CN1199569C - Double engineering bacterium biological pesticide and its production method - Google Patents

Abstract

The present invention relates to a bacterial agent of Bacillus thuringiensis (Bt)double-effect engineering bacteria, which is cloned by genes, and a production method for biological insecticide of the double-effect engineering bacteria. The bacterial agent uses a Bacillus thuringiensis 4.0718 strain and the subspecies of Bacillus thuringiensis, etc. as parasitifer, spider poison protein genes of fusion protein genes with a strong initiating sequence and a domain II sequence are integrated to Bt bacterium DNA to carry out stable heredity by constructing an adjustable and controllable expression vector and construct gene engineering bacteria capable of generating spider poison protein and Bt poison protein, and the double-effect biological insecticide is produced by fermentation. The titer of the bacterial agent is more than 6000 iu /mug, and an insecticidal rate of the larvae of lepidoptera, diptera and coleoptera is from 90 to 96%; the bacterial agent has the advantages of green insecticide, no residues and safety for people and livestock, and the bacterial agent is favourable to protect ecological environment.

Description

A kind of double engineering bacterium biological pesticide and production method thereof

Technical field the present invention relates to a kind of bacillus thuringiensis,Bt through gene clone (Bacillusthuringiensis is called for short Bt) double engineering bacterium, and the production method of double engineering bacterium biological pesticide.

The use of the long-term a large amount of chemical pesticides of background technology, pest resistance strengthens, and causes the heavy damage of ecotope, and direct harm humans health; Simultaneously agricultural exports is returned goods because of the remains of pesticide problem and is directly caused enormous economic loss, China in 1998 because of the residue of pesticide problem with a toll of 7,400,000,000 dollars.Since the eighties, Bt is subjected to increasing attention as the application of farming, woods, sanitary insect pest insecticide, especially its to have desinsection toxicity stronger, insecticidal spectrum is wide, insect is difficult to produce resistance, residual life is short, nuisanceless, to the person poultry safety, be easy to important feature such as suitability for industrialized production, therefore obtained important society, economy, ecological three big benefits.But the Bt insecticide still exists a series of problem: (1) insecticidal spectrum relative narrower; (2) insecticidal effect and speed are killed effect and are starkly lower than chemical pesticide; (3) therefore desinsection longevity of residure weak point exists certain limitation in the use.

In recent years, because the fast development of technique for gene engineering, existing scientist adopts engineered method to open up the new bio field of pesticides.Chinese patent 86108756 discloses a kind of recombinant virus that comprises the dna segment of 2 kinds of nucleopolyhedrosis viruss that Japanese preceding field is advanced to make up, because the host insect difference of two kinds of viruses, thereby can put out and prevent and treat two kinds of insects by the insecticide of its production, enlarged insecticidal spectrum.Notification number is the patent of applications such as the Australian MEH person of outstanding talent of CN-1044298A steps on, and is by utilizing the funnel-web spider toxin gene to make up engineered plant with Pest Control, having obtained certain effect.The Michael J A Dan of the U.S. was adopting preferred codon in the unifacial leaf of high expressed or dicotyledon protein in 1989, in plant, expressed the Bt toxin, therefore realized directly pest-resistant new method of plant, the patent announcement of this method number is CN-1044298A.Though with insect baculovirus as the killing gene carrier with to cultivate the research work of aspects such as genetically modified plants more extensive, on using, be subjected to the restriction of himself characteristic always.Because baculoviral can not be expressed the foreign protein of q.s at short notice mostly, therefore, makes insect stop to get food and needs 2-3 days at least, generally needed 4～14 days just can kill the host, and the food of getting of insect has caused remarkable harm to crop in the meantime.In addition, the host range of baculoviral is narrow relatively, so its application is subjected to suitable restriction.Because of the pollution problem of fears are entertained that genetically modified food, transgene agricultural product is ordered to forbid to sell and study by government department in developed countries such as European market and Japan, Thailand.The production of genetically modified crops at present and development are in the world still among heated argument, but overwhelming majority of countries has been taked resistance.Advocate genetically modified food research with developed countries such as the U.S., Canada, seldom sell, be mainly used in and sell to third world countries but in fact they are domestic.

