CN107344961A - A kind of method for extracting No. 0/1 microspecies differential protein peptide fragment of tobacco black shank bacterium and the otherness peptide fragment of identification extraction - Google Patents
A kind of method for extracting No. 0/1 microspecies differential protein peptide fragment of tobacco black shank bacterium and the otherness peptide fragment of identification extraction Download PDFInfo
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- CN107344961A CN107344961A CN201710535445.3A CN201710535445A CN107344961A CN 107344961 A CN107344961 A CN 107344961A CN 201710535445 A CN201710535445 A CN 201710535445A CN 107344961 A CN107344961 A CN 107344961A
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- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention provides a kind of authentication method of No. 0 and No. 1 biological strain otherness peptide fragment of tobacco black shank, and 6 distinctive peptide fragments are according to said method identified, wherein, No. 0 microspecies peptide fragment has SEQ ID NO：Amino acid sequence shown in 13, No. 1 microspecies peptide fragment have SEQ ID NO：Amino acid sequence shown in 46.Belong to pathogenic identification technology field.This method comprises the following steps：(1) total protein of tobacco black shank bacterium is extracted；(2) SDS polyacrylamide gel electrophoresises are carried out with the albumen obtained in step (1)；(3) the running gel figure obtained according to step (2) picks out differential band；(4) band for obtaining (3) digests, and extracts peptide hydrolysis, carries out LC MS analyses；(5) mass spectral results are used with Mascot2.2 software retrieval associated databases, obtains differential protein peptide fragment.
Description
Technical field
The present invention relates to pathogenic identification technology field, and it is No. 0/1 small to be specifically related to a kind of extraction tobacco black shank bacterium
The method of species diversity albumen peptide fragment and the otherness peptide fragment of identification extraction.
Background technology
Tobacco black shank is that a kind of global crushing soil passes oomycetes disease.Black shank bacterium (Phytophthora
Parasitica var.nicotianae) it is a kind of facultative parasite, pyrophilous high humidity, at 28-30 DEG C, humidity reaches temperature
When more than 80%, created favorable conditions to the propagation of germ sporangium and the release of zoospore, be now best suitable for the black shin of tobacco
The prevalence of disease causes cigarette strain Large Scale Death with occurring.Tobacco rolls into a ball a phase to prosperous long-term intercurrent disease as its host plant at it
Peak the phase, its root of main harm and basal part of stem.
The black shank bacterium biological strain reported in the world has 4 kinds, two biological strains in China at least No. 0 and No. 1, when
It is preceding still using No. 0 microspecies as dominant races.Different Races of Phytophthora Parasitica Var. Nicotianae show difference in terms of pathogenicity, i.e., different cigarettes
Grass product kind is different to the anti-perceptual shape of different biological strains, as NC1071 and L8 kinds are disease-resistant for No. 0 microspecies, and for 1
Number microspecies are then susceptible；Florida301 kinds are disease-resistant for No. 0 and No. 1 biological strain；H21 and small gold 1025 are for No. 0
It is susceptible with No. 1 biological strain, therefore find differential protein peptide fragment and be particularly important.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of extraction No. 0/1 microspecies differential protein of tobacco black shank bacterium
The method of peptide fragment and the otherness peptide fragment of identification.
In order to solve the above technical problems, the present invention provides a kind of extraction No. 0/1 microspecies differential protein of tobacco black shank bacterium
The method of peptide fragment, it is characterised in that comprise the following steps：
The first step：Extract the total protein of black shank bacterium；
Second step：SDS- polyacrylamide gel electrophoresises are carried out with the albumen obtained in step 1；
3rd step：The running gel figure obtained according to step 2 picks out differential band；
4th step：The band that 3 are obtained digests, and extracts peptide hydrolysis, carries out LC-MS analyses；
5th step：Mass spectral results are used with Mascot2.2 software retrieval associated databases, obtains differential protein peptide fragment.
Wherein, extract solution is prepared in step 1, the mixing of 2 μ L protease inhibitors is added in every 500 μ L mycoprotein extract solutions
Thing, 4 μ L protein stabilisers, mix rearmounted standby on ice.
Wherein, 10% separation gel and 4% concentration glue are prepared in step 2,10% ammonium persulfate (AP) should be eventually adding, mix
Encapsulating immediately afterwards；When room temperature is higher, before AP is added, glue can be placed on to 4 DEG C of precoolings, then add AP, stood after mixing
That is encapsulating.
Wherein, deposition condition is in step 2：The initial value for setting electric current is 10-20mA, after sample enters separation gel,
Electric current is arranged to 20-40mA；Or constant pressure 60-80V when starting, treat that sample is thicker into separating, 100V is changed to, when hectograph bottom
When spacing with dye front is 0.5cm, electrophoresis terminates.
