CN109929866A - The biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck - Google Patents
The biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck Download PDFInfo
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Abstract
The present invention relates to agromicrobiology technology and the technical fields of biological prevention, more particularly to the biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck, the present invention provides one plant of biocontrol microorganisms (2R, 3R) -2,3- butanediol dehydrogenase (butanediol dehydrogenase, 2,3-bdh) bacterium is knocked outGJ11△bdhMethod for preventing and treating tobacco weather-fleck.Different material is acted on into tobacco root, is simulated by indoor climate spot, discovery 3-hydroxy-2-butanone can prevent and treat weather spot disease.In Enshi tobacco weather-fleck diseased region screen to obtain one plant of production 3-hydroxy-2-butanone Bei Laisi bacillus (Bacillus velezensis)GJ11, simulated by weather spot, which has lower control efficiency to tobacco weather spot.By knocking out 2, the 3-bdh gene of Bei Laisi bacillus, it is found that the 3-hydroxy-2-butanone yield of the bacterium significantly improves, which significantly improves the prevention and treatment of tobacco weather spot.It is detected, is found by Physiology and biochemistryGJ11△bdhFermentation liquid can be improved the activity of the antioxidase of tobacco, reduce the activity keto concentration as caused by ozone in plant.
Description
Technical field
The present invention relates to agromicrobiology technology and the technical fields of biological prevention, more particularly to one plant of tobacco
The biocontrol bacterial strain Nei Shengbeilaisi bacillus of weather spot disease.
Background technique
Tobacco weather fleck is when tobacco plant is in seedling stage or Adult plant, since long term survival is same with ozone in low temperature
When existing environment in, caused by blade spot disease.The disease is in regularity distribution, and blade tip, phyllopodium, leaf central part tissue are more
It concentrates, mostly along vein two sides tissue development.Scab just be needle point size water stain shape canescence or brown dot, after be extended to
The big spot of diameter l-3 mm subcircular, medial necrosis, surrounding chlorosis, multiple scabs are fused into bulk withered spot, vein two sides when serious
Scab in irregular shape it is shrivelled, mesophyll necrosis, leaf abscission.Scab is in perforated on maturescent middle and lower part or bottom leaf,
Occur many scattered round spots fine crushing on blade face, about 5 mm of size, the later period also perforates, but scab edge is without auburn boundary
Limit, is different from shothole disease.Moreover, because the period and occurrence condition that disease occurs are different, disease symptom is also different, such as day shift
Blackspot type etc. after type, foxiness type, ring spot type, dust type, the brown point-type of necrosis, the brown point-type of non-necrosis, climax leaves foxiness type, rain.?
In above-mentioned Damage Types, hickie type is most common type.
Ozone enters blade interior by two kinds of approach, directly penetrates into leaf epidermis angle and stomatal conduction absorbs, but with
The latter is main path.In stomatic chamber, ozone can stimulate membrane lipid peroxidatio and damage membrane flow by " ozone decomposition "
Property directly reacted with plasma membrane, influence the permeability of water and ion on film, cellular integration caused to be lost, cell inclusion leaks to
Space between cells, dirty-green or water stain shape is presented in the aggrieved area of blade at this time.After effect in several hours, ozone passes through plasma membrane and enters cell
Device, Chloroplast are damaged, and the state of oxidation of chlorophyll a and chlorophyll b is destroyed, and especially affect Photosystem I I
(PSII) activity loses photosynthetic capacity；Or ozone enter after cell can be spontaneous be converted into active oxygen (ROS),
Such as superoxide radical (O2-), hydroxyl (OH-) and hydrogen peroxide (H2O2), raised ROS level may cause to promote oxygen in the cell
Change environment, cause the modification to structural protein and activated protein, especially ozone and derivative ROS can be present in ammonia
The sulfydryl reaction of exposure on base acid, mainly cysteine, tryptophan, tyrosine, methionine and histidine, this may be straight
Connect the vigor for reducing plant.Research has shown that plant, which is exposed in raised level of ozone, to damage plant itself, especially
It is that ozone can inhibit the photosynthesis of plant, accelerate aging, hinders growth and reduce crop yield.With superoxipe ion and hydroxyl
Base free radical is compared, H2O2Be it is metastable, can as a kind of signaling molecule of intercellular communication, in guard cell,
H2O2It is capable of the ion channel of active cell film, stomata is caused to be closed.
Chemical reagent also adjust by a kind of method as prevention and treatment tobacco weather-fleck, many antioxidants, stomata and growth
Section agent, antidotal agent etc. have proved that the disease can be effectively prevented.There is large-scale tobacco weather spot prior to the country in foreign countries
Point disease, uses the Agro-chemicals control disease for the first time.And then a large amount of chemical reagent prevention and treatment experiments, identification have also been carried out all over China
Many potent agents.Such as: synergy Bordeaux mixture, ethylene allophanamide (EDU), superoxide dismutase (SOD) etc..But
The fact that have to and face is that there are a degree of influences on farmland soil property for chemical reagent, and abuse chemical reagent or even can make
At environmental pollution.So still needing to scholars on the road of prevention and treatment tobacco weather mottle disease probed into and continuing to explore the way forward.
Endophyte is increasingly taken seriously in biological control with its unique endogeny, and endophyte is widely present in plant
In vivo, theoretical research and Development volue have been to be concerned by more and more people, and have vital meaning in biological control.
Summary of the invention
In order to solve the above technical problems, the present invention, which provides one kind, can generate the prebiotic of the substance for preventing and treating tobacco weather fleck
Bacterium, the bacterium are easy to industrialized production；The microbial bacterial agent is environmentally safe nuisanceless, has good development prospect and application latent
Power.
The biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck of the invention, experimentation include
Following steps:
(1) screening and identification of tobacco weather spot biocontrol bacteria:
A. raw and rhizosphere bacteria separation in tobacco: 0.5 g root, stem and leaf are weighed respectively, is carried out respectively with 75% ethanol solution
30 s are sterilized, twice with aseptic water washing, impregnate 1 min with 0.1% mercuric chloride respectively, aseptic water washing three times, is separately added into 5 ml
Sterile water grinding sufficiently, after gradient dilution, is respectively coated on LB plate, 37 DEG C of 48 h of culture, different thin of picking colony form
Bacterium isolates and purifies, and -20 DEG C of glycerol tubes of bacterial strain of purifying are saved；
B. control efficiency of the 3-hydroxy-2-butanone to tobacco weather spot: choosing the tobacco of 5 ~ 7 leaves, distinguishes to 50 mL concentration of tobacco pouring root
For the 3-hydroxy-2-butanone solution of 2 g/L, 4 g/L and 6 g/L, incubated at room temperature three days；Water 50 mL at the 4th day, simulation climate spot
The generation of disease；
C. the screening of high yield 3-hydroxy-2-butanone bacterium: the bacterium screened is inoculated in LB culture medium, 37 DEG C, 180 rpm live overnight
Change, 1% activation bacterium solution of switching is in 37 DEG C of LB liquid medium of 8% glucose, and 180 rpm cultivate 48 h, with the side of gas-chromatography
Method detects 3-hydroxy-2-butanone concentration, screens out the bacterial strain that a plant height produces 3-hydroxy-2-butanoneGJ11；
d. GJ11To the control efficiency of tobacco weather spot: pickingGJ11Single bacterium is fallen in fluid nutrient medium, and 37 DEG C, 180 rpm mistakes
Night activation, 1% activation bacterium solution of switching is in the LB liquid medium containing 8% glucose, and 37 DEG C, 180 rpm cultivate 48 h；Selection 5 ~
The tobacco in 7 leaf periods, to 50 mL fermentation liquid of tobacco pouring root, 50 mL clear water of pouring root is cultivated three days at room temperature as control, the
Water 50 mL at four days, is put into the generation of simulation climate spot disease in incubator；
E. bacterial strain 16sDNA gene sequencing is identified: 16sDNA gene amplification product being sequenced after purification, passes through NCBI data
Library carries out BLAST comparison, using the phylogenetic tree of MEGA5.2 software building Antagonistic Fungi, identifies bacterial strainGJ11Strain；
(2) high yield 3-hydroxy-2-butanone engineering bacteriaGJ11△bdhBuilding:
a. T2-bdhThe building of expression vector: the PCR amplification that carry out to bdh gene L arm and R arm pieces section simultaneously runs glue verifying, by phase
It closes segment and carries out purification and recovery, and using L arm and R arm as template, carry out SOE-PCR, construction of recombinant vector, Escherichia coli convert,
The screening of recon and recombinant plasmid verifying；
b. GJ11△bdhThe building of bacterial strain: conversion GJ11-bdh bacterial strain is selected, 10 mL is seeded to and contains Km resistance (20 μ g/
ML in LB culture medium), 42 DEG C of cultures, until bacterium solution is muddy, dilution applies Km resistance (20 μ g/mL) 37 DEG C of plate cultures, chooses single bacterium
It falls, extracts its total DNA, separately design single-swap verifying primer, L arm single-swap, R arm single-swap takes corresponding primer, total to extract
DNA is template, and 20 μ L systems carry out PCR amplification, and agarose gel electrophoresis selects single exchange strains；
Select single exchange strains WH1-dan-bdh, in 5 mL non-resistant LB culture mediums, 28 DEG C, 200 rpm cultivate 24 h, it is dilute
Painting plate is released, single colonie is chosen and puts bacterium respectively to LB plate and Km resistance (20 μ g/mL) plate, picking resistance loses bacterial strain, extracts
Total DNA, respectively with 100 bp sequence of L arm upstream for F primer, with 100 bp sequence of R arm downstream for R primer, 20 μ L amplification systems
Amplification carries out Blast comparison in NCBI to sequencing primer bdh-dan-LF, bdh-dan-RR；
(3)GJ11WithGJ11△bdh3-hydroxy-2-butanone yield and weather spot preventive effect variation:
PickingGJ11WithGJ11△bdhSingle bacterium is fallen in LB liquid medium, and 37 DEG C, 180 rpm are activated overnight, and 1% switching is in containing
The liquid LB of 8% glucose, 37 DEG C, 180 rpm are cultivated, every 3-hydroxy-2-butanone content of 12 h sample detection；
The tobacco for choosing for 5 ~ 7 leaf periods, to 50 mL of fermentation liquid of above-mentioned 48 h of tobacco pouring root, clear water processing is as control, in room
Water at culture three days, the 4th day 50 mL under temperature, is put into the generation of simulation climate spot disease in incubator；
(4)GJ11△bdhFermentation liquid influences active oxygen: the same step of the processing mode of tobacco (3), during processing every 3
H takes 8 leaves with punch；
A. blade the detection of hydrogen peroxide: is soaked in 1 mg/mL(pH3.8 being formulated by 0.01M phosphate buffer)
DAB solution 8h removes chlorophyll with ethyl alcohol: 95 DEG C of glycerol (9:1 V/V), 5 min；10 times of microscopies, whether there is or not H for observation2O2It generates, palm fibre
The representative of color spot point has H2O2It generates；
B. the detection of superoxipe ion: blade is soaked in the 0.01M phosphate buffer containing 0.01%NBT, dark impregnates 60
Min, production object stablize dark green spot, expose 2 h, ethyl alcohol: 95 DEG C of glycerol (9:1V/V), 5 min remove chlorophyll, 10 times
Microscopy, whether there is or not dark green spots generation, dark green spots representatives superoxipe ion generation for observation；
(5)GJ11△bdhThe influence of fermentation broth on tobacco correlation enzyme activity:
a. GJ11△bdhThe influence of fermentation broth on tobacco SOD enzyme activity: SOD enzyme activity, an enzyme activity are measured using NBT photoreduction met hod
Unit definition is enzyme amount required when the reduction of NBT to be suppressed to control half, observes SOD enzyme activity situation of change；
b. GJ11△bdhThe influence of fermentation broth on tobacco POD enzyme activity: guaiacol method measures POD activity, and observation POD enzyme activity becomes
Change situation；
c. GJ11△bdhThe influence of fermentation broth on tobacco CAT enzyme activity: ultraviolet absorption method measures CAT enzyme activity, and observation CAT enzyme activity becomes
Change situation.
The biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck of the invention, a of the step (1)
In Step d, ozone treatment condition are as follows: ozone ventilating mode is that every 20 min leads to 5 min ozone, the model of ozone generator
MF-Y type mobile ozone generator.
The biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck of the invention, the c of the step (1)
In step, GC conditions method detection parameters are as follows: gal promise FFAP capillary column (mm of 30 m × 0.32 ID, 0.33 μm
Film), flame ionization detector, carrier gas N2, flow is 1.0 mL/min, 30 mL/min of H flow, air mass flow 300
ML/min, Splitless injecting samples, sample volume 1 μ L, 200 DEG C of sample injector temperature, 280 DEG C of detector temperature；Column oven temperature program:
40 DEG C of 1 min of holding are warming up to 60 DEG C of 1.5 min of holding with 6 DEG C/min, are warming up to 160 DEG C with 15 DEG C/min and keep 2.5
min。
The biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck of the invention, a of the step (2)
In step, amplification system is 50 μ L, using primer bdh L- BamHI, bdh L-R as primer amplification bdh gene L arm, bdh R-
F, bdhR- XbaI expands the R arm of bdh, amplification condition: 94 DEG C of initial denaturations 5 min, 94 DEG C of 30 s, Tm of denaturation annealing 30 s, 72
DEG C 45 s extend 30 circulations, and 10 min of rear 72 DEG C of polishings runs glue verifying；
50 μ L amplification systems:
1 μ L of DNA profiling
1 μ L of F primer
1 μ L of R primer
dNTP 1 µL
Buffer 5 µL
0.5 μ L of Pfu enzyme
ddH2O 40.5 µL
SOE-PCR condition are as follows: using L arm and R arm as template, using bdh L- BamHI, bdhR- XbaI as primer, 50 μ L systems
Cyclic amplification amplification condition: 94 DEG C of initial denaturations 5 min, 94 DEG C of 30 s, Tm of denaturation annealing 30 s, 72 DEG C, 90 s extension 30 follow
Ring, 5 min of rear 72 DEG C of polishings；
50 μ L amplification systems:
0.5 μ L of L arm
0.5 μ L of R arm
bdh L- BamHI 1 µL
bdhR- XbaI 1 µL
dNTP 1µL
Buffer 5 µL
0.5 μ L of high-fidelity MIX
ddH2O 40.5µL
Escherichia coli step of converting are as follows: take the 100 μ L competent cells prepared, 10 μ L connection products are added, gently mix
Even, ice bath 30 min, 42 DEG C of 90 s of thermal shock are transferred quickly in ice bath, ice bath 2-3 min, and 300 μ L LB culture mediums are added,
37 DEG C are slowly incubated for 45 min, and appropriate culture medium is taken to apply Km(50 μ L/mL) resistant panel, while doing and not adding the feminine gender of product right
According to the positive control with empty T2 (2) carrier；
The screening of recon and recombinant plasmid verification step are as follows: take and 1 mL resistance containing Km (50 μ are added in 2 mL sterile centrifugation tubes
L/mL) LB culture medium selects corresponding single bacterium respectively and drops down onto 37 DEG C of cultures in centrifuge tube, is marked, until bacterium solution is muddy, takes
0.5 mL glycerol tube saves, and another 0.5 mL boils 5 min, 2 μ L is taken to carry out bacterium colony PCR verifying in 10 μ L amplification systems；
According to bacterium colony PCR as a result, cultivating corresponding transformant, plasmid is extracted again, and carry out in 20 μ L systems to its respective segments
PCR verifying；
20 μ L amplification systems:
0.4 μ L of Plasmid DNA
F 0.4 µL
R 0.4 µL
2×mix 10 µL
ddH2O 8.8 µL.
The product of amplification is sent to the raw work sequence verification in Shanghai.
The biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck of the invention, the b of the step (2)
In step,
L arm single-swap: F primer is 100 bp of L arm upstream or so, and R primer is 100 bp of junction fragment downstream on T2(2) plasmid；
R arm single-swap: F primer swims 100 bp for junction fragment upstream on T2(2) plasmid, and R primer is 100 bp of R arm downstream；
Homologous double-crossover experimental procedure are as follows: single exchange strains WH1-dan-bdh is selected, in 5 mL non-resistant LB culture mediums, 28
DEG C, 200 rpm cultivate 24 h, dilution applies plate, choose single colonie and put bacterium respectively to LB plate and Km resistance (20 μ g/mL) plate,
Picking resistance loses bacterial strain, extracts total DNA, respectively with 100 bp sequence of L arm upstream for F primer, with 100 bp sequence of R arm downstream
For R primer, the amplification of 20 μ L amplification systems.
The biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck of the invention, a of the step (5)
Experimental procedure are as follows: willGJ11△bdhFerment 36 h, 50 mL of test group tobacco seedlings pouring root in the LB culture medium containing 8% glucose
Fermentation liquid, 50 mL clear water of control group tobacco seedlings pouring root, three repetitions of every group of processing；After incubated at room temperature 3 days, it is put into 10 DEG C of culture
Simulation climate spot occurs in case, primary every 3 h sampling, 6 min/20 min of ozone flux；
Weigh 0.2 g of blade to be put into pre-cooling mortar, the 0.05 mol/L PBS(pH 7.8 for adding 1 mL to be pre-chilled) and 0.2 g PVP
Ice bath grinding is slurried, and after the completion of grinding, 0.05 mol/L PBS(pH 7.8 is added) mortar is rinsed, make 2 mL of final volume；4
DEG C, 8000 r/min are centrifuged 15 min, and supernatant is thick zyme extract；
SOD enzyme activity is measured using NBT photoreduction met hod, when an enzyme-activity unit is defined as the reduction of NBT being suppressed to control half
Required enzyme amount.
The biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck of the invention, the b of the step (5)
Experimental procedure are as follows: the pretreatment of tobacco seedlings, the acquisition of sample is with the preparation method of enzyme solution with consistent in SOD enzymatic determination；
0.05 mol/L PBS(pH 7.0 is added in the small centrifuge tube of 4 mL) 1 mL, 1 mL, 1% guaiacol and the thick enzyme of 1 mL
Liquid is put into 5 min of water-bath in 30 DEG C of thermostat water baths after mixing, after the reaction was completed, then 1 mL is added into pipe
0.3% H2O2, reaction solution is poured into cuvette rapidly after mixing, OD value is measured at 470 nm and is increased speed, with distilled water school
Zero；Every group is done 3 parallel, reduction errors；
The active calculation method of POD is as follows:
POD activity (Δ OD g-1•min-1)=(Δ OD470×VT)/(FW×t×V1)
T in formula: reaction time
VT: sample liquids total volume；
V1: measurement amount of samples；
FW: sample fresh weight.
The biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck of the invention, c in the step (5)
Experimental procedure are as follows: the pretreatment of tobacco seedlings, the acquisition of sample is with the preparation method of enzyme solution with consistent in SOD enzymatic determination；
It takes 0.2mL crude enzyme liquid in test tube, buffer 1.5mL is successively added into test tube, distilled water 1mL adds after 25 DEG C of preheatings
Enter the H of 0.3mL0.3%2O2, timing immediately, and pour into quartz colorimetric utensil rapidly, light absorption value, Mei Geyi are measured under 240nm
Minute reading is primary, total 3min, 3 repetitions of experimental setup；Reduce by 0.1 enzyme amount with A240 in 1min as 1 enzyme-activity unit
(U), it presses examination and calculates CAT activity:
Catalase activity (Ug-1·min-1)=(△ A240 × VT0.1 × V of)/(1×t×FW)
T in formula: reaction time；
VT: sample liquids total volume；
V1: measurement amount of samples；
FW: sample fresh weight.
Compared with prior art the invention has the benefit that by different antioxidant irrigation tobacco roots, detection
Its control efficiency to tobacco weather spot finally found that 3-hydroxy-2-butanone has preferable control efficiency to tobacco weather spot, in weather
The plant cigarette district domain of spot morbidity is separated to the bacillus that a plant height produces 3-hydroxy-2-butanoneGJ11, through 16SDNA gene sequencing and construct
Phylogenetic tree identification, the bacterial strain be Bei Laisi bacillus (Bacillus velezensis), by knocking out Bei Laisi gemma
BacillusbdhGene, discoveryGJ11△bdh3-hydroxy-2-butanone yield improve 2 times compared with wild mushroom in 84h, control efficiency improves 4
Times, up to 60%, the microbial bacterial agent is environmentally safe nuisanceless, has good development prospect and application potential.
