CN106591348A - Streptomycete recombinant plasmid pIJ8600-attM, and construction method and applications thereof - Google Patents

Streptomycete recombinant plasmid pIJ8600-attM, and construction method and applications thereof Download PDF

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CN106591348A
CN106591348A CN201610697539.6A CN201610697539A CN106591348A CN 106591348 A CN106591348 A CN 106591348A CN 201610697539 A CN201610697539 A CN 201610697539A CN 106591348 A CN106591348 A CN 106591348A
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attm
streptomycete
pij8600
recombinant plasmid
dna
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林岚
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Southeast University
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    • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
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Abstract

The invention discloses a streptomycete recombinant plasmid pIJ8600-attM, and a construction method and applications thereof. The recombinant plasmid is prepared by inserting a functional esterase gene attM segment into an escherichia coli-streptomycete shuttle plasmid pIJ8600 by adopting restriction enzyme digestion and DNA connection principle. The plasmid is used to degrade streptomycete quorum sensing molecule gamma-butyrolactone; so the plasmid can be used to control actinomycete (pathogen of animals and plants) based on quorum sensing and can also be used to inhibit secondary metabolites or biomembranes generated by some actinomycetes in the air and water. The plasmid can be applied to gene expression based on homologous recombination of streptomycete after butyrolactone degradation, compared with other physical and chemical methods, the cost is low, there is no residue, and the method is environment-friendly.

Description

Streptomycete recombinant plasmid pIJ8600-attM and its construction method and its application
Technical field
The present invention relates to technical field of bioengineering, and in particular to a kind of streptomycete recombinant plasmid pIJ8600-attM and its Construction method and its application.
Background technology
Quorum sensing (quorum sensing, QS) mechanism is used to regulate and control some biology having a social nature by microorganism Behavior, makes single celled bacterium, actinomyces embody the feature of multicellular organism, such as bacterial flora trip, bioluminescence, biomembrane shape Into, toxin/antibiotic synthesis, pathogenic generation and host's symbiosis etc..QS is in bacterium, the phase of actinomyces and eucaryote host Mutual relation (symbiosis, Nei Sheng cause a disease) and microorganism Secondary Metabolic Regulation of Callus aspect play very important effect.Therefore causing The field extensive applications such as bacteria-treating, biological membrane removal, toxin suppression.QS operations depend on microbial cell density up to one Determine QS signaling molecules after threshold value to produce, so as to activate related gene transcription and expression.The QS signals point in gramnegative bacterium Son is N- acyl homoserine lactones classes, and QS signaling molecules are oligopeptides in gram-positive bacterium, and in actinomyces such as strepto- In bacterium, the chemical nature of QS signaling molecules is gamma-butyrolacton.
There is group's sense in nature simultaneously and (quorum quench, QQ) phenomenon is quenched, mainly received by Reverse transcriptase QS Body protein or degraded QS signaling molecules and play a role.At present group's sense be quenched enzyme (QQ enzymes) research bacterium QS is related to it is more, And the rarely seen research for streptomycete QS.AttM genes are first at Agrobacterium tumefaciems (Agrobacterium tumefaciens) Find in C58 bacterial strains, its expression product is fat splitting enzyme AttM, refers to document：" Carlier A, Chevrot R, Dessaux Y, and Faure D.The assimilation ofγ-butyrolactone in Agrobacterium tumefaciens C58interferes with the accumulation of the N-acyl-homoserine lactone signal.Mol.Plant-Microbe Interact.2004,17(9):951–957.”.AttM has following activity：Decompose Gamma-butyrolacton generates gamma-hydroxybutyric acid, and the latter is degraded to again other products and enters tricarboxylic acid cycle, refers to document： “Uroz S,Dessaux Y,Oger P.Quorum sensing and quorum quenching:The Yin and Yang of bacterial communication.ChemBioChem.2009,10(205):205–216.”；Therefore, fat splitting enzyme AttM Can be used to target the pathogenic of interference streptomycete, have wide application prospects in terms of the animals and plants disease control caused by streptomycete.
So far, caused by containment streptomycete the moist building of animals and plants disease such as potato scab and suppression The secondary metabolites such as valinomycins aspect that Streptomyces Griseoflavus are produced in room air, propose both at home and abroad using chemistry, physics, Various solution routes such as biological method.Wherein biological method has nontoxic, noresidue advantage, and the biology based on QQ Prevention and controls have the characteristics of not affecting the nascent metabolism of pathogenic streptomycete, so avoid endurance strain from occurring.
