CN104894149B - A kind of chitinase and its encoding gene to the high virulence of Caenorhabditis elegans - Google Patents
A kind of chitinase and its encoding gene to the high virulence of Caenorhabditis elegans Download PDFInfo
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- CN104894149B CN104894149B CN201510124341.4A CN201510124341A CN104894149B CN 104894149 B CN104894149 B CN 104894149B CN 201510124341 A CN201510124341 A CN 201510124341A CN 104894149 B CN104894149 B CN 104894149B
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Abstract
The invention belongs to agriculture technical field of microbial genetic engineering, and in particular to a kind of chitinase and its encoding gene to the high virulence of Caenorhabditis elegans.The gene source that the present invention clones is in pseudomonad.The amino acid sequence of nucleotide sequence of the present invention comprising chitinase gene and its coding.Escherichia coli (Escherichia coli) DH5 α/6p pachi comprising the gene plasmid are deposited in China typical culture collection center (CCTCC), and deposit number is CCTCC NO：M2015139.By biological function verification, it was demonstrated that the desinsection chitinase has toxicity to Caenorhabditis elegans, can reach toxic effect by the worm's ovum and cuticula of Caenorhabditis elegans of degrading.The invention discloses application of the chitinase in terms of biological and ecological methods to prevent plant disease, pests, and erosion nematode.
Description
Technical field
The invention belongs to agriculture technical field of microbial genetic engineering, and in particular to a kind of to the high virulence of Caenorhabditis elegans
Chitinase and its encoding gene.
Background technology
Caenorhabditis elegans (Caenorhabditis elegans) is because its genetic background understands, individual configurations are simple, raw
History living is short, completion etc. has been sequenced in genome, in heredity and Developmental Biology, behavior and Neurobiology, aging and life-span, the mankind
Genetic disease, the interaction of pathogen and living organism, drug screening, the emergency reaction of animal, environmental and letter
Number conduction etc. field be used widely (Kaletta etc., 2006).
Pseudomonad, abundant chitinase can be produced in its bio-metabolic process, can will be several in organism
Fourth matter composition is hydrolyzed to oligosaccharides or monose, so as to provide required energy substance (Wang Zhiwei etc., 2006) for its growth.Separately
Outside, the main foreigner tourists as rhizosphere microorganism, pseudomonad be widely used in biological control (Yang Haijun etc.,
2004).Its caused micromolecular compound (such as cyanide) and hydrolase (such as serine protease) have very to nematode
Strong toxicity (Singh etc., 2010).
Chitinase, mainly there are 18 families and the class of 19 family two, wherein most 19 families come from plant, and 18 families
Widely exist in different kind organism (Henrissat etc., 1993).Chitinase is mainly by typical catalytic domain and land
Two parts form, and also have a small number of chitinases from fungi to be only made up of catalytic domain.Chitinase can hydrolyze chitin production
Raw chitin oligo saccharide and 2-Acetamido-2-deoxy-D-glucose, and be widely present in the biology of nature, play and focus in biological control
The effect wanted.
Chitin, it is the linear polymer connected by N- acetyl-β-D Glucosamines by β-Isosorbide-5-Nitrae-glycosidic bond,
It is one of most abundant natural polymer, is distributed widely in animal, plant and microbial body.There are some researches show chitin
Matter is the important composition composition of nematode worm's ovum and cuticula, and vital angle is play in the growth and breeding of nematode
Color.During a certain amount of chitin or chitosan are manured into soil, it can induce and largely breed life using the thalline of the material
It is long, so as to effectively control the propagation (Radwan etc., 2011) of plant pest.
At present, the site of action and the mechanism of action on chitinase are reported few.There are some researches show chitinase energy
The cell membrane of fungi is enough dissolved, participates in the formation of the sprouting of spore, aqtocytolysis and spore, and some fungies can be assisted
Parasitism infects host.In addition, chitinase can hydrolyze the chitin on insect midgut funnel, perforation is formed so as to cause to enclose
Eat film shaping delay, passage provided for other intestinal endotoxins such as crystalline protein, cooperate with raising insecticidal activity (Smirnoff,
1974).Influence for nematode, present invention demonstrates that, chitinase can hydrolyze the chorion and cuticula of nematode, so as to failure line
The institutional framework of worm, causes Dissolve things inside to exosmose.
The content of the invention
There are the several of high virulence to Caenorhabditis elegans (wild type N2, similarly hereinafter) object of the present invention is to provide a kind of
Fourth matter enzyme and its encoding gene, the gene are obtained by designing primer Direct Cloning from pseudomonas aeruginosa gene group.
Full length gene 1452bp (such as sequence table SEQ ID NO:In 1 corresponding to 1-1452b bases shown in sequence), encode 483 amino
Acid (its protein such as sequence table SEQ ID NO encoded:Shown in 2).The gene sequencing result is through in Genbank databases
BLASTN is compared, it is found that it is nearest with the chitinase gene affiliation of pseudomonas aeruginosa, show that the gene most has
It is probably a chitinase gene.Collect the nematode chitinase that kills reported and build phylogenetic analysis, with the gene code
Amino acid sequence similarity highest also only have 40%, it was demonstrated that the gene is a new nematicidal protein gene.