Detailed description of the invention
Control efficiency of Fig. 1 3-hydroxy-2-butanone to tobacco weather spot；
Fig. 2GJ11Gas chromatogram；
Fig. 3GJ11Phylogenetic tree；
Fig. 4 GJ11WithGJ11△bdh3-hydroxy-2-butanone change of production；
Fig. 5GJ11WithGJ11△bdhTo the control efficiency of tobacco weather-fleck；
Fig. 6GJ11△bdhRemoving of the fermentation liquid to active oxygen；
Fig. 6 AGJ11△bdhTo H in tobacco body2O2Influence；
Fig. 6 BGJ11△bdhTo O in tobacco body2-Influence；
Fig. 7 GJ11△bdhThe influence of fermentation broth on tobacco correlation enzyme activity；
Fig. 7 A GJ11△bdhInfluence to tobacco SOD enzyme；
Fig. 7 B GJ11△bdhInfluence to tobacco POD enzyme；
Fig. 7 C GJ1△bdhThe influence of 1 pair of tobacco CAT enzyme.
Specific embodiment
With reference to embodiment, the embodiment of the present invention is furthur described in detail.Following embodiment is used for
Illustrate the present invention, but is not intended to limit the scope of the invention.
The biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck of the invention, experimentation is such as
Under:
(1) screening and identification of tobacco weather spot biocontrol bacteria:
A. raw and rhizosphere bacteria separation in tobacco: 0.5 g root, stem and leaf are weighed respectively, is carried out respectively with 75% ethanol solution
30 s are sterilized, twice with aseptic water washing, impregnate 1 min with 0.1% mercuric chloride respectively, aseptic water washing three times, is separately added into 5 ml
Sterile water grinding sufficiently, after gradient dilution, is respectively coated on LB plate, 37 DEG C of 48 h of culture, different thin of picking colony form
Bacterium isolates and purifies, and -20 DEG C of glycerol tubes of bacterial strain of purifying are saved；
B. control efficiency of the 3-hydroxy-2-butanone to tobacco weather spot: choosing the tobacco of 5 ~ 7 leaves, distinguishes to 50 mL concentration of tobacco pouring root
For the 3-hydroxy-2-butanone solution of 2 g/L, 4 g/L and 6 g/L, incubated at room temperature three days；Water 50 mL at the 4th day, simulation climate spot
The generation of disease；Ozone ventilating mode is that every 20 min leads to 5 min ozone, the model MF-Y type mobile ozone of ozone generator
Generator records disease index and control efficiency after 12 h of ozone treatment, sees Fig. 1, and wherein a is that clear water is handled to tobacco weather spot
Influence, b is that 3-hydroxy-2-butanone is handled to the control efficiency of tobacco weather spot；
C. the screening of high yield 3-hydroxy-2-butanone bacterium: the bacterium screened is inoculated in LB culture medium, 37 DEG C, 180 rpm live overnight
Change, 1% activation bacterium solution of switching is in 37 DEG C of LB liquid medium of 8% glucose, and 180 rpm cultivate 48 h, with the side of gas-chromatography
Method detects 3-hydroxy-2-butanone concentration, obtains the bacterial strain that a plant height produces 3-hydroxy-2-butanoneGJ11, see Fig. 2, wherein GC conditions are as follows: Jia Nuo
FFAP capillary column (mm of 30 m × 0.32 ID, 0.33 μm of Film), flame ionization detector, carrier gas N2, flow
For 1.0 mL/min, 30 mL/min of H flow, 300 mL/min of air mass flow, Splitless injecting samples, 1 μ L of sample volume, sample injector temperature
200 DEG C, 280 DEG C of detector temperature of degree；Column oven temperature program: 40 DEG C of 1 min of holding are warming up to 60 DEG C of holdings with 6 DEG C/min
1.5 min are warming up to 160 DEG C of 2.5 min of holding with 15 DEG C/min；
d. GJ11To the control efficiency of tobacco weather spot: pickingGJ11Single bacterium is fallen in fluid nutrient medium, and 37 DEG C, 180 rpm mistakes
Night activation, 1% activation bacterium solution of switching is in the LB liquid medium containing 8% glucose, and 37 DEG C, 180 rpm cultivate 48 h；Selection 5 ~
The tobacco in 7 leaf periods, to 50 mL fermentation liquid of tobacco pouring root, 50 mL clear water of pouring root is cultivated three days at room temperature as control, the
Water 50 mL at four days, is put into the generation of simulation climate spot disease in incubator；Ozone ventilating mode is that every 20min leads to 5min
Ozone records disease index and control efficiency after handling 12 h, sees Fig. 6；
E. bacterial strain 16sDNA gene sequencing is identified: 16sDNA gene amplification product being sequenced after purification, passes through NCBI data
Library carries out BLAST comparison, using the phylogenetic tree of MEGA5.2 software building Antagonistic Fungi, identified bacterial strainGJ11For Bei Laisi
Bacillus (Bacillus velezensis), see Fig. 3；
(2) high yield 3-hydroxy-2-butanone engineering bacteriaGJ11△bdhBuilding:
a. T2-bdhThe building of expression vector:
<1>L arm and R arm pieces section PCR amplification: the PCR amplification carried out to bdh gene L arm and R arm pieces section, amplification system are 50 μ
L, using primer bdh L- BamHI, bdh L-R as primer amplification bdh gene L arm, bdh R-F, bdhR- XbaI expand the R of bdh
Arm, amplification condition: 94 DEG C of initial denaturations 5min, 94 DEG C of denaturation 30s, Tm annealing 30s, 72 DEG C of 45s extend 30 circulations, rear 72 DEG C of benefits
Neat 10min runs glue verifying；
50 μ L amplification systems:
1 μ L of DNA profiling
1 μ L of F primer
1 μ L of R primer
dNTP 1µL
Buffer 5µL
0.5 μ L of Pfu enzyme
ddH2O 40.5µL
<2>SOE-PCR: carrying out purification and recovery to associated clip, respectively using L arm and R arm as template, using L arm and R arm as template,
Using bdh L- BamHI, bdhR- XbaI as primer, 50 μ L system cyclic amplification amplification conditions: 94 DEG C of initial denaturation 5 min, 94
DEG C denaturation 30 s, Tm anneal 30 s, 72 DEG C, 90 s extend 30 circulation, 5 min of rear 72 DEG C of polishings；
50 μ L amplification systems:
0.5 μ L of L arm
0.5 μ L of R arm
bdh L- BamHI 1 µL
bdhR- XbaI 1 µL
dNTP 1µL
Buffer 5 µL
0.5 μ L of high-fidelity MIX
ddH2O 40.5µL
<3>construction of recombinant vector: the extracting of T2 (2) plasmid: the small extraction reagent kit of plasmid
Recovery purifying rear connecting piece section is learnt from else's experience in digestion and T2 (2) plasmid carries out BamHI and Xbal double digestion respectively, is used
Buffer is 0.5 × K, and 37 DEG C of digestions are stayed overnight in 50 μ L systems；
50 μ L digestion systems:
DNA 20 µL
10×K 2.5 µL
BamHI 1 µL
XbaI 1 µL
ddH2O 25.5 µL
37 DEG C of digestions are stayed overnight
Purification and recovery is carried out using PCR product purification and recovery kit after double digestion；
Segment and carrier are mixed in 45 DEG C of water-bath 5min, are inserted into ice immediately after by not enzyme and buffer before enzyme downlink connection,
Ligase and buffer is added, joint efficiency can be improved；
25 μ L enzyme disjunctor systems:
5 μ L of carrier
15 μ L of segment
Buffer 2.5 µL
1 μ L of T4 ligase
ddH2O 1.5 µL
Can 4 DEG C of enzymes even overnight, 16 DEG C or 22 DEG C connection 1-3 h；
<4>Escherichia coli convert: the preparation of bacillus coli DH 5 alpha competence and the conversion of thermal shock method, reference molecule biologic operation hand
Volume takes the 100 μ L competent cells prepared, and 10 μ L connection products are added, mix gently, 30 min of ice bath, 42 DEG C of heat
90 s are hit, are transferred quickly in ice bath, 300 μ L LB culture mediums are added in ice bath 2-3 min, and 37 DEG C are slowly incubated for 45 min, take
Appropriate culture medium applies Km(50 μ L/mL) resistant panel, while doing the positive of the negative control and sky T2 (2) carrier that do not add product
Control；
<5>it the screening of recon and recombinant plasmid verifying: takes and 1 mL resistance containing Km (50 μ L/ is added in 2 mL sterile centrifugation tubes
ML) LB culture medium selects corresponding single bacterium respectively and drops down onto 37 DEG C of cultures in centrifuge tube, is marked, until bacterium solution is muddy, takes
0.5 mL glycerol tube saves, and another 0.5 mL boils 5 min, 2 μ L is taken to carry out bacterium colony PCR verifying in 10 μ L amplification systems；Root
According to bacterium colony PCR as a result, cultivating corresponding transformant, plasmid is extracted again, and PCR in 20 μ L systems is carried out to its respective segments and is tested
Card；
20 μ L amplification systems:
0.4 μ L of Plasmid DNA
F 0.4 µL
R 0.4 µL
2×mix 10 µL
ddH2O 8.8 µL.
