CN1167790C - High-toxicity gene engineering bacteria WG001 of Bacillus thuringiensis and its production process and product - Google Patents

Abstract

The present invention relates to construction, production technology and a product of bacillus thuringiensis gene engineering bacteria, which belongs to the technical field of microbial pesticides. The present invention is characterized in that recombinant plasmids pBMB1808 containing crylAa10-P20 genes are transferred into a high-toxicity strain YBT-1520 of cotton bollworms to obtain a strain of high-toxicity gene engineering bacteria WG001, and the strain of high-toxicity gene engineering bacteria WG001 is suitable for producing microbial pesticides (a kind of bacillus thuringiensis is preserved in CCTCC in 13rd September, 2001, and the preservation number is M201033). The strain carries with crylAa, crylAb and crylAc insecticidal crystal protein genes. Compared with natural strains, the yield of the insecticidal crystal proteins of WG001 is enhanced by more than one time, and the yield of parasporal crystals is increased by 2.1 times. In addition, the insecticidal crystal proteins of WG001 have good prevention effect on diamondback moths and cotton bollworms.

Description

Thuringiensis gene engineering bacteria WG001 and production technique and product

Technical field

The invention belongs to the microbial pesticide technical field, be specifically related to be used for the seed selection of the high virulence thuringiensis gene engineering bacterial strain of microbial pesticide, its production technique and products thereof exploitation.

Background technology

The production of insecticide Bacillus thuringiensis be unable to do without bacterial strain, and bacterial strain performance quality directly affects quality.Be separated to first bacterial strain of Tribactur from 1901 in the last hundred years, worldwide separation screening arrives the Tribactur wild strain of a large amount of excellent propertys and is used as the production bacterial classification, not only improve fermentation titer and insecticidal toxicity greatly, and widened its insecticidal activity spectrum greatly.But, production and application practice over past ten years also prove, also have some drawbacks with natural bacterial strain inevitably as the thuringiensis praeparatum preparation of producing bacterium, outstanding behaviours exists: specificity is too strong, and is relatively poor to the control effect of aged insect and the compound generation of various pests; Lasting period is shorter; The use repeatedly of big area unitary agent can cause the generation of insect-resistant etc.Obviously, be difficult to overcome these drawbacks by further separating new bacterial strain from nature.Therefore, from the middle and late eighties,, make up the direction that the recombinant bacterial strain with premium properties becomes domestic and international microbial pesticide development along with to Tribactur molecular biology and genetic further investigation.Engineering bacteria commodity preparation is gone into operation in succession as MVP, Foil.Gone through since the nineties to register or enter the later stage field test have 10 surplus kind of genetic recombination sterilant.

With the first-generation product of Tribactur wild-type production, though widespread use, and obtained the better prevention effect, and remain further raising aspect tiring in fermentation and quality product, thus can improve output, reduce cost, improve prevention effect, further the broadened application scale.

Summary of the invention

The objective of the invention is to overcome the prior art deficiency, the genetic engineering bacterium that is used to produce microbial pesticide by gene engineering method seed selection from Tribactur, use this genetic engineering bacterium fermentative production microbial pesticide, to improve the fermentation level and the quality product of microbial pesticide.

The present invention is achieved through the following technical solutions:

Feature of the present invention comprises the screening method of thuringiensis gene engineering strain WG001, the liquor of deep liquid fermentation process and thuringiensis praeparatum preparation and the preparation of powder product.

Starting strain of the present invention is to use the patented strain YBT-1520 (Chinese invention patent that insects such as bollworm, small cabbage moth is had high virulence, the patent No. is ZL 95110049.1) do recipient bacterium, with accessory protein gene P20 and the insecticidal crystalline gene Cry1Ac10 and the carrier reorganization of dissociating, be transferred in the recipient bacterium, filter out a kind of efficient gene engineering bacterium WG001.By to Research on Process, develop genetic engineering bacterium pesticide product of new generation.

Below concrete ins and outs of the present invention are further described:

A kind of engineering strain of producing microbial pesticide, have cry1Aa, cry1Ab and cry1Ac insecticidal crystalline gene, it is characterized in that described bacterial strain is Tribactur (Bacillus thuringiensis) WG001, CCTCC M 201033.

