CN108441458A - Effectively genetic engineering bacillus amyloliquefaciens of prevention gray mold and preparation method thereof and its application - Google Patents
Effectively genetic engineering bacillus amyloliquefaciens of prevention gray mold and preparation method thereof and its application Download PDFInfo
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- CN108441458A CN108441458A CN201711172205.8A CN201711172205A CN108441458A CN 108441458 A CN108441458 A CN 108441458A CN 201711172205 A CN201711172205 A CN 201711172205A CN 108441458 A CN108441458 A CN 108441458A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2442—Chitinase (3.2.1.14)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01014—Chitinase (3.2.1.14)
Abstract
Effectively genetic engineering bacillus amyloliquefaciens of prevention gray mold and preparation method thereof and its application, it belongs to molecular biology field.The present invention passes through genetic engineering, expand Parasitism broom bacterium chitinase gene ech42, and connect solution expression vector pHT43, it is electroporated by setting to build recombinant expression carrier pHT43/ech42, recombinant expression carrier pHT43/ech42 described in step 1 is imported in bacillus amyloliquefaciens, the shock voltage is 12.5kv/cm or 15kv/cm or 17.5kv/cm.The enzyme activity of 8h pHT43 ech42/BA bacterial strains improves about 54.3% than wild-type strain BA enzyme activity in chitinase activity detection of the present invention, and in terms of preventing gray mold, protection effect is still 28.9% or so after 20 days for spray bacterium, with lasting biocontrol effect.
Description
Technical field
The invention belongs to molecular biology fields；More particularly to a kind of genetic engineering solution starch bud of effective prevention gray mold
Born of the same parents bacillus and preparation method thereof and its application.
Background technology
Mycophyta disease is to restrict the principal element of high-yield plant, and the prevalence of fungal disease makes crops cause extremely sternly
The underproduction of weight, the invasion of the fungal diseases such as grey mold, leaf mold, root-rot, early epidemic can be often subject to for this crop of tomato, are caused
A large amount of underproduction of tomato.The main means of prevention of prevention gray mold is chemical prevention at present, and chemical prevention pollutes the environment, pesticide
The problems such as residual, also endangers the health of human body；By this mode of breeding for disease resistance, to prevent gray mold, the phase reaches thoroughly anti-
It controls, but there is presently no the antigen-like materials for finding gray mold, therefore gray mold and infeasible is prevented by breeding for disease resistance；Mesh
It is preceding by biological control, using some environmentally friendly microorganisms come to prevent fungal disease be a kind of best solution, both
The clean hygiene of crops is also ensured in the deficiency that chemical prevention can be filled up.
Parasitism broom bacterium can prevent the harm of pathogen as other biocontrol microorganisms by various disease resistance mechanisms, substantially
5 aspects can be divided into be prevented：(1) Competition, (2) bacteriolysis, (3) antibiosis, (4) hyperparasitism, (5)
Induce plant disease-resistant.Wherein bacteriolysis mainly produces this chitin hydrolase of chitinase, true to dissolve cause of disease
The cell wall of bacterium causes disease fungus plasmalemma to rupture, to inhibit the growth and breeding of disease fungus.
Bacillus amyloliquefaciens are widely distributed in nature, can prevent fungal disease by a variety of antifungal mechanisms,
Mainly have：(1) antibacterial substance is generated.Bacillus amyloliquefaciens are mainly inhibited to the growth of pathogen mycelia by generating
Substance come the effect of exercising, antibacterial substance mainly has Antagonistic protein and lipopeptide antibiotic etc..(2) induced resistance.Using short of money
Antimicrobial treatment fruit can cause host to generate morphologic change, while cause physiological acoustic signals, and then cause to post
The variation etc. of main vegetable protein group and disease-resistant related gene expression.(3) living space and nutriment are competed with pathogen.
(4) it is directly acted on pathogen.Other than generating the secondary metabolites such as antibiotic, Antagonistic Fungi also will produce a series of to cause of disease
Bacterium inhibits the extracellular hydrolase to play an important role, such as chitinase.
Have currently on the market and has much been commercialized, biological pesticide made of the antagonistic microbe by using pathogen
The product for carrying out controlling plant diseases is being sold, such as bacillus subtilis, bacillus licheniformis, Pseudomonas fluorescens, wax gemma
Bacillus, Trichoderma, bacillus thuringiensis, bacillus amyloliquefaciens etc., these biocontrol microorganisms are during controlling plant diseases
Achieve good effect.So that becoming possibility by biocontrol microorganisms to prevent pathogen.
Invention content
It is an object of the present invention to provide a kind of genetic engineering bacillus amyloliquefaciens of effective prevention gray mold and its preparations
Method and its application.
The invention is realized by the following technical scheme：
One plant of genetic engineering bacillus amyloliquefaciens for effectively preventing gray mold, this plant of entitled solution starch brood cell's bar of bacterium classification
Bacterium (Bacillus amyloliquefaceins) is transferred to pink glutinous broom bacterium chitinase gene in this plant of bacterium, in 2017
It was preserved in Chinese microorganism strain preservation administrative center (CGMCC) on June 29, deposit number is CGMCC No.14366, preservation
Address is the institute 3 of Chaoyang District, Beijing City Beichen Lu 1.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold of the present invention, including it is as follows
Step：
Step A, by genetic engineering, Parasitism broom bacterium chitinase gene ech42 is expanded, and connect expression vector
PHT43, structure recombinant expression carrier pHT43/ech42；
Step B, electroporated by setting, the recombinant expression carrier pHT43/ech42 described in step 1 is imported into solution starch
In bacillus, the shock voltage is 12.5kv/cm or 15kv/cm or 17.5kv/cm.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold of the present invention, step A packets
Include following steps：
Step A1, it is cloned by PCR and obtains C.rosea chitinase genes ech42；
Step A2, by the connection of the step A1 chitinase gene ech42 and carrier T obtained；
Step A3, the chitinase gene ech42 that step 2 obtains is connect with expression vector pHT43.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold of the present invention, in step A1
The gene order that C.rosea chitinase ech42 genes (DQ523687) are found by GenBank detects soft with signal peptide
Part is detected signal peptide sequence, and should not be containing the restriction enzyme site introduced on primer, with primmer 5 in clone gene
Software Design primers：
Primer ech42E：5 '-TATCTAGACGTGCCACTCCTCGTATGGAGG-3 ', digestion point Xba I；
ech42B：5 '-CCCCCCGGGGGAGAGGCTGTTCTTGAT-3 ', digestion point Sma I；
PCR reaction mixture be：Template 1 μ l, 10 μM of 1 μ l of sense primer ech42E, 10 μM of downstream primer
4 μ l, EasyTaq archaeal dna polymerases of ech42B 1 μ l, 10 × EasyTaq buffer solution, 5 μ l, 2.5mM dNTPs 0.5 μ l, it is ultrapure
37.5 μ l of water；
PCR reaction conditions are：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 0.5min, 68 DEG C of annealing 0.5min, 72 DEG C extend
1min, 35 cycles；After last 72 DEG C extend 5min, preserved at 4 DEG C.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold of the present invention, in step A2
The recycling of pcr amplification product is carried out with purifying with reference to kit StarPrep Gel Extraction Kit；By the purpose of recycling
Gene ech42 is attached with carrier T, and reaction system is pMD18-T carriers 1 μ l, target gene ech42 4 μ l, 5 μ of solution I
l；It will be accredited as the plasmid vector extracted in positive bacterium solution from bacterium solution PCR, carry out single endonuclease digestion and double digestion identification, it is restricted interior
Restriction endonuclease sites Xba I, the Sma I introduced during enzyme cutting selection PCR, digestion system are：10 times of Tango buffer solutions
2 μ l, plasmid 10 μ l, Xba I 1 μ l, Sma I 1 μ l, 6 μ l of no enzyme water.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold of the present invention, in step A3
Double digestion is carried out to pMD18-T-ech42 and pHT43 expression vectors using Xba I, Sma I respectively, later by expression vector and