The product of amplification is sent to the raw work sequence verification in Shanghai；
b. GJ11△bdhThe building of bacterial strain:
<1>single-swap: conversion GJ11-bdh bacterial strain is selected, the LB culture medium that 10 mL contain Km resistance (20 μ g/mL) is seeded to
In, 42 DEG C of cultures, until bacterium solution is muddy, dilution applies Km resistance (20 μ g/mL) 37 DEG C of plate cultures, chooses single colonie, it is total to extract it
DNA separately designs single-swap verifying primer:
L arm single-swap: F primer is 100 bp of L arm upstream or so, and R primer is 100 bp of junction fragment downstream on T2(2) plasmid；
R arm single-swap: F primer swims 100 bp for junction fragment upstream on T2(2) plasmid, and R primer is 100 bp of R arm downstream；
<2>homologous double-crossover: single exchange strains have Km resistance, after Homo~logous exchange, in fact it could happen that single crossover homologous arm is again
Secondary single-swap and reply wild type, it is also possible to another homology arm occurs single-swap and knocks out lower target fragment, and two kinds of bacterial strains are equal
It is sensitive to Km, by filtering out Km sensitive strain, so that it is determined that knock-out bacterial strain；
Select single exchange strains WH1-dan-bdh, in 5 mL non-resistant LB culture mediums, 28 DEG C, 200 rpm cultivate 24 h, it is dilute
Painting plate is released, single colonie is chosen and puts bacterium respectively to LB plate and Km resistance (20 μ g/mL) plate, picking resistance loses bacterial strain, extracts
Total DNA, respectively with 100 bp sequence of L arm upstream for F primer, with 100 bp sequence of R arm downstream for R primer, 20 μ L amplification systems
Amplification；
<3>it is sequenced: after PCR amplification, sending to the raw work sequencing in Shanghai, sequencing primer bdh-dan-LF, bdh-dan-RR,
NCBI Blast is compared；
(3)GJ11WithGJ11△bdh3-hydroxy-2-butanone yield and weather spot preventive effect variation:
PickingGJ11WithGJ11△bdhSingle bacterium is fallen in LB liquid medium, and 37 DEG C, 180 rpm are activated overnight, and 1% switching is in containing
The liquid LB of 8% glucose, 37 DEG C, 180 rpm culture every 3-hydroxy-2-butanone content of 12 h sample detection, is shown in Fig. 4；
The tobacco for choosing for 5 ~ 7 leaf periods, to 50 mL of fermentation liquid of above-mentioned 48 h of tobacco pouring root, clear water processing is as control, in room
Water at culture three days, the 4th day 50 mL under temperature, is put into the generation of simulation climate spot disease in incubator；Ozone ventilating mode
Lead to 5 min ozone for every 20 min, record disease index and control efficiency after handling 12 h, see Fig. 5, wherein a is clear water processing
Influence to tobacco weather-fleck, b areGJ11The control efficiency of fermentation broth on tobacco weather spot disease, c areGJ11△bdhHair
Control efficiency of the zymotic fluid to weather spot disease；
(4)GJ11△bdhFermentation liquid influences active oxygen: the processing mode of tobacco is used every 3 h during processing with (3)
Punch takes 8 leaves；
A. blade the detection of hydrogen peroxide: is soaked in 1 mg/mL(pH3.8 being formulated by 0.01M phosphate buffer)
DAB solution 8h removes chlorophyll with ethyl alcohol: 95 DEG C of glycerol (9:1V/V), 5 min；10 times of microscopies, brown spot representative have H2O2It produces
It is raw, see Fig. 6 A；
B. the detection of superoxipe ion: blade is soaked in the 0.01M phosphate buffer containing 0.01%NBT, dark impregnates 60
Min, production object stablize dark green spot, expose 2 h, ethyl alcohol: 95 DEG C of glycerol (9:1V/V), 5 min remove chlorophyll, 10 times
Microscopy does not have dark green spots in figure, illustrates that no superoxipe ion generates, sees Fig. 6 B；
(5)GJ11△bdhThe influence of fermentation broth on tobacco correlation enzyme activity:
a. GJ11△bdhThe influence of fermentation broth on tobacco SOD enzyme activity:
<1>willGJ11△bdhFerment 36 h in the LB culture medium containing 8% glucose, 50 mL of test group tobacco seedlings pouring root fermentation
Liquid, 50 mL clear water of control group tobacco seedlings pouring root, three repetitions of every group of processing；After incubated at room temperature 3 days, it is put into 10 DEG C of incubator
Simulation climate spot occurs, primary every 3 h sampling, 6 min/20 min of ozone flux, since sampling will lead to ozone concentration drop
It is low, so ozone flux increases herein；
<2>weigh 0.2 g of blade to be put into pre-cooling mortar, the 0.05 mol/L PBS(pH 7.8 for adding 1 mL to be pre-chilled) and 0.2 g
The grinding of PVP ice bath is slurried, and after the completion of grinding, 0.05 mol/L PBS(pH 7.8 is added) mortar is rinsed, make final volume 2
mL；4 DEG C, 8000 r/min are centrifuged 15 min, and supernatant is thick zyme extract；
SOD enzyme activity is measured using NBT photoreduction met hod, when an enzyme-activity unit is defined as the reduction of NBT being suppressed to control half
Required enzyme amount；
4 mL centrifuge tubes are taken, two is taken as control group, reagent is added according to table 1, after mixing, an effective masking foil of control,
Shading is covered completely, is put with other each pipes and is reacted 20 min in the sunlight, 25 ~ 35 DEG C of reaction temperature；
1 reaction system of table
| Reagent name | Dosage (mL) | Final concentration |
| 0.05 mol/L PBS | 1.5 | |
| 130 mmol/L Met | 0.3 | 13 mmol/L |
| 750 μmol/L NBT | 0.3 | 75 μmol/L |
| 20 μm of ol/L riboflavin | 0.3 | 2 μmol/L |
| 100 μmol/L EDTA-Na2Solution | 0.3 | 10 μmol/L |
| Enzyme solution | 0.1 | Control is replaced with 0.1 mL of buffer PBS |
| Distilled water | 0.5 | |
| Total volume | 3.3 |
Shading pipe returns to zero as blank, the colorimetric estimation OD value at 560 nm, and every group is done three parallel laboratory tests, reduces error；
Activity indicates that calculation method is as follows with every gram of fresh weight enzyme unit:
A in formula0: the absorption value of irradiation control tube；
As: the absorbance value of sample cell；
Vt: sample liquid total volume (mL)；
V1: amount of samples (mL) when measurement；
Fw: sample fresh weight (g)；
Enzyme activity situation of change is as shown in Figure 7 A；
b. GJ11△bdhThe influence of fermentation broth on tobacco POD enzyme activity: guaiacol method is used in the active measurement of POD；In peroxidating
