CN109180790A - Brevibacillus laterosporus A60 albumen exciton PeBL2 and its encoding gene and application - Google Patents
Brevibacillus laterosporus A60 albumen exciton PeBL2 and its encoding gene and application Download PDFInfo
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- CN109180790A CN109180790A CN201811041405.4A CN201811041405A CN109180790A CN 109180790 A CN109180790 A CN 109180790A CN 201811041405 A CN201811041405 A CN 201811041405A CN 109180790 A CN109180790 A CN 109180790A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/12—Leaves
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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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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Abstract
The invention discloses a kind of Brevibacillus laterosporus A60 albumen exciton PeBL2 and its encoding gene and applications, and amino acid sequence is as shown in SEQ ID NO:2 in sequence table.Protein of the present invention can induce plant defense response, improve plant resistance to environment stress, and related plant disease is tobacco gray mold.PeBL2 is not only to disclose Brevibacillus laterosporus A60 bacterial strain inducing plant resistance to environment stress mechanism to provide theoretical basis, and provide new way to improve the disease resistance of plant.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to a kind of Brevibacillus laterosporus A60 exciton PeBL2 and its
Encoding gene and application.
Background technique
Having 70% in corps diseases is to cause huge loss to agricultural production as caused by disease fungus.Excessively
It relies on chemical prevention bring agricultural product quality and safety, the mankind and Environmental security and has been subjected to global extensive concern, therefore, seek
Safely and effectively biological control strategy becomes the important research content for ensureing crop production safety.
Biocontrol bacteria has been applied to the biological control of diseases and pests of agronomic crop as a kind of important biological pesticide, such is thin
Bacterium not only has directly kill and ecological niche Competition to target disease, moreover it is possible to resist invading for disease by inducing plant resistance
Dye reduces chemical pesticide usage amount to mitigate the generation of disease.In recent years, it is immune that plant identification is separated from biocontrol bacteria
Exciton has been to be concerned by more and more people, and identifying for biocontrol bacteria exciton can not only be to illustrate biocontrol bacteria mechanism of action to mention
For theoretical basis, while exciton can also be developed to the biological control for being applied to corps diseases at protein immunization inducer, tool
There is wide value of exploiting and utilizing.
Summary of the invention
The object of the present invention is to provide a kind of Brevibacillus laterosporus secretory protein PeBL2 that can be improved plant resistance to environment stress
And its encoding gene and application.
A kind of Brevibacillus laterosporus A60 exciton PeBL2, amino acid sequence such as SEQ ID NO:2 institute in sequence table
Show.
Brevibacillus laterosporus A60 exciton PeBL2 of the present invention is improving the application in plant resistance to environment stress.
Application of the present invention, wherein the plant is this life cigarette.
A kind of PeBL2 gene, nucleotides sequence are classified as one of what follows:
(1) polynucleotide sequence of the protein as shown in SEQ ID NO:2 is encoded；
(2) with above-mentioned polynucleotide sequence according to the complementary polynucleotide sequence of basepairing rule.
PeBL2 gene of the present invention, wherein its polynucleotide sequence is as shown in SEQ ID NO:1.
A kind of recombinant vector contains PeBL2 gene of the present invention.
Recombinant vector of the present invention, wherein the carrier is in pET28 vector multiple cloning site insertion institute of the present invention
The recombinant vector for the PeBL2 gene stated.
A kind of host cell of genetic engineering, contains recombinant vector of the present invention.
The host cell of genetic engineering of the present invention, wherein the host cell is to contain recombination of the present invention
The e. coli bl21 (DE3) of carrier.
Protein of the invention refers to from Brevibacillus laterosporus (Brevibacillus laterosporus) A60
Culture solution, the supernatant of culture solution or the protein of thallus.
Brevibacillus laterosporus A60 exciton PeBL2 difference from prior art of the present invention is:
Brevibacillus laterosporus A60 exciton PeBL2 protein of the present invention can significantly improve plant against fungal disease
Resistance, 10 μM of His-PeBL2 are inoculated with the pathogen of Botrytis cinerea (Botrytis cinerea) after handling this life tobacco leaf 3d, lesion diameter
Highest inhibiting rate can respectively reach 40.2%.