This is killed into Caenorhabditis elegans gene cloning to pGEX-6p-1 expression vectors (purchased from U.S. GE Healthcare public affairs
Department) on, as recombinant plasmid transformed Escherichia coli Escherichia coli BL21 (DE3) (being purchased from Invitrogen companies),
The fusion protein that size is 79.0kDa is obtained through induced expression, purifying obtains size as the pure of 53.0kDa after removing GST labels
Albumen.And the albumen is used for raw survey Caenorhabditis elegans, the results showed that：Its half lethal concentration LC50For 387.3 ± 31.7 μ g/
ml。
In order to realize the above object present invention employs following technical scheme：
Bacterial strain (disengaging time of the present invention from state Key Laboratory of Agricultural Microbiology preservation：
In September, 2012；Separately point：Wuhan City, Hubei Province Hua Zhong Agriculture University Lion Rock) in screening obtain one plant to beautiful hidden bar line
Worm has the candidate strain of high virulence (Laboratories Accession numbering is 0364).
The genomic DNA of the candidate strain is extracted, and using it as template, with 16S universal primer 1492R (5-
GGTTACCTTGTTACGACTT-3) and 27F (5-AGAGTTTGATCCTGGCTCAG-3) enters performing PCR amplification, the identified bacterial strain
Most possible is pseudomonas aeruginosa (Pseudomonas aeruginosa).
The active ingredient of the bacterial strain is subjected to soda acid and heat treatment respectively, tentatively infers that the active ingredient is most likely to be
Protein matter.
The original strain is inoculated into LMZ culture medium (compositions respectively：0.2% (mass volume ratio) gelatin, 0.8% albumen
Peptone, 0.1% yeast extract, 0.05% ammonium sulfate, 0.001% ferrous sulfate, 0.05% magnesium sulfate, pH7.0), chitin training
Support base (composition：0.5% peptone, 0.25% yeast extract, 0.5% sodium chloride, 0.2% tobacco brown spot pathogen, pH7.0), ox
Milk culture medium (composition：0.5% peptone, 0.25% yeast extract, 0.5% sodium chloride, 1% skimmed milk power, pH7.0), training
Support 2d after fermentation liquid and be used for raw survey Caenorhabditis elegans, thus it is speculated that chitinase caused by the bacterial strain has certain pass with killing nematode
System.
The chitinase gene in pseudomonas aeruginosa is found in NCBI genome database, designs primer from the bacterium
Clone has obtained total length 1452bp target gene (nucleotide sequence such as SEQ ID NO in the genomic DNA of strain:Shown in 1), and
And the gene is loaded on expression vector pGEX-6p-1, obtain recombinant vector pGEX-6p-pachi conversion e. coli bl21s
(DE3), through induced expression, purifying, destination protein has been obtained.The albumen is named as pachi by us, and the sequence of its protein is such as
SEQ ID NO:Shown in 2.
Recombinant vector pGEX-6p-pachi is converted into Escherichia coli (Escherichia coli) DH5 α, we will obtain
The Escherichia coli of restructuring are named as bacillus coli DH 5 alpha/6p-pachi, Escherichia coli DH5 α/6p-pachi, in
Deliver the China typical culture collection center preservation of Chinese Wuhan Wuhan Universitys within 03 20th, 2015, its preserving number is
CCTCC NO：M2015139.
More detailed technical scheme is shown in《Embodiment》Content.
Brief description of the drawings
Sequence table SEQ ID NO:1 is the chitinase to Caenorhabditis elegans with high virulence that present invention clone obtains
The nucleotide sequence (corresponding to 1-1452bp base shown in sequence) and its corresponding amino acid sequence (1-1452bp) of gene,
Wherein 1-1452bp is that code area is CDS).
Sequence table SEQ ID NO:2 be the present invention high virulence chitinase gene coding protein sequence, molecular weight
For 53.0kDa.
Fig. 1：The technology of the present invention flow chart.
Fig. 2：Nematode life cycle schematic diagram.
Fig. 3：Nematode death identification figure.A figures are living nematode displaing micro picture in wherein Fig. 3；B figures are that dead nematode shows in Fig. 3
Micro- picture.
Fig. 4：Original plasmid pGEX-6p-1 collection of illustrative plates.
Fig. 5：The recombinant plasmid pGEX-6p-pachi collection of illustrative plates that the present invention is built.
Fig. 6：Chitinase pachi is expressed and the SDS-PAGE analysis charts of purifying.To the explanation of each swimming lane in Fig. 6：Swimming lane M
It is standard protein sample (being purchased from Fermentas companies, magnitude range 14.4-116kDa)；Swimming lane 1 is Escherichia coli DE3/pGEX-
6p-1 does not induce broken cell supernatant；Swimming lane 2 is Escherichia coli DE3/pGEX-6p-1 induction broken cell supernatants；Swimming lane 3 is big
Enterobacteria DE3/pGEX-6p-pachi does not induce broken cell supernatant；Swimming lane 4 is that Escherichia coli DE3/pGEX-6p-pachi is lured
Lead broken cell supernatant；Swimming lane 5 is to purify and cut off the pachi protein liquids after GST labels.
Fig. 7：Standard protein concentration curve.
Fig. 8：2-Acetamido-2-deoxy-D-glucose standard curve.
Fig. 9：Chitinase pachi is to Caenorhabditis elegans fatal rate.
Figure 10：Chitinase pachi suppresses Caenorhabditis elegans breeding schematic diagram.