The content of the invention
In order to overcome the deficiencies in the prior art, the present invention to provide a kind of streptomycete recombinant plasmid pIJ8600-attM And its construction method is applied with it, the recombinant plasmid can be applicable to be based on for the degraded of streptomycete QS signaling molecules gamma-butyrolacton The preventing and treating of the animals and plants lumpy jawl clams substance of QQ, while producing secondary metabolism for some actinomyces in the environment such as air, water body The suppression of thing.
For achieving the above object, the technical solution used in the present invention is：
A kind of recombinant plasmid, the plasmid is based on Escherichia coli-streptomycete shuttle plasmid pIJ8600, using restricted Digestion and DNA catenation principles, insert functional fat splitting enzyme gene attM fragments built-up；It includes and derives from The DNA replication dna starting point (ori) of pUC18 plasmids, be easy in Escherichia coli and streptomycete screen apramycin (Apra) resist Property mark aac (3) IV, be easy to engage DNA and be transferred to the transfer starting point (oriT), the multiple limits that come from RK2 plasmids of recipient cell The MCS (MCS) of property restriction enzyme site processed composition, it is close to being induced by thiostrepton for MCS (MCS) upstream The strong promoter tipAp of driving and positioned at MCS (MCS) downstream transcription terminator tfd in case rotation stop record it is logical Read；It also includes fat splitting enzyme gene attM as above.
The plurality of restriction enzyme site includes NdeI, XbaI, BamHI, BglII.
The nucleotide sequence of the fat splitting enzyme gene attM is shown in DNA sequence data storehouse Genbank, and accession number U59485 should Full length gene is 771bp, and G+C contents are 57%, encode the fat splitting enzyme albumen of 256 amino acid.
A kind of construction method of recombinant plasmid, Jing PCR are expanded from Agrobacterium tumefaciems Agrobacterium tumefaciens Fat splitting enzyme gene attM coding region sequences (CDS) that C58 bacterial strains are obtained enters with the pIJ8600 carriers after NdeI/BglII double digestions Row connection, obtains the recombinant plasmid pIJ8600-attM for carrying attM, the attM coded sequences of insertion be placed in tipAp promoters it Under, expressed by the driving of thiostrepton (Thio).
The construction method is concretely comprised the following steps：
Step a, the clone of attM genetic fragments：
Using reverse transcription-pcr (RT-PCR) technology from Agrobacterium tumefaciems (Agrobacterium tumefaciens) C58 Middle amplification is obtained；Glue reclaim purpose band is cut with the agarose gel electrophoresis of mass concentration 1%, wherein, agarose gel electrophoresis is adopted DNA gel QIAquick Gel Extraction Kit is used, it is in 10M Tris-HCl buffer solutions, to store in -20 DEG C that DNA is dissolved in pH for 8.0, concentration；
Step b, restricted digestion：
The PCR primer (i.e. attM genetic fragments) of above-mentioned purifying carries out Nde I and Bgl II double digestions, digestion
The agarose gel electrophoresis of product Jing mass concentrations 1%, cut glue reclaim；
PIJ8600 plasmids Nde I and Bgl II double digestions；The agarose gel electrophoresis of digestion products Jing 0.8%, cut glue return Receive；
Step c, the pIJ8600 plasmids of digestion and the connection of attM genes：
The attM genetic fragments of digestion carry out DNA and are connected with the pIJ8600 plasmids of same digestion, and 16 DEG C are incubated overnight；
Step d, converts bacillus coli DH 5 alpha cloning host：
Above-mentioned full dose coupled reaction product is proceeded in E.coli DH5 α by chemical transformation；
Step e, bacterium colony PCR and sequence verification：
Transformant 10 on picking LB/Apra flat boards ,-the 8h of shaken cultivation 6, take 1 μ l bacterium solutions carries out bacterium colony PCR as template Identification；
Jing bacterium colonies PCR identifies that correct transformant carries out the sequencing of inserted gene attM.
A kind of above-mentioned recombinant plasmid pIJ8600-attM answering in the degraded of streptomycete QS signaling molecule gamma-butyrolactons With.