Under object enzyme (POD) catalysis, H2O2Guaiacol is oxidized to dark brown product；This product has maximum light absorption value at 470 nm,
Therefore the activity of peroxidase can be measured by the absorbance change surveyed under 470 nm；
The pretreatment of tobacco seedlings, the acquisition of sample is with the preparation method of enzyme solution with consistent in SOD enzymatic determination；
0.05 mol/L PBS (pH 7.0), 1 mL, 1 mL, 1% guaiacol and the thick enzyme of 1 mL are added in the small centrifuge tube of 4 mL
Liquid is put into 5 min of water-bath in 30 DEG C of thermostat water baths after mixing, after the reaction was completed, then 1 mL is added into pipe
0.3% H2O2, reaction solution is poured into cuvette rapidly after mixing, OD value is measured at 470 nm and is increased speed, with distilled water school
Zero；Every group is done 3 parallel, reduction errors；
The active calculation method of POD is as follows:
POD activity (Δ OD g-1•min-1)=(Δ OD470×VT)/(FW×t×V1)
T in formula: reaction time；
VT: sample liquids total volume；
V1: measurement amount of samples；
FW: sample fresh weight；
POD enzyme activity variation such as Fig. 7 B；
c. GJ11△bdhThe influence of fermentation broth on tobacco CAT enzyme activity: it is measured using ultraviolet absorption method；Hydrogen peroxide is in 240 nm
There is a strong light absorption value at place, catalase can peroxynitrite decomposition hydrogen so that the value of reaction solution absorbance A 240 reduces pass through detection
The pace of change of A240 can detecte out the variation of hydrogen peroxide enzyme activity；
The pretreatment of tobacco seedlings, the acquisition of sample is with the preparation method of enzyme solution with consistent in SOD enzymatic determination；
It takes 0.2 mL crude enzyme liquid in test tube, 1.5 mL of buffer, 1 mL of distilled water, 25 DEG C of preheatings is successively added into test tube
Afterwards, the H of 0.3 mL0.3% is added2O2, timing immediately, and pour into quartz colorimetric utensil rapidly, light absorption value is measured under 240 nm,
It was read every one minute once, totally 3 min, 3 repetitions of experimental setup；Reduce by 0.1 enzyme amount with A240 in 1 min as 1 enzyme
Unit (U) living presses examination and calculates CAT activity:
Catalase activity (Ug-1·min-1)=(△ A240 × VT0.1 × V of)/(1×t×FW)
T in formula: reaction time；
VT: sample liquids total volume；
V1: measurement amount of samples；
FW: sample fresh weight；
CAT enzyme activity changes into Fig. 7 C.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, several improvements and modifications, these improvements and modifications can also be made
Also it should be regarded as protection scope of the present invention.
Claims (8)
1. the biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck, which is characterized in that its experimentation packet
Include following steps:
(1) screening and identification of tobacco weather spot biocontrol bacteria:
A, raw and rhizosphere bacteria separation in tobacco: 0.5 g root, stem and leaf are weighed respectively, is disappeared respectively with 75% ethanol solution
30 s of poison, twice with aseptic water washing, respectively with 0.1% mercuric chloride impregnate 1 min, aseptic water washing three times, be separately added into 5 ml without
The grinding of bacterium water sufficiently, after gradient dilution, is respectively coated on LB plate, 37 DEG C of 48 h of culture, the different bacterium of picking colony form
It isolates and purifies, -20 DEG C of glycerol tubes of bacterial strain of purifying is saved；
B, control efficiency of the 3-hydroxy-2-butanone to tobacco weather spot: choosing the tobacco of 5 ~ 7 leaves, distinguishes to 50 mL concentration of tobacco pouring root
For the 3-hydroxy-2-butanone solution of 2 g/L, 4 g/L and 6 g/L, incubated at room temperature three days, the 4th day whens, water 50 mL, simulation climate spot
The generation of disease；
C, the screening of high yield 3-hydroxy-2-butanone bacterium: being inoculated with the bacterium screened in LB culture medium, and 37 DEG C, 180 rpm are activated overnight,
For 1% activation bacterium solution of switching in 37 DEG C of LB liquid medium of 8% glucose, 180 rpm cultivate 48 h, are examined with the method for gas-chromatography
3-hydroxy-2-butanone concentration is surveyed, the bacterial strain that a plant height produces 3-hydroxy-2-butanone is screened outGJ11；
d、GJ11To the control efficiency of tobacco weather spot: pickingGJ11Single bacterium is fallen in fluid nutrient medium, and 37 DEG C, 180 rpm mistakes
Night activates, and 1% activation bacterium solution of switching is in the LB liquid medium containing 8% glucose, and 37 DEG C, 180 rpm, 48 h of culture, selection 5 ~
The tobacco in 7 leaf periods, to 50 mL fermentation liquid of tobacco pouring root, 50 mL clear water of pouring root is cultivated three days at room temperature as control, the
Water 50 mL at four days, is put into the generation of simulation climate spot disease in incubator；
E, bacterial strain 16sDNA gene sequencing is identified: 16sDNA gene amplification product being sequenced after purification, passes through NCBI data
Library carries out BLAST comparison, using the phylogenetic tree of MEGA5.2 software building Antagonistic Fungi, identifies bacterial strainGJ11Strain；
(2) high yield 3-hydroxy-2-butanone engineering bacteriaGJ11△bdhBuilding:
a、T2-bdhThe building of expression vector: the PCR amplification that carry out to bdh gene L arm and R arm pieces section simultaneously runs glue verifying, by phase
It closes segment and carries out purification and recovery, and using L arm and R arm as template, carry out SOE-PCR, construction of recombinant vector, Escherichia coli convert,
The screening of recon and recombinant plasmid verifying；
b、GJ11△bdhThe building of bacterial strain: conversion GJ11-bdh bacterial strain is selected, 10 mL is seeded to and contains Km resistance (20 μ g/
ML in LB culture medium), 42 DEG C of cultures, until bacterium solution is muddy, dilution applies Km resistance (20 μ g/mL) 37 DEG C of plate cultures, chooses single bacterium
It falls, extracts its total DNA, separately design single-swap verifying primer, L arm single-swap, R arm single-swap takes corresponding primer, total to extract
DNA is template, and 20 μ L systems carry out PCR amplification, and agarose gel electrophoresis selects single exchange strains；
