell, and picking positive transformant SM/pEX18PAT will be in respectively the OH11 (OD of logarithmic phase
600=0.5) carries out amphiphilic with SM/pEXl8GMPAT and engage, at dull and stereotyped (100 μ g/mL Sm+200 μ g/mL Gm) the top sieve menu recon of LA.Choose 1O single recon respectively after blank LA substratum was rule rear 2 days, again line in LA (without the Nacl) substratum that contains 10% sucrose, cultivating single bacterium colony (the producing solubility melanochrome) preliminary judgement that grows out after 2-3 days is engineering strain OHl1PA for " hmgA-disruption mutant ".
With sterilizing toothpick the engineering strain OH11PA parallel scribing of preliminary judgement is inoculated in LB flat board (100 μ g/mL Sm), LB dull and stereotyped (100 μ g/mL Sm+200 μ g/mL Gm), screening can't be grown in LB dull and stereotyped (100 μ g/mL Sm+200 μ g/mL Gm) simultaneously in LB dull and stereotyped (100 μ g/mL Sm) growth, and produce the bacterium colony of black haloing in periphery of bacterial colonies, after these bacterial strain activation, extract DNA, utilize the Auele Specific Primer PBAT3/PBAT4 of aiiA further the doubtful mutant strain of screening to be carried out the PCR checking, with positive mutant strain called after OHl1PA.
2 results show:
By pcr amplification, as shown in Figure 2, successfully obtained fusion fragment (1434bp), hmgA-F (420bp) and the hmgA-R (430bp) of PB500-aiiA.By the enzymatic ligation, successfully hmgA-F, hmgA-R and PB500-aiiA are cloned among the suicide vector PEXl8GM subsequently, are built into recombinant vectors pEX18PAT (Fig. 3; Fig. 4).By conjugation, pEXl8PAT is imported among the OH11, produce the melanic bacterium colony of solubility (being that hmgA exchanges zygote for the first time) in LA dull and stereotyped (100 μ g/mLSm+200 μ g/mL Gm) screening.Zygote after blank LB line, is lined on the 2YT flat board of 10% sucrose again,, will produce the melanic single bacterium colony of solubility and choose after 2 days 22 ℃ of cultivations, checking is to the susceptibility of Gm.As shown in Figure 5, grow out single bacterium colony all to the Gm sensitivity from the LA flat board that contains 10% sucrose, and produce solubility melanochrome at the LA flat board.Extract the DNA that produces the melanic bacterial strain of solubility, utilize the Auele Specific Primer PAT-1/PAT-2 of aiiA that doubtful double exchange mutant strain is verified, find that all doubtful mutant strains all can amplify the specific band (Fig. 6) of a 0.75kb, conform to expection, show that aiiA successfully has been incorporated in the hmgA gene of OH11 bacterial strain, engineering strain OH11PA successfully constructs.
Embodiment 2: the unmarked engineering strain of the molten bacillus of product enzyme that contains PB500-aiiA is used the biological and ecological methods to prevent plant disease, pests, and erosion of bacterial soft rot
1 materials and methods
1.1 test materials
Used agrobacterium tumefaciens (Agrobacterium tumefaciens) is so kind as to give by Life Science College professor Zhu Jun of Agricultural University Of Nanjing in this test.The molten bacillus of product enzyme is separated by the contriver with Chinese cabbage soft rot bacteria and preserves.Carrier pME6863 is so kind as to give by professor Molina of Swiss Federal Institute of Technology.Microbial culture is used the LB substratum, and culture temperature is 30 ℃, and the rotating speed of liquid culture is 225rpm/min.Microbiotic gentamicin (Gm), Streptomycin sulphate (Sm), spectinomycin (Spe), tsiklomitsin (Tc), 5-bromo-4-chloro-3-indoles-β-D-galactoside (X-gal), the chemical reagent such as SDS, NaCO3 are all purchased Nanjing assistant research fellow biotech firm.