A kind of method for preparing thuringiensis gene engineering bacteria WG001 and products thereof prepares according to following steps:

(1) with the WG001 inoculation on the test tube slant substratum, cultivate for 30 ℃ and activated in 24 hours, transfer then in eggplant bottle inclined-plane, cultivated 72 hours for 30 ℃, be to produce and use slant strains;

(2) will add sterilized water in the cultured eggplant bottle slant strains, make every milliliter of suspension that contains 0.2-1.0 hundred million viable cell, change over to then in the 500ml serum bottle, syringe needle or the inoculated tube of bacterium beyond the Great Wall went out, bottle is put in 80 ℃ of water-baths insulation 15-20 minute bacteria suspension is heat-treated and with pressure differential method this bacteria suspension is inserted seeding tank, this jar medium pH is 6.5-7.5 during inoculation;

(3) press seeding tank volumetrical 60-80% and drop into substratum, this substratum prepares according to following proportioning: soybean cake powder 3.0-5.0%, add α-Dian Fenmei 0.05 gram of 2000IU/g with every gram W-Gum, at 80 ℃, liquefaction W-Gum 1.0-5.0%, the yeast powder 0.1-1.0% for preparing under the condition, corn steep liquor 1.0-4.0%, peptone 0.1-0.5%, inorganic salt 0.1-2.0%, bubble enemy 0.05-0.2%, all raw materials all needed the 80-100 mesh sieve, be 7.5-9.0 before making the pH sterilization, the sterilization back is 6.5-7.5, with 0.1mPa; 121 ℃ high pressure steam sterilization kept 30 minutes, be cooled to 30-35 ℃ after, insert the bacteria suspension that activated inclined-plane lawn is made;

(4) press fermentor tank volume 60-70% and drop into substratum, this substratum prepares according to following proportioning: soybean cake powder 3.0-5.0%, add α-Dian Fenmei 0.005 gram of 2000IU/g with every gram W-Gum, liquefaction W-Gum 1.0-5.0%, the yeast powder 0.1-1.0% that under 80 ℃ of conditions, prepares, corn steep liquor 1.0-4.0%, peptone 0.1-0.5%, inorganic salt 0.1-2.0%, bubble enemy 0.05-0.2%, all raw materials all needed the 80-100 mesh sieve, be 7.5-9.0 before making the pH sterilization, the sterilization back is 6.5-7.5; With 0.1mPa, 121 ℃ high pressure steam stirs under the 200-250rpm condition, keeps sterilization in 30 minutes, is cooled to after 30-33 ℃ and moves into seed liquor by 1% of the volume that feeds intake;

(5) seed tank culture, at temperature 30-32 ℃, tank pressure 0.03-0.05mPa, air flow is pressed V/V1: 0.6-1: 0.8, stir under the 200-250rpm condition and cultivated 6-12 hour;

(6) fermentor cultivation, at temperature 28-32 ℃, tank pressure 0.03-0.08mPa, air flow 1 is pressed V/V1: 0.6-1: 1.2, stir under the 200-250rpm condition and cultivated 24-48 hour, there is brood cell's capsule of 20-40% to break with microscopy, just can put jar;

(7) fermented liquid liquorization: fermented liquid pH is transferred to 4.5-6.5 with HCl, according to the fermented liquid level, adopt whizzer that fermented liquid is concentrated 0.5-1 doubly, add 3-10% NaCl, 0.1-0.5% Sodium Benzoate and No. 100 emulsifying agents of 1-3% farming breast then, through check, toxicity evaluation is reached more than the 8000IU/ μ l, be the liquid dosage form biotic pesticide after the packing;

(8) in concentrated broth, add No. 100 emulsifying agents of an amount of diatomite or lime carbonate and 1-3% farming breast, spray-dried operation, the preparation water content is controlled at below 4%, and through check, make toxicity evaluation reach 16000IU/mg respectively and more than the 32000IU/mg, be the powder-type biotic pesticide after the packing.