The Bt bacterium can produce crystalline protein, Lepidoptera (Lepidopteran), diptera (dipteran) and coleoptera (coleopteran) larva had toxic action, this kind crystal toxalbumin is degraded into the toxicity core fragment under the alkali condition of larva enteron aisle, cause perforation to form ion channel on the function cells film, cause insect death.

Tiger line toxin (Huwentoxin is called for short HWTX) is the polypeptide class neurotoxin of separation and purification from the venom of China's rare spider kind Selenocosmiahuwena (Selenocosmia Huwena), has now named two kinds of toxin, i.e. HWTX-I and HWTX-II.Its total number of atnino acid is respectively 33 and 74.Toxicity test shows, the nerve by the glutamic acid mediation of the blocking-up insect that these two kinds of toxin can both be special-muscle conduction and cause paralysis, death.Lei Shi ravenizhu toxin (Raventoxin-I, Raventoxin-II) be the two class polypeptide neurotoxins that belong to separation and purification the thick poison of novel species Lei Shi ravenizhu (Macrothele raveni) from recent findings and the different imitative spider section ravenizhu named, the amino acid residue number is respectively 43 and 33, nervous activity to insect has the strong inhibition effect, can cause death.America agatoxin (agatoxins) is the polypeptide class neurotoxin of separation and purification from America Criny spider (Agelenopsis aperta) poison gland.One group of toxin (u-agatoxins I-VI) is wherein arranged, and it has comprised 6 kinds of toxin, can cause that the irreversible neural paralysis of insect causes death.Australia agatoxin (atracotoxins) is the polypeptide class neurotoxin of separation and purification from the spider venom of Australian Agelena (Atrax), δ-ACTX-HVIa wherein, Atracotoxin-HVIC, Omega-actx-hvla three toxoids have special neural inhibitory action to insect.The special neurotoxin (α-Latroinsectotoxin of insect in latrodectus mactans (Latrodectus Mactaustredisimguttatus) toxin family, δ-Latroinsectotoxin) is the polypeptide neurotoxins of purifying from this kind spider venom, can cause a large amount of releases of insect neurotransmitter and paralysis, death rapidly.

Summary of the invention the present invention is intended to utilize the new bacterial strain 4.0718 of bacillus thuringiensis,Bt efficient insecticide (the Bacillus thuringiensis subsp.Kurstaki of seed selection, country's culture presevation number: CCTCCNO:M200016) and Su Yun gold subspecies (Bacillus thuringiensis subsp.thuringiensis), Kustak subspecies (Bacillus thuringiensis subsp.kurstaki), galleria mellonella waxmoth subspecies (Bacillus thuringiensis subsp.galleriae), Wuhan subspecies (Bacillus thuringiensis subsp.wuhanensis), China's subspecies (Bacillusthuringiensis subsp.chinensis) are as recipient bacterium, the special neurotoxin gene of the insect of above-mentioned multiple spider is cloned and realizes high expressed with suitable carriers, construct the new bacterial strain that the new bio insecticide is produced in a strain, the development excess of export is imitated, wide spectrum, the insecticide of safety is to overcome problem that the Bt insecticide exists and current gene engineering pesticide plant an open question still.

Double engineering bacterium biological pesticide, be a kind of Bt of generation toxalbumin and the Thuricide engineering bacterium preparation that after gene clone, produces spider poison protein, the living spores content of the Thuricide engineering bacterium of said preparation is at 8,000,000,000-10,000,000,000/ml, tiring in that 6000 international units/more than the μ g, preparation reaches 90-96% to the larva killing rate of Lepidoptera, diptera and coleoptera of preparation.

The preparation method of double engineering bacterium biological pesticide, be made up of the structure of Thuricide engineering bacterium and production two parts of Thuricide engineering bacterium agent:

(1) structure of bacillus thuringiensis,Bt double engineering bacterium

1. make up the controllable express carrier

---recipient bacterium is bacillus thuringiensis,Bt 4.0718 and Su Yun gold subspecies, Kustak subspecies, galleria mellonella waxmoth subspecies, Wuhan subspecies and Chinese subspecies.