Wherein, mass spectrometry parameters in step 4：200 DEG C of capillary temperature, detection mode：Cation.
Present invention also offers the amino acid sequence for identifying No. 0/1 microspecies differential protein peptide fragment of tobacco black shank bacterium
SEQ ID NO：1-6.Wherein SEQ ID NO：1-3 is No. 0 microspecies specific proteins peptide fragment, SEQ ID NO：4-6 is No. 1 small
Species specificity albumen peptide fragment.
Brief description of the drawings
Fig. 1 is SEQ ID NO：1VHNIEATFGEEEFVGKK mass spectrograms (No. 0 microspecies)
Fig. 2 is SEQ ID NO：2TLAEFQNNPVDR mass spectrograms (No. 0 microspecies)
Fig. 3 is SEQ ID NO：3FGENTANTVLTK mass spectrograms (No. 0 microspecies)
Fig. 4 is SEQ ID NO：4 KGNDDDDEGGNYSSYSGDM (Oxidation) GGGVHGSR mass spectrograms are (No. 1 small
Kind)
Fig. 5 SEQ ID NO：5ITFDESQVTYEELLK mass spectrograms (No. 1 microspecies)
Fig. 6 SEQ ID NO：6FNDIVLR mass spectrograms (No. 1 microspecies)
Embodiment
The technical problems to be solved by the invention are to provide a kind of extraction No. 0/1 microspecies differential protein of tobacco black shank bacterium
The method of peptide fragment and the otherness peptide fragment of identification.
In order to solve the above technical problems, the present invention provides a kind of extraction No. 0/1 microspecies differential protein of tobacco black shank bacterium
The method of peptide fragment, it is characterised in that comprise the following steps：
The first step：Extract the total protein of black shank bacterium；
Second step：SDS- polyacrylamide gel electrophoresises are carried out with the albumen obtained in step 1；
3rd step：The running gel figure obtained according to step 2 picks out differential band；
4th step：The band that 3 are obtained digests, and extracts peptide hydrolysis, carries out LC-MS analyses；
5th step：Mass spectral results are used with Mascot2.2 software retrieval associated databases, obtains differential protein peptide fragment.
Wherein, extract solution is prepared in step 1, the mixing of 2 μ L protease inhibitors is added in every 500 μ L mycoprotein extract solutions
Thing, 4 μ L protein stabilisers, mix rearmounted standby on ice.
Wherein, 10% separation gel and 4% concentration glue are prepared in step 2,10% ammonium persulfate (AP) should be eventually adding, mix
Encapsulating immediately afterwards；When room temperature is higher, before AP is added, glue can be placed on to 4 DEG C of precoolings, then add AP, stood after mixing
That is encapsulating.
Wherein, deposition condition is in step 2：The initial value for setting electric current is 10-20mA, after sample enters separation gel,
Electric current is arranged to 20-40mA；Or constant pressure 60-80V when starting, treat that sample is thicker into separating, 100V is changed to, when hectograph bottom
When spacing with dye front is 0.5cm, electrophoresis terminates.
Wherein, mass spectrometry parameters in step 4：200 DEG C of capillary temperature, detection mode：Cation.
Present invention also offers the amino acid sequence for identifying No. 0/1 microspecies differential protein peptide fragment of tobacco black shank bacterium
SEQ ID NO：1-6.Wherein SEQ ID NO：1-3 is No. 0 microspecies specific proteins peptide fragment, SEQ ID NO：4-6 is No. 1 small
Species specificity albumen peptide fragment.
Embodiments of the present invention are described in detail using embodiment and accompanying drawing below, how skill is applied to the present invention whereby
Art means solve technical problem, and the implementation process for reaching technique effect can fully understand and implement according to this.
No. 0/1 biological strain protein extraction of tobacco black shank bacterium and the differential protein peptide fragment of identification
The first step：Bacterial strain is No. 0 and No. 1 biological strain of Phytophthora nicotianae Breda Phytophthora nicotianae, in
Plant protection research department of Dohanykutato Intezet of Academy of Agricultural Sciences of state isolates and purifies preservation.The solid oat medium in 28 DEG C of insulating boxs
Activation 7 days.
Second step：Extract black shank bacterium albumen：Extract solution is prepared, 2 μ L are added in mycoprotein extract solution cold every 500 μ L
Protease inhibitor cocktail, 4 μ L protein stabilisers, mix rearmounted standby on ice；100mg black shank bacterium mycelia is taken to be ground with liquid nitrogen
Mill, add mycoprotein extract solution；After mixing, 15-20min is vibrated under the conditions of 4 DEG C；At 4 DEG C, under the conditions of 14000rpm from
Heart 15min；Supernatant is quickly sucked to the clean centrifuge tube of another precooling, you can obtain black shank bacterium total protein；By above-mentioned albumen
- 80 DEG C of refrigerators are sub-packed in after extract is quantitative to save backup.