Described in the present invention fermentation tank culture medium also can adopt following formulation, its prescription is: soybean cake powder 3.5-5.0%, add α-Dian Fenmei 0.005 gram of 2000IU/g with every gram W-Gum, liquefaction W-Gum 2.0-5.0%, the yeast powder 0.1-0.5% that under 80 ℃ of conditions, prepares, corn steep liquor 1.0-3.0%, peptone 0.2-0.5%, inorganic salt 0.1-1.0%, bubble enemy 0.05-0.2%, all raw materials all needed the 80-100 mesh sieve, making the preceding pH of sterilization is 7.5-9.0, and the sterilization back is 6.5-7.5.

In preparation process of the present invention, when the fermentation of fermentor tank entered logarithmic phase, its air flow was pressed V/V 1: 1.0-1: 1.2 adjust.

The biology of WG001 bacterial strain and hereditary property are described:

Biological characteristics: thalline direct rod shape, nourishing body is chain or Dan Sheng, long 3-5 micron, wide 1-1.2 micron, Gram-positive, the brood cell is cylindric or sub-elliptical, and end is given birth to partially, and brood cell's capsule does not expand, bacterium colony circle on beef-protein medium, edge-smoothing, the plentiful wax shape that is of lawn, growth temperature 10-45 ℃, optimum growth temperature 26-32 ℃, appropriate pH 6.8-7.4, facultative suspicion oxygen is grown in containing the beef peptone substratum of 7%NaCl, at ribose, glucose, fructose, produce acid in maltose and the Zulkovsky starch substratum, the clark and Lubsreaction positive, parasporal crystal is long, wide all than the parasporal crystal increase by 32% of YBT-1520, be two pyramids.In fermentation culture, can vigorous growth in the substratum that with multiple agricultural byproducts is raw material, produce a large amount of parasporal crystals, to the toxicity evaluation of insect between 4000IU/mg-6000IU/mg.

Genetics characteristic: the present invention is to be the genetic engineering bacterium that starting strain obtains with the natural bacterial strain YBT-1520 of Tribactur, and its flagellar antigen serotype is H3ab, and parasporal crystal is made up of 130KDa and 65KDa crystallin.Through succeeding transfer culture, auxiliary gene can be in engineering bacteria stable existence, express normal, to the growth moment-less influence of recipient bacterium.

Embodiment

Structure and the preservation of embodiment 1 thuringiensis gene engineering bacteria WG001:

Starting strain of the present invention is to use patented strain YBT-1520 to high virulence of insect such as bollworm, small cabbage moths (referring to the Chinese invention patent specification sheets, patent No. ZL 95110049.1) do recipient bacterium, with accessory protein gene P20 and the insecticidal crystalline gene cry1Ac10 and the carrier reorganization of dissociating, be transferred in the recipient bacterium, screening obtains efficient engineering strain WG001 of the present invention.

Obtaining on the basis of supper toxic strain, first rear clone cry2Ab, cry1Ac, 2 cry1Ab, cyt1A, cry1c, 2 cry1Ea totally eight insecticidal crystalline genes.To wherein cry2Ab, cry1Ac and 2 cry1Ea totally 4 genes carried out complete sequence analysis, the result shows cry1Ac10 and has four bases different to deliver the highest cry1Ac gene of homology, this gene is accepted by GenBank, be numbered: AJ002514, simultaneously named first gene of China that this is named by this council by international thuringiensis gene.

Use the new PCR clone technology of a cover that the applicant sets up, successfully from Israel subclass, cloned accessory protein gene P20, and confirmed that P20 is to lepidopteran specific gene cry1Ab with to the synergism of wild strain insecticidal crystal protein