The digestion glue recovery product of chitinase gene ech42 is attached, and linked system is 5 μ l, ech42 12.8 μ l of pHT43, and 10
2 μ l, T4DNA ligase of × T4DNA ligase buffer solutions, 0.2 μ l.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold of the present invention, step B packets
Include following steps：
Step B1, wild type bacillus amyloliquefaciens BA Electroporation-competent cells are prepared；
Step B2, electroporated, it is thin that recombinant expression carrier pHT43/ech42 is imported into bacillus amyloliquefaciens BA competence
Born of the same parents；
Step B3, positive restructuring bacillus amyloliquefaciens are screened, the plasmid in bacillus amyloliquefaciens is extracted.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold of the present invention, step B1 packets
Include following steps：
Step B1-1, the bacillus amyloliquefaciens BA bacterial strains for preserving laboratory, take out from -70 DEG C of refrigerators, wait melting
Picking bacterium solution afterwards carries out three sections of scribing line on LB solid mediums, is inverted and is incubated overnight in 37 DEG C of constant incubators；
Step B1-2, the BA single bacterium colonies grown after overnight with yellow pipette tips picking, in 5mlLB fluid nutrient mediums,
It is incubated overnight in 150rpm, 37 DEG C of earthquake incubators；
Step B1-3, in superclean bench, 2.5ml overnight cultures is taken out with sterile blue pipette tips, are added to 40ml
In fresh bacillus amyloliquefaciens growth medium, 37 DEG C, 200rpm shaken cultivations to OD600=0.85-0.95 are described
The formula of bacillus amyloliquefaciens growth medium is peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, sorbierite 91g/
L；
Step B1-4, after OD600 values reach, immediately by bacterium solution carry out ice-water bath 10min, by bacterium solution be divided in 1.5ml from
In heart pipe, then in refrigerated centrifuge, 5000g, 4 DEG C of centrifugation 5min, each centrifuge tube centrifugation twice, collect solution starch brood cell
Bacillus thalline；
Step B1-5, turn culture medium with the electricity without containing ion and mixing resuspension is carried out to the thalline of enrichment, draw 1ml every time
So that thalline is gathered in tube bottom in 4 DEG C of centrifugation 10min of 8500rpm in centrifuge tube, and by cryogenic freezing centrifuge, adopt
Rinsing 4 times is carried out with such step, the formula that the electricity without containing ion turns culture medium is sorbierite 90g/L, mannitol
92.5g/L, glycerine 100ml/L；
Step B1-6, after washing, turn culture medium with 60 μ l electricity of sterile yellow pipette tips addition into each centrifuge tube, obtain
Wild type bacillus amyloliquefaciens BA Electroporation-competent cells, spare, it is sorbierite 90g/L that the electricity, which turns culture medium, sweet
Reveal alcohol 92.5g/L, glycerine 100ml/L.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold of the present invention, step B2 packets
Include following steps：
Step B2-1, by freshly prepared wild type bacillus amyloliquefaciens BA Electroporation-competent cells or it is stored in ice
Competence in case is taken out from refrigerator, wait for competence bacterium solution melt postposition on ice with 10 μ l recombinant plasmid pHT43/ech42 mixings
Ice-water bath 2min afterwards；
Step B2-2, the mixture after step B2-1 ice baths is transferred in the electric shock cup of 1mm, and electric shock cup is put into
In BIO-RAD GenePulser Xcell, the electrotransformation parameter of five gradients is set：Be respectively 12.5kv/cm, 15kv/cm,
17.5kv/cm、20kv/cm、21kv/cm.It after setting parameter, shocks by electricity immediately to electric shock cup, electric shock resistance value is 200
Ω, the time constant 4.5-5.0ms of electric shock；
Step B2-3, it uses blue electron gun head to draw 1ml incubations after step B2-2 electric shocks immediately to be added in 37 DEG C of recovery medium
To mixing in electric shock cup, it is transferred in the sterile centrifugation tube marked after mixing, centrifuges 37 DEG C of effective incubator, 200rpm shakes
Culture 3-4h is swung, the formula of the recovery medium is peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, sorbierite
90g/L, mannitol 70g/L；
Step B2-4, after step B2-3 shaken cultivations, centrifuge tube is centrifuged into 1min in 8000g, 900 μ l are drawn with pipette tips
Supernatant simultaneously discards, and thalline is resuspended remaining liquid, and is coated in the LB solid mediums containing chloramphenicol, in 37 DEG C
It is incubated overnight in constant incubator.
The application of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold of the present invention, is preventing gray mold
In application.
The genetic engineering bacillus amyloliquefaciens of effective prevention gray mold of the present invention, pass through the hand of genetic engineering
Section expands Parasitism broom bacterium chitinase gene ech42, and connects expression vector pHT43, successfully builds recombinant expression carrier
pHT43/ech42.The genetic engineering solution starch brood cell of wild mushroom and effective prevention gray mold of the present invention after IPTG induces 8h
Bacillus dissolves cell wall with lysozyme, extracts protein liquid, carries out SDS-PAGE electrophoresis, having containing pHT43/ech42
There is purpose band at 42KD in the genetic engineering bacillus amyloliquefaciens of effect prevention gray mold.
The genetic engineering bacillus amyloliquefaciens of effective prevention gray mold of the present invention, the N- acetylaminos Portugal of measurement
Grape Standard for Sugars curve uses DNS reducing sugar methods, respectively with the N-acetylglucosamine titer of different final concentrations in OD540
The lower light absorption value for surveying each pipe, drafting are illustrated in fig. 3 shown below standard curve.It can see N-acetylglucosamine content and light absorption value
In the relationship of once linear function, obtained standard curve is y=0.5154x+0.0036, and the related coefficient of each point and curve is
R2=0.9783 shows that correlation is very high.
The genetic engineering bacillus amyloliquefaciens and wild mushroom chitin enzyme activity of effective prevention gray mold of the present invention
Property detection comparison, find effectively prevention gray mold genetic engineering bacillus amyloliquefaciens pHT43-ech42/BA induced through IPTG
2h, 4h, it 8h, 12h, is sampled respectively for 24 hours, while wild bacillus amyloliquefaciens BA is induced without IPTG, culture is corresponding
Time after measuring chitinase through DNS reducing sugar methods, effectively prevents genetic engineering bacillus amyloliquefaciens and the open country of gray mold
The enzyme activity of raw bacterium is not unalterable, and pHT43-ech42/BA bacterial strains exist in 8h chitinase enzyme activity highests, BA bacterial strains
12h chitinase enzyme activity highests.The enzyme activity of 8h pHT43-ech42/BA bacterial strains improves about than wild-type strain BA enzyme activity
54.3%, in terms of preventing gray mold, the genetic engineering bacillus amyloliquefaciens pHT43/ech42 for effectively preventing gray mold is wilder
Raw bacterium is significantly increased, and protection effect is still 28.9% or so after 20 days for spray bacterium, with lasting biocontrol effect.
Description of the drawings
Fig. 1 is the structure of the present invention effectively genetic engineering bacillus amyloliquefaciens recombinant vector of prevention gray mold；
Fig. 2 is that the destination protein electrophoresis of the present invention effectively genetic engineering bacillus amyloliquefaciens of prevention gray mold examines figure
Piece；
Fig. 3 is the N-acetylglucosamine standard curve that the present invention measures；
Fig. 4 is the genetic engineering bacillus amyloliquefaciens and wild mushroom chitinase activity of the present invention effectively prevention gray mold
Detect correlation curve；
Fig. 5 is that the genetic engineering bacillus amyloliquefaciens of the present invention effectively prevention gray mold are sprayed and sprayed with wild mushroom and water
Spray tomato to prevent the gray mold state of an illness comparison photo, wherein BA, ech42 and ck respectively represent spray wild mushroom and effectively
Prevent genetic engineering bacillus amyloliquefaciens, the water of gray mold；
Fig. 6 is that the genetic engineering bacillus amyloliquefaciens of the present invention effectively prevention gray mold spray kind handled with wild mushroom
Eggplant is to preventing gray mold disease index block diagram；
Fig. 7 is that the genetic engineering bacillus amyloliquefaciens of the present invention effectively prevention gray mold spray kind handled with wild mushroom
Eggplant compares block diagram to preventing gray mold control effect.
