CN101382520B - Classification and determination method for bacillus thuringiensis strains by mass-spectrometric technique - Google Patents
Classification and determination method for bacillus thuringiensis strains by mass-spectrometric technique Download PDFInfo
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Abstract
The invention discloses a method for carrying out classification and determination on bacillus thuringiensis strains by using the mass-spectrometric technique, which comprises the following steps: (1) the bacillus thuringiensis strains are cultured until crystals and spores are completely released from mother cells, and purification is carried out to the mixture of the spores/parasporal crystals of the strains; (2) by imbedding the crystallin mixture generated by the bacillus thuringiensis strains into the polyacrylamide gel block and combining with LC-MS/MS, the enzymolysis compound peptide in the gel is analyzed to determine the composition of the protoxin in the crystalline; and (3) the gene order of the determined new protein is surmised according to the mass-spectrum analysis result, and clone and expression are carried out to the corresponding new genes, thereby providing a basis for the phyletic classification of the bacillus thuringiensis strains.
Description
Technical field
The present invention relates to a kind of bacillus thuringiensis strains classification authentication method, specifically, relate to a kind of method of utilizing mass-spectrometric technique that bacillus thuringiensis strains is classified and identified.
Background technology
Thuringiensis (Bacillus thuringiensis is called for short Bt) is a kind of gram-positive bacterium of extensive distribution, belongs to the wax Bacillus.Insecticidal crystal protein is the main constituent of thuringiensis at the produced simultaneously parasporal crystal of brood cell's formation, and 10 purpose insects such as Lepidoptera in the Arthropoda (Lepidoptera), coleoptera (Coleoptera), Diptera (Diptera) and protozoa (Protozoa), Platyhelminthes (Platghelminthes), Nemathelminthes agriculture and forestry injurious insects such as (Nemathelminthes) are had special toxic action.Parasporal crystal enters the parent toxin that dissolving takes place behind the alimentary canal of sensitive insect and discharge 27-140kD.Under the effect of midgut proteinase, parent toxin is activated as the toxic polypeptide of 23-70kD.Toxin and midgut BBM bubble (BBMV, Brush border membrane vesicle) is had an effect and form the duct on cell membrane subsequently, destroys the osmotic equilibrium of cell, causes lysis, finally causes larva death.
From Schnepf etc. first from the Bt bacterial strain clone cry gene so far, existing more than 400 kind of insecticidal crystal protein is in the news.1989, H
Fte and Whiteley are according to the homology of the amino acid sequence of 42 insecticidal crystal proteins having reported at that time and the difference of insecticidal spectrum, the HW categorizing system has been proposed, be about to the Bt insecticidal crystal protein and be divided into two big classes, crystal protein gene family (crystal protein genes), be Cry albumen, comprise CryI, CryII, CryIII, CryIV and extracellular dissolubility crystalline protein family (cytolytic protein), i.e. Cyt albumen.Along with going deep into of Bt molecular biology research, new cry gene is constantly separated, the clone, and some Cry albumen is fit to original HW system, and Cry albumen but more then is not suitable for this categorizing system.For addressing this problem, nineteen ninety-five, the insecticidal crystal protein and the unnamed gene council that form by N.Crickmore etc. in the invertebrate pathology annual meeting, have been set up, the council has proposed new categorizing system thus, promptly all only according to the difference of ICPs amino acid sequence homology, and consider that no longer the difference of insecticidal spectrum names from the crystalline protein with insecticidal activity of thuringiensis.Homology adopts arabic numeral to name, as Cry1, Cry2 at the insecticidal crystal protein below 45%; Homology adopts the capitalization English letter name, as Cry1A, Cry1B between 45%-78%; Homology adopts the name of small letter English alphabet, as Cry1Ac, Cry1Ab between 78%-95%; Homology is also used arabic numeral more than 95%, as Cry1Aa1, Cry1Aa2.The gene of coded insect-killing crystalline protein is with corresponding italic and the name of small letter initial, as cry1Ac1.To 2007, the insecticidal crystalline gene kind of Bt extended to 56 groups, more than 100 subgroup.At present, this system is also continuing expansion.