Microorganism Deposit Information
Brevibacillus laterosporus (Brevibacillus laterosporus) A60 bacterial strain of the present invention is this experiment
The high activity biocontrol bacterial strain that room is isolated from Hainan Changjiang's soil is protected on January 9th, 2012 in Chinese microorganism strain
Hiding administration committee's common micro-organisms center (CGMCC) has carried out preservation, number of registering on the books CGMCC No:5694.Address: north
The institute 3 of the Chaoyang District Jing Shi North Star West Road 1, phone 010-64807355.
With reference to the accompanying drawing to Brevibacillus laterosporus A60 exciton PeBL2 protein of the invention and its encoding gene
It is described further with application.
Detailed description of the invention
Fig. 1 is the chromatogram that crude protein passes through cation exchange column HiTrap SP FF in the present invention；A, B represents 2 and washes
De- peak；
Fig. 2A is to carry out reversed phase column chromatography to eluting peak B using Agilent 1200HPLC high performance liquid chromatography in the present invention
Map, wherein A, B, C, D, E, F represent five eluting peaks；
Fig. 2 B is the exciton Activity determination result of different eluting peaks；
Fig. 3 A is the SDS-PAGE map of purifying protein PeBL2 in the present invention；M represents Marker；
Fig. 3 B is the Activity determination of purifying protein PeBL2；
Fig. 4 is the qualitative detection figure that His-PeBL2 handled and compareed tobacco leaf reactive oxygen species；A left side is control, and the right side is
Processing；
Fig. 5 is that the experiment of infecting that His-PeBL1 induces this life cigarette to inhibit the pathogen of Botrytis cinerea (Botrytis cinerea) is tied
Fruit figure.
Specific embodiment
1 Brevibacillus laterosporus A60 of embodiment culture and the preparation of fermentating metabolism liquid
Using LB culture medium (peptone 10g, yeast powder 5g, sodium chloride 10g are dissolved in distilled water 1000ml, adjust pH to
7.5).Picking activation single bacterium falls within and carries out shaking flask shake culture in the LB culture medium of 2L, and condition of culture is 30 DEG C, 180rpm is cultivated
Greater than 48h.4600rpm centrifugation removal somatic cells, supernatant are used for Protein Separation.
The preparation and activity monitoring of 2 crude protein of embodiment
The gained supernatant filter membrane (Whatman product) of 0.45mm is filtered twice, until there is no thallus.It carries out later
Ammonium sulfate precipitation is slowly added to ammonium sulfate and makes up to 80% saturation degree, and 4 DEG C are stirred overnight.12000rpm, centrifugation in 20 minutes are received
Collection precipitating.Sediment is resuspended with 25m mol/L MES-NaOH (pH 6.2), recycles bag filter desalination, bag filter choosing
Take molecular cut off in 8000D-12000D.Use distilled water as dialyzate first, dialysis uses 25m mol/L afterwards three times
MES-NaOH (pH 6.2) buffer, then dialysis 2-3 times is carried out, dialysis needs interval about 4h to replace a dialyzate every time, protects
It is consistent to be able to maintain salinity for solution inside and outside card bag filter, while also having achieved the purpose that replace buffer.After the completion of dialysis,
10000rpm, centrifugation 10min remove the precipitating generated in dialysis procedure, obtain crude protein solution.
The monitoring of bioactivity: with growth 2 months, this life cigarette with 6-8 piece leaf was material, with 25mM, pH's 6.2
Mes-NaOH buffer is control, and crude protein solution concentration is 10 μm of ol/L, using 1mL without the syringe of syringe needle, by 50 μ
LMes-NaOH buffer, 50 μ L crude protein solution are injected into blade respectively from the leaf back side, with Tris-Hcl (PH7.8)
Solution is as control.See whether that necrotic plaque is formed for 24 hours.
As a result: injecting the blade of crude protein solution, waterlogging spot occur after injection site 6h, blade becomes at injection after 12h
It is transparent more thinner, for 24 hours after typical allergic reaction can be presented, there is necrotic plaque to be formed.Control has no any reaction, and normal
Blade is the same.
3 crude protein Proteins In Aqueous Solutions exciton of embodiment isolate and purify and biological activity determination
(1) ion-exchange chromatography separates
It uses(GE Healthcare) the protein purification instrument of explore 10 to resulting crude protein carry out into
The purifying of one step.Sample passes through cation seperation column HiTrap SP FF cation exchange column (GE Healthcare), each loading first
40ml, equilibrium liquid are the MES (pH6.2) of 25mM, the MES (pH6.2) and 1M NaCl, flow velocity 2ml/min that eluent is 20mM.