---plasmid pXLZ401 makes up, with the gentle method extracting of Triton X-100 bacterial strain 4.0718 total plasmids, after the PstI/BamHI double digestion, obtain the genetic fragment about 4Kb, use the agarose gel electrophoresis separation and purification, in addition with PstI/BamHI double digestion shuttle vector pHT3101, obtain having the chain dna of two different cohesive ends, use the alkaline phosphatase dephosphorylation, the genetic fragment about the 4Kb of separation and purification is mixed with dephosphorylized carrier pHT3101, use T ₄Dna ligase connects, and produces plasmid pXLZ401.

---obtaining of ICPs gene strong promoter and SD sequence, plasmid pXLZ401 electricity is transformed a strain do not have the crystal mutant strain, by erythromycin plate screening transformant, select to produce the transformant of crystal by gram stain microscopy, on the LB medium, activate, cultivate transformant, extract plasmid with TritonX-100, through agarose gel electrophoresis separation and purification plasmid pXLZ401, and use the pstI/BamHI double digestion, pass through CsCl density gradient centrifugation behind the double digestion, the Glass-milk method obtains to have the fragment of complete ICPs open reading frame, will with computer software sequence be analyzed behind the sequencing fragment, determines its initiating sequence and SD sequence.

---the structure of plasmid pXLZ402, above-mentioned fragment is by the total length initiating sequence before the pcr amplification initiation codon ATG, and hold at 5 ' end and 3 ' and to add ECoRI, SalI site respectively, purified, use the ECoRI/SalI double digestion, carrier pHT3101 uses the ECoRI/SalI double digestion equally, uses the alkaline phosphatase dephosphorylation, both mix, and use T ₄Dna ligase couples together both under the condition that ATP exists, and forms plasmid pXLZ402, Transformed E .coli DH _{5 α}Middle amplification.

2. the chemosynthesis of the special insect specific neurotoxin gene of spider and domain II sequence obtains

---the chemosynthesis of spider virus gene, adopt chemical synthesis process according to spider virus gene sequence, synthesize the toxin polypeptide full-length gene, hold at the 5 ' end and 3 ' of gene to have ECoRI, XbaI site respectively, add terminator TGA in addition at 3 ' end simultaneously.

---act on the obtaining of domain II sequence of insect midgut cell-membrane receptor among the ICPs, in Gene Bank, search the domain II sequence of existing ICPs, pass through sequence analysis software, array is conserved region relatively, and with the order-checking fragment relatively, determine its domain II border according to conserved region, obtain domain II genetic fragment with chemosynthesis and PCR method, have SalI, ECoRI restriction enzyme site at 5 ' end and 3 ' end respectively, add initiation codon ATG in addition at 5 ' end, can obtain domain II section by PCR.

---the structure of plasmid pXLZ403, with alkaline lysis method of extracting plasmid pXLZ402, behind the SalI/XbaI double digestion, use the alkaline phosphatase dephosphorylation, after in addition acquired domain II sequence being cut with the ECoRI enzyme, with chemosynthesis 5 ' hold the toxin gene that has the ECoRI site to mix, use T ₄Dna ligase connects, and behind the junction fragment purifying, mixes with XbaI enzyme cutting and with dephosphorylized chain plasmid pXLZ402, uses T ₄Dna ligase connects, and forms plasmid pXLZ403, transforms the plasmid-free mutant strain, by the dull and stereotyped transformant of selecting of erythromycin.

The antigen-4 fusion protein gene that 3. will have initiating sequence is incorporated into Bt bacterium DNA and goes up genetic stability

---the structure of plasmid pXLZ404, wild type is changeed Cuo Tn5401 two ends remove the border that transposase is discerned with exonuclease, delete transposase with the method that enzyme is cut, the purifying rest segment connects with the T4DNA ligase, is inserted among the pHT3101, form plasmid pXLZ404, change E.coli DH over to _{5 α}Middle amplification.