Figure 11：Chitinase pachi is to Caenorhabditis elegans growth effect, worm's ovum and cuticula degraded schematic diagram.Wherein
A figures are chitinase pachi to Caenorhabditis elegans growth effect in Figure 11；B figures are that chitinase pachi degradeds are elegant in Figure 11
The schematic diagram of beautiful hidden rhabditida worm's ovum；C figures are the signals of chitinase pachi degraded Caenorhabditis elegans cuticula in Figure 11
Figure.
Embodiment
Embodiment 1：Supper toxic strain screens
By the bacterial strain (disengaging time of this Laboratories Accession：In September, 2012；Separately point：Wuhan City, Hubei Province Central China agricultural
University's Lion Rock) respectively streak inoculation obtain LB (Media Components：1% (mass volume ratio, similarly hereinafter) peptone, 0.5% yeast
Extract, 1% sodium chloride, 1.5% agar powder) on flat board, after 37 DEG C of constant incubator culture 12-14 hours, flat board is grown
Single bacterium colony, picking single bacterium colony are inoculated into LB liquid medium (composition：1% (mass volume ratio, similarly hereinafter) peptone, 0.5% yeast
Extract, 1% sodium chloride), 37 DEG C culture two days later, by different extension rates (1 ×, 3 ×, 5 ×, 10 ×, 20 ×) dilution.
By cultured Caenorhabditis elegans, (cultural method is shown in the step 2) aseptic water washing of embodiment 6, stands, and removes
Supernatant, addition sterile water wash is clean to polypide, then with M9 buffer solutions (1L solution：Sodium chloride 5.0g, potassium dihydrogen phosphate
3.0g, disodium hydrogen phosphate 6g, bitter salt 0.25g) it is resuspended, obtain nematode liquid.A clean 96 sterile orifice plates are taken, per hole
The μ l of nutrient solution 100 of above-mentioned different extension rates are added, while add 5 μ l Caenorhabditis elegans liquid (about 40), if three
Parallel laboratory test.And negative control (sod cultivation is same as above) is used as with E. coli OP50 (Brenner, 1974), used
Sealed membrane is by 96 pore plate by sealing, in 20 DEG C of cultures two days later, at inverted microscope (model XSD-1B, purchased from Olympus companies)
Lower observation, the stiff nematode (as shown in Figure 3) for death of nematode.
From this laboratory (disengaging time：In September, 2012；Separately point：Wuhan City, Hubei Province Hua Zhong Agriculture University lion
Mountain) preservation 300 plants of bacterial strains in screening obtain 6 plants of bacterial strains to Caenorhabditis elegans with high virulence (result see the table below 1) and (compile
Number be respectively 0364,2258, CL4, F39-2, J3-4, J3-15).
16 plants of candidate strains of table are to Caenorhabditis elegans toxic effect
Embodiment 2：The classification of active material judges
Screen to obtain in above-described embodiment 16 plants of bacterial strains are inoculated into LB liquid medium respectively, and (composition is shown in embodiment
1LB fluid nutrient mediums), 37 DEG C of cultures two days later, prepare clasmatosis liquid：1ml nutrient solution is added in 1.5ml centrifuge tube
(OD600~1.2), 12000rpm centrifuges 1min, and supernatant is transferred into another clean 1.5ml centrifuge tube as fermentation
Supernatant, thalline with 400 μ l PBSs (pH7.4,140mM sodium chloride, 2.7mM potassium chloride, 10.0mM disodium hydrogen phosphates,
1.8mM dipotassium hydrogen phosphates) it is resuspended, in the sonicator (DN of JY92- II, purchased from the limited public affairs of Ningbo Toshiba biotechnology share
Department) in smudge cells, obtain clasmatosis liquid.By the sod cultivation of embodiment 1, given birth to respectively with fermented supernatant fluid and clasmatosis liquid
Survey line worm.As a result such as table 2 below.
The candidate strain fermented liquid supernatant of table 2 and clasmatosis liquid are to Caenorhabditis elegans toxic effect
According to above-mentioned table 2, collect respectively in each bacterial strain has higher active composition (such as 0364 to Caenorhabditis elegans
The clasmatosis liquid of bacterial strain, the fermented liquid supernatant of 2258 bacterial strains, other bacterial strains are identical).
Each active ingredient of above-mentioned collection is heat-treated respectively：Each active ingredient is respectively at thermostat water bath (model
SHHW, purchased from Shanghai east star building materials testing equipment Co., Ltd) in 60 DEG C and 100 DEG C processing 10min, then take out cold on ice
But, raw survey line worm (sod cultivation is with embodiment 1), as a result such as table 3 below, therefore select active ingredient lethal to Caenorhabditis elegans
Rate is high and thermally sensitive bacterial strain (numbering 0364) carries out soda acid processing, and using the bacterial strain as candidate strain.
Influence of the Temperature Treatment of table 3 to each active ingredient activity
Soda acid processing：The clasmatosis liquid of candidate strain adjusts pH with 1mol/L hydrochloric acid and 1mol/L sodium hydroxides respectively
(3,4,5,6,7,8,9,10,11), 1mol/L is used respectively after being then put into 4 DEG C of refrigerators (being purchased from Qingdao company of Haier) processing 12h
Hydrochloric acid and 1mol/L sodium hydroxides adjust pH to neutrality, raw survey line worm (sod cultivation is with embodiment 1).As a result such as table 4 below.