It carries out linear elution (0%-100%, 30min), Protein elution peak (see Fig. 1) is acquired, by each Protein elution peak component
The detection of bioactivity is carried out after desalination, the results showed that, protein peak B can cause the allergic reaction of this life cigarette.
(2) reverse phase chromatography
Utilize Agilent 1200HPLC high-efficient liquid phase chromatogram purification eluting peak B, Waters Atlantis T3C18 reverse phase
Column (2.1 × 150mm, 3.5m, 40 DEG C), equilibration buffer are the ultrapure water containing 1% trifluoroacetic acid, and elution buffer is
100% acetonitrile.Gradient: 20min, water 80%-0%；Acetonitrile 20%-100%, washes needle loading, a 80ul, flow velocity:
1.0ml/min.Loading is repeated several times and collects each component, reversed phase chromatography map is shown in Fig. 2A.Five main peaks will be obtained, by embodiment
The monitoring method of bioactivity carries out raw survey in 2, and there is obvious exciton activity at the peak D.As a result see Fig. 2 B.
The identification of 4 albumen exciton of embodiment
B5 adhesive tape is cut to be analyzed by mass spectrometry.Use Triple TOF 5600TOF MS analyzer (Applied
Biosystems the active segment collected after preparative HPLC) is analyzed, and data are obtained with MS scan pattern, scanning range is
250-2000(m/z).Use time-of-flight mass spectrometry instrument 5800MALDI-TOF/TOF, AB SCIEX) further analysis, swashs
Light source is neodymium, and 355 nanometers: YAG laser acceleration voltage of wavelength is 2kV, using positive ion mode and automatic data acquisition data, one
Rank scanning of the mass spectrum range is 800-4000 dalton, selects signal-to-noise ratio to analyze greater than 50 two stages parent ion mass spectrums (MS/MS), often
A illustration clicks selection 8A parent ion, and second order ms (MS/MS) accumulation superposition 2500, collision energy 2kV CID is closed.It will obtain
Sequence information in NCBI bacillus brevis (40021) database retrieval, marking sequence is carried out to the confidence level of result.Wherein divide
The highest albumen of number is the hypothesis albumen (accession number ERM16658.1) from Brevibacillus laterosporus.By this protein elicitor
It is named as PeBL2 (Protein elicitor Brevibacillus Laterosporus 2).Its amino acid sequence is shown in sequence
SED ID NO:2 in table.The albumen theoretical molecular weight is 17.2kDa.
The clone of 5 protein elicitor PeBL2 encoding gene of embodiment
PeBL2 albumen is made of 156 amino acid, theoretical molecular weight 17.22kD, isoelectric point 9.66.It is surveyed according to mass spectrum
Sequence result and the degeneracy design primer of codon are PeBL2 forward direction 5 '-TCAACCACATCCTCCGTACAGC-3 ' and PeBL1
Reversed 5'-TGCTACGAACCAGAGAAGCCA-3', as shown in SEQ ID NO:3-4.And with Brevibacillus laterosporus A60 gene
Group is template, and PCR amplification goes out the DNA fragmentation of 471bp, is named as PeBL2 gene, the sequence of the gene such as SEQ ID NO:
Shown in 1.
The prokaryotic expression of 6 PeBL2 gene of embodiment and purifying
(1) building of expression vector
This experiment uses pET28His TEV/LIC carrier, design protein elicitor gene PeBL2 removal signal peptide
Specific primer, forward primer: 5 '-CGGGATCC TCAACCACATCCTCCGTACAGC-3 ', reverse primer: 5 '-
CCCTCGAGTGCTACGAACCAGAGAAGCCA-3 ', as shown in SEQ ID NO:5-6.Expand (94 DEG C of overall length PeBL2 gene
3min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 35cycles；72℃10min；4℃∝).Reaction condition: 22 DEG C of incubations
30min；75 DEG C of 20min terminate reaction.T4 polymerase processed 2 μ l of carrier and 1 μ l of PCR product is taken to mix, 22 DEG C of connections
5min is added 1 μ l 25mM EDTA, 22 DEG C of 5min and terminates reaction.The product connected conversion Trans1-T1 competence is thin
Born of the same parents.Picking positive colony shakes bacterium and extracts plasmid, digestion and sequence verification.