The present invention is a recipient bacterium with the natural bacterial strain YBT-1520 of Tribactur, transforming the recombinant plasmid pBMB1808 that will contain the cry1Ac10-P20 gene by electricity is transformed in this bacterial strain, obtain transformant WG001 with the dull and stereotyped 28 ℃ of screenings of the erythromycin of 25 μ g/ml, extract plasmid, by plasmid map, Southern hybridization and polyacrylamide gel electrophoresis checking transformant.Make PCR with the specificity primer, qualification result shows that the transformant plasmid is amplified out the These positive bands of 583bps.Plasmid with WG001 goes back to bacillus coli DH 5 alpha once more in addition, obtains to contain pBMB1808 intestinal bacteria transformant again.These presentation of results pBMB1808 has been transferred in the natural bacterial strain.The WG001 bacterial classification through the LB activation of spending the night, 1% is transferred in 20mlPM erythromycin substratum earlier then, and 28 ℃ of thalline brood cells that are cultured to more than 90% come off, and quantitative sampling is done polyacrylamide gel electrophoresis (SDS-PAGE) analysis.The result shows that the expression amount of insecticidal crystal protein of the 130KDa of WG001 obviously improves, and bio-imaging system density scanning quantitative analysis shows that the expression amount of WG001 is 2.14 times of YBT-1520.

The method that the recombinant plasmid pBMB1808 electricity of the described cry1Ac10-P20 of containing gene is transformed into natural bacterial strain YBT-1520 comprises:

With 272mM sucrose, the solution of 15% glycerine preparation at the mid-50-80 μ of 0.4cm electricimpulse cup l competent cell, adds plasmid 5 μ l (about 1 μ g) as damping fluid.Electricimpulse adopts 25 μ F, 200 Ω, and the condition electric shock of 6.25-9KV/cm is once.

Described screening and authentication step of carrying out transformant by plasmid map, PCR and polyacrylamide gel electrophoresis (SDS-PAGE) comprises:

With 1% peptone, 0.5% yeast powder, 1%NaCl, the nutrient solution of the pH7.2 activated spawn of spending the night is again with activation bacterium liquid inoculation (1/100) LB nutrient solution.Treat thalli growth to mid-log phase, collect thalline, (10mM Tris.Cl, 1mM EDTA pH8.0) wash bacterium once to TE.Thalline is suspended from 100 μ l solution I (50mM Glucose, 25mM Tris.Cl (pH8.0), 10mM EDTA), adds 5-10 μ l N,O-Diacetylmuramidase (20mg/ml), and later operation is undertaken by the alkaline process plasmid extraction.

The method that described colibacillary plasmid extraction follows conventional lines.

The condition of described PCR is: 94 ℃ 1 minute, 49 ℃ 1 minute, 72 ℃ 1 minute, 25 circulations.

Described DNA-RNA hybridizing method comprises: the plasmid electrophoresis adopts 0.55% sepharose, and Hybond membrane uses nitrocellulose filter (Hybond), specifically changes film step and hybridization and colour developing according to a conventional method.

Described polyacrylamide gel electrophoresis method comprises: L ₈200 μ l are centrifugal for the substratum fermented liquid, and precipitation respectively washes twice with 1MNaCl and sterilized water, and throw out is suspended from the 1ml sterilized water again, getting 100 μ l mixes with equivalent sample-loading buffer (2X), handled 3 minutes in the boiling water, get 10-20 μ l point sample, solution preparation and other operations are according to a conventional method.

The spawn culture of embodiment 2 Tribactur engineering bacteria WG001, go down to posterity and preservation:

With the WG001 bacterial strain on the beef-protein medium inclined-plane in 30 ℃ of cultivations, can do short term storage to this bacterial strain at 4 ℃; With freeze pipe or the phase preservation of sand pipe range, as original strain.Producing bacterial classification needs to transfer from original strain, is inoculated on the beef-protein medium inclined-plane.

The production technique of embodiment 3 Tribactur engineering bacteria WG001 sterilants

The WG001 bacterial strain is divided into liquid submerged fermentation and preparation two portions as the zymotechnique of biotic pesticide: liquid submerged fermentation and preparation process are as follows:

One, strain preparation:

1, original strain:

In the virulence height of Hua Zhong Agriculture University microbial pesticide chamber, the Tribactur WG001 bacterial strain of stable performance is an initial strain as original strain with freeze pipe or sand tube preservation.

2, slant strains:

Slant culture based formulas: extractum carnis 0.5%, peptone 1.0%, NaCl0.5%, agar 2.0%, pH value 7.2.With substratum branch pack into test tube or eggplant bottle, with 0.1mPa, 121 ℃ of moist heat sterilizations 30 minutes treat that temperature is reduced to about 50 ℃ to put into the inclined-plane that if temperature is too high, water of condensation is many, the inclined-plane can be put 37 ℃ and cultivate one day, as asepsis growth, can use.