Specific implementation mode
Specific implementation mode one：
One plant of genetic engineering bacillus amyloliquefaciens for effectively preventing gray mold, this plant of entitled solution starch brood cell's bar of bacterium classification
Bacterium (Bacillus amyloliquefaceins) is transferred to pink glutinous broom bacterium chitinase gene in this plant of bacterium, in 2017
It was preserved in Chinese microorganism strain preservation administrative center (CGMCC) on June 29, deposit number is CGMCC No.14366, preservation
Address is the institute 3 of Chaoyang District, Beijing City Beichen Lu 1.
The genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment, the structure of recombinant vector
As shown in Fig. 1, by the means of genetic engineering, Parasitism broom bacterium chitinase gene ech42 is expanded, and connect expression vector
PHT43 successfully builds recombinant expression carrier pHT43/ech42.
The genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment, the inspection of destination protein electrophoresis
It is as shown in Fig. 2 to test picture：Wherein M- protein moleculars quality standard；1- induces the solution starch bud containing pHT43/ech42 through IPTG
Born of the same parents bacillus；2- induces the bacillus amyloliquefaciens containing pHT43/ech42 without IPTG；Solution starch brood cell's bar that 3- is induced through IPTG
Bacterium；The bacillus amyloliquefaciens that 4- is induced without IPTG.From the wild mushroom that can be derived that in figure after IPTG induces 8h and effectively
The genetic engineering bacillus amyloliquefaciens of prevention gray mold dissolve cell wall with lysozyme, extract protein liquid, carry out
SDS-PAGE electrophoresis, electrophoresis result such as attached drawing 2, the genetic engineering solution starch of effective prevention gray mold containing pHT43/ech42
There is purpose band at 42KD in bacillus.
The genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment, the N- acetyl ammonia of measurement
Base glucose standard curve is as shown in Fig. 3：Using DNS reducing sugar methods, respectively with the N- acetamido glucoses of different final concentrations
Standard for Sugars liquid surveys the light absorption value of each pipe at OD540, and drafting is illustrated in fig. 3 shown below standard curve.It can see N- acetylaminos Portugal
Grape sugared content and light absorption value are in the relationship of once linear function, and obtained standard curve is y=0.5154x+0.0036, each point with
The related coefficient of curve is R2=0.9783, shows that correlation is very high.
The genetic engineering bacillus amyloliquefaciens and wild mushroom chitin of effective prevention gray mold described in present embodiment
Enzyme assay correlation curve is as shown in Fig. 4：The effectively genetic engineering bacillus amyloliquefaciens pHT43- of prevention gray mold
Ech42/BA through IPTG induction 2h, 4h, 8h, 12h, be sampled respectively for 24 hours, while wild bacillus amyloliquefaciens BA without
IPTG is induced, and cultivates the corresponding time, after measuring chitinase through DNS reducing sugar methods, as a result such as attached drawing 4.It can be seen that having
The genetic engineering bacillus amyloliquefaciens of effect prevention gray mold and the enzyme activity of wild mushroom are not unalterable, pHT43-
Ech42/BA bacterial strains are in 8h chitinase enzyme activity highests, and BA bacterial strains are in 12h chitinase enzyme activity highests.8h pHT43-
The enzyme activity of ech42/BA bacterial strains improves about 54.3% than wild-type strain BA enzyme activity.
The genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment are sprayed to be sprayed with wild mushroom
Grant water spray tomato to prevent gray mold state of an illness potted plant experiment steps are as follows：
(1) it will be grown in tomato seedling of the gardening head of a station to 5 leaf, 1 heart, be put into homemade plastic greenhouse, selected in the same size
Tomato seedling, be randomly divided into four groups, 31 seedlings of each group need slow seedling 5 days or so after being put into canopy, keep seedling suitable
Answer new environment；
(2) by the gene work of the original strain cultivated in 37 DEG C of constant-temperature shaking incubators and effective prevention gray mold
Journey bacillus amyloliquefaciens dilute 10 times respectively, for spraying seedling.100ml is added originally into the tomato for cover with grey mold
Water fully shakes up, and the bacteria suspension of tomato the pathogen of Botrytis cinerea is made；
(3) protection effect is divided into 3 groups of processing：Spray original strain BA, effective genetic engineering for preventing gray mold
Bacillus amyloliquefaciens, CK (distilled water)；
(4) three groups of processing spray the pathogen of Botrytis cinerea again after spraying biocontrol bacterial strain (step is 3.) after waiting for for 24 hours
Bacteria suspension；
(5) disease index is measured within the 15th and 20 day after spraying biocontrol bacterial strain.
The genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment are sprayed to be sprayed with wild mushroom
It grants water and sprays, wherein BA, pHT43/ech42 and ck as shown in Fig. 5 to the comparison photo for preventing the gray mold state of an illness in tomato
Genetic engineering bacillus amyloliquefaciens, the water for spraying wild mushroom and effectively preventing gray mold are respectively represented, pot experiment exists respectively
Determine corresponding disease index and preventive effect within 15th day, 20 days.In terms of preventing gray mold, the gene work of gray mold is effectively prevented
Journey bacillus amyloliquefaciens pHT43/ech42 is significantly increased compared with wild mushroom, spray bacterium after 20 days protection effect still 28.9% a left side
The right side, disease index are as shown in table 1：
1 potted plant experiment state of an illness contrast table of table
The genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment spray at wild mushroom
The tomato of reason is as shown in Fig. 6 to preventing gray mold disease index block diagram, and attached drawing 7 is that present embodiment effectively prevents gray mold
Genetic engineering bacillus amyloliquefaciens spray the tomato that is handled with wild mushroom and compare block diagram to preventing gray mold control effect,
From attached drawing 6 and attached drawing 7, the biocontrol effect that can be will become apparent from present embodiment is more preferable, the tomato pair of specific different disposal
It is as shown in table 2 to prevent gray mold control effect：
The 2 potted plant experiment state of an illness of table prevents Contrast on effect table
Specific implementation mode two：
The preparation of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold according to specific implementation mode one
Method includes the following steps：
Step A, by genetic engineering, Parasitism broom bacterium chitinase gene ech42 is expanded, and connects solution starch brood cell's bar
Bacterium expression vector pHT43 builds recombinant expression carrier pHT43/ech42,
Step B, electroporated by setting, the recombinant expression carrier pHT43/ech42 described in step 1 is imported into solution starch
In enzyme bacillus, the shock voltage is 12.5kv/cm or 15kv/cm or 17.5kv/cm.