The processing of the soda acid of table 4 is to candidate strain clasmatosis liquid activity influence
Its active ingredient (clasmatosis liquid) of candidate strain (numbering 0364) is handled through Overheating Treatment and soda acid, finds the work
Sexual element is all more sensitive to temperature and soda acid, and the initial guess active material is particularly likely that protein matter.
In addition, candidate strain (numbering 0364) is inoculated into LMZ culture medium (compositions respectively：0.2% (mass volume ratio) is bright
Glue, 0.8% peptone, 0.1% yeast extract, 0.05% ammonium sulfate, 0.001% ferrous sulfate, 0.05% magnesium sulfate, pH
7.0), chitin culture medium (composition：0.5% peptone, 0.25% yeast extract, 0.5% sodium chloride, 0.2% colloid chitin
Matter, pH 7.0) and milk culturemedium (composition：0.5% peptone, 0.25% yeast extract, 0.5% sodium chloride, 1% degreasing
Milk powder, pH 7.0), 37 DEG C of cultures two days later, according to above-mentioned clasmatosis liquid and preparation method thereof, obtain the bacterium of each medium culture
Clasmatosis liquid, and dilute different multiples (1 ×, 2 ×), raw survey line worm (sod cultivation is with embodiment 1).It the results are shown in Table 5.
The candidate strain (numbering 0364) is grown in chitin culture medium, has higher fatal rate to Caenorhabditis elegans.Therefore,
Infer that chitinase caused by the bacterial strain may take part in infection processs of the bacterial strain to nematode.
Influence of the different culture media culture of table 5 to candidate strain activity
Embodiment 3：The identification of candidate strain and the clone of target gene
(1) extraction of candidate strain STb gene：
By the candidate strain (numbering 0364), LB liquid medium (composition is shown in embodiment 1LB fluid nutrient mediums) is inoculated into,
After 37 DEG C of culture 2d, 2ml bacterium solution is taken in centrifuge tube, collects thalline；
With 200 μ l TE buffer (formulas：50mmol/Tris-HCl pH8.0,10mmol/L EDTA, pH is adjusted to 8.0)
Thalline is resuspended, and adds the μ g/ml lysozymes of 50 μ l 100 (lysozyme is purchased from Sigma companies), 37 DEG C of water-bath 1h；
Add 500 μ l bacterial lysate (lysate compositions：20% (mass volume ratio, similarly hereinafter) dodecyl sodium sulfate
(SDS), 5% disodium ethylene diamine tetraacetate (EDTA2Na), 5% sodium acetate (NaAc), 3% sodium citrate, 5% (volume：Volume,
Similarly hereinafter) TrionX-100,5%Tween20,50%Tris-Cl (50mM)), after 70 DEG C of water-bath 10min, take out and cooled down in 4 DEG C,
Add 150 μ l 5mol/L sodium chloride solutions；
Add isometric phenol：Chloroform：Isoamyl alcohol (volume ratio 25：24：1), mix, 12000rpm centrifugation 10min, take
Supernatant, repeat the step for 2 times；
Supernatant is taken, the isopropanol for adding 2/3 volume is gently mixed, and 30min, 12000rpm centrifugations are stood in -20 DEG C
10min；
Supernatant is abandoned, retains precipitation, the ethanol for adding 70% washs 2 times, after drying at room temperature, is dissolved in 50 μ l ddH2In O,
Be stored in -40 DEG C it is standby.
(2) the 16S gene magnifications of candidate strain：
The bacteria total DNA extracted using step 1 is template, using 16S universal primer 1492R (5-
GGTTACCTTGTTACGACTT-3) expanded with 27F (5-AGAGTTTGATCCTGGCTCAG-3),
PCR system is as follows：
PCR conditions：94 DEG C of pre-degeneration 4min；94 DEG C, 30s；52 DEG C, 30s；72 DEG C, 90s, 30 circulations；72 DEG C of extensions
10min, 15 DEG C, 5min.
By PCR primer with glue reclaim kit (being purchased from border biological gene Science and Technology Ltd. of Beijing village ally) after purification,
It is sequenced in Bo Hong bio tech ltd, as a result compares and learn through Blast, the candidate strain and Pseudomonas
Aeruginosa (pseudomonas aeruginosa) affiliation is nearest.
(3) target gene expands：
According to the nucleotide sequence of the pseudomonas aeruginosa chitinase genes of NCBI Genebank warehouse publications
(Accession Number：CP006831 primer, forward primer F (5-) are designed
CCGGAATTC), ATGATCAGGATCGACTTTTCCCAGTTGCA-3 comprising the restriction enzyme sites of EcoR I (see under forward primer F
Dashed part)；Reverse primer R (5-CCGCTCGAGTCAGCGCAGCGGCCGCC-3), comprising the restriction enzyme sites of Xho I (see reversely
Primer R underscore part).Primer is synthesized by Bo Hong bio tech ltd.
PCR is expanded
PCR system：
PCR conditions：94 DEG C of pre-degeneration 4min；94 DEG C, 30s；60 DEG C, 30s；72 DEG C, 90s, 30 circulations；72 DEG C of extensions
10min, 15 DEG C, 5min.