The cultivation of slant strains: initial strain WG001 is inoculated on the test tube slant substratum, and 30 ℃ of cultivations activated in 24 hours.Transfer from the test tube slant then in eggplant bottle inclined-plane, cultivated 72 hours for 30 ℃, the visual inspection lawn is plentiful, surperficial oyster white, and no plaque, microscopic examination does not have assorted bacterium, and 90% above brood cell comes off and can be used for producing.Use slant strains for producing, in refrigerator, preserve and be no more than a week at most.

Two, zymotechnique:

1, seed tank culture:

The seed tank culture based formulas is: soybean cake powder 3.0-5.0%, liquefaction W-Gum 1.0-5.0%, yeast powder 0.1-1.0%, corn steep liquor 1.0-4.0%, peptone 0.1-0.5%, inorganic salt 0.1-2.0%, bubble enemy 0.05-0.2%, with the α-Dian Fenmei that the amount of W-Gum 0.5% is added 2000IU/g, all raw materials all needed the 80-100 mesh sieve, and pH is 7.5-9.0 before the sterilization.Press seeding tank volumetrical 60-80% and drop into substratum, then high pressure steam is directly fed fermentor tank and make a jar temperature reach 121 ℃, pressure is 0.1mPa, keeps 30 minutes, be cooled to 30-35 ℃ after, insert the bacteria suspension that activated slant strains is made.Sterilization back pH is 6.5-7.5, is incubated 10 minutes in the sterilization process under 80 ℃ of conditions.

The substratum of seeding tank also can adopt following prescription: soybean cake powder 3.5-5.0%, liquefaction W-Gum 2.0-5.0%, yeast powder 0.1-0.5%, corn steep liquor 1.0-3.0%, peptone 0.2-0.5%, inorganic salt 0.1-1.0%, bubble enemy 0.05-0.2%, the α-Dian Fenmei of adding 2000IU/g with the amount of W-Gum 0.5%, all raw materials all needed the 80-100 mesh sieve, pH is 7.5-9.0 before the sterilization, and sterilization back pH is 6.5-7.5, is incubated 10 minutes in the sterilization process under 80 ℃ of conditions;

The inoculation of seeding tank: in cultured eggplant bottle slant strains, add sterilized water, make every milliliter of bacteria suspension that contains 0.2-1.0 hundred million viable cell, change in 500 milliliters of serum bottles, beyond the Great Wall through the sterilization syringe needle or inoculated tube, and bind with rope and to put in 80 ℃ of water-baths insulation 15-20 minute, to kill the free phage and to activate the brood cell.Through heat treated brood cell's suspension, insert seeding tank with pressure differential method.Seed tank culture base pH value is 7.0 during inoculation.

Seed tank culture: at temperature 30-32 ℃, tank pressure 0.03-0.05MPa, air flow 1: 0.6-1: 0.8 (V/V) stirs under the 200-250rpm condition and cultivated 6-12 hour.Grow up to nourishing body through whole sprouting of microscopic examination brood cell, bacterium shape is normal, and no living contaminants, nutrient solution contain hundred million/milliliter of viable count 0.6-1.0, can make qualified seed liquor and use.

2, fermentor cultivation:

Fermentor cultivation based formulas: soybean cake powder 3.0-5.0%, liquefaction W-Gum 1.0-5.0%, yeast powder 0.1-1.0%, corn steep liquor 1.0-4.0%, peptone 0.1-0.5%, inorganic salt 0.1-2.0%, bubble enemy 0.05-0.2%, the α-Dian Fenmei of adding 2000IU/g with the amount of W-Gum 0.5%, all raw materials all needed the 80-100 mesh sieve, pH is 7.5-9.0 before the sterilization, and sterilization back pH is 6.5-7.5, is incubated 10 minutes in the sterilization process under 80 ℃ of conditions.