Specific implementation mode three：
The genetic engineering bacillus amyloliquefaciens of effective prevention gray mold according to specific implementation mode one or two
Preparation method, step A include the following steps：
Step A1, it is cloned by PCR and obtains C.rosea chitinase genes ech42；
Step A2, by the connection of the step A1 chitinase gene ech42 and carrier T obtained；
Step A3, the chitinase gene ech42 that step 2 obtains is connect with expression vector pHT43.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment, step
The gene order for finding C.rosea chitinase ech42 genes (DQ523687) in A1 by GenBank, is examined with signal peptide
Software is surveyed, signal peptide sequence is detected, and is not used containing the restriction enzyme site introduced on primer in clone gene
5 software Design primers of primmer：
Primer ech42E：5 '-TATCTAGACGTGCCACTCCTCGTATGGAGG-3 ', digestion point Xba I；
ech42B：5 '-CCCCCCGGGGGAGAGGCTGTTCTTGAT-3 ', digestion point Sma I；
PCR reaction mixture be：Template 1 μ l, 10 μM of 1 μ l of sense primer ech42E, 10 μM of downstream primer
4 μ l, EasyTaq archaeal dna polymerases of ech42B 1 μ l, 10 × EasyTaq buffer solution, 5 μ l, 2.5mM dNTPs 0.5 μ l, it is ultrapure
37.5 μ l of water；
PCR reaction conditions are：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 0.5min, 68 DEG C of annealing 0.5min, 72 DEG C extend
1min, 35 cycles；After last 72 DEG C extend 5min, preserved at 4 DEG C.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment, step
The recycling of pcr amplification product is carried out with purifying with reference to kit StarPrep Gel Extraction Kit in A2；It will recycling
Target gene ech42 be attached with carrier T, reaction system is pMD18-T carriers 1 μ l, target gene ech42 4 μ l, molten
5 μ l of liquid I；It will be accredited as the plasmid vector extracted in positive bacterium solution from bacterium solution PCR, carries out single endonuclease digestion and double digestion identification, limit
Restriction endonuclease sites Xba I, the Sma I introduced during property restriction endonuclease selection PCR processed, digestion system are：10 times of Tango
2 μ l of buffer solution, Plasmid DNA 10 μ l, Xba I 1 μ l, Sma I 1 μ l, no 6 μ l of enzyme water water.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment, step
The recycling of PCR product glue is carried out with reference to kit (StarPrep Gel Extraction Kit) in A2, before carrying out glue recycling,
Need to prepare one the rubber knife piece of cleaning sterile, ultraviolet gel analysis instrument, 1% Ago-Gel of preparation, and the electricity more renewed
Swimming liquid (1 × TAE), steps are as follows：
(1) glue is cut：After preparing offset plate, point sample carries out electrophoresis, and electrophoresis about 20min or so takes out offset plate under ultraviolet lamp
It is rapid to cut target gene fragment, the part that blob of viscose edge is free of DNA is removed as possible, cuts blob of viscose as much as possible for convenience of colloidal sol
Broken, operation is quick under ultraviolet lamp, to reduce the ultraviolet damage waited to DNA；
(2) blob of viscose quality is weighed：1.5ml centrifuge tubes weight is weighed in advance and is removed the peel, and the DNA fragmentation cut is passed through repeatedly
Cutting makes it shred in pipe, and the higher the better for the DNA content of blob of viscose, should not be overweight；
(3) colloidal sol：By 1：1 ratio is added 1 μ l sol solutions (MB) per 1mg glue, is incubated for 55 DEG C in water-bath, per 1-2
Minute carries out gentle inversion to centrifuge tube, and blob of viscose is made to be come into full contact with sol solutions, wants gentle when reverse, to prevent DNA fragmentation disconnected
It splits；
(4) DNA is combined：Liquid after colloidal sol is cooled to room temperature, liquid is transferred to the centrifugal adsorbing column with collecting pipe
On, room temperature static 1-2min is put into after trim in centrifuge, and 12000rpm centrifuges 1min, to ensure the highest rate of recovery, centrifugation
The liquid in collecting pipe is carefully sucked in adsorption column again afterwards, and is put into collecting pipe, 12000rpm centrifuges 1min again, from
By the waste liquid reject in collecting pipe after the heart；
(5) pillar is cleaned：Using the cleaning solution (MW) that absolute ethyl alcohol is added, pipette measures 600 μ l, is added to containing
On the adsorption column of DNA sample, supercentrifuge 10000rpm centrifuges 1min, the waste liquid of tube bottom is outwelled, again by pillar insertion tube
In, wait for centrifugation next time；
(6) pillar is cleaned again：(MW) that 600 μ l are equally measured with liquid-transfering gun, is added in pillar, same with above-mentioned steps
Sample rotating speed is centrifuged, and tube bottom waste liquid is outwelled after centrifugation, pillar is turned back in pipe, carries out the empty from empty to be thrown away from end of 4min
Pillar is dried in the air in dry place, carries out the volatilization of absolute ethyl alcohol by collecting pipe, and to prevent influencing postorder digestion, enzyme even operates；
(7) it elutes：It takes a clean 1.5ml centrifuge tube of sterilizing and marks, adsorption column is placed in it, and to
It is 65 DEG C of deionization aqua sterilisas that 55 μ l temperature are added on the adsorbed film in centrifugal adsorbing column center, is placed at room temperature for 1min, 12000rpm
Centrifuge 1min.Liquid in centrifuge tube is carefully sucked in adsorption column after centrifugation and crosses column centrifugation again to improve the rate of recovery；
(8) it stores：It discards adsorption column, and glue recycling post-fragment is stored in spare in -20 DEG C of refrigerators.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment, step
The preparation of E. coli DH5 α competence in A2, steps are as follows：
(1) the E.coli DH5 α original strains being stored in -70 DEG C of refrigerators are picked with connecing collarium, is crossed using three sections
Mode is crossed on LB solid plates.Scribed tablet is put into constant incubator and is cultivated at 37 DEG C of temperature；
(2) single bacterium colony that E.coli DH5 α have been grown on rear observation tablet overnight, by the way of connecing bacteria liquid culture,
With sterile yellow pipette tips, the single bacterium colony on picking tablet, and expanded in 5ml LB liquid mediums, 37 DEG C of shaking tables shake
It swings overnight, E.coli DH5 α have grown into the logarithmic growth later stage at this time；
(3) bacterium solution after 1ml shake cultures is drawn, is transferred in the fresh LB culture mediums of 50ml, 37 DEG C of constant temperature of 180rpm
After shaking table shake culture 2 hours, the OD600 values of a bacterium solution are surveyed every half an hour, until OD600=0.5 or so stops shaking bacterium；
(4) bacterium solution of culture is dispensed into 1.5ml centrifuge tubes, often pipe plus 1250 μ l of bacterium solution, the bacterium solution after packing are placed on
Ice bath 10min is carried out on ice, and the centrifuge tube equipped with bacterium solution, which is filled to 4 DEG C of 3000g on refrigerated centrifuge, carries out centrifugation 10min richnesses
Collect E.coli DH5 α；
(5) supernatant is outwelled after centrifuging, the 0.1mol/L CaCl2 solution after 500 μ l ice baths is drawn with blue electron gun head, to bacterium
Body is resuspended.After thalline after resuspension carries out ice bath 20min, 10min is centrifuged in 4 DEG C of rotating speed 3000g of temperature again；
(6) centrifuge tube after centrifugation, can significantly observe that Escherichia coli are enriched in tube bottom, discard supernatant, if not
It abandons totally available yellow or white pipette tips is sucked out, the CaCl2 containing 15% glycerine that the 0.05mol/L of 200 μ l is then added is molten
Liquid, the step will carry out on ice, and to prevent influencing the changing effect of competence, last ice bath 2min is put into -70 DEG C of refrigerators,
It is on-demand when conversion.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment, step
Connection product converts E. coli DH5 α competence in A2, and steps are as follows：
(1) competent cell for taking out packing storage in -70 DEG C of refrigerators with gloves, thaws at ambient temperature,
And it is put on ice for immediately；
(2) using white pipette tips, total range is 10 μ l, and all above-mentioned steps are completely carried out to the production of carrier T connection
Object is transferred in competent escherichia coli cell, is squeezed into after connection product to take advantage of a situation and is carried out mixing, and ice water to mixed liquor with rifle
Bathe 30min；
(3) by the bacterium solution after ice bath, it is immediately placed in thermal shock 90s in 42 DEG C of metal baths, it is cold on ice that thermal shock finishes rapid placement
But 3min；
(4) in superclean bench, the fresh LB liquid mediums of 1ml are added into the centrifuge tube after thermal shock (without anti-
Raw element), 37 DEG C of shaking table shake cultures, 1 hour (concussion duration can be appropriately extended) makes Escherichia coli normal growth, and express matter
The antibiotics resistance gene deposited in grain；
(5) high speed centrifugation will be carried out on centrifuge after centrifuge tube trim, so that thalline is deposited, 1ml is discarded in super-clean bench
Supernatant, with blue electron gun head it is soft remaining 200 μ l supernatants are subjected to suspension mixing with thalline, the painting to sterilize on alcolhol burner is used in combination
Remaining bacteria suspension is coated on the LB solid mediums containing Amp by cloth stick, is inverted in 37 DEG C of constant incubators after bacterium solution is dry
Overnight incubation；
(6) it needs to carry out control when converting, connection product, Qi Tacao is replaced with the aseptic double-distilled water of same volume
Make identical, is respectively coated on the LB solid mediums containing Amp and the LB solid mediums without Amp, as a contrast, with true
Determine the vigor of Amp antibiotic and the vigor of competence.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment, step
The authentication step of recon is as follows in A2, and table 1 is bacterium solution PCR reaction system tables：
(1) single bacterium colony grown in tablet is simply counted and is marked, 4 are then selected from single bacterium colony
It is a, it is seeded to progress shaking table culture in fluid nutrient medium and stays overnight, should enhance to recombination added with Amp antibiotic in fluid nutrient medium
The screening effect of son；
(2) 1 μ l bacterium solutions is taken to carry out PCR after being incubated overnight, system is 20 μ l, as shown in table 2-5, amplification condition and upper phase
Together；
(3) after PCR instrument EP (end of program), 5 μ l bacterium solutions PCR products of extraction are mixed with the loading buffer of 1 μ l, point sample
In prepared 1% Ago-Gel, whether the electrophoresis under 180v voltage conditions, observation contains at Marker respective straps
Bacterium solution can be carried out the plasmid extraction work of next step with target gene ech42 segments of the same size if being shown as positive by having
Make.