Select single exchange strains WH1-dan-bdh, in 5 mL non-resistant LB culture mediums, 28 DEG C, 200 rpm cultivate 24 h, it is dilute
Painting plate is released, single colonie is chosen and puts bacterium respectively to LB plate and Km resistance (20 μ g/mL) plate, picking resistance loses bacterial strain, extracts
Total DNA, respectively with 100 bp sequence of L arm upstream for F primer, with 100 bp sequence of R arm downstream for R primer, 20 μ L amplification systems
Amplification carries out Blast comparison in NCBI to sequencing primer bdh-dan-LF, bdh-dan-RR；
(3)GJ11WithGJ11△bdh3-hydroxy-2-butanone yield and weather spot preventive effect variation:
PickingGJ11WithGJ11△bdhSingle bacterium is fallen in LB liquid medium, and 37 DEG C, 180 rpm are activated overnight, and 1% switching is in containing
The liquid LB of 8% glucose, 37 DEG C, 180 rpm are cultivated, every 3-hydroxy-2-butanone content of 12 h sample detection；
The tobacco for choosing for 5 ~ 7 leaf periods, to 50 mL of fermentation liquid of above-mentioned 48 h of tobacco pouring root, clear water processing is as control, in room
Water at culture three days, the 4th day 50 mL under temperature, is put into the generation of simulation climate spot disease in incubator；
(4)GJ11△bdhFermentation liquid influences active oxygen: the same step of the processing mode of tobacco (3), during processing every 3
H takes 8 leaves with punch:
A, blade the detection of hydrogen peroxide: is soaked in 1 mg/mL(pH3.8 being formulated by 0.01M phosphate buffer)
DAB solution 8h, with ethyl alcohol: 95 DEG C of glycerol (9:1 V/V), 5 min remove chlorophyll, and 10 times of microscopies, whether there is or not H for observation2O2It generates, palm fibre
The representative of color spot point has H2O2It generates；
B, the detection of superoxipe ion: blade is soaked in the 0.01M phosphate buffer containing 0.01%NBT, dark impregnates 60
Min, production object stablize dark green spot, expose 2 h, ethyl alcohol: 95 DEG C of glycerol (9:1V/V), 5 min remove chlorophyll, 10 times
Microscopy, whether there is or not dark green spots generation, dark green spots representatives superoxipe ion generation for observation；
(5)GJ11△bdhThe influence of fermentation broth on tobacco correlation enzyme activity:
a、GJ11△bdhThe influence of fermentation broth on tobacco SOD enzyme activity: SOD enzyme activity, an enzyme activity are measured using NBT photoreduction met hod
Unit definition is enzyme amount required when the reduction of NBT to be suppressed to control half, observes SOD enzyme activity situation of change；
b、GJ11△bdhThe influence of fermentation broth on tobacco POD enzyme activity: guaiacol method measures POD activity, and observation POD enzyme activity becomes
Change situation；
c、GJ11△bdhThe influence of fermentation broth on tobacco CAT enzyme activity: ultraviolet absorption method measures CAT enzyme activity, and observation CAT enzyme activity becomes
Change situation.
2. the biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck as described in claim 1, feature
It is, in a and Step d of the step (1), ozone treatment condition are as follows: ozone ventilating mode is that logical 5 min of every 20 min are smelly
Oxygen, the model MF-Y type mobile ozone generator of ozone generator.
3. the biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck as claimed in claim 2, feature
It is, in the step c of the step (1), GC conditions method detection parameters are as follows: gal promise FFAP capillary column (30 m ×
0.32 mm ID, 0.33 μm of Film), flame ionization detector, carrier gas N2, flow is 1.0 mL/min, H flow 30
ML/min, 300 mL/min of air mass flow, Splitless injecting samples, sample volume 1 μ L, 200 DEG C of sample injector temperature, detector temperature 280
DEG C, column oven temperature program: 40 DEG C of 1 min of holding are warming up to 60 DEG C of 1.5 min of holding with 6 DEG C/min, with 15 DEG C/min heating
To 160 DEG C of 2.5 min of holding.
4. the biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck as claimed in claim 3, feature
It is, in a step of the step (2), amplification system is 50 μ L, is expanded by primer of primer bdh L- BamHI, bdh L-R
Increase bdh gene L arm, bdh R-F, bdhR- XbaI expand the R arm of bdh, amplification condition: 94 DEG C of 5 min of initial denaturation, 94 DEG C of denaturation
30 s, Tm annealing, 30 s, 72 DEG C of 45 s extend 30 circulations, and 10 min of rear 72 DEG C of polishings runs glue verifying；
50 μ L amplification systems:
1 μ L of DNA profiling
1 μ L of F primer
1 μ L of R primer
dNTP 1 µL
Buffer 5 µL
0.5 μ L of Pfu enzyme
ddH2O 40.5 µL
SOE-PCR condition are as follows: using L arm and R arm as template, using bdh L- BamHI, bdhR- XbaI as primer, 50 μ L systems
Cyclic amplification amplification condition: 94 DEG C of initial denaturations 5 min, 94 DEG C of 30 s, Tm of denaturation annealing 30 s, 72 DEG C, 90 s extension 30 follow
Ring, 5 min of rear 72 DEG C of polishings；
50 μ L amplification systems:
0.5 μ L of L arm
0.5 μ L of R arm
bdh L- BamHI 1 µL
bdhR- XbaI 1 µL
dNTP 1µL
Buffer 5 µL
0.5 μ L of high-fidelity MIX
ddH2O 40.5µL
Escherichia coli step of converting are as follows: take the 100 μ L competent cells prepared, 10 μ L connection products are added, gently mix
Even, ice bath 30 min, 42 DEG C of 90 s of thermal shock are transferred quickly in ice bath, ice bath 2-3 min, and 300 μ L LB culture mediums are added,
37 DEG C are slowly incubated for 45 min, and appropriate culture medium is taken to apply Km(50 μ L/mL) resistant panel, while doing and not adding the feminine gender of product right
According to the positive control with empty T2 (2) carrier；
The screening of recon and recombinant plasmid verification step are as follows: take and 1 mL resistance containing Km (50 μ are added in 2 mL sterile centrifugation tubes
L/mL) LB culture medium selects corresponding single bacterium respectively and drops down onto 37 DEG C of cultures in centrifuge tube, is marked, until bacterium solution is muddy, takes
0.5 mL glycerol tube saves, and another 0.5 mL boils 5 min, 2 μ L is taken to carry out bacterium colony PCR verifying in 10 μ L amplification systems；
According to bacterium colony PCR as a result, cultivating corresponding transformant, plasmid is extracted again, and carry out in 20 μ L systems to its respective segments
PCR verifying；
20 μ L amplification systems:
0.4 μ L of Plasmid DNA
F 0.4 µL
R 0.4 µL
2×mix 10 µL
ddH2O 8.8 µL.