Thuringiensis becomes the main production bacterial strain of present biological pesticide because containing abundant insecticidal crystal protein, the evaluation of bacterial strain and insecticidal proteins is necessary (finding new bacterial strain, new albumen).It is carried out proteome analysis, help to study the associated protein that in the thuringiensis sweat crystalline protein is formed with material impact.The Bt bacterial strain is main active insecticidal components with the insecticidal crystal protein of its generation, helps to predict all-sidedly and accurately its insecticidal activity so identify the crystalline protein composition of Bt bacterial strain.Mainly be that (High-Performance Liquid Chromatography HPLC) carries out by Western blotting and high performance liquid chromatography at present to the evaluation of Bt insecticidal crystal protein.But adopt liquid chromatography and Western blotting to identify that all there is the shortcoming of length consuming time in Bt parent toxin composition, need provide standard items as liquid chromatography, therefore the prerequisite that detects is the pure sample product that make up the expression strain of destination protein and obtain this albumen, and Western blotting also needs to obtain the destination protein of purifying, and animal such as immune rabbit, obtain antiserum.In addition, because the Bt parent toxin has sequence homology more than 78% three grades of levels, also limited the application of classic methods such as liquid chromatography and Western blotting on parent toxin is identified.
Along with the development of mass-spectrometric technique, the advantage of mass spectrometry method is applied to the proteome analysis of each biological tissue gradually.2002, Cry1Aa, Cry1Ab, Cry1Ac and the Cry2Aa parent toxin that the HD1-Dipel bacterial strain produces, the Cry1Ac that the HD73 bacterial strain produces are analyzed and identified to utilization SDS-PAGE such as Ranasinghe series connection ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOFMS); Crystalline protein components such as the Cry1Hb that the am2 bacterial strain produces, Cry1Db parent toxin have shown the advantage of mass-spectrometric technique on the Bt parent toxin is identified for the first time.2006, employing SDS-PAGE such as Lee formed in conjunction with the crystalline protein that ESI-Q-TOF has analyzed the Btkonkukian subspecies, though identified two kinds of memebrane proteins, do not identify insecticidal crystal protein.
Though mass spectrometry method is applied to the evaluation of different strains crystalline protein, memebrane protein gradually, but only be confined to the analysis in the proteomics field, there is not further gene order according to the new albumen of mass spectrum results presumption, and in conjunction with the clone of new gene, express the equimolecular biological method, for the genealogical classification of bacillus thuringiensis strains provides foundation, and then provide technical support for the production of biological pesticide.
Summary of the invention
The object of the present invention is to provide a kind of new method of utilizing mass-spectrometric technique that bacillus thuringiensis strains is classified and identified, this method can identify quickly and accurately with the closely-related crystalline protein of Bt bacterial strain insecticidal activity to be formed, thereby can classify to the Bt bacterial strain more quickly and accurately, determine to insecticidal properties from identification of strains, somatotype, can settle at one go.
The objective of the invention is to be achieved through the following technical solutions, it may further comprise the steps: (1) is cultivated bacillus thuringiensis strains and is discharged fully from mother cell up to crystal and brood cell, and bacterial strain brood cell/parasporal crystal potpourri is purified; (2) the crystalline protein potpourri that produces by the embedding bacillus thuringiensis strains is in the polyacrylamide gel piece, analyzes the compound peptide section of enzymolysis in the glue in conjunction with LC-MS/MS, identifies the composition of parent toxin in the crystalline protein; (3) gene order of the new albumen that identifies according to the mass spectrum results presumption, and corresponding new gene cloned, expresses, thus foundation provided for the genealogical classification of bacillus thuringiensis strains.
The inventive method can identify quickly and accurately with the closely-related crystalline protein of Bt bacterial strain insecticidal activity to be formed, thereby more quickly and accurately the Bt bacterial strain is classified.Determine to insecticidal properties from identification of strains, somatotype, can settle at one go, thereby the production that can be the high-quality biological pesticide provides powerful technical support.