By PCR primer with glue reclaim kit (being purchased from border biological gene Science and Technology Ltd. of Beijing village ally) after purification,
It is sequenced in Bo Hong bio tech ltd, as a result compares and learn through Blast, the gene is with coming from Pseudomonas
Aeruginosa chitinase gene affiliation is nearest, and the DNA sequence dna total length of the gene is 1452bp, using AUG as starting
Codon, terminator codon UGA, 483 amino acid are encoded, and the chitinase is named as pachi.The chitinase
Belong to the family of glycosyl hydrolase 18, contain typical catalytic domain CHA, bonding pad FN3, land CBD.With it is other reported kill
The amino acid sequence of nematode chitinase is compared, and highest only has 40% similitude (being shown in Table 6), and it is one new to show the gene
Kill nematode chitinase.
The pachi of table 6 kills nematode chitinase homology with having reported
(4) PCR primer and plasmid pGEX-6p-1 digestions：
EcoR I-Xho I (being purchased from TAKARA) double digestion system is selected in digestion, and digestion system is as follows：Enzyme cutting buffering liquid
10μl；EcoR I, 3 μ l；Xho I, 3 μ l；PCR purified products (about 3 μ g)；Add water to 100 μ l.Mix after 37 DEG C of water-bath 12h,
With glue reclaim kits.Plasmid pGEX-6p-1 digestions system and condition are same as above.
(5) endonuclease bamhi is connected with carrier：
Linked system：5 × ligase buffer solution (is purchased from TAKARA companies), 2 μ l；Double digestion PCR primer, 3 μ l are (about
200ng)；Double digestion carrier, 1 μ l (about 40ng)；T4 ligases, 0.5 μ l；Add water to 10 μ l.After mixing, 16 DEG C of incubated overnights.
(6) recombinant plasmid transformed E. coli DH5 α：
Take the μ l of enzyme connect product thing 1 and 50 μ l competent escherichia coli cell E.coli DH5 α to mix, suck the 1mm electricity of precooling
In revolving cup (being purchased from BIO-RAD companies), electroporated (voltage 12.5kv/cm) (electroporation model MicroPulser, is purchased from
BIO-RAD companies), 500 μ l LB fluid nutrient mediums (composition is shown in embodiment 1LB fluid nutrient mediums) are added, 37 DEG C of recovery 1h, are applied
It is distributed in and contains 100 μ g/ml ampicillin/LB plates, after 37 DEG C of insulation 14h, single bacterium colony that picking is grown is inoculated in containing 100 μ g/
In ml ampicillin LB fluid nutrient mediums (composition is shown in embodiment 1LB fluid nutrient mediums), 37 DEG C of culture 12h.
(7) plasmid extraction：
The centrifuge tube of a 2ml is taken, collects 2ml bacterium solutions, centrifuging and taking thalline；
Bacterium is resuspended with 250 μ l solution I (25mM Tris-Cl (pH8.0), 10mM ethylenediamine tetra-acetic acids, 50mM glucose)
Body, 250 μ l solution II (200mM sodium hydroxides, 1% (mass volume ratio) dodecyl sodium sulfate (SDS)) are added, are turned upside down
2~3 times, 350 μ l solution III (3M potassium acetates, 5M acetic acid) are added, are mixed, 12000rpm centrifugation 10min, supernatant is taken, adds
The isopropanol of 2/3 volume is gently mixed, and 30min, 12000rpm centrifugations 10min are stood in -20 DEG C；Supernatant is abandoned, retains precipitation, adds
The ethanol for entering 70% washs 2 times, after drying at room temperature, is dissolved in 50 μ l ddH2In O, be stored in -20 DEG C it is standby.
(8) recombinant plasmid transformed E. coli BL21 (DE3)：
The μ l of plasmid 1 extracted and 30 μ l competent escherichia coli cell E.coli BL21 (DE3) are taken to mix, suction is pre-
In cold 1mm electricity revolving cup (being purchased from BIO-RAD companies), electroporated (voltage 12.5kv/cm) (electroporation model
MicroPulser, purchased from BIO-RAD companies), adding 500 μ l LB fluid nutrient mediums, (composition is shown in embodiment 1LB Liquid Cultures
Base), it is coated on containing 100 μ g/ml ampicillin/LB plates (composition is shown in embodiment 1LB flat boards), after 37 DEG C are incubated 14h, picking
The single bacterium colony grown, it is inoculated in containing (composition is shown in embodiment 1LB Liquid Cultures in 100 μ g/ml ampicillin LB fluid nutrient mediums
Base), after 37 DEG C are cultivated 12h, take 800 μ l bacterium solutions and the μ l of glycerine 200 of sterilizing to mix, be stored in -80 DEG C.
(9) microbial characteristic of bacterial strain：
The Escherichia coli that above-mentioned steps 6 obtain, for the bacillus pumilis of two sections of blunt circles, 0.5 × 1~3 microns of size, all heights
Amphitrichous, it can move, it is impossible to produce gemma, good, Gram-negative is coloured to general alkali dyestuff.Bacterium colony surface
Smooth, protrusion is opaque, is circular colonies.By the bacillus coli DH 5 alpha/6p-pachi, Escherichia coli DH5 α/
6p-pachi sent Chinese Wuhan Wuhan Universitys China typical culture collection center on 03 20th, 2015, and deposit number is
CCTCC NO：M2015139.