The application of the recombinant plasmid pIJ8600-attM includes：
(1) non-pathogenic streptomycete (non-pathogenic Streptomycetes) is converted, is then returned and is connected to quick Sense host, to carry the non-pathogenic streptomycete of pIJ8600-attM plasmids as medium, plays group's sense quenching effect, precisely destroys Cause of disease streptomycete it is pathogenic；The cause of disease streptomycete is scab streptomycete；
(2) carrying the non-pathogenic streptomycete of pIJ8600-attM plasmids is used in the environment such as air, water body, some unwrapping wire Bacterium produces the removing of poisonous secondary metabolites；The actinomyces are Streptomyces Griseoflavus, and it is that figured silk fabrics ammonia is mould that it produces poisonous secondary metabolites Plain valinomycin.The invention has the beneficial effects as follows：The invention provides the carrier pIJ8600-attM of a New function, should Carrier is, based on Escherichia coli-streptomycete shuttle plasmid pIJ8600, using restricted digestion and catenation principle, to insert work( The fat splitting enzyme gene attM fragments of energy property are built-up.The subsequent use of the plasmid degraded butyrolactone is homologous heavy based on streptomycete The gene expression of group, with low cost compared to other physics, chemical method, noresidue, environmental friendliness.
Description of the drawings
Fig. 1 is toxin TXT (A) 5-9,5 ' -9 difference in TLC detection Streptomyces scabies fermentation broth extracts Represent in S.scabies attM transformants (S.scabies/pIJ8600-attM), S.scabies control group 5d shaking flasks and add Enter thiostrepton (Thio) and harvest, extract toxin TXT in 9d, Std. represents TXT standard items；(B) under ultraviolet device (249nm) The detached toxin TXT of detection Jing TLC technologies, is respectively from left to right S.scabies attM transformant (S.scabies/ PIJ8600-attM), S.scabies control groups (unconverted) and TXT standard items；
Fig. 2 is the constructing technology route map of pIJ8600-attM recombinant plasmids；
Fig. 3 is that impacts of the fat splitting enzyme gene attM conversion Streptomyces scabies to the latter toxin producing TXT is real Test conceptual scheme.
Specific embodiment
The present invention is further described below.
The invention provides a kind of recombinant plasmid pIJ8600-attM, the plasmid is worn based on Escherichia coli-streptomycete Shuttle plasmid pIJ8600, using restricted digestion and DNA catenation principles, inserts functional fat splitting enzyme gene attM fragments It is built-up；It includes and derives from the DNA replication dna starting point (ori) of pUC18 plasmids, is easy to be screened in Escherichia coli and streptomycete Apramycin (Apra) resistance marker aac (3) IV, be easy to engage DNA be transferred to recipient cell come from RK2 plasmids turn Move starting point (oriT), the MCS (MCS) of multiple restriction enzyme sites composition, be close on MCS (MCS) The strong promoter tipAp driven by thiostrepton induction of trip and the tanscription termination positioned at MCS (MCS) downstream Sub- tfd in case rotation stop record read over (bibliography " Sun J., Kelemen G.H., Fern á ndez-Abalos J.M.andBibb M.J.Green fluorescent protein as a reporter for spatial and temporal gene expression in Streptomyces coelicolor A3(2).Microbiology 1999, 145:2221–2227.”)；It also includes fat splitting enzyme gene attM as above.
The plurality of restriction enzyme site includes NdeI, XbaI, BamHI, BglII.
The nucleotide sequence of the fat splitting enzyme gene attM is shown in DNA sequence data storehouse Genbank, and accession number U59485 should Full length gene (from initiation codon to terminator codon) is 771bp, and G+C contents are 57%, encode the solution ester of 256 amino acid Zymoprotein.
Above-mentioned recombinant plasmid pIJ8600-attM, construction method be：Contain NdeI and BglII digestions in PCR amplifications end The complete fat splitting enzyme gene attM fragments of site sequence, are connected to Escherichia coli-streptomycete shuttle plasmid pIJ8600 On (being given by Britain John Inns research centers Mervyn doctors J.Bibb) NdeI and BglII restriction enzyme sites, restructuring is built Plasmid pIJ8600-attM, Transformed E .coli DH5 α bacterial strains (are purchased from Takara companies), carry out plasmid replication and maintain.It is positive Transformant leads to this recombinant plasmid pIJ8600-attM after Sangon Biotech's sequence verification is correct Cross across category conjugation that to be transferred to streptomycete Streptomyces scabies intracellular.Because pIJ8600 is the integration of streptomycete Type expression plasmid, carries integrase gene int φ C31 and temperate bacteriophage attachment site attP on plasmid, thus can with receive There is homologous recombination and express in somatic chromosomal DNA.