(2) inducing expression
Expression vector obtained in step (1) is transformed into e. coli bl21 (DE3) with heat shock method.Plasmid extract and
The method of conversion expression host strain is the same as (1).Correct recombinant strains will be verified to be activated overnight, 1mL is taken to be incubated overnight liquid
It is added to (1% inoculum concentration) in the 100mL LB liquid medium containing 100 μ g/mL kanamycins, 37 DEG C, 200r/min oscillation
It is 0.6~0.8 that 2~3h of culture, which is cultivated to OD600,.It is added inducer IPTG (final concentration of 0.2mmol/L), 16 DEG C, 220r/
Min continues shaken cultivation 16h, inducing expression destination protein.Thalline were collected by centrifugation is added buffer.After ultrasonication thallus, 4
DEG C high speed centrifugation collects supernatant, obtains recombinant protein liquid.
(3) purifying of recombinant protein
It utilizesThe purifying of explorer10 protein purification instrument progress recombinant protein.Select Histrap HP
Column column carries out affinity chromatography, first balances affinity column with cleaning buffer solution (50mM Tris, 200mM Nacl, PH7.8)；It takes
The recombinant protein liquid 40mL sample introduction obtained in step (2), flow velocity 2mL/min, after elution to baseline, elution buffer (50mM
Tris, 200mM Nacl, 500mM imidazoles, PH7.8) it is eluted, collect eluting peak；Utilize Hitrap Desalting
Column carries out desalination removal of impurities to sample.Each component carries out SDS-PAGE detection, as a result sees Fig. 3 A.
As a result: obtaining the amalgamation and expression albumen (His-PeBL2 recombinant protein) of purifying.Recombinant protein (His-PeBL1) energy
Enough cause this life Tobacco Leaves that allergic reaction occurs and as a result sees Fig. 3 B with native protein activity having the same.
7 recombinant protein of embodiment (His-PeBL1) causes oxidative burst defense reaction on this life cigarette
Oxidative burst refers to the accumulation of reactive oxygen species, and reactive oxygen species include H2O2.This experiment passes through monitoring H2O2Product
Tire out the process to illustrate oxidative burst.The Genes For Plant Tolerance that oxidative burst (Reactive Oxygen Species, ROS) is induced in exciton
Very important effect is played in sick defense response, is participated in regulation stomatal movement, is promoted Apoptosis, directly or indirectly
The pathogen for killing invasion, can activate on transcriptional level as second messenger and regulate and control various defense response genes in plant
Expression.Method in this life Tobacco Leaves processing method such as the monitoring of 2 bioactivity of embodiment, His-PeBL2 concentration are 4 μM, place
8h sampling is managed, using Tris-HCL buffer as negative control.The tomato leaf cut is placed in containing 0.01%Triton-X-
100 and 1mg/mL nitro 3 in the solution of 3'- diaminobenzidine (DAB), penetrates into solution 5min with low vacuum pressure, and will
Blade is incubated at room temperature overnight.The decoloration in alcohol lactophenol (95% ethyl alcohol: lactic acid: phenol, 2:1:1), until leaf is free of
Then chlorophyll is rinsed with water.By observing the reddish brown precipitation generated in blade, to detect the H of generation2O2。
As a result: thering is apparent brown precipitate matter to occur with this life Tobacco Leaves of His-PeBL2 processing 8h, and compare without brown
Color generates, and illustrates that His-PeBL2 can cause this life Tobacco Leaves H2O2Accumulation, as a result see Fig. 4.
8 PeBL2 of embodiment induces this life cigarette that the pathogen of Botrytis cinerea (Botrytis cinerea) is inhibited to infect experiment
First on the right side of 30ul His-PeBL2 injection tobacco leaf, every plant of processing three pieces leaf is control, every place with buffer
Reason repeats three plants.It is placed in 28 DEG C of incubators and cultivates 3 days after inoculation Botrytis cinerea bacteria cake, observe incidence, take a picture and measure
Bacteria cake diameter on each processing blade, calculates inhibiting rate.
As a result: the blade of His-PeBL2 processing, slowly, inhibiting rate reaches 40.2% to the control of bacteria cake growth fraction.As a result see figure
5。
Embodiment described above only describe the preferred embodiments of the invention, not to model of the invention
It encloses and is defined, without departing from the spirit of the design of the present invention, those of ordinary skill in the art are to technical side of the invention
The various changes and improvements that case is made should all be fallen into the protection scope that claims of the present invention determines.