The product of amplification is sent to the raw work sequence verification in Shanghai.
5. the biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck as claimed in claim 4, feature
It is, in the b step of the step (2),
L arm single-swap: F primer is 100 bp of L arm upstream or so, and R primer is 100 bp of junction fragment downstream on T2(2) plasmid；
R arm single-swap: F primer swims 100 bp for junction fragment upstream on T2(2) plasmid, and R primer is 100 bp of R arm downstream；
Homologous double-crossover experimental procedure are as follows: single exchange strains WH1-dan-bdh is selected, in 5 mL non-resistant LB culture mediums, 28
DEG C, 200 rpm cultivate 24 h, dilution applies plate, choose single colonie and put bacterium respectively to LB plate and Km resistance (20 μ g/mL) plate,
Picking resistance loses bacterial strain, extracts total DNA, respectively with 100 bp sequence of L arm upstream for F primer, with 100 bp sequence of R arm downstream
For R primer, the amplification of 20 μ L amplification systems.
6. the biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck as claimed in claim 5, feature
It is, the experimental procedure of a of the step (5) are as follows: willGJ11△bdhFerment 36 h in the LB culture medium containing 8% glucose,
50 mL fermentation liquid of test group tobacco seedlings pouring root, 50 mL clear water of control group tobacco seedlings pouring root, three repetitions of every group of processing,
After incubated at room temperature 3 days, it is put into simulation climate spot in 10 DEG C of incubator and occurs, ozone flux 6 primary every 3 h sampling
min/20 min；
Weigh 0.2 g of blade to be put into pre-cooling mortar, the 0.05 mol/L PBS(pH 7.8 for adding 1 mL to be pre-chilled) and 0.2 g PVP
Ice bath grinding is slurried, and after the completion of grinding, 0.05 mol/L PBS(pH 7.8 is added) mortar is rinsed, make 2 mL of final volume,
4 DEG C, 8000 r/min are centrifuged 15 min, and supernatant is thick zyme extract；
SOD enzyme activity is measured using NBT photoreduction met hod, when an enzyme-activity unit is defined as the reduction of NBT being suppressed to control half
Required enzyme amount.
7. the biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck as claimed in claim 6, feature
It is, the experimental procedure of the b of the step (5) are as follows: the pretreatment of tobacco seedlings, the acquisition of sample and the same SOD of the preparation method of enzyme solution
It is consistent in enzymatic determination；
0.05 mol/L PBS(pH 7.0 is added in the small centrifuge tube of 4 mL) 1 mL, 1 mL, 1% guaiacol and the thick enzyme of 1 mL
Liquid is put into 5 min of water-bath in 30 DEG C of thermostat water baths after mixing, after the reaction was completed, then 1 mL is added into pipe
0.3% H2O2, reaction solution is poured into cuvette rapidly after mixing, OD value is measured at 470 nm and is increased speed, with distilled water school
Zero,
Every group is done 3 parallel, reduction errors.
8. the biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck as claimed in claim 7, feature
It is, the experimental procedure of c in the step (5) are as follows: the pretreatment of tobacco seedlings, the acquisition of sample and the same SOD of the preparation method of enzyme solution
It is consistent in enzymatic determination；
It takes 0.2mL crude enzyme liquid in test tube, buffer 1.5mL is successively added into test tube, distilled water 1mL adds after 25 DEG C of preheatings
Enter the H of 0.3mL0.3%2O2, timing immediately, and pour into quartz colorimetric utensil rapidly, light absorption value, Mei Geyi are measured under 240nm
Minute reading is primary, total 3min, 3 repetitions of experimental setup, reduces by 0.1 enzyme amount with A240 in 1min as 1 enzyme-activity unit
(U).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112251379A (en) * | 2020-10-22 | 2021-01-22 | 宜宾五粮液股份有限公司 | Acid-resistant acetoin-producing Bacillus belgii DQA21 and application |
| CN116925950B (en) * | 2022-12-30 | 2024-05-14 | 四川农业大学 | Biological control strain for pepper leaf spot disease and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107586737A (en) * | 2017-09-18 | 2018-01-16 | 华中农业大学 | The Bei Laisi bacillus of one plant of anti-yellowing cucurbit wilt and its Microencapsulated Slow microbial inoculum |
| CN107779420A (en) * | 2017-06-27 | 2018-03-09 | 湖北省烟草公司恩施州公司 | A kind of Nei Shengbeilaisi bacillus of two plants of antagonism tobacco bacterial wilts and its application |
| CN108587974A (en) * | 2018-05-15 | 2018-09-28 | 湖南农业大学 | A kind of preparation method of Bei Laisi bacillus, application and its zymotic fluid |
| CN109370939A (en) * | 2018-11-06 | 2019-02-22 | 上海交通大学 | One plant of Bei Laisi bacillus and its separation method and application |
-
2019
- 2019-02-15 CN CN201910115554.9A patent/CN109929866A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107779420A (en) * | 2017-06-27 | 2018-03-09 | 湖北省烟草公司恩施州公司 | A kind of Nei Shengbeilaisi bacillus of two plants of antagonism tobacco bacterial wilts and its application |
| CN107586737A (en) * | 2017-09-18 | 2018-01-16 | 华中农业大学 | The Bei Laisi bacillus of one plant of anti-yellowing cucurbit wilt and its Microencapsulated Slow microbial inoculum |
| CN108587974A (en) * | 2018-05-15 | 2018-09-28 | 湖南农业大学 | A kind of preparation method of Bei Laisi bacillus, application and its zymotic fluid |
| CN109370939A (en) * | 2018-11-06 | 2019-02-22 | 上海交通大学 | One plant of Bei Laisi bacillus and its separation method and application |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112251379A (en) * | 2020-10-22 | 2021-01-22 | 宜宾五粮液股份有限公司 | Acid-resistant acetoin-producing Bacillus belgii DQA21 and application |
| CN112251379B (en) * | 2020-10-22 | 2022-03-15 | 宜宾五粮液股份有限公司 | Acid-resistant acetoin-producing Bacillus belgii DQA21 and application |
| CN116925950B (en) * | 2022-12-30 | 2024-05-14 | 四川农业大学 | Biological control strain for pepper leaf spot disease and application thereof |
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