Embodiment 4：Induction, expression, purifying and the analysis of albumen
(1) expression of recombinant protein：
The single bacterium colony that the conversion of the step 8 of picking above-described embodiment 3 is grown, is inoculated in containing 100 μ g/ml ampicillin LB liquid
In culture medium (composition is shown in embodiment 1LB fluid nutrient mediums), after 37 DEG C of culture 12h, with 1% (volume：Volume) inoculum concentration extremely
1L contains in 100 μ g/ml ampicillin LB fluid nutrient mediums (composition is shown in embodiment 1LB fluid nutrient mediums), and 37 DEG C of cultures 2~
3h, treat OD600Reach 0.6, add isopropyl-β-D-thiogalactoside (IPTG) to final concentration of 0.1mM, 18 DEG C of induction 12h
Afterwards.Thalline is collected by centrifugation, is washed 2~3 times with PBS (composition is shown in embodiment 2PBS buffer solutions), then it is heavy with 50ml PBS
It is outstanding, then with high pressure cell cracker (being purchased from THERMO companies of the U.S.) smudge cells.Broken liquid is in 4 DEG C, 12000rpm/min
10min (centrifuge model Avanti J-E, purchased from BACKMAN companies) is centrifuged, takes supernatant, the above-mentioned centrifugation of repetition 2~3 times.
Supernatant is collected, protein expression situation is detected with SDS-PAGE.
(2) purifying of albumen：
Destination protein is purified using GST affinity purifications system, concrete operation step is following, and (all operations are entered at 4 DEG C
OK)：
Fill post：1ml column material GSH Sepharose 4B (being purchased from Amersham-Pharmacia companies) are taken in chromatographic column
In, eluted with the PBS of 5 column volumes, it is clean to alcohol washes, activate column material；
With reference to：Clasmatosis liquid supernatant is slowly added into pillar, pillar flow control is in 0.5ml/min；
Elution：Foreign protein, pillar stream are eluted with the PBS (composition is shown in embodiment 2PBS buffer solutions) of precooling (4 DEG C)
Speed control is in 1ml/min.With Bradford reagents (being purchased from Shanghai biotechnology Co., Ltd) detection foreign protein elution feelings
Condition；
Enzymolysis：The PBS for the HRV 3CP (being purchased from Pharmacia companies) and 1ml that 10 μ l concentration are 10unit/ μ l is taken to buffer
Liquid is mixed, and adds chromatographic column, and 16h is digested in 4 DEG C；
Collect albumen：The albumen after enzymolysis is collected in 1.5ml centrifuge tube.
(3) detection of purifying protein：
Albumen is detected using 12% SDS-PAGE.Formula is as follows：
5% concentration glue：
12% separation gel：
Detection method：The protein sample for taking 40 μ l to purify, 10 μ l albumen sample-loading buffers of addition (5 ×, it is purchased from TAKARA public affairs
Department), boiling water bath 10min, the μ l of loading 10.As a result Fig. 4 is seen.
(4) determination of protein concentration：
Albumen is carried out using Bradford protein quantifications detection kit (being purchased from Shanghai biotechnology Co., Ltd)
Matter quantifies.Concrete operations are as follows：
Standard protein sample (being purchased from Shanghai biotechnology Co., Ltd) is taken to be diluted to 0 μ g/ml, 0.01 μ g/ml with PBS,
9 ladders such as 0.02 μ g/ml, 0.04 μ g/ml, 0.06 μ g/ml, 0.08 μ g/ml, 0.10 μ g/ml, 0.15 μ g/ml, 0.20 μ g/ml
Degree；
In 96 hole elisa Plates, the μ l of standard protein sample 20 per hole after the above-mentioned dilution of addition, each concentration is repeated 4 times, each hole
200 μ l Bradford Reagent are added, are mixed, after room temperature places 10min, (Thermo is purchased from ELIASA
Scientific companies) measure A595Reading.
Mark song (see Fig. 7) is drawn, the protein concentration for calculating sample is 3mg/ml.
Embodiment 5：Chitinase activity determines
(1) tobacco brown spot pathogen is prepared：
5g solids chitin (being usually off-white powder or sheet, purchased from Aladdin companies) is weighed, is added to 250ml
In triangular flask, 100ml concentrated hydrochloric acid is added, is stirred on magnetic stirring apparatus (78-1 types, purchased from big-and-middle instrument plant of Community of Jin Tan County city)
Mix 12 hours and be completely dissolved to chitin, the lysate is added in the 1L sterilized waters of precooling, (be purchased from 4 DEG C of constant temperature refrigerators
Qingdao company of Haier) in stand 12 hours, precipitation is collected by centrifugation, and be resuspended with sterilized water, then centrifuge, so repeatedly 7-10
Secondary, the pH to re-suspension liquid is 7.0, that is, obtains tobacco brown spot pathogen.
(2) chitinase activity determines：
1.5ml centrifuge tubes are taken, add chitinase and 150 μ l above-described embodiments 5 that the purifying of 3 μ l above-described embodiments 4 obtains
The tobacco brown spot pathogen that step 1 obtains, mix, (model SHHW, set in 40 DEG C of thermostat water baths purchased from Shanghai east star building materials testing
Standby Co., Ltd) 1 hour is incubated, taking out 12000rpm centrifugations, (centrifuge model Centrifuge 5415D, are purchased from
Eppendorf companies) 1min, the μ l of Aspirate supernatant 120, DNS nitrite ions (the 1L solution of addition isometric (120 μ l)：Tartaric acid
Potassium sodium 182.0g, 3,5- dinitrosalicylic acid 6.3g, sodium hydroxide 21.0g, phenol 5.0g, anhydrous sodium sulfite 5.0g), and in
100 DEG C of reaction 5min, after cooling, 200 μ l reaction solutions are drawn into 96 clean hole elisa Plates, (are purchased from ELIASA
Thermo Scientific companies) measure A540Reading.Above-mentioned every group of experiment set 3 it is parallel.