Sequence table
<110>Plant Protection institute, Chinese Academy of Agricultral Sciences
<120>Brevibacillus laterosporus A60 albumen exciton PeBL2 and its encoding gene and application
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 471
<212> DNA
<213>Brevibacillus laterosporus (Brevibacillus laterosporus)
<400> 1
atgcgtaaac aatctaaaaa aatgattgta agtcttttgt ctacgtcact attggctgta 60
tctctagcag ttcctgtttc agcatcaacc acatcctccg tacagcaggc tgtagtagta 120
ccgaacagtg aggtcggggc acaacaggta gggacctatg gatggaagac tcatattaca 180
aaagaaggtg tacaaatagt cattaaacac attagaaaag ggggcgctac gttagaaaga 240
gcgattagtc agctatctcc tacggcggct caatccttta aaagatggtc taataaaata 300
gcagatcatc tggagggaat catcaaagga tgggaacaaa aagaagaata cgctattgtt 360
tatctaaaag atcaactaag gaactttttg aaggccaact cgaatctttc tgatggaaca 420
atccaggaga ttgttgttgc tatagagtgg cttctctggt tcgtagcata g 471
<210> 2
<211> 156
<212> PRT
<213>Brevibacillus laterosporus (Brevibacillus laterosporus)
<400> 2
Met Arg Lys Gln Ser Lys Lys Met Ile Val Ser Leu Leu Ser Thr Ser
1 5 10 15
Leu Leu Ala Val Ser Leu Ala Val Pro Val Ser Ala Ser Thr Thr Ser
20 25 30
Ser Val Gln Gln Ala Val Val Val Pro Asn Ser Glu Val Gly Ala Gln
35 40 45
Gln Val Gly Thr Tyr Gly Trp Lys Thr His Ile Thr Lys Glu Gly Val
50 55 60
Gln Ile Val Ile Lys His Ile Arg Lys Gly Gly Ala Thr Leu Glu Arg
65 70 75 80
Ala Ile Ser Gln Leu Ser Pro Thr Ala Ala Gln Ser Phe Lys Arg Trp
85 90 95
Ser Asn Lys Ile Ala Asp His Leu Glu Gly Ile Ile Lys Gly Trp Glu
100 105 110
Gln Lys Glu Glu Tyr Ala Ile Val Tyr Leu Lys Asp Gln Leu Arg Asn
115 120 125
Phe Leu Lys Ala Asn Ser Asn Leu Ser Asp Gly Thr Ile Gln Glu Ile
130 135 140
Val Val Ala Ile Glu Trp Leu Leu Trp Phe Val Ala
145 150 155
<210> 3
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tcaaccacat cctccgtaca gc 22
<210> 4
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tgctacgaac cagagaagcc a 21
<210> 5
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cgggatcctc aaccacatcc tccgtacagc 30
<210> 6
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
ccctcgagtg ctacgaacca gagaagcca 29
Claims (9)
1. a kind of Brevibacillus laterosporus A60 albumen exciton PeBL2, it is characterised in that: SEQ in amino acid sequence such as sequence table
Shown in ID NO:2.
2. Brevibacillus laterosporus A60 albumen exciton PeBL2 described in claim 1 is improving answering in systemic resistance of plant
With.
3. PeBL2 according to claim 2 is improving the application in systemic resistance of plant, it is characterised in that: the plant
For this life cigarette.
4. a kind of PeBL2 gene, which is characterized in that its nucleotides sequence is classified as one of what follows:
(1) polynucleotide sequence of the protein as shown in SEQ ID NO:2 is encoded；
(2) with above-mentioned polynucleotide sequence according to the complementary polynucleotide sequence of basepairing rule.
5. PeBL2 gene according to claim 4, it is characterised in that: its polynucleotide sequence such as SEQ ID NO:1 institute
Show.
6. a kind of recombinant vector, it is characterised in that: contain PeBL2 gene described in claim 4 or 5.
7. recombinant vector according to claim 6, it is characterised in that: the carrier is in pET30 vector multiple cloning site
It is inserted into the recombinant vector of PeBL2 gene described in claim 4 or 5.
8. a kind of host cell of genetic engineering, it is characterised in that: contain recombinant vector described in claim 6 or 7.
9. the host cell of genetic engineering according to claim 8, it is characterised in that: the host cell is containing having the right
It is required that the e. coli bl21 of recombinant vector described in 6 or 7.
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