---the structure of plasmid pXLZ405, extract plasmid pXLZ403, pXLZ404 respectively with the gentle method of TritonX-100, the recombinant fragment of separation and purification pXLZ403, be inserted on the site of having deleted transposase among the pXLZ404, cut except that the Bt replicon among the plasmid pXLZ404 with enzyme, produce plasmid pXLZ405, electric conversion, particle gun or metal ion revulsion Transformed E .coli DH _{5 α}Middle amplification, the dull and stereotyped transformant of selecting of ampicillin.

---plasmid pXLZ405 is incorporated on the chromosome of bacterial strain 4.0718, wild type transposons Tn5401 is changed on the chromosome of bacillus thuringiensis,Bt 4.0718, produce new bacterial strain, plasmid pXLZ405 electricity is transformed new bacterial strain, improved Tn5401 and total length fusion are incorporated among the wild type transposons Tn5401 that is present on the chromosome, with the dull and stereotyped transformant of selecting of erythromycin.

(2) production of Su Yun bacterium bacillus engineering microbial inoculum

1. actication of culture, the engineering bacteria bacterial classification of preservation are transferred in flat bottle medium behind slant culture, under 28～35 ℃ of conditions, cultivate 30～48h, are made into spore liquid, and the inoculum concentration with 5% is used for the seeding tank inoculation.

2. seeding tank fermentation, spore liquid is inserted through sterilizing and being equipped with in the first class seed pot of culture fluid, the feeding filtrated air is cultivated, obtain the first class seed pot zymocyte liquid, by 5%～8% inoculum concentration the first order seed zymotic fluid is inserted in the secondary seed jar culture fluid, feed filtrated air and stir culture, obtain secondary seed jar zymocyte liquid.

3. fermentation tank culture, the inoculum concentration by 5%～8% inserts secondary seed jar zymotic fluid in the fermentation tank, and under 25 ℃～30 ℃ temperature condition, feed filtrated air and cultivate 42-48h,

4. concentrate, ferment tank through ultrafiltration and concentration, is added Synergistic additives with bacterium liquid after finishing, bottling.

Down in detail the present invention is being described in detail in conjunction with the accompanying drawings.

Fig. 1 plasmid pHT3101 structure chart;

ICPs gene strong promoter and SD sequence are obtained schematic diagram on Fig. 2 bacterial strain 4.0718 plasmids;

Fig. 3 spider toxin gene expression vector establishment schematic diagram;

Fig. 4 fusion is incorporated into schematic diagram on the Bt bacterium chromosome;

Fig. 5 economic benefits and social benefits disinsection engineering bacteria fermentation manufacturing technique schematic flow sheet;

(1) structure of inducible expression vector

1. F-strain

F-strain of the present invention is Dipel (the Bacillus thuringiensis of seed selection Subsp.kurstaki) efficient insecticide novel bacterial Hu4.0718 (national culture presevation number: CCTCC NO: M200016), synteny has 6 plasmids not of uniform size, its 1～No. 3 plasmid carry cry1Aa, The genes such as cry1Ab, cry1Ac, cry1Ax, cry1Cb, cry2Ac are to Lepidoptera and dipteral insect Very strong toxicity is arranged. Recipient bacterium also has Su Yun gold subspecies (Bacillus thuringiensis subsp. Thuringiensis), Kurstaki (Bacillus thuringiensis subsp. Kurstaki), galleria mellonella waxmoth subspecies (Bacillus thuringiensis subsp.galleriae), Wuhan Subspecies (Bacillus thuringiensis subsp.wuhanensis), Chinese subspecies (Bacillus Thuringiensis subsp.chinensis).