1.2 extraction and the detection of AHL class signaling molecule
Method with ethyl acetate extraction is extracted AHL class signaling molecule from Chinese cabbage soft rot bacteria (Pectobacterium carotovorumsubsp.carotovorum).Tested bacteria is carried out LB cultivate (100mL), when inoculum concentration reaches OD
600nm=0.6 o'clock, collect each inoculum, collect supernatant after centrifugal (12,000rpm, 10min).Afterwards, culture supernatant is with ethyl acetate extraction twice, and in Rotary Evaporators with the extraction liquid rotary evaporation, in-70 ℃ of preservations, for subsequent use after concentrated 200 times.
The qualitative checking method of AHL is as follows: add 180 μ L AT substratum (2 μ g/mL Tc+100 μ g/mL Spe+100 μ g/mL Gm+300 μ g/mL X-gal) in 96 orifice plates, afterwards with 20 μ L 10
7The hypersensitization of cfu/mL detects bacterial strain JZA1 and joins in the above-mentioned 180 μ L AT substratum, add at last 100 μ L, 1% water agar (60 ℃), after with pipettor nutrient solution evenly being mixed, 96 orifice plates are put into 28 ℃ of incubators cultivate, observe the results of hydrolysis of X-gal behind the 10-12h.If the X-gal hydrolysis becomes blue (being that aperture is blueness), show to detect in the liquid to have certain density AHL, color is more blue, discloses the concentration that detects AHL in the liquid higher; Without blue, show that the AHL that detects in the liquid is by degradable in the aperture.
The detection by quantitative of AHL adopts the beta-galactosidase enzymes method: will detect bacterial strain JZA1 in 1: 100 ratio and join in the AT substratum, and add respectively the concentrated culture supernatant (10%) of the bacterial strain of participating in the experiment, and be cultured to OD
600Be 0.2～1.0; In the 2mL centrifuge tube, add Z-buffer, 0.5%SDS, CHCl
3And the above-mentioned cultivation bacterium of 0.2mL liquid, fully vibration.Add chromogenic substrate ONPG, timing adds 1M NaCO to the solution displaing yellow
3Termination reaction is calculated Miller Units, detectionofβ-galactosidaseactivity.
1.3 the Degrading experiment of AHL class signaling molecule
In 5mL liquid LB substratum, add the concentrated AHL class signaling molecule of 5 μ L, be made into the LB-AHL liquid nutrient medium.Afterwards, with the inoculum (OD of 10 μ L OH11 and OH11PA
600nm=0.6) adds in the LB-AHL nutrient solution, after the incubated overnight, utilize AHL class signaling molecule hypersensitization to detect the concentration that bacterial strain JZA1 detects AHL in the incubated overnight, estimate the OH11A bacterial strain to the degradation capability of AHL by comparing the middle AHL concentration difference (activity of beta-galactosidase enzymes) of incubated overnight liquid and blank nutrient solution (LB-AHL).
1.4 germ and biocontrol microorganisms separates again in the stripped biological and ecological methods to prevent plant disease, pests, and erosion test of soft rot of Chinese cabbage and the incidence tissue
Clean the blade of fresh Chinese cabbage with tap water, afterwards, the alcohol with 70% carries out surface sterilization.After the alcohol volatilization of blade surface, blade is cut into the dice of 10 * 4cm with the sterilization knife blade.The product molten bacillus of enzyme and Bacteria erwinia are cultivated 48h in the LB nutrient solution after, bacterial concentration is adjusted into 10
7CFU/mL will produce afterwards the molten bacillus bacterium of enzyme liquid and mix in 1: 1 by volume with Bacteria erwinia bacterium liquid, and place 4h in 28 ℃ of incubators.Subsequently, prick the aperture that 2mm is dark with sterilizing toothpick at the blade surface of sterilizing, the 5 μ L product molten bacillus of enzyme and Bacteria erwinia mixed bacteria liquid are inoculated in the aperture, postvaccinal leaf piece is placed in the culture dish that contains aseptic filter paper (sterilized water is moistening), and after in 28 ℃ of incubators, placing 12h, observe the occurring degree of leaf piece.Adopt the right-angled intersection method to measure longest diameter and the shortest diameter of scab, mean value is effective diameter the most, utilizes formula π * (diameter/2)
2Calculate onset area, and the evaluation index of conduct morbidity severity.