The construction method is concretely comprised the following steps：
Step a, the clone of attM genetic fragments：
Using reverse transcription-pcr (RT-PCR) technology from Agrobacterium tumefaciems (Agrobacterium tumefaciens) C58 Middle amplification is obtained；Glue reclaim purpose band is cut with the agarose gel electrophoresis of mass concentration 1%, wherein, agarose gel electrophoresis is adopted DNA gel QIAquick Gel Extraction Kit is used, it is in 10M Tris-HCl buffer solutions, to store in -20 DEG C that DNA is dissolved in pH for 8.0, concentration；
Step b, restricted digestion：
The PCR primer (i.e. attM genetic fragments) of above-mentioned purifying carries out Nde I and Bgl II (purchased from New EnglandInc) double digestion, the agarose gel electrophoresis of digestion products Jing mass concentrations 1%, cuts glue reclaim；
PIJ8600 plasmids (8.1kb sizes) Nde I and Bgl II double digestions；The Ago-Gels of digestion products Jing 0.8% Electrophoresis, cut glue reclaim；
Step c, the pIJ8600 plasmids of digestion and the connection of attM genes：
The attM genetic fragments of digestion carry out DNA and are connected with the pIJ8600 plasmids of same digestion, and 16 DEG C are incubated overnight；
Step d, converts bacillus coli DH 5 alpha cloning host：
Above-mentioned full dose coupled reaction product is proceeded in E.coli DH5 α by chemical transformation；
Step e, bacterium colony PCR and sequence verification：
Transformant 10 on picking LB/Apra flat boards ,-the 8h of shaken cultivation 6, take 1 μ l bacterium solutions carries out bacterium colony PCR as template Identification；
Jing bacterium colonies PCR identifies that correct transformant send Sangon Biotech (Shanghai) Co., Ltd. to carry out inserted gene The sequencing of attM.
The present invention carries out understanding esterase gene expression product AttM to phytopathogen-scab streptomycete (Streptomyces scabies) produces the influence research of poison, shows that AttM albumen can suppress the toxin of S.scabies Thaxtomin (TXT) synthesizes, and contains the generation of shot hole.In view of AttM PD streptomycete QS signaling molecule γ-Ding Nei Ester, therefore with the application potential of preventing and treating animals and plants streptomycosis substance.
Recombinant plasmid pIJ8600-attM carries integrase gene int φ C31 and temperate bacteriophage attachment site AttP, can occur homologous recombination and stably express with recipient cell (such as streptomycete) chromosomal DNA.Purposes is as follows：(I) by it Conversion non-pathogenic streptomycete (non-pathogenic Streptomycetes), then returns and is connected to sensitive host, to carry The non-pathogenic streptomycete of pIJ8600-attM plasmids is medium, plays group's sense and (QQ) effect is quenched, and precisely destroys cause of disease strepto- Bacterium such as S.scabies's is pathogenic；(II) the non-pathogenic streptomycete for carrying pIJ8600-attM plasmids can be additionally used in environment In such as water body, some actinomyces (such as Streptomyces Griseoflavus) produce poisonous secondary metabolites (such as：Valinomycins valinomycin) Removing.
Below in conjunction with specific embodiment, the method for operating of the present invention can be expanded on further.However, those skilled in the art Member is easy to understand, and the content described by embodiment is only used for describing the present invention in detail, and should not also without limitation on the present invention's Scope.
Embodiment 1：The structure of pIJ8600-attM recombinant plasmids
The clone of a.attM genetic fragments：
Concrete operations are as follows：28 DEG C culture Agrobacterium tumefaciems C58 bacterial strains in YEB fluid nutrient mediums (YE 5g/l, Tryptone 10g/l, NaCl 5g/l, sucrose 5g/l, MgSO4.7H2O, pH 7.0) to exponential phase (OD560=0.5), adopt The total serum IgE of Agrobacterium tumefaciems C58 is extracted with bacterium total serum IgE Rapid extraction kit (being purchased from Shanghai Sheng Gong companies), with without RNase DNase I process, remove possible DNA pollution.Then, cDNA synthesis adopts PrimeScriptTMII 1st Strand CDNA synthetic agent box (be purchased from Takara companies) is carried out, then with cDNA as template, specific primer it is (positive：P1 5’- GGAATTCCATATGCTTCAGTCGGGTAC-3 ' and reversely：P2 5’-GAAGATCTTTACGCGTAAAATTCGGGA-3 ', under NdeI, BglII restriction enzyme site is respectively at line) carry out PCR and amplify attM genetic fragments.PCR amplification system (50 μ l) such as table 1：
Table 1
PCR cycle parameter：94 DEG C of 4min, 94 DEG C of 35s, 57 DEG C of 35s, 72 DEG C of 1min (circulation 29 times), 72 DEG C of extension 5min.