Fermentor tank also can adopt following prescription: soybean cake powder 3.5-5.0%, liquefaction W-Gum 2.0-5.0%, yeast powder 0.1-0.5%, corn steep liquor 1.0-3.0%, peptone 0.2-0.5%, inorganic salt 0.1-1.0%, bubble enemy 0.05-0.2%, the α-Dian Fenmei of adding 2000IU/g with the amount of W-Gum 0.5%, all raw materials all needed the 80-100 mesh sieve, pH is 7.5-9.0 before the sterilization, and sterilization back pH is 6.5-7.5, is incubated 10 minutes in the sterilization process under 80 ℃ of conditions.

Fermentation culture: press fermentor tank volume 60-70% and drop into substratum, make fermentation jar temperature reach 121 ℃ by high pressure steam, pressure is 0.1mPa, keeps sterilization in 30 minutes, is cooled to after 30-33 ℃ the 1-100% immigration seed liquor by the volume that feeds intake.At temperature 28-32 ℃, tank pressure 0.03-0.08MPa, air flow 1,0.6-1: 1.2 (V/V) stir under the 200-250rpm condition and cultivated 24-48 hour, have brood cell's capsule of 20-40% to break with microscopy, just can put jar.

Fermentation control: transfer to about 8.0 before the basal culture medium sterilization; PH value after the sterilization is about 7.0-7.2, makes brood cell's germination rate reach the highest.PH drops to 5.8-6.0 after thalline begins to grow, the pH value about 8.5 when putting jar of ging up again subsequently.Ventilation: at earlier fermentation, the bacterium number is low, and oxygen consumption is few; Enter logarithmic phase, oxygen consumption increases, and general requirement per minute fermention medium volume reaches 1: 1.0 to 1: 1.2 with the ratio of the volume of bubbling air.After the brood cell begins to form, reduce though bacterial metabolism is active, therefore oxygen must keep bigger air flow in the fermentation later stage to the material impact that is formed with of brood cell's maturation and parasporal crystal.Temperature: check temperature value at any time, controlled temperature changes at 30 ± 2 ℃.

3, fermented product aftertreatment and packing:

Fermented liquid adds auxiliary agent and makes liquor product or the spray-dried pulvis of making after acidifying and an amount of concentrating.

Fermented liquid liquorization: fermented liquid pH is transferred to 4.5-6.5 with HCl, according to the fermented liquid level, adopt whizzer that fermented liquid is concentrated 0.5-1 doubly, add 3-10%NaCl, 0.1-0.5% Sodium Benzoate and No. 100 emulsifying agents of 1-3% farming breast (" farming breast " then, model is 100 types, and packing specifications is 500 milliliters/bottle, and the manufacturer is Jiangsu Hai'an Petrochemical Plant.The characteristic that No. 100, the farming breast is that soluble in water and multiple solvent is stablized in acid ﹠ alkali liquid.Be used to prepare the Multiple Pesticides emulsifying agent, as the biological pesticide of microorganisms such as organophosphorus, organochlorine pesticide and Tribactur preparations etc.), through check, toxicity evaluation is reached more than the 8000IU/ μ l, be the liquid dosage form biotic pesticide after the packing.

The fermentation liquor powder thinner: fermented liquid through the fluid-type continuous centrifuge concentrate reach finite concentration after, adds fillers such as an amount of diatomite or lime carbonate, be wetting agent with newborn No. 100 emulsifying agents of 1-3% farming, squeeze in the header tank of spraying drying cat head.When inlet temperature reaches 200-220 ℃, begin spraying, control flows into the speed of centrifugal pan slurries, make institute's drying tower middle level temperature remain on 65-70 ℃ like this, make institute's exsiccant preparation water content below 4%, and, make toxicity evaluation reach 16000IU/mg or more than the 32000IU/mg, be the powder-type biotic pesticide after the packing through check.

Insecticide Bacillus thuringiensis pulvis of the present invention by every bag 250 the gram, 500 the gram, 1000 the gram or bigger, with aluminium foil bag or plastic bag packaging; Suspension agent and finish are used plastic bottle packing by every bottle of 250ml, 500ml, 1000ml, according to following quality standard configuration and check.