Embodiment
The invention will be further described below in conjunction with embodiment, but protection scope of the present invention can not be thought and is confined to following embodiment.Concerning the person of an ordinary skill in the technical field, under the basic premise that does not break away from the present invention's design, can also make some simple deductions or be equal to replacement, these are equal to alternative and still will be regarded as within protection scope of the present invention.
1, Bt strain culturing
With the bacterial strain rinsed with sterile water, evenly coat the LB solid plate, to be inverted for 30 ℃ and to cultivate 8h, picking list bacterium colony continues the line purifying 1 time.Choose single bacterium colony behind the 8h and connect LB inclined-plane or dull and stereotyped the cultivation.Second day switching 5mL LB shake-flask culture carries out culture presevation behind the 8h, and presses 2% inoculum concentration switching 50mL G-Tris nutrient culture media.30 ℃, 2000rpm cultivates 60h, discharges fully from mother cell up to crystal and brood cell.
2, the purification of Bt bacterial strain brood cell/parasporal crystal potpourri
With the Bt inoculation in the LB fluid nutrient medium, shaken cultivation (30 ℃ 200rpm) behind the 8h, are transferred in the fresh G-Tris fluid nutrient medium of 50mL and shake (30 ℃ of bottle shaken cultivation, 200rpm) about 60h, from mother cell, discharge fully up to crystal and brood cell.Bt4.0718, HD1, the strictness of HD73 bacterial strain are by identical amount operation repetitive.Isopyknic nutrient solution 10, the centrifugal 10min of 000rpm, collecting precipitation, the NaCl washing of 0.5M is once; The precipitation of centrifugal acquisition suspends with physiological saline, 4 ℃ of ultrasonic Treatment suspending liquid 3 times; Suspending liquid after the ultrasonic Treatment is centrifugal and with physiological saline washing 2 times, and 5% acetone and distilled water respectively wash twice, and the gained precipitation is the brood cell/parasporal crystal potpourri of purification.This potpourri branch is filled in the Ep pipe of 1.5mL, places the vacuum freeze drying instrument to carry out drying, gained dry powder is deposited standby for 4 ℃.
3, the SDS-PAGE of Bt bacterial strain parasporal crystal analyzes
Get a crystalline protein working sample, suspend with an amount of ultrapure water, add equal-volume 2 * SDS sample-loading buffer, 100 ℃ are boiled 5min, 12, the centrifugal 5min of 000rpm gets supernatant and carries out the SDS-PAGE analysis, and concentrated gum concentration is 5%, working voltage is 50V, resolving gel concentration is 10%, and working voltage is 100V, dyes with Coomassie brilliant blue R-250 after electrophoresis finishes.Gel after the decolouring scans with gel scanner, utilizes relative mobility, molecular weight and the protein concentration of Gel-Pro analyzer computed in software albumen.The compound method that concentrates glue and separation gel is as follows:
| 5% concentrates glue | Volume (mL) | 10% separation gel | Volume (mL) |
| Water | 5.500 | Water | 5.000 |
| 30% acrylamide | 1.300 | 30% acrylamide | 5.900 |
| 0.5M?Tris-HCl，pH6.8 | 1.000 | 1.5M?Tris-HCl，pH8.8 | 3.800 |
| 10％SDS | 0.080 | 10％SDS | 0.150 |
| 10％APS | 0.080 | 10％APS | 0.150 |
| TEMED | 0.008 | TEMED | 0.009 |
4, the embedding of Bt crystalline protein sample
Get the brood cell/parasporal crystal potpourri of the Bt bacterial strain of 1 purifying, adding an amount of ultrapure water suspends, and the 2 * embedding protein sample that adds equivalent is handled damping fluid, mix, boiling water bath 5min, the centrifugal 5min of 13200rpm draws in supernatant to the new Ep pipe, is made into the albumen biased sample of 1-5 μ g/ μ L concentration.
Get 20 μ g albumen biased samples in 1 new 0.5mL Ep pipe, the acrylamide 20 μ L of adding 30%, replenish ultrapure water to cumulative volume 48 μ L, mix, the 10%APS that adds the fresh configuration of 1 μ L, 1 μ L TEMED stirs, and gets 25 μ L potpourri branches and is filled in another 0.5mL Ep pipe, room temperature is placed 1h, makes 12% acrylamide blob of viscose.