The PCR primer for obtaining cuts glue reclaim with 1% agarose gel electrophoresis.
B. restricted digestion：
The PCR primer (i.e. attM genetic fragments) of above-mentioned purifying carries out Nde I and Bgl II (purchased from New EnglandInc) double digestion.Digestion system (50 μ l) is as shown in table 2：
Table 2
Gently mix, in 37 DEG C 4-5h are incubated.
The agarose gel electrophoresis of digestion products Jing 1%, cut glue reclaim.
PIJ8600 plasmids (plasmid size 8.1kb) Nde I and Bgl II double digestions (refer to above-mentioned reaction condition)；Enzyme Cut the agarose gel electrophoresis of product Jing 0.8%, cut glue reclaim.
C. the connection of the pIJ8600 plasmids of digestion and attM genes：
The attM genetic fragments of digestion carry out DNA and are connected with the pIJ8600 plasmids of same digestion, coupled reaction system (10 μ L) as shown in table 3：
Table 3
16 DEG C are incubated overnight.
D. bacillus coli DH 5 alpha cloning host is converted
Above-mentioned full dose coupled reaction product is proceeded in E.coli DH5 α by chemical transformation.Concrete operations are as follows：-80 The E.coli DH5 α competent cell thawed on ice of DEG C preservation, the μ l of full dose connection product 10 are added to containing 50 μ l E.coli In the Ep pipes of DH5 α, finger flicks Ep pipes and is allowed to mix；Take out after ice bath 30min, 42 DEG C of water-bath 90s, then be moved back to 2min on ice； 450 μ l LB liquid are added, 200rpm shaking tables are in 37 DEG C of recovery 1h；150,350 μ l recovery bacterium solutions are taken respectively, and coating contains 50 μ g/ml The LB flat boards of Apra, 37 DEG C of 12-15h of incubation observe the formation of clone, and picking white transformant carries out bacterium colony PCR checkings.
E. bacterium colony PCR and sequence verification
Transformant 10 on picking LB/Apra flat boards, in 5ml LB/Apra liquid, 37 DEG C, 200rpm culture 6- 8h.1 μ l bacterium solutions are taken as template, forward primer P in PCR primer3(5 '-CTTGCACCTCACGTCACGTGAG-3 ') with The design of ptipA promoter sequences, reverse primer P on pIJ8600 plasmids4(5 '-TTGAAGTTCGCCCAGGCGTCAG-3 ') basis The end sequence of attM genes 3 ' is designed.
Bacterium colony PCR reaction systems (10 μ l) such as table 4：
Table 4
PCR cycle parameter：94 DEG C of 4min, 94 DEG C of 35s, 57 DEG C of 35s, 72 DEG C of 1min (circulation 29 times), 72 DEG C of extension 5min.
The agarose gel electrophoresis of PCR primer Jing 1%, band meets expection.
Jing bacterium colonies PCR identifies that correct transformant send Sangon Biotech (Shanghai) Co., Ltd. to carry out inserting gene attM Sequencing.
Embodiment 2：Checking recombinant plasmid pIJ8600-attM conversion scab streptomycetes produce the inhibition of poison to the bacterium
Recombinant plasmid pIJ8600-attM containing oriT must could pass through under helper plasmid pUZ8002 is assisted Engagement branch mode is imported in acceptor scab streptomycete, is realized across category DNA transmission.Carry the Escherichia coli of pUZ8002 ET12567, is a kind of Methylation deficient bacterial strain, can be used as the donor of engagement transfer；Scab streptomycete be acceptor, recombinant plasmid Carriers of the pIJ8600-attM as fat splitting enzyme gene attM.
A. recombinant plasmid transformed Escherichia coli ET12567 (pUZ8002)
Escherichia coli ET12567 (pUZ8002) is activated：E.coli ET12567 (pUZ8002) bacterial strains are by Britain John Inns research centers Mervyn doctors J.Bibb give, in this laboratory with glycerine conservation in -80 DEG C.In the μ containing kanamycins 25 Activate after the bacterium on the LB flat boards of g/ml and the μ g/ml of chloramphenicol 25, prepare E.coli ET12567 (pUZ8002) competence thin Born of the same parents, concrete grammar is compiled with reference to J. Pehanorm Brookers, D.W. Russells《Molecular Cloning:A Laboratory guide》(third edition).