Three, quality test:

1, biological value check:

Fermentation intermediate product and product quality inspection, method by " the agricultural industry criteria NY293-95 of People's Republic of China (PRC) thuringiensis praeparatum preparation " regulation, do to carry out biological assay with diamondback moth larvae for the examination insect, adopt the standard substance [Cs3ab-1999 of the applicant's prepared in laboratory, toxicity evaluation 20000IU/mg (international unit/milligram)], standard substance and testing sample are carried out serial dilution, the infection liquid and the pickles artificial diet of different concns are mixed and made into the infection feed, infect three age diamondback moth larvae, check the larval mortality under the different concns, calculate the LC50 value of testing sample and standard substance with statistical technique, by the toxicity evaluation of following formula calculation sample:

2, crystallin assay (SDS-PAGE method):

(1) gets 200 μ l fermented liquids and add 800 μ l water or former powder dilution certain multiple, with 30 seconds of ultrasonication;

(2) get 100 μ l diluents to another Eppendorf pipe, and add 0.5mol/1NaOH25 μ l, room temperature was placed 5 minutes;

(3) add 65 μ l, 3 * laemmli sample buffer (850 μ l, 3 * laemmli+150 μ l mercaptoethanol);

(4) add a cover back 100 ℃ of water-baths 5 minutes;

(5) at a high speed (1000rpm) centrifugal 3 minutes;

(6) add in supernatant 10 μ l to the 10%SDA-PAGE gel grooves;

(7) add 10 μ l, 15 μ l, 20 μ l standard proteins respectively in specified control gel groove.

(8) electrophoresis under the 120V-150V condition;

(10) with behind the coomassie brilliant blue staining gel, it is clear to background to decolour with destainer again;

(11) with visible light gel is scanned, and alike with Image-Master software processes figure.

(12) calculate the crystalline content of sample according to the integrated value reference standard of crystallin absorption peak;

3, detected result:

1, fermented liquid technical indicator: through biological assay, fermented liquid to the toxicity evaluation of small cabbage moth between 4000-6000IU/ μ l, to the toxicity evaluation of bollworm between 3500-5500IU/ μ l; Protein content is between 3.5-5.5g/l.

2, technical target of the product:

Suspension agent: toxicity evaluation is more than the 8000IU/ μ l;

Finish: toxicity evaluation is more than the 8000IU/ μ l;

Wettable powder: toxicity evaluation is more than the 16000IU/mg;

Efficient wettable powder: toxicity evaluation is more than the 32000IU/mg;

Former powder: toxicity evaluation is about 60000IU/mg.

Embodiment 4 product of the present invention field effects for example

The control cotton and the efficient Tribactur engineering bacteria of the vegetables pest WG001 that utilize p20 to make up, its control small cabbage moth effect improves 2.65 times than the product Dipel of U.S. Abbott company, improves 3.1 times than the effect of chemical pesticide 20% Belmark EC.Prevention effect is greater than 85%-90%.The prevention effect of bollworm is improved 20% than Dipel, and suitable with the virulence of 800 times of diluents of 40% acephatemet, prevention effect reaches 80%-91%.

Prevention effect to bollworm

Compared with the prior art, thuringiensis gene engineering strain WG001 of the present invention compares with the natural bacterial strain that sets out, and insecticidal crystal protein output is enhanced about more than once, and parasporal crystal increases 2.1 times, and small cabbage moth and bollworm are all had good prevention effect.

Claims (4)

1, a kind of engineering strain of producing microbial pesticide, have cry1Aa, cry1Ab and cry1Ac insecticidal crystalline gene, it is characterized in that described bacterial strain is Tribactur (Bacillus thuringiensis) WG001, CCTCC M 201033.

2, a kind of method for preparing thuringiensis gene engineering bacteria WG001 and products thereof, its feature may further comprise the steps:

(1) with the WG001 inoculation on the test tube slant substratum, cultivate for 30 ℃ and activated in 24 hours, transfer then in eggplant bottle inclined-plane, cultivated 72 hours for 30 ℃, be to produce and use slant strains;

(2) will add sterilized water in the cultured eggplant bottle slant strains, make every milliliter of suspension that contains 0.2-1.0 hundred million viable cell, change over to then in the 500ml serum bottle, syringe needle or the inoculated tube of bacterium beyond the Great Wall went out, bottle is put in 80 ℃ of water-baths insulation 15-20 minute bacteria suspension is heat-treated and with pressure differential method this bacteria suspension is inserted seeding tank, this jar medium pH is 6.5-7.5 during inoculation;