1 bacterium solution PCR reaction systems of table
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment, step
The extracting method of escherichia coli plasmid is extracted with reference to escherichia coli plasmid with reference to kit (StarPrep Plasmid in A2
Miniprep Kit) it carries out, steps are as follows：
(1) prepare：With reference to the related description of kit, by absolute ethyl alcohol and RNase A be added mixing in respective mother liquor,
It marks, is for use；
(2) thalline is collected：4 centrifuge tubes totally to sterilize are taken respectively, are marked, and are separately added into 1.5ml and are incubated overnight
Above-mentioned bacterium solution, in 12000rpm centrifuge 1min, discard supernatant liquid after centrifugation, if tube bottom enrichment bacterium solution it is less, can rejoin
Supernatant is abandoned in 1.5ml bacterium solutions, again centrifugation (centrifugation number is depending on bacterial concentration size)；
(3) suspension thalline：250 μ l cell suspending liquids (S1) are added in the centrifuge tube after receipts bacterium, repeatedly with blue pipette tips
Piping and druming, makes the thalline of enrichment adequately be suspended in S1 solution；
(4) cracking and neutralization：250 μ l (S2) are added into the centrifuge tube after above-mentioned steps, lid upper tube cap is gently run immediately
Inverse makes it be uniformly mixed, and places after waiting for bacterium solution to clarify, and 350 μ l (S3) and gently mixing are added, and is put in centrifuge high
Speed centrifugation 15min, can observe the white solid matter of bottom of the tube after centrifugation；
(5) DNA films combine：Centrifuge tube after centrifugation is taken out, supernatant is carefully sucked out with blue pipette tips, is transferred to mark
In the centrifugal adsorbing column for the insertion collecting pipe remembered, 12000rpm room temperatures centrifuge 1min, to ensure maximum organic efficiency, will receive
Liquid in collector sucks in adsorption column again, and turns back in collecting pipe.12000rpm room temperatures centrifuge 1min again, discard collection
Waste liquid in pipe；
(6) cleaning of adsorption column：It is combined to said gene and 500 μ l (WB) is added in the adsorption column finished, lid upper tube cap, in
High speed centrifugation in centrifuge discards waste liquid in pipe after centrifugation.500 μ l (WB) of equivalent are added into adsorption column again, it is same to turn
Speed centrifuges again, after centrifugation, adsorption column is uncapped and is not added with any liquid, is put into centrifuge and carries out unloaded centrifugation 2min,
It can be placed on dry place after centrifugation and about 15min be dried, next-step operation can be carried out；
(7) plasmid is eluted：It is put into clean centrifuge tube after adsorption column is dried, and is added above adsorption column
55 μ l sterile deionized waters can be stored at room temperature 2min to improve the rate of recovery in 60 DEG C of water-baths.At room temperature in centrifuge
12000rpm centrifuges 1min, collects Plasmid DNA.To ensure organic efficiency, the liquid containing Plasmid DNA after centrifugation is inhaled again
Go out to be put into and carries out secondary centrifuging on silica gel absorption film；
(8) plasmid is preserved：Centrifugal adsorbing column is discarded, and plasmid solution is stored in -20 DEG C of refrigerators, it is on-demand.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment, step
Plasmid enzyme restriction detection positive colony sub-step is in A2：
(1) it will be accredited as the plasmid vector extracted in positive bacterium solution from bacterium solution PCR, carries out single endonuclease digestion and double digestion mirror
Fixed, restriction enzyme selects the restriction endonuclease sites (Xba I, Sma I) introduced during PCR.It is limited with reference to Thermo
Property digestion enzyme specification processed is selected as 20 μ l systems carry out (single endonuclease digestion system is identical with this) in the following table 2：
2 digestion system of table
To ensure digesting efficiency, centrifuge tube is put in 37 DEG C of one nights of incubation in metal bath.
(2) second days successively by the plasmid and loading of plasmid and double digestion after the plasmid directly extracted, single endonuclease digestion
Buffer is mixed, and point plus 5 μ the l 180v in 1% Ago-Gel carry out electrophoresis 15min.In ultraviolet after electrophoresis
It is observed under lamp, the case where three swimming lanes, point has the Article 2 that first swimming lane of non-digested plasmid can have single endonuclease digestion plasmid than point
Swimming lane on the lower, point have the swimming lane of double digestion plasmid can show two cleaning bands, one at upper one under, if under
DNA small fragments coincide with target gene size, then digestion is accredited as the positive.
(3) sequencing of carrier T is recombinated：Bacterium solution PCR and single double digestion are examined into the correct large intestine bar for carrying carrier T of connection
Bacterium bacterium solution is sent to sequencing company and carries out the measurement of sequence, after obtaining sequencing result, spliced, and pass through the comparison on NCBI
Service, is compared splicing result.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment, step
Sequencing is finished the Escherichia coli of the DH5 α containing pMD18-T-ech42 respectively and is expressed containing pHT43 by the extraction of plasmid in A3
The Escherichia coli of carrier are seeded in the LB culture mediums containing Amp antibiotic, progress shaking table culture, with reference to above-mentioned after shaking table culture
Escherichia coli plasmid extracting method carries out plasmid extraction operation to bacterium solution respectively.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment, plasmid
The double digestion method of carrier carries out double digestion to pMD18-T-ech42 and pHT43 expression vectors respectively using Xba I, Sma I,
34 DEG C of one nights of incubation in metal bath, digestion system are 50 μ l, system such as the following table 3：
3 50 μ l digestion systems of table
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment, step
After glue recycling target gene and expression vector endonuclease bamhi are digestion in A3, by digestion products o'clock in 1% Ago-Gel
In, and glue recycling is carried out with reference to the method for recycling with the purifying of pcr amplification product, the segment (ech42) containing target gene
Swimming lane glue recycles lower end target gene, the carrier segments of the swimming lane glue recycling upper end containing expression vector pHT43.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment, step
Double digestion is carried out to pMD18-T-ech42 and pHT43 expression vectors using Xba I, Sma I respectively in step A3 in A3, later
The digestion glue recovery product of expression vector and chitinase gene ech42 are attached, linked system is 5 μ l of pHT43,
12.8 μ l, 10 × T4DNA Ligase Buffer of ech42,2 μ l, T4DNA Ligase, 0.2 μ l.