The conversion of recombinant plasmid：The μ l of E.coli ET12567 (pUZ8002) competent cell 50 are taken in the 1.5ml of precooling In Ep pipes, 1 μ l recombinant plasmids pIJ8600-attM is added to mix, on ice after 30min, 42 DEG C of heat shock 90s, then be moved back to and stand on ice 2min；Add 450 μ l LB culture mediums, 37 DEG C of 200rpm recovery 1h；150-250 μ l recovery bacterium solutions are taken respectively, and coating contains A Bo Draw the μ g/ml of mycin 50, the resistance LB flat board of the μ g/ml of the kanamycins 25 and μ g/ml of chloramphenicol 25.37 DEG C, observation gram after 14-16h Longzi is formed, and only pIJ8600-attM transformants could grow in above-mentioned resistant panel.
, there is 25 single bacterium colonies, as E.coli ET12567 (pIJ8600- in two resistance LB flat boards of transformation experiment attM/pUZ8002)。
B. Escherichia coli shift with scab streptomycete across category DNA
E.coli ET12567 (pIJ8600-attM/pUZ8002) can be by engaging between Escherichia coli-Streptomyces, mat Proceeded in acceptor streptomycete S.scabies by the oriT carried on pIJ8600, φ C31 conformabilities position on pIJ8600 carriers Point, can be by attM gene integrations to S.scabies chromosomes；Additionally, pIJ8600 does not contain streptomycete replication origin, category In streptomycete suicide vector, if can not be incorporated on the chromosome of acceptor streptomycete, can lose because of not reproducible, refer to Document：" Chen Wenqing explains sub- ox, Zheng Yinghua. expression of the Vitreoscilla hemoglobin gene in streptomyces fradiae and its to thalline Growth and the impact of tylosin synthesis. Chinese antibiotic magazine 2004,29 (9):516–520.”.
(1) activation of scab streptomycete and product spore：From -80 DEG C of taking-up S.scabies glycerine bacterial classifications, oese peek microlitre Bacterium solution lines TSA flat boards (tryptone 17g/l, soy peptone 3g/l, glucose 2.5g/l, NaCl 5g/l, K2HPO4 On 2.5g/l, agar 1.8%, pH 7.3 ± 0.2), 30 DEG C of 4-5d of culture grow to bacterium colony.
S.scabies is in product spore culture medium ISP for inoculation4(soluble starch 10g/l, K2HPO41g/l, MgSO4·7H2O 1g/l, NaCl 1g/l, (NH4)2SO42g/l, CaCO32g/l, FeSO4·7H2O0.001g/l, MnCl2·4H2O 0.001g/ L, agar 1.8-2%) and MS (mannitol 20g/l, analysis for soybean powder 20g/l, agar 1.8-2%), 28 DEG C of culture 7-8d are to there is spore Formed.
(2) preparation of scab streptomycete spore suspension：Spore suspension is scraped from flat board in appropriate 2 × YT fluid nutrient mediums To spore final concentration 2 × 10 in (tryptone 16g/l, yeast extract 10g/l, NaCl 5g/l, pH 7.0)8Individual/ml.
Mating experiment：By donor E.coli ET12567 (pIJ8600-attM/pUZ8002) cultivate in LB liquid to OD6000.4-0.6, the centrifugation of 5ml bacterium solutions is taken, precipitation is resuspended in 500 μ l LB that (bacterial population is about 10 after being washed twice with LB8 It is individual)；The μ l of S.scabies spore liquids 500 are taken in 50 DEG C of heat shock 10min；By isopyknic E.coli bacterium solutions and S.scabies spores Liquid is mixed, and most of supernatant is sucked after centrifugation, and bacterial sediment is suspended in residual liquid, is then coated containing 10mmol/L MgCl2(step advantageously forms joint element) TSA flat boards, 30 DEG C of 16-20h of culture.With containing 500 μ g/ml nalidixic acid (purposes Be suppress Escherichia coli Growth) and 1000 μ g/ml thiostreptons 1ml sterilized water submergence flat board (bibliography “Phornphisutthimas S,Sudtachat N,Bunyoo C,et al.Development of an intergeneric conjugal transfer system for rimocidin–producing Streptomyces rimosus.Lett.Appl.Microbiol.2010,50(5):530-536. "), continue to cultivate 3-4d, that is, joint element occur.