2. the obtaining of ICPs gene strong promoter and SD sequence on bacterial strain 4.0718 plasmids

Fig. 2 is the schematic diagram of seeking ICPs gene strong promoter and SD sequence from bacterial strain 4.0718 plasmids. By the gentle method extracting of TritonX-100 bacterial strain 4.0718 total plasmids, at the PstI/BamHI double digestion Laggard row agarose gel electrophoresis carries out separation and purification to the genetic fragment about clone's 4Kb. PHT3101 (structure is seen Fig. 1) is a kind of shuttle vector that can both copy at Bt and E.coli, carries Two kinds of replicons of Bt, E.coli, performance erythromycin resistance in Bt, performance ammonia benzyl green grass or young crops in E.coli The mycin resistance. Use the PstI/BamHI double digestion, obtain the chain dna with two different cohesive ends, Use the alkaline phosphatase dephosphorylation, can prevent himself cyclisation and add heavily to the difficulty of the work, with an above-mentioned 4kb left side Right fragment is mixed with dephosphorylized carrier, connects with the T4DNA ligase, produces plasmid pXLZ401. Electricity transforms a strain does not have the crystal mutant strain, by erythromycin plate screening transformant, sees by microscope simultaneously Examine and detect the transformant with complete ICPs ORFs that can produce the prismatic crystal. Cultivate at LB Activation transformant 24h inserts in the LB fluid nutrient medium on the base inclined-plane, and under 30 ℃, 200rpm cultivates 18-24h extracts plasmid by the TritonX-100 method. Separation, purifying behind agarose gel electrophoresis The purpose plasmid. Can obtain with complete ICPs ORFs with PstI/BamHI double digestion plasmid Fragment. To with computer software sequence be analyzed behind the sequencing fragment, determine its initiating sequence and SD Sequence.

3. the structure of plasmid pXLZ402

By fragment sequence design primer, add the monoclonal site at the primer two ends, make and pass through pcr amplification Before the ATG upstream of total length initiating sequence with ECoRI site, downstream with the SalI site, with PCR Use the ECoRI/SalI double digestion behind the product purification, carrier pHT3101 is used ECoRI/SalI equally Double digestion, and use the alkaline phosphatase dephosphorylation. Both are mixed, use T₄Dna ligase couples together both under the condition that ATP exists, and forms plasmid pXLZ402, Transformed E .coli DH_5αMiddle amplification.

(2) chemical synthesis of the special insect specific neurotoxin gene of spider and domain II sequence obtains

1. the gene order of the many kinds of special neurotoxins of spider insect is known, (sees according to spider virus gene sequence Illustrate 14 pages～23 pages), adopt the synthetic toxin polypeptide full-length gene of chemical synthesis process, and make it 5 ' end is with the EcoRI site, and 3 ' end is with the XbaI site, and it is close to be added with in addition termination at 3 ' end simultaneously Numeral TGA.

2.ICPs in act on the obtaining of domain II sequence of insect midgut cell-membrane receptor

In Gene Bank, search the domain II sequence of existing ICPs, pass through sequence analysis software The array such as (such as Jellyfish) is conserved region relatively, and with the order-checking fragment relatively, determine it according to conserved region Domain II border. According to border sequence design primer, it is prominent after avoiding the toxin fragment to insert frameshit to take place In the situation about becoming, the domain II gene that makes amplification at 5 ' end with ATG, simultaneously respectively 5 ' end, 3 ' end is with SalI, ECoRI restriction enzyme site. Can obtain domain II section by PCR.

3. the structure of plasmid pXLZ403

Fig. 3 is spider toxin expression vector establishment schematic diagram, by alkaline lysis method of extracting plasmid pXLZ402, Behind the SalI/XbaI double digestion, use the alkaline phosphatase dephosphorylation; The domain that will produce by PCR After the II sequence is cut with the ECoRI enzyme, with chemical synthesis 5 ' end is with the toxin gene in ECoRI site Mix, use T₄Dna ligase connects, and mixes with XbaI enzyme cutting and with dephosphorylized chain plasmid pXLZ402 behind the junction fragment purifying, uses T₄Dna ligase connects, and forms plasmid pXLZ403, transforms The plasmid-free mutant strain is by the dull and stereotyped transformant of selecting of erythromycin.

(3) insecticidal function checking

1.ELISA detect the expression product of toxin gene

With the target spider toxin immune rabbit of purifying, obtain to be connected with horseradish peroxidase behind the antibody, Form the enzyme len antibody; After the bacterium of cultivating processed with ultrasonic wave, with transferring film behind the total protein electrophoresis, with anti-Body hybridization, wash-out, colour developing have determined whether the toxin expression.