Specific implementation mode four：
The genetic engineering bacillus amyloliquefaciens of effective prevention gray mold according to specific implementation mode one or two
Preparation method, step B include the following steps：
Step B1, wild type bacillus amyloliquefaciens BA Electroporation-competent cells are prepared；
Step B2, electroporated, it is thin that recombinant expression carrier pHT43/ech42 is imported into bacillus amyloliquefaciens BA competence
Born of the same parents；
Step B3, positive restructuring bacillus amyloliquefaciens are screened, the plasmid in bacillus amyloliquefaciens is extracted.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment, step
B1 includes the following steps：
Step B1-1, the bacillus amyloliquefaciens BA bacterial strains for preserving laboratory, take out from -70 DEG C of refrigerators, wait melting
Picking bacterium solution afterwards carries out three sections of scribing line on LB solid mediums, is inverted and is incubated overnight in 37 DEG C of constant incubators；
Step B1-2, the BA single bacterium colonies grown after overnight with yellow pipette tips picking, in 5mlLB fluid nutrient mediums,
It is incubated overnight in 150rpm, 37 DEG C of earthquake incubators；
Step B1-3, in superclean bench, 2.5ml overnight cultures is taken out with sterile blue pipette tips, are added to 40ml
In fresh bacillus amyloliquefaciens growth medium, 37 DEG C, 200rpm shaken cultivations to OD600=0.85-0.95 are described
The formula of bacillus amyloliquefaciens growth medium is peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, sorbierite 91g/
L；
Step B1-4, after OD600 values reach, immediately by bacterium solution carry out ice-water bath 10min, by bacterium solution be divided in 1.5ml from
In heart pipe, then in refrigerated centrifuge, 5000g, 4 DEG C of centrifugation 5min, each centrifuge tube centrifugation twice, collect solution starch brood cell
Bacillus thalline；
Step B1-5, turn culture medium with the electricity without containing ion and mixing resuspension is carried out to the thalline of enrichment, draw 1ml every time
So that thalline is gathered in tube bottom in 4 DEG C of centrifugation 10min of 8500rpm in centrifuge tube, and by cryogenic freezing centrifuge, adopt
Rinsing 4 times is carried out with such step, the formula that the electricity without containing ion turns culture medium is sorbierite 90g/L, mannitol
92.5g/L, glycerine 100ml/L；
Step B1-6, after washing, turn culture medium with 60 μ l electricity of sterile yellow pipette tips addition into each centrifuge tube, obtain
Wild type bacillus amyloliquefaciens BA Electroporation-competent cells, spare, it is sorbierite 90g/L that the electricity, which turns culture medium, sweet
Reveal alcohol 92.5g/L, glycerine 100ml/L.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment, step
B2 includes the following steps：
Step B2-1, by freshly prepared wild type bacillus amyloliquefaciens BA Electroporation-competent cells or it is stored in ice
Competence in case is taken out from refrigerator, wait for competence bacterium solution melt postposition on ice with 10 μ l recombinant plasmid pHT43/ech42 mixings
Ice-water bath 2min afterwards；
Step B2-2, the mixture after step B2-1 ice baths is transferred in the electric shock cup of 1mm, and electric shock cup is put into
In BIO-RAD GenePulser Xcell, the electrotransformation parameter of five gradients is set：Be respectively 12.5kv/cm, 15kv/cm,
17.5kv/cm、20kv/cm、21kv/cm.It after setting parameter, shocks by electricity immediately to electric shock cup, electric shock resistance value is 200
Ω, the time constant 4.5-5.0ms of electric shock；
Step B2-3, it uses blue electron gun head to draw 1ml incubations after step B2-2 electric shocks immediately to be added in 37 DEG C of recovery medium
To mixing in electric shock cup, it is transferred in the sterile centrifugation tube marked after mixing, centrifuges 37 DEG C of effective incubator, 200rpm shakes
Culture 3-4h is swung, the formula of the recovery medium is peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, sorbierite
90g/L, mannitol 70g/L；
Step B2-4, after step B2-3 shaken cultivations, centrifuge tube is centrifuged into 1min in 8000g, 900 μ l are drawn with pipette tips
Supernatant simultaneously discards, and thalline is resuspended remaining liquid, and is coated in the LB solid mediums containing chloramphenicol, in 37 DEG C
It is incubated overnight in constant incubator.
The preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in present embodiment, step
The screening of B3 positive recombinants includes the following steps with identification：
(1) single bacterium colony after picking is incubated overnight be added in the LB culture mediums of chloramphenicol, 37 DEG C, 180rpm shaken cultivations
Overnight；
(2) bacterium solution PCR：With reference to the method that positive soldier of Lv Lu Yan et al. detects positive restructuring bacillus amyloliquefaciens, prepare
10mg/ml lysozyme solns, being added to 1ml cultures has in the bacterium solution of bacillus amyloliquefaciens, until final concentration of 0.4mg/ml, shakes
37 DEG C of oscillation 30min of bed make lysozyme play one's part to the full, and dissolve bacillus amyloliquefaciens cell wall, and bacterium is boiled in boiling water
Liquid is put into -20 DEG C of refrigerators and freezes 5min, cell wall is made fully to rupture immediately after, and plasmid is dissolved out, and 1 μ l is then taken to split
It solves liquid and carries out bacterium solution PCR detections, PCR system is 20 μ l.Bacterium solution PCR is verified as positive bacterium solution, carries out carrying for subsequent plasmid
It takes；
(3) extraction Yu the digestion detection of plasmid：The extraction of plasmid is carried with reference to escherichia coli plasmid in bacillus amyloliquefaciens
Only lysozyme is added to final concentration 0.4mg/ml, 37 DEG C of shaking table after cell suspending liquid (S1) is added in the method taken thereto
Oscillation 30min makes lysozyme play one's part to the full.Subsequent step is identical as front plasmid extraction.After extracting plasmid, confrontation
Grain carries out single endonuclease digestion and double digestion identification, and digestion system is 20 μ l, and then point sample is in 1% Ago-Gel, after electrophoresis
Observation result is carried out in ultraviolet gel imaging system.
Specific implementation mode five：
The genetic engineering bacillus amyloliquefaciens of effective prevention gray mold according to specific implementation mode one or two
Preparation method, the induced expression and its SDS-PAGE electrophoresis following steps of chitinase：
(1) preparation of reagent
Table 4 concentrates the formula of glue and separation gel
(2) destination protein is prepared
The bacillus amyloliquefaciens BA for being transferred to recombinant plasmid pHT43/ech42 is seeded to 2 bottles of 50ml with yellow pipette tips to contain
Have in the LB culture mediums of chloramphenicol, 37 DEG C of constant-temperature shaking incubators, 180rpm carries out shaken cultivation, is detected every half an hour
OD values are suspended shaking table culture, are added into the bacillus amyloliquefaciens BA of pHT43/ech42 until when OD600 is 0.6-0.8
The IPTG of final concentration of 1mmol/L, 37 DEG C, the bacteriolyze of final concentration of 0.4mg/ml is added into bacterium solution after 180rpm inductions 8h
Enzyme cracks bacillus amyloliquefaciens.After bacterium solution fades to clarification, bacterium solution is divided in spare in 1.5ml centrifuge tubes.Point
Before sample, 16 μ l bacterium solutions are taken out, 4 μ l reduced form 5 × SDS sample-loading buffers are added, 10min is boiled after mixing, albumen sample is finished,
It can directly put to polyacrylamide gel and suffer.