C. the zygosporic checking of scab streptomycete
(1) extraction of streptomycete joint element genomic DNA：Toothpick picking streptomycete joint element, proceeds to TSB and cultivates a couple of days. 2ml cultures are taken, 12000 × g centrifugation 4min collect mycelium, are resuspended in SET buffer (75mM NaCl, 25mM EDTA PH 8.0,10mM Tris-HCl pH 7.5) in 500 μ l, add 20 μ l lysozymes (50mg/ml) solution, 37 DEG C of insulations 30- 60min to solution becomes viscous, centre concussion 2-3 times.100 μ l 10%SDS are added, 55 DEG C incubate 1.5h and clarify to solution, middle Concussion is several times.The μ l of 3mol/l NaAc solution 20 are added, is turned upside down and be cooled to several times 37 DEG C.12000 × g is centrifuged 10min, takes Supernatant.Add isopyknic phenol:Chloroform:Isoamyl alcohol (25:24:1, PH7.5) mix, 12000 × g centrifugation 10min.Take supernatant, Repeat to use phenol/chloroform/isoamyl alcohol extraction, until centre occurs without albumin layer.2.2 times of volume absolute ethyl alcohols are added, are mixed, Supernatant is abandoned after 12000 × g centrifugation 5min, is washed twice with 70% ethanol, during 50 μ l ultra-pure waters are dissolved in after drying at room temperature, put- 20 DEG C standby.
(2) joint element PCR checkings：
As template, with P3, P4 is primer to streptomycete joint element STb gene with extraction, does PCR checkings.Joint element verifies PCR Reaction system (25 μ l systems) such as table 5：
Table 5
PCR cycle parameter：94 DEG C of 4min, 94 DEG C of 35s, 57 DEG C of 35s, 72 DEG C of 1min (circulation 29 times), 72 DEG C of 5min.
The agarose gel electrophoresis of PCR primer Jing 1% checks whether the expected band of appearance.If have meeting expected band, For positive findings, as attM transformants.
(3) TXT detections in S.scabies attM engagements transformant
I S.scabies attM transformants are inoculated into oatmeal fluid nutrient medium (Oatmeal broth, OMB, swallow by () Flour immersion liquid 20g/l, trace salting liquid * 1ml/l, pH 7.2, trace salting liquid * are prepared：FeSO4.7H2O 0.1g, MnCl2.4H2O 0.1g,ZnSO4.7H2O 0.1g, are dissolved in 100ml dH2O), 29-30 DEG C of shaken cultivations of 250rpm, with unconverted S.scabies as control.
(ii) in the shaking flask of S.scabies transformants and control group culture 5d, 6d, 7d, 10 μ g/ml are separately added into (final concentration) thiostrepton (Thio) is induced, and continues to cultivate to the 9th day.
(iii) extraction of extract of crude toxin：
Scab streptomycete is cultivated to 9d, collects nutrient solution, and 8000rpm centrifugations, take supernatant, with equal-volume acetic acid second at 4 DEG C Ester is extracted, and then the ethyl acetate phase of the sample rotary evaporated to dryness that reduces pressure at 55 DEG C is dry, obtains CE, be placed in 4 DEG C it is standby With.
(iv) thin-layer chromatography (Thin-layer chromatography, TLC) of toxin TXT is determined
Above-mentioned crude extract 0.5mL chloroforms dissolve, used as TLC loading samples.Solvent is chloroform in chromatography cylinder：Methyl alcohol [9:1 (v/v)], about 0.5-1cm of height.With pencil on the tlc plate at the 1cm of bottom gently standardized horizontal line as point sample position, Then capillary syring pipette samples point sample, spot diameter is less than 2mm.After sample solvent volatilization, the TLC plates for putting sample are put Enter in chromatography cylinder and open up layer with ascending method, until solvent is risen to from the top of plate about 1cm；TLC plates are taken out, Convenienct pencil mark is found Go out solvent advanced position, dry (after to be deployed dose of volatilization), observe the position of spot and take pictures, as shown in Figure 1.
The above is only the preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should It is considered as protection scope of the present invention.