(3) press seeding tank volumetrical 60-80% and drop into substratum, this substratum prepares according to following proportioning: soybean cake powder 3.0-5.0%, α-Dian Fenmei 0.05 gram that adds 2000IU/g with every gram W-Gum, at 80 ℃, the liquefaction W-Gum 1.0-5.0% for preparing under the condition, yeast powder 0.1-1.0%, corn steep liquor 1.0-4.0%, peptone 0.1-0.5%, inorganic salt 0.1-2.0%, bubble enemy 0.05-0.2%, all raw materials all needed the 80-100 mesh sieve, be 7.5-9.0 before making the pH sterilization, the sterilization back is 6.5-7.5, with 0.1mPa, 121 ℃ high pressure steam sterilization, kept 30 minutes, after being cooled to 30-35 ℃, insert the bacteria suspension that activated inclined-plane lawn is made;

(4) press fermentor tank volume 60-70% and drop into substratum, this substratum prepares according to following proportioning: soybean cake powder 3.0-5.0%, add α-Dian Fenmei 0.005 gram of 2000IU/g with every gram W-Gum, liquefaction W-Gum 1.0-5.0%, the yeast powder 0.1-1.0% that under 80 ℃ of conditions, prepares, corn steep liquor 1.0-4.0%, peptone 0.1-0.5%, inorganic salt 0.1-2.0%, bubble enemy 0.05-0.2%, all raw materials all needed the 80-100 mesh sieve, be 7.5-9.0 before making the pH sterilization, the sterilization back is 6.5-7.5; With 0.1mPa, 121 ℃ high pressure steam stirs under the 200-250rpm condition, keeps sterilization in 30 minutes, is cooled to after 30-33 ℃ and moves into seed liquor by 1% of the volume that feeds intake;

(5) seed tank culture, at temperature 30-32 ℃, tank pressure 0.03-0.05mPa, air flow is pressed V/V1: 0.6-1: 0.8, stir under the 200-250rpm condition and cultivated 6-12 hour;

(6) fermentor cultivation, at temperature 28-32 ℃, tank pressure 0.03-0.08mPa, air flow is pressed V/V1: 0.6-1: 1.2, stir under the 200-250rpm condition and cultivated 24-48 hour, there is brood cell's capsule of 20-40% to break with microscopy, just can put jar;

(7) fermented liquid liquorization: fermented liquid pH is transferred to 4.5-6.5 with HCl, according to the fermented liquid level, adopt whizzer that fermented liquid is concentrated 0.5-1 doubly, add 3-10%NaCl, 0.1-0.5% Sodium Benzoate and No. 100 emulsifying agents of 1-3% farming breast then, through check, toxicity evaluation is reached more than the 8000IU/ μ l, be the liquid dosage form biotic pesticide after the packing;

(8) in concentrated broth, add No. 100 emulsifying agents of an amount of diatomite or lime carbonate and 1-3% farming breast, spray-dried operation, the preparation water content is controlled at below 4%, and through check, make toxicity evaluation reach 16000IU/mg or more than the 32000IU/mg, be the powder-type biotic pesticide after the packing.

3, method according to claim 2, it is characterized in that, described fermentor cultivation based formulas is: soybean cake powder 3.5-5.0%, add α-Dian Fenmei 0.005 gram of 2000IU/g with every gram W-Gum, liquefaction W-Gum 2.0-5.0%, the yeast powder 0.1-0.5% that under 80 ℃ of conditions, prepares, corn steep liquor 1.0-3.0%, peptone 0.2-0.5%, inorganic salt 0.1-1.0%, bubble enemy 0.05-0.2%, all raw materials all needed the 80-100 mesh sieve, making the preceding pH of sterilization is 7.5-9.0, and the sterilization back is 6.5-7.5.

4, method according to claim 2 is characterized in that, enters logarithmic phase at ferment tank, and its air flow is pressed V/V 1: 1.0-1: 1.2 adjust.