(3) gel is prepared, steps are as follows：
(3-1) cleans glass plate：Four pieces of glass plates with glue are cleaned up with tap water, then are washed away with distilled water
It one time, is placed on dry place and is dried.
(3-2) is good with gel maker card by glass plate, puts on the top of the shelf, fills distilled water, detects whether leakage.
After (3-3) observes no leakage, 10% separation gel will be prepared and carry out encapsulating, two boards will encapsulating, use rifle after encapsulating
Distilled water is gently added in head, and distilled water is filled it up with, and can observe whether distilled water liquid level declines, to prevent the hair of leak-stopping liquid situation
It is raw.
After (3-4) about 30min, separation gel has solidified, and at this time outwells the distilled water on upper layer, prepares 5% concentration glue,
It is inserted into comb after encapsulating, tries not to generate bubble during inserting comb.Glue is concentrated after about 30min to be solidified, at this time
Point sample can be carried out into glue hole.
(4) point sample
Prepared offset plate is removed into pedestal, is put into electrophoresis tank, and 1 × Tirs- glycine electrophoresis is added into slot and delays
Fliud flushing, liquid feeding will influence electrophoretic effects, liquid level adds to dipped glue hole softly to prevent generating bubble in offset plate bottom.Electrophoresis liquid adds
After good, albumen Marker (5 μ l) is sequentially added and the protein sample (20 μ l) that boils that treated.
(5) electrophoresis
When bromophenol blue is shown in upper layer concentration glue, electrophoresis is carried out using the voltage of 80v；Albumen run to separation gel with
When concentrating the interface of glue, it can be collapsed into a line, select 120v voltages to carry out electrophoresis at this time.
(6) it dyes
Bromophenol blue can represent smaller protein band as a kind of indicator during running glue, when this blue bands
Run to electrophoresis apparatus can be closed at the about 1cm of glass plate bottom and stop electrophoresis, after the clean blade excision of upper layer concentration glue, will under
Layer separation calymma enters in prepared coomassie brilliant blue staining liquid, is dyed.Separation gel is taken out after 1h, with weak destainer into
Row boiling decoloring is put into gel imaging system, carries out imaging preservation, finally by offset plate until showing clearly band on offset plate
It is put into preserve and be preserved in liquid.
Specific implementation mode six：
The genetic engineering bacillus amyloliquefaciens of effective prevention gray mold according to specific implementation mode one or two
Preparation method, DNS reducing sugar methods survey chitinase enzyme activity, and DNS reducing sugar methods are exactly dinitrosalicylic acid system, the original of this method
Reason, which is dinitrosalicylic acid (DNS), redox reaction occurs with reduced sugar, and can be contained according to reduced sugar after boiling
Amount number show the colors of the different depths, we using this principle by chitinase enzymolysis product and dinitrosalicylic acid into
Row reaction, carries out colorimetric, you can obtain the vigor size of chitinase, include the following steps in microplate reader after boiling：
(1) making of standard curve
It is separately added into corresponding reagent by the reagent content in table 5,10min is boiled after mixing well, it is then cold with flowing
Water cools down immediately, and each pipe is settled to 10ml, and blank is done with distilled water, and every pipe is measured under 540nm wavelength with microplate reader
Light absorption value, and make a record, often pipe is done repeats three times, seeks average value three times.It is horizontal with N-acetylglucosamine content
Coordinate, light absorption value are ordinate, draw N-acetylglucosamine standard curve.
The making of table 5- acetylglucosamine standard curves
(2) induced expression of recombinant chitinase
The bacillus amyloliquefaciens BA for being transferred to recombinant plasmid pHT43/ech42 is seeded to 2 bottles of 100ml with yellow pipette tips
In LB liquid medium containing chloramphenicol, while being inoculated with the original bacillus amyloliquefaciens bacterial strain (BA) without exogenous plasmid
In 100ml LB liquid mediums in 37 DEG C of constant-temperature shaking incubators, 180rpm carries out shaken cultivation, and one is detected every half an hour
Lower OD values, until when OD600 is 0.6-0.8, pause shaking table culture adds into the bacillus amyloliquefaciens containing recombinant plasmid
Enter the IPTG of final concentration of 1mmol/L, 37 DEG C, 180rpm induces 2h, 4h, 8h, 12h, is sampled respectively for 24 hours respectively.
(3) measurement of recombinant chitinase enzyme activity
The bacterium solution of different times after IPTG is induced centrifuges 1min with centrifuge 8000rpm, draws 0.5ml supernatants respectively
For liquid in (it is control that control boils the supernatant after inactivating with 100 DEG C) in test tube, the glue for adding 1.0mL 1% (w/v) is several
Ding Zhi, 37 DEG C of incubation 1h in water-bath, it is to be restored to room temperature after water-bath, 1.5mL DNS reagents are added thereto, and fast
100 DEG C of speed boils 10min, after cooled down immediately with tap water, centrifuge 8000rpm centrifuges 5min, with distillation
Reaction solution is settled to 10ml by water, takes supernatant in microplate reader, and wavelength is that absorbance is measured at 540nm, and each processing repeats
Three times.
Sequence table
<110>Northeast Agricultural University
<120>Effectively genetic engineering bacillus amyloliquefaciens of prevention gray mold and preparation method thereof and its application
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1218
<212> DNA
<213>Parasitism broom bacterium（Bionectria ochroleuca）
<400> 1
cgtgccactc ctcgtatgga ggacctggct tccactgacc tttctacgcg tgcaactgga 60
tccgtcaatg ctgtttactt cactaactgg ggcatttacg gacgcaactt ccagcctgcg 120
gatcttccag cttcaagcat ttcgcatgtc ctctattctt tcatgaacct ccgagcggat 180
ggcacggttt actcgggtga cacctacgcc gacttggaga agcattactc tgacgactca 240
tggaatgata tcggaacaaa cgcctatggt tgtgtcaagc agctctacaa gctcaagaag 300
gcaaaccgct cgctcaagat catgctgtcc attggtggct ggacttggtc gaccaacttt 360
cccgctgctg cctccaccga ggctacccgt gctacatttg ccaagaccgc tgttgaattc 420
atgaaggatt ggggttttga cggcattgac gtcgactggg agtaccccgc cagtgagaca 480
gaagccaaca acatggtcct ccttcttcag cgagttcgcc aggagctcga ctcgtactcc 540
gcaacatatg ctaatggcta tcacttccaa ctttccattg ccgctcccgc aggacctgac 600
cattacaagg ttcttaagct agcccagctc ggttccgtcc tcgacaacat caacctcatg 660
gcctacgact atgcaggttc ctgggacagt gtcagcggtc atcaagccaa cctgtatcct 720
agcacatcaa accccagctc aactccattc agcaccaagg ctgcggtcga cgcatacatc 780
gcagctggcg tccctgcaag caagatcatt ctaggtatgc ccatctacgg cagagctttt 840
gtgggaaccg acggaccagg caagccctac tccactatcg gcgaaggcag ctgggagagc 900
ggaatctggg actacaaagt ccttcccaag gccggcgcaa ccgtgattac cgactccgcg 960
gccggtgcta cctacagtta cgactccagc agcaggacca tgatctcata cgataccccc 1020
gatatggtcc gcacaaaggt ctcatatgct aagggtctcg gtctcggagg cagcatgttc 1080
tgggaggcat cagccgacaa gaccggctct gactcgctta tcggcactgc cctcagcagc 1140
atgggcagcc ttgacagcac ccagaactgc ctcagctacc ccaactccaa gttcgacaat 1200
atcaagaaca gcctctcc 1218
Claims (10)
1. one plant of genetic engineering bacillus amyloliquefaciens for effectively preventing gray mold, it is characterised in that：This plant of entitled solution of bacterium classification
Bacillus amylobacter Bacillus amyloliquefaceins are transferred to pink glutinous broom bacterium chitinase gene in this plant of bacterium,
It is preserved in Chinese microorganism strain preservation administrative center (CGMCC), deposit number CGMCC on June 29th, 2017
No.14366。
2. a kind of preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold described in claim 1,
It is characterized in that：Include the following steps：
Step A, by genetic engineering, Parasitism broom bacterium chitinase gene ech42 is expanded, and connect expression vector pHT43, structure
Build recombinant expression carrier pHT43/ech42；
Step B, electroporated by setting, the recombinant expression carrier pHT43/ech42 described in step 1 is imported into solution starch brood cell
In bacillus, the shock voltage is 12.5kv/cm or 15kv/cm or 17.5kv/cm.