5, the in-situ enzymolysis of Bt crystalline protein sample
The polyacrylamide blob of viscose that is embedded with the crystalline protein sample for preparing is immersed in the immobile liquid spends the night, changed 2 times immobile liquid in second day again.The blob of viscose that proteopexy is good is cut into the 2-3 fritter, carries out trypsase original position enzymolysis.
6, LC-MS/MS analyzes Bt bacterial strain crystalline protein in-situ enzymolysis peptide section
(1) ESI-tc-MS/MS analyzes the composition of parent toxin in the Bt crystalline protein
The peptide section that enzymolysis is good is dissolved in 33 μ L and contains in the aqueous solution of 0.1% formic acid, and the centrifugal 15min of 13200rpm gets in the sample bottle of 30 μ L to ESI-Trap, carries out CapLC-MS/MS and analyzes.
Electron spray-quadrupole rod-flight time tandem mass spectrometer (Electrospray ionization quadruple time-of-flight of Britain Micromasss company is selected in mass spectrophotometry for use, be called for short ESI Q-TOF) carry out, be equipped with capillary liquid chromatography instrument (CapLC) and receive liter (Nano) spraying source.All mensuration are all carried out under positive ion mode.Series connection fragment with Glu-fib is proofreaied and correct, and quality error is scholar 0.1u.Atomization gas is a nitrogen, and collision gas is argon gas, and the source temperature is 80 ℃, and taper hole voltage is 45V, spray nozzle voltage 3000V, and the MCP detector voltage is 2700V.The MS of instrument and the conversion of MS/MS are by the control of Automated Data Dependent Acquisition (DDA) pattern in the instrument Control Software.An automatic sample handling system equipment equipment C18 desalination pre-column (5mmlong * 300 μ m i.d.) and a C18 capillary column (150mmlong * 75 μ m i.d.).Preceding 5min behind sample introduction is by the pre-column desalination and concentrate flow velocity 250nL/min.The later time is carried out the separation of peptide section by gradient elution through the C18 capillary column, and flow velocity is 200nL/min.Mobile phase A liquid is the aqueous solution that contains 0.1% formic acid/4.9%ACN/95%, and B liquid is for containing 0.1% formic acid/4.9%H
2O/95%ACN (v/v/v).Sample is used the desalination of C liquid in pre-column after, analytical column carries out gradient elution in order to following gradient: 5-40min B liquid concentration rises to 50% from 5%, and 40-50min B liquid concentration rises to 95%, 50-60min from 50%, B liquid concentration remains on 95%, 60-70min A liquid equilibrium analysis post.Peptide section after the separation directly enters ion gun, once selects the peptide section of three maximum intensitys (above the thresholding of setting and with 2 or 2 above positive charges) to carry out the MS/MS analysis.
(2) database search of protein is identified
The data input base (NCBInr) that ESI-Q-TOF obtains uses the tandem mass spectrometry data search function (MS/MS Ions Search or Sequence query) in the Mascot software to search for (http://www.matrixscience.com).The search of database selects the fixing selected parameter of database search of modifying of following parameters as follows: fixedly be modified to Carbamidomethyl (Cys), nickase is Trypsin, permission maximum not restriction enzyme site (Missed cleavages) number is 1, variablely be modified to the methionine oxidation, the quality tolerance of peptide section is 1.2Da, and the quality tolerance of fragmention is 0.6Da.The score of peptide section (Individual ion scores) surpasses the peptide section that is regarded as accurate evaluation of its threshold value (threshold), and the confidence level that has is greater than 95%.
PTS for first coupling is the highest, and mates the candidate albumen that many scores surpass the peptide section of threshold value, does not carry out artificial inspection, just thinks that this protein has obtained identifying accurately.For the candidate albumen of score second, whether it mates the new peptide section that has 1 above high score albumen not mate through manual detection, thereby determines result's choice.If the candidate albumen coupling that PTS is lower has the polypeptide that does not occur more than 1 or 1 in the higher albumen of score, think that then this protein is accurately identified, otherwise given up.Carry out having in the Bt bacterial strain evaluation of the parent toxin of high homology according to this principle.