(3) 2-Acetamido-2-deoxy-D-glucose Specification Curve of Increasing：
Weigh 0.05g 2-Acetamido-2-deoxy-D-glucose solid, add 5ml sterilized waters, glass bar is stirred to solid dissolving, i.e.,
Concentration be 10mg/ml 2-Acetamido-2-deoxy-D-glucose mother liquor, dilute by different proportion (concentration 0.2mg/ml, 0.4mg/ml,
0.6mg/ml, 0.8mg/ml, 1.0mg/ml, 1.2mg/ml), chromogenic reaction is the same as above-mentioned steps 2DNS chromogenic reactions.Drafting standard
Curve such as Fig. 8.
Enzyme activity defines：At 40 DEG C of temperature, pH7.0 reaction conditions, and in the case of substrate saturation, this is several in 1 minute
The enzyme amount that the degraded of fourth matter enzymatic tobacco brown spot pathogen is produced needed for 1mg 2-Acetamido-2-deoxy-D-glucoses is defined as an enzyme activity list
Position (1U).
It is 0.023U to be computed obtaining the chitinase activity.
Embodiment 6：Chitinase is to Caenorhabditis elegans semilethal rate LC50The measure bred with suppression
(1) cracking and synchronization of Caenorhabditis elegans：
There is the flat board for the Caenorhabditis elegans adult largely become pregnant with 2ml aseptic water washings, stood in 2ml centrifuge tubes,
Treat that Caenorhabditis elegans sinks to ttom of pipe, suck supernatant, add 2ml sterilized waters and be resuspended, stand.Repeat to treat beautiful hidden bar line 2~3 times
Worm cleans up.Add 1ml lysate (compositions：3.5ml ddH2O, 0.5ml 5M sodium hydroxides, 1ml 4-6% hypochlorous acid
Sodium).Treat that nematode is completely dissolved, after worm's ovum all discharges (general 5-10min), worm's ovum is collected by centrifugation in 800rpm, and is buffered with M9
Liquid (composition：0.5% (mass volume ratio, similarly hereinafter) sodium chloride, 0.3% potassium dihydrogen phosphate, 0.6% disodium hydrogen phosphate, 0.025%
Magnesium sulfate) cleaning 3~5 times, to cleaning up.Add 1ml M9 to be resuspended, be put into 20 DEG C of hatchings.
(2) culture of Caenorhabditis elegans：
Picking E. coli OP50 (Brenner, 1974) single bacterium colony, it is inoculated in containing 100 μ g/ml ammonia benzyl moulds
In plain LB fluid nutrient mediums, after 37 DEG C of culture 12h, 200 μ l bacterium solutions are taken to be coated on NGM solid medium (compositions：(0.3% chlorination
Sodium, 0.25% peptone, 1.5% agar powder) sterilizing after add 1mg/ml cholesterol (be dissolved in 100% alcohol) 1ml,
1mol/l calcium chloride 21ml, 1mol/l magnesium sulfate 41ml, 1mol/l kaliumphosphate buffer (pH6.5) 25ml) flat board, by hatching
Caenorhabditis elegans is inoculated on flat board, and 20 DEG C of cultures can obtain L4 larvas in 2~3 days.
(3) semilethal rate LC50Measure：
With the children of aseptic water washing Caenorhabditis elegans L4 (see L4 larva in the history of life of Fig. 2 Caenorhabditis elegans) phases
Worm, stand, remove supernatant, addition sterile water wash is clean to polypide, then with M9 buffer solutions (1L solution：Sodium chloride 5.0g,
Potassium dihydrogen phosphate 3.0g, disodium hydrogen phosphate 6g, bitter salt 0.25g) it is resuspended.A clean 96 sterile orifice plates are taken, per hole
(concentration is 980 μ g/ml, 730 μ g/ml, 640 μ g/ml, 520 μ g/ml, 250 μ g/ml, 150 μ to the various concentrations added after dilution
G/ml, 75 μ g/ml, 0 μ g/ml) chitinase 1 serving 00 μ l, 5 μ l L4 larvas (about 40), each sample set 4 it is parallel.Will
96 orifice plates are sealed with sealed membrane and place 2d after 20 DEG C.In inverted light microscope (model model XSD-1B, purchased from Olympus
Company) under observe, it is impossible to movable nematode is designated as death.Suitable concentration gradient is selected to draw out complete fatal rate curve
(Fig. 9), and LC is gone out by schematic calculation50For 387.3 ± 31.7 μ g/ml.