3. the preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold according to claim 2,
It is characterized in that：Step A includes the following steps：
Step A1, it is cloned by PCR and obtains C.rosea chitinase genes ech42；
Step A2, by the connection of the step A1 chitinase gene ech42 and carrier T obtained；
Step A3, the chitinase gene ech42 that step 2 obtains is connect with expression vector pHT43.
4. the preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold according to claim 3,
It is characterized in that：The gene order of C.rosea chitinase ech42 genes (DQ523687) is found in step A1 by GenBank,
With signal peptide inspection software, signal peptide sequence is detected, and not contain the digestion introduced on primer in clone gene
Site, with 5 software Design primers of primmer：
Primer ech42E：5 '-TATCTAGACGTGCCACTCCTCGTATGGAGG-3 ', digestion point Xba I；
ech42B：5 '-CCCCCCGGGGGAGAGGCTGTTCTTGAT-3 ', digestion point Sma I；
PCR reaction mixture be：Template 1 μ l, 10 μM of sense primer ech42E 1 μ l, 10 μM of downstream primer ech42B 1
5 μ l, 2.5mM dNTPs of μ l, 10 × EasyTaq buffer solution, 4 μ l, EasyTaq archaeal dna polymerases 0.5 μ l, 37.5 μ of ultra-pure water
l；
PCR reaction conditions are：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 0.5min, 68 DEG C of annealing 0.5min, 72 DEG C of extension 1min,
35 cycles；After last 72 DEG C extend 5min, preserved at 4 DEG C.
5. the preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold according to claim 3,
It is characterized in that：The recycling of pcr amplification product and purifying reference kit StarPrep Gel Extraction Kit in step A2
It carries out；The target gene ech42 of recycling is attached with carrier T, reaction system is 1 μ l of pMD18-T carriers, target gene
4 μ l of ech42,5 μ l of solution I；The plasmid vector extracted in positive bacterium solution will be accredited as from bacterium solution PCR, carry out single endonuclease digestion and
Double digestion identifies that restriction enzyme selects restriction endonuclease sites Xba I, the Sma I introduced during PCR, digestion system
For：10 times of Tango buffer solutions 2 μ l, pMD18-T/ech42 10 μ l, Xba I 1 μ l, Sma I 1 μ l, 6 μ l of no enzyme water.
6. the preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold according to claim 3,
It is characterized in that：Double digestion is carried out to pMD18-T/ech42 and pHT43 expression vectors using Xba I, Sma I respectively in step A3, it
The digestion glue recovery product of expression vector and chitinase gene ech42 are attached afterwards, linked system is 5 μ l of pHT43,
12.8 μ l, 10 × T4DNA ligase buffer solutions of ech42,2 μ l, T4DNA ligase, 0.2 μ l.
7. the preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold according to claim 2,
It is characterized in that：Step B includes the following steps：
Step B1, wild type bacillus amyloliquefaciens BA Electroporation-competent cells are prepared；
Step B2, electroporated, recombinant expression carrier pHT43/ech42 is imported into bacillus amyloliquefaciens BA competent cells；
Step B3, positive restructuring bacillus amyloliquefaciens are screened, the plasmid in bacillus amyloliquefaciens is extracted.
8. the preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold according to claim 7,
It is characterized in that：Step B1 includes the following steps：
Step B1-1, the bacillus amyloliquefaciens BA bacterial strains for preserving laboratory, take out from -70 DEG C of refrigerators, are chosen after melting
Bacterium solution is taken, three sections of scribing line are carried out on LB solid mediums, is inverted and is incubated overnight in 37 DEG C of constant incubators；
Step B1-2, the BA single bacterium colonies grown after overnight with yellow pipette tips picking, in 5mlLB fluid nutrient mediums, 150rpm, 37
It is incubated overnight in DEG C earthquake incubator；
Step B1-3, in superclean bench, 2.5ml overnight cultures is taken out with sterile blue pipette tips, are added fresh to 40ml
Bacillus amyloliquefaciens growth medium in, 37 DEG C, 200rpm shaken cultivations to OD600=0.85-0.95, the Xie Dian
The formula of powder bacillus growth medium is peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, sorbierite 91g/L；
Step B1-4, after OD600 values reach, bacterium solution is subjected to ice-water bath 10min immediately, bacterium solution is divided in 1.5ml centrifuge tubes
In, then in refrigerated centrifuge, 5000g, 4 DEG C of centrifugation 5min, each centrifuge tube centrifugation twice, collect bacillus amyloliquefaciens
Thalline；
Step B1-5, turn culture medium with the electricity without containing ion and mixing resuspension carried out to the thalline of enrichment, every time draw 1ml in from
In heart pipe, and thalline is set to be gathered in tube bottom, using this in 4 DEG C of centrifugation 10min of 8500rpm by cryogenic freezing centrifuge
Kind step carries out rinsing 4 times, and the formula that the electricity without containing ion turns culture medium is sorbierite 90g/L, mannitol
92.5g/L, glycerine 100ml/L；
Step B1-6, after washing, turn culture medium with 60 μ l electricity of sterile yellow pipette tips addition into each centrifuge tube, obtain wild
Type bacillus amyloliquefaciens BA Electroporation-competent cells, spare, it is sorbierite 90g/L, mannitol that the electricity, which turns culture medium,
92.5g/L, glycerine 100ml/L.
9. the preparation method of the genetic engineering bacillus amyloliquefaciens of effective prevention gray mold according to claim 7,
It is characterized in that：Step B2 includes the following steps：
Step B2-1, it by freshly prepared wild type bacillus amyloliquefaciens BA Electroporation-competent cells or is stored in refrigerator
Competence from refrigerator take out, after competence bacterium solution melt postposition on ice with ice after 10 μ l recombinant plasmid pHT43/ech42 mixings
Water-bath 2min；
Step B2-2, the mixture after step B2-1 ice baths is transferred in the electric shock cup of 1mm, and electric shock cup is put into BIO-
In RAD GenePulser Xcell, the electrotransformation parameter of five gradients is set：Be respectively 12.5kv/cm, 15kv/cm,
17.5kv/cm、20kv/cm、21kv/cm.It after setting parameter, shocks by electricity immediately to electric shock cup, electric shock resistance value is 200
Ω, the time constant 4.5-5.0ms of electric shock；
Step B2-3,1ml incubations are drawn with blue electron gun head immediately after step B2-2 electric shocks to be added to electricity in 37 DEG C of recovery medium
Mixing in cup is hit, is transferred to after mixing in the sterile centrifugation tube marked, 37 DEG C of effective incubator, 200rpm shaken cultivations are centrifuged
3-4h, the formula of the recovery medium are peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, sorbierite 90g/L, sweet
Reveal alcohol 70g/L；
Step B2-4, after step B2-3 shaken cultivations, centrifuge tube is centrifuged into 1min in 8000g, 900 μ l supernatants are drawn with pipette tips
And discard, thalline is resuspended remaining liquid, and is coated in the LB solid mediums containing chloramphenicol, in 37 DEG C of constant temperature incubations
It is incubated overnight in case.
10. a kind of one of claim 1-9 any one of them effectively prevents the genetic engineering bacillus amyloliquefaciens of gray mold
Application, it is characterised in that：Application in preventing gray mold.
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