3rd step：Carry out SDS- polyacrylamide gel electrophoresises：
(1) vertical slab electrophoresis groove is installed：Operated by operation instructions.
(2) prepared with glue and gel slab
The separation gel of table 1 and concentration glue composition
| Separation gel (10%) | Concentrate glue (4%) | |
| dd H2O | 4.2mL | 3.1mL |
| Buffer | 2.5mL | 1.25mL |
| Acr+Bis 30.8% | 3.2mL | 0.7mL |
| 10%SDS | 100μL | 50μL |
The preparation of separation gel：10ml 10% separation gel is prepared by table 1, after mixing with pipettor by coagulant liquid add to it is long,
In slit between short glass plate, about 5cm (to comb tooth bottom about at 1cm)；Appropriate dd H are taken with 1mL pipettor2O, edge
Long glass wooden partition is slowly injected into, and about 3-4mm is high, to carry out water seal, makes the upper surface of separation gel smooth.After about 30min, gel with
There is the different boundary of refractive index in water seal interlayer, then it represents that gel polymerize completely, the water for the sealing that inclines, then is blotted with filter paper bar.
Pay attention to：AP should be eventually adding, encapsulating immediately after mixing；When room temperature is higher, before AP is added, glue can be placed on to 4 DEG C in advance
It is cold, AP is then added, encapsulating immediately after mixing.
Glue is concentrated to prepare：Prepare 4% concentration glue 5mL and be well mixed, concentration glue is added to separation gel (now with pipettor
It has polymerize) on, glass plate top edge distance about 0.5cm stops glue, should when sample slot template (comb) is inserted in concentration glue
Gently insert, not exert oneself too much.Gel polymerisation after about 30min, then 20-30min is placed, make gel " aging ".Carefully take out sample
Product slot template, moisture unnecessary in groove is sucked with fillet filter paper, and offset plate is removed from pouring the adhesive container and is fixed on vertical electrophoresis
On groove, buffer solution Tris- glycine pH8.3 is poured into upper and lower slot electrode and exceedes short slab 0.5cm, you can prepare sample-adding.
(3) processing of sample and sample-adding
The processing of sample：After sample liquid and sample buffer are mixed in equal volume, 3- is incubated in 100 DEG C of boiling water baths
5min, take out cooling sample-adding.
Sample-adding：Only add a kind of sample protein matter at the spill of each sample cell, if mixed protein must be known
Its relative molecular weight, injection volume is determined according to the thickness of the concentration of sample-adding protein and gel, general injection volume is 10-
25 μ L (or 5-15 μ g albumen).If sample is diluter, applied sample amount can be increased.The bubble in sample cell is removed with syringe needle.
During sample-adding, the syringe needle of micro syringe is gently added to the bottom of loading slot as far as possible, and syringe needle can not be by the glue surface of Baltimore groove
Stave, this step operates in electrode buffer.
(4) deposition condition：The initial value for setting electric current is 10-20mA, and after sample enters separation gel, electric current is arranged to
20-40mA；Or constant pressure 60-80V when starting, treat that sample is thicker into separating, be changed to 100V.When hectograph bottom and dye front
When spacing is 0.5cm, electrophoresis terminates.
(5) gel is peeled off and fixed：When terminating electrophoresis, gel version is taken out, and extracts the edge sealing pad of half out, should during extraction
Slowly, should not glue stave, then gently take out glass plate again, obtain complete gel.Carefully removed from following glass plate
Gel, gel is cut one jiao as sample-adding and indicated, gel slab is placed in big culture dish, fixer is added to culture dish
In, slowly rotate rotary shaker 2h.
(6) dyeing and decolouring：Dyeing liquor is added into culture dish, is taken out after about 1h, it is (fixed when should repeatedly be rinsed
Liquid), then (destainer) is decolourized, terminate to decolourize when seeing clearly protein band.
(7) mapped according to relative mobility, calculate sample molecular weight subunit.
4th step：The differential band of each regional black shank bacterium albumen is found according to electrophoretogram, carries out Mass Spectrometer Method
5th step：The protein band that 4 are obtained carries out FASP enzymolysis → extraction peptide hydrolysis → ESI mass spectrums → software data
Analysis → identification protein.