The dull and stereotyped antagonistic activity of pathogenic fungi or oomycetes is measured
2, the result shows:
Because bacterial strain OH11PA produces solubility melanochrome in LB-AHLA (containing 2% (v/v) signaling molecule AHLA among the LB), so that culture supernatant becomes black, this concentration of detecting AHLA in the nutrient solution for later use JZA1 brought difficulty, because melanochrome has disturbed X-gal in the aperture to be hydrolyzed the observation of rear blueness.In order to overcome this problem, we have screened the material that the energy check melanin produces.We find that OH11PA does not then produce melanochrome when substratum contains 2% glucose, disclose glucose and can suppress OH11PA generation melanochrome (Fig. 7).Therefore, in follow-up AHLA Degrading experiment, we have additionally added again final concentration in original LB-AHLA cultivates be 2% glucose, establishment OH11PA produce melanochrome.Therefore, use the LB-AHLA-glucose substratum, and detect bacterial strain JZA1 in conjunction with the AHL hypersensitization, can estimate the degradation efficiency of the external source signal molecule of OH11PA AHLA.As shown in Figure 8, whether blue according to each aperture in the qualitative test of AHL, can find the signaling molecule AHLA that bacterial strain OH11PA can significantly degrade and be produced by A.tumefaciens R10, aperture is substantially not aobvious blue.Under same operational condition, the wild-type OH11 AHLA that then can't degrade causes aperture to present obvious blue.In order to determine that further OHl1PA is to the degradation efficiency of AHLA, we have carried out the detection by quantitative of AHL, as shown in Figure 9, by measuring the activity of beta-galactosidase enzymes in the nutrient solution, find the activity (Miller unite) of beta-galactosidase enzymes in contrast culture liquid (AHLA and LB mixed solution): 1579.1 ± 111.0.Simultaneously, the activity of beta-galactosidase enzymes in the mixed-culture medium of OH1 1PA and AHLA (Miller unite) is: 149.3 ± 7.8, show that most AHLA is degraded by OH1 1PA, and confirm that further OH11PA can be to the degradation efficiency of AHLA.In addition, in OH11 and AHLA mixed-culture medium, the activity of beta-galactosidase enzymes (Miller unite): 1448.5 ± 34.7, active similar to beta-galactosidase enzymes in the contrast culture liquid further shows the OH11 AHLA that can not degrade.
Isolated test shows, bacterial strain OH11PA can significantly reduce pathogenic on Chinese cabbage of Bacteria erwinia.As shown in figure 10, behind the inoculation 12h, on the Chinese cabbage blade, the vaccination of inoculating separately Bacteria erwinia becomes rotten on every side, and rotten area reaches 1.44 ± 0.07cm
2Simultaneously, the rotten area around the vaccination of OH11 and Bacteria erwinia co-inoculation has also reached 1.51 ± 0.13cm
2, show that OH11 can not reduce pathogenic on Chinese cabbage of Bacteria erwinia.Yet under same operational condition, the area that rots around the vaccination of OH11PA and Bacteria erwinia co-inoculation is minimum, and 0.38 ± 0.07cm is only arranged
2, show that OH11PA reduces pathogenic on Chinese cabbage of germ.
Claims (3)
1. a strain can prevent and treat the unmarked engineering strain structure of the molten bacillus of product enzyme of phytobacterial disease.
2. the engineering bacteria claimed in claim 1 of encoding is used the degraded of the colony induction signaling molecule AHL of bacterial soft rot bacterium generation.
3. the engineering bacteria claimed in claim 1 of encoding is used the biological and ecological methods to prevent plant disease, pests, and erosion of bacterial soft rot.
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| CN107119066B (en) * | 2017-05-06 | 2020-05-22 | 中国海洋大学 | Heterogenous expression quorum sensing enzyme quenching method |
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Application publication date: 20130227 |