(4) suppress to breed measure：
A clean 96 sterile orifice plates are taken, (concentration is 150 μ g/ml, 100 μ g/ to the various concentrations added per hole after dilution
Ml, 75 μ g/ml, 50 μ g/ml, 30 μ g/ml, 0 μ g/ml) chitinase 1 serving 00 μ l, 5 μ l L4 larvas (3~5), 5 μ l's is big
Enterobacteria E.coli OP50 (are resuspended, to whole OD in PBS600~0.6), each sample set 4 it is parallel.96 orifice plates are sealed
Film sealing places 2d after 20 DEG C.Observed under inverted microscope (model XSD-1B, purchased from Olympus companies), count larva
Hatching number.Select suitable concentration gradient to draw out and completely breed suppression curve (Figure 10).And by the way that half suppression is calculated
Concentration IC processed50For 106.1 ± 4.3 μ g/ml.
Embodiment 7：Measure that chitinase suppresses to the larval growth of Caenorhabditis elegans and to worm's ovum and cuticula
Degraded measure
(1) measure suppressed to Caenorhabditis elegans larval growth：
(the Caenorhabditis elegans worm's ovum that the step 1) of embodiment 6 is collected, being resuspended in 1ml M9 buffer solutions, (1L is molten as described above
Liquid：Sodium chloride 5.0g, potassium dihydrogen phosphate 3.0g, disodium hydrogen phosphate 6.0g, bitter salt 0.25g) in, it is placed in 20 DEG C of constant temperature
Incubator (being purchased from Tianjin Stettlen Instrument Ltd.) is incubated 1d, treats that egg hatch obtains L1 larvas.Take one clean sterile
96 orifice plates, per hole add dilution after various concentrations (300 μ g/ml, 250 μ g/ml, 200 μ g/ml, 150 μ g/ml, 100 μ g/
Ml, 50 μ g/ml, 0 μ g/ml) chitinase 1 serving 00 μ l, 5 μ l L1 larvas (about 40), 5 μ l E. coli
OP50 (is resuspended, to whole OD in PBS600~0.6), each sample set 4 it is parallel.96 orifice plates are sealed after 20 with sealed membrane
DEG C place 2d.In flying-spot microscope (model IX73, purchased from Olympus companies) 40 × lower shooting nematode picture (see in Figure 11
A figure).
(2) the degraded measure to worm's ovum：
(the Caenorhabditis elegans worm's ovum that the step 1) of embodiment 6 is collected, is resuspended in M9 buffer solutions (1L solution as described above：Chlorine
Change sodium 5.0g, potassium dihydrogen phosphate 3.0g, disodium hydrogen phosphate 6g, bitter salt 0.25g) in, take a clean 96 sterile holes
Plate, the μ g/ml of 100 μ l 200 chitinase is added per hole, 5 μ l worm's ovums (about 40), 20 DEG C of insulations are placed in, respectively at 4 hours
With sampling observation in 8 hours, and worm's ovum figure is shot with flying-spot microscope (model IX73, purchased from Olympus companies) 40 × lower
Piece (see the B figures in Figure 11)
(3) the degraded measure of Stratum Corneum：
(the Caenorhabditis elegans L4 phases that the step 2) of example 6 is collected are (see the history of life of Fig. 2 Caenorhabditis elegans as described above
Middle L4 larva) larva, it is resuspended in 200 μ l M9 buffer solution (1L solution：Sodium chloride 5.0g, potassium dihydrogen phosphate 3.0g, phosphoric acid hydrogen
Disodium 6g, bitter salt 0.25g) in, a clean 96 sterile orifice plates are taken, the several of the μ g/ml of 100 μ l 1000 are added per hole
Fourth matter enzyme, 5 μ l Caenorhabditis elegans worms skins (about 40), is placed in 20 DEG C of insulations, and observation is sampled after 12 hours, and aobvious with scanning
Micro mirror (model IX73, purchased from Olympus companies) 40 × lower shooting worm's ovum picture (see the C figures in Figure 11).
Leading reference
1. Wang Zhi is big etc., microorganism chitinase progress biotechnology communications, and 2006,17:439-442；
2. poplar sea monarch etc., the biological control Effect study Chinese Ecological Agriculture journal of pseudomonad, 2004,3；
3. Brenner S(1974)The genetics of Caenorhabditis elegans Genetics 77:
71-94
Henrissat B,Bairoch A(1993)New families in the classification of
glycosyl hydrolases based on amino acid sequence similarities Biochem j 293:
781-788；
4. Kaletta T,Hengartner MO(2006)Finding function in novel targets:
C.elegans as a model organism Nature reviews Drug discovery 5:387-398doi:
10.1038/nrd2031；
5. Radwan MA,Farrag SAA,Abu-Elamayem MM,Ahmed NS(2011)Extraction,
characterization,and nematicidal activity of chitin and chitosan derived from
shrimp shell wastes Biology and Fertility of Soils 48:463-468
doi:10.1007/s00374-011-0632-7；
6. Singh N,Kumar S,Bajpai VK,Dubey RC,Maheshwari DK,Kang SC(2010)
Biological control of Macrophomina phaseolina by chemotactic fluorescent
Pseudomonas aeruginosa PN1and its plant growth promotory activity in chir-
pine Crop Protection 29:1142-1147doi:10.1016/j.cropro.2010.04.008；
7. Smirnoff W(1974)Three years of aerial field experiments with<i>
Bacillus thuringiensis</i>plus chitinase formulation against the spruce
budworm Journal of Invertebrate Pathology 24:344-348。
Claims (1)
- It is 1. a kind of such as SEQ ID NO：The protein sequence of chitinase gene pachi codings shown in 2 is killing beautiful hidden bar line Application in worm.
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