(1) FASP is digested：
Sample segment is taken in 100 μ L 25mM NH4HCO3, concussion dissolving.0.22 μm of membrane filtration, add 30 μ L SDT
95 DEG C of (4%SDS, 100mM Tris, 100mM DTT, pH 7.6), boil 5 minutes, after cooling, add 200 μ L UA (8M
Urea, 100mM Tris, pH8.5), it is transferred in super filter tube, 14000g centrifugation 15min, this step is repeated 3 times.Add 100 μ
L50mM IAA, room temperature lucifuge are reacted 30 minutes.100 μ LUA solution, 14000g centrifugation 30min are added, this step is repeated 3 times.
The HCO 3 of 100 μ L 25mM NH 4 are added, 14000g is centrifuged 30 minutes, and this step is repeated 3 times.Add 4 μ g Trypsin, 37 DEG C
React 20h.Renew centrifuge tube, centrifugation 14000g 15min；Add 40 μ L25mM NH4HCO3, 14000g 15min are centrifuged, are received
Collect filtrate.The redissolution of the formic acid of 40 μ L 0.1% is added after peptide fragment is lyophilized.
(2) mass spectral results database retrieval is identified
Peptide fingerprint and tandem mass spectrum data are integrated and pass through research tool Proteomics Discovery 1.4
(Thermo) retrieved for database Uniprot Phytophthora parasitica, searching algorithm Sequest
HT, parent ion error：20ppm, daughter ion error：0.8Da, leak enzyme site：2.Obtain differential protein peptide fragment amino acid sequence
And mass spectrogram is shown in Fig. 1-6.
All above-mentioned this intellectual properties of primarily implementation, the not this new product of implementation of setting limitation other forms
And/or new method.Those skilled in the art will utilize this important information, the above modification, to realize similar execution feelings
Condition.But all modifications or transformation belong to the right of reservation based on new product of the present invention.
The above described is only a preferred embodiment of the present invention, being not the limitation for making other forms to the present invention, appoint
What those skilled in the art changed or be modified as possibly also with the technology contents of the disclosure above equivalent variations etc.
Imitate embodiment.But it is every without departing from technical solution of the present invention content, the technical spirit according to the present invention is to above example institute
Any simple modification, equivalent variations and the remodeling made, still fall within the protection domain of technical solution of the present invention.
Claims (6)
- A kind of 1. method for extracting No. 0/1 microspecies differential protein peptide fragment of tobacco black shank bacterium, it is characterised in that this method includes Following steps：The first step：Extract the total protein of black shank bacterium；Second step：SDS- polyacrylamide gel electrophoresises are carried out with the albumen obtained in step 1；3rd step：The running gel figure obtained according to step 2 picks out differential band；4th step：The band that 3 are obtained digests, and extracts peptide hydrolysis, carries out LC-MS analyses；5th step：Mass spectral results are used with Mascot2.2 software retrieval associated databases, obtains differential protein peptide fragment.
- 2. the method for extraction No. 0/1 microspecies differential protein peptide fragment of tobacco black shank bacterium, its feature exist as claimed in claim 1 In：Extract solution is prepared in step 1,2 μ L protease inhibitor cocktails, 4 μ L albumen are added in every 500 μ L mycoprotein extract solutions Stabilizer, mix rearmounted standby on ice.
- 3. the method for extraction No. 0/1 microspecies differential protein peptide fragment of tobacco black shank bacterium as claimed in claim 1 or 2, it is special Sign is：10% separation gel and 4% concentration glue are prepared in step 2,10% ammonium persulfate (AP) should be eventually adding, after mixing immediately Encapsulating；When room temperature is higher, before AP is added, glue can be placed on to 4 DEG C of precoolings, then add AP, encapsulating immediately after mixing.
- 4. the method for the extraction No. 0/1 microspecies differential protein peptide fragment of tobacco black shank bacterium as described in claims 1 to 3, it is special Sign is：Deposition condition is in step 2：The initial value for setting electric current is 10-20mA, after sample enters separation gel, by electric current It is arranged to 20-40mA；Or constant pressure 60-80V when starting, treat that sample is thicker into separating, 100V is changed to, when hectograph bottom and dyestuff When the spacing in forward position is 0.5cm, electrophoresis terminates.
- 5. the method for extraction No. 0/1 microspecies differential protein peptide fragment of tobacco black shank bacterium, its feature exist as claimed in claim 1 In：Mass spectrometry parameters in step 4：200 DEG C of capillary temperature, detection mode：Cation.
- 6. one kind extraction No. 0/1 microspecies differential protein peptide fragment of tobacco black shank bacterium, it is characterised in that identify tobacco black shank The amino acid sequence of No. 0/1 biological strain differential protein peptide fragment of bacterium is respectively SEQ ID NO：1-6.Wherein SEQ ID NO：1- 3 be No. 0 microspecies specific proteins peptide fragment, SEQ ID NO：4-6 is No. 1 microspecies specific proteins peptide fragment.
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