Claims (9)

1. a kind of recombinant plasmid, it is characterised in that：The plasmid is, based on Escherichia coli-streptomycete shuttle plasmid pIJ8600, to adopt With restricted digestion and DNA catenation principles, functional fat splitting enzyme gene attM fragments are inserted built-up；It is comprising next Come from pUC18 plasmids DNA replication dna starting point (ori), be easy in Escherichia coli and streptomycete screen apramycin (Apra) resistance marker aac (3) IV, be easy to engage DNA and be transferred to the transfer starting point for coming from RK2 plasmids of recipient cell (oriT), the MCS (MCS) of multiple restriction enzyme sites composition, be close to MCS (MCS) upstream by Thiostrepton induction drive strong promoter tipAp and positioned at MCS (MCS) downstream transcription terminator tfd with Prevent that what is transcribed to read over；It also includes fat splitting enzyme gene attM as above.
2. recombinant plasmid according to claim 1, it is characterised in that：The plurality of restriction enzyme site includes NdeI, XbaI,BamHI,BglII。
3. recombinant plasmid according to claim 1, it is characterised in that：The nucleotide sequence of the fat splitting enzyme gene attM is shown in DNA sequence data storehouse Genbank, accession number U59485, the full length gene is 771bp, and G+C contents are 57%, encode 256 ammonia The fat splitting enzyme albumen of base acid.
4. a kind of construction method of recombinant plasmid according to claim 1, it is characterised in that：Jing PCR are expanded from crown gall agriculture Fat splitting enzyme gene attM coding region sequences (CDS) that bacillus Agrobacterium tumefaciens C58 bacterial strains are obtained with PIJ8600 carriers after NdeI/BglII double digestions are attached, and obtain the recombinant plasmid pIJ8600-attM for carrying attM, insert The attM coded sequences for entering are placed under tipAp promoters, are expressed by the driving of thiostrepton.
5. the construction method of recombinant plasmid according to claim 4, it is characterised in that：The concrete steps of the construction method For：
Step a, the clone of attM genetic fragments：
Expanded from Agrobacterium tumefaciems (Agrobacterium tumefaciens) C58 using reverse transcription-pcr technology and obtained；With The agarose gel electrophoresis of mass concentration 1% cuts glue reclaim purpose band, wherein, agarose gel electrophoresis is reclaimed using DNA gel Kit, it is in 10MTris-HCl buffer solutions, to store in -20 DEG C that DNA is dissolved in pH for 8.0, concentration；
Step b, restricted digestion：
The PCR primer of above-mentioned purifying carries out Nde I and Bgl II double digestions, the Ago-Gel of digestion products Jing mass concentrations 1% Electrophoresis, cut glue reclaim；
PIJ8600 plasmids Nde I and Bgl II double digestions；The agarose gel electrophoresis of digestion products Jing 0.8%, cut glue reclaim；
Step c, the pIJ8600 plasmids of digestion and the connection of attM genes：
The attM genetic fragments of digestion carry out DNA and are connected with the pIJ8600 plasmids of same digestion, and 16 DEG C are incubated overnight；
Step d, converts bacillus coli DH 5 alpha cloning host：
Above-mentioned full dose coupled reaction product is proceeded in E.coli DH5 α by chemical transformation；
Step e, bacterium colony PCR and sequence verification：
Transformant 10 on picking LB/Apra flat boards ,-the 8h of shaken cultivation 6, take 1 μ l bacterium solutions carries out bacterium colony PCR mirror as template It is fixed；
Jing bacterium colonies PCR identifies that correct transformant carries out the sequencing of inserted gene attM.
6. a kind of recombinant plasmid according to claim 1 in the degraded of streptomycete QS signaling molecule gamma-butyrolactons should With.
7. purposes according to claim 6, it is characterised in that：Including：
(1) non-pathogenic streptomycete (non-pathogenic Streptomycetes) is converted, is then returned and is connected to sensitive place It is main, to carry the non-pathogenic streptomycete of pIJ8600-attM plasmids as medium, group's sense quenching effect is played, precisely destroy cause of disease Streptomycete it is pathogenic；
(2) carrying the non-pathogenic streptomycete of pIJ8600-attM plasmids is used in the environment such as air, water body, some actinomyces institutes Produce the removing of poisonous secondary metabolites.
8. purposes according to claim 7, it is characterised in that：In the purposes (1), the cause of disease streptomycete is scab chain Mould.
9. purposes according to claim 7, it is characterised in that：In the purposes (2), the actinomyces are sallow strepto- Bacterium, it is valinomycins valinomycin that it produces poisonous secondary metabolites.
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