The protein of identifying compares by the Swiss-Prot/TrEMBL in website http://www.ncbi.nlm.nih.gov/Entrez and the http://www.expasy.org/sprot/.
7, the cloning and expression of new protein gene sequence
For the new albumen of accurate evaluation, be foundation with its polypeptid acid sequence of identifying, oppositely infer its gene order according to codon, and as template design primer.Total DNA carries out pcr amplification to this bacterial strain, and clone PCR products, obtains the gene order of new albumen.According to this sequence full length sequence in later stage that further increases, and express, further accurately identify new albumen, determine the classification position of the new bacterial strain of bacillus thuringiensis.
Claims (1)
1. the method for utilizing mass-spectrometric technique that bacillus thuringiensis strains is classified and identified, it is characterized in that, may further comprise the steps: (1) is cultivated bacillus thuringiensis strains and is discharged fully from mother cell up to crystal and brood cell, and bacterial strain brood cell/parasporal crystal potpourri is purified; (2) the crystalline protein potpourri that produces by the embedding bacillus thuringiensis strains is in the polyacrylamide gel piece, analyze the compound peptide section of enzymolysis in the glue in conjunction with LC-MS/MS, identify the composition of parent toxin in the crystalline protein: the peptide section that enzymolysis is good is dissolved in 33 μ L and contains in the aqueous solution of 0.1% formic acid, the centrifugal 15min of 13200rpm, get in the sample bottle of 30 μ L to ESI-Trap, carry out CapLC-MS/MS and analyze; All mensuration are all carried out under positive ion mode; Series connection fragment with Glu-fib is proofreaied and correct, and quality error is scholar 0.1u, and atomization gas is a nitrogen, and collision gas is argon gas, and the source temperature is 80 ℃, and taper hole voltage is 45V, spray nozzle voltage 3000V, and the MCP detector voltage is 2700V; The MS of instrument and the conversion of MS/MS are by the control of the Automated Data Dependent Acquisition pattern in the instrument Control Software; An automatic sample handling system equipment C18 desalination pre-column of equipment and a C18 capillary column; Preceding 5min behind sample introduction is by the pre-column desalination and concentrate flow velocity 250nL/min; The later time is carried out the separation of peptide section by gradient elution through the C18 capillary column, and flow velocity is 200nL/min; Mobile phase A liquid is the aqueous solution that contains 0.1% formic acid/4.9%ACN/95%, and B liquid is for containing 0.1% formic acid/4.9%H
2O/95%ACN (v/v/v); Sample is used the desalination of C liquid in pre-column after, analytical column carries out gradient elution in order to following gradient: 5-40min B liquid concentration rises to 50% from 5%, and 40-50min B liquid concentration rises to 95%, 50-60min from 50%, B liquid concentration remains on 95%, 60-70minA liquid equilibrium analysis post; Peptide section after the separation directly enters ion gun, once selects the peptide section of three maximum intensitys to carry out the MS/MS analysis; (3) for the new albumen of accurate evaluation, be foundation, oppositely infer its gene order according to codon with its polypeptid acid sequence of identifying, and as template design primer; Total DNA carries out pcr amplification to this bacterial strain, and clone PCR products, obtains the gene order of new albumen; According to this sequence full length sequence in later stage that further increases, and express, further accurately identify new albumen, determine the classification position of the new bacterial strain of bacillus thuringiensis.
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| EP1781701A2 (en) * | 2004-08-20 | 2007-05-09 | Wyeth | Progesterone receptor structure |
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| EP1781701A2 (en) * | 2004-08-20 | 2007-05-09 | Wyeth | Progesterone receptor structure |
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| 丁学知等.苏云金芽孢杆菌CB4?7D 菌株晶体毒素性质的研究.《农业生物技术学报》.2005,第13卷(第3期),365-371. * |
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