2. verify the correctness that toxin is expressed

By the HPIC purified fusion protein, whether the order-checking of C end can be determined to express correct.

3. indoor insecticidal test

(4) will be incorporated into the antigen-4 fusion protein gene of initiating sequence the upper genetic stability of Bt bacterium DNA

1. the structure of plasmid pXLZ404.

Fig. 4 is incorporated into schematic diagram on the chromosome with whole antigen-4 fusion protein gene. Wild type is turned to Cuo The border of removing transposase identification is processed by exonuclease in the Tn5401 two ends, deletes with the method that enzyme is cut Transposase inserts among the pHT3101 then, produces plasmid pXLZ404, changes E.coli DH over to_5αMiddle amplification.

2. the structure of plasmid pXLZ405

Extract plasmid pXLZ403 by the gentle method of TritonX-100, recombinant fragment is separated, insert among the plasmid pXLZ404 behind the purifying and deleted on the site of transposase, and cut except that the Bt replicon among the plasmid pXLZ404, produce plasmid pXLZ405 with enzyme.

3. plasmid pXLZ405 is incorporated on the chromosome of bacterial strain 4.0718

The wild type transposons changes on the chromosome of Bt4.0718 bacterial strain, plasmid pXLZ405 electricity is transformed bacterial strain 4.0718, improved Tn5401 and total length fusion can be incorporated among the wild type transposons Tn5401 that has been present on the chromosome, and lose transfer ability, with the dull and stereotyped transformant of selecting of erythromycin.

4. functional verification

Determine that by southern hybridization, ELISA clone spider virus gene is present on the chromosome and can expresses, insecticidal test determines that the desinsection of engineered strain tires.

(5) fermenting and producing

Fig. 5 is an economic benefits and social benefits disinsection engineering bacteria zymotechnique schematic flow sheet, mainly comprises actication of culture, I and II seeding tank fermented and cultured and production fermentation tank culture.

1. actication of culture

With the high-efficiency broad spectrum disinsection engineering bacteria streak inoculation of preservation on solid seed culture medium inclined-plane, the flat bottle that medium will be housed before the inoculation was sterilized 30 minutes under 121 ℃ of conditions, 30～48h is cultivated in the inoculation back under 28～35 ℃ of conditions, be made into spore liquid, and the inoculum concentration with 5% is used for the seeding tank inoculation.

2. seeding tank fermentation

With the first class seed pot sterilization, sterilization again is cooled to 30 ℃ behind the medium of packing into, and spore liquid is inserted in the culture fluid earlier, feeds filtrated air and cultivates, and can get the first class seed pot zymocyte liquid; With secondary seed jar sterilization 30min under 121 ℃, the sterilization again behind the medium of packing into is cooled to 30 ℃, and the first class seed pot zymotic fluid is inserted the secondary seed jar by 5%～8% inoculum concentration, feed filtrated air and stirring and cultivate, can get secondary seed jar zymocyte liquid.

3. production fermentation tank culture

Earlier with the fermentation tank sterilization, sterilization again behind the medium of packing into, pressurize is cooled to 25 ℃～30 ℃, by 5%～8% by kind of amount secondary seed jar zymotic fluid is inserted in the fermentation tank and cultivate, passes through filtrated air.

4. concentrate

After fermentation cylinder for fermentation finishes, bacterium liquid is concentrated, adds Synergistic additives, bottling subsequently by ultrafiltration, can put on market.

5. sterilize and condition of culture

High pressure steam sterilization is adopted in above-mentioned sterilization, and promptly at 121 ℃, the 30min that sterilizes under the pressure 0.3-0.5kg/cm2 condition after the fermentation tank inoculation, cultivated 36～48 hours throughput 1～2Vols/vol/min, oxygen content 30～40% under 28～35 ℃ of conditions.The seed tank culture formula of liquid: beef extract 0.3～0.8%, peptone 0.7～1.2%, glucose 0.1～0.6%, NaCl 0.1～0.6%, MgSO ₄7H ₂O0.01～0.06%, K ₂HPO ₄0.01%～0.06%, MnSO ₄0.02%～0.08%, pH value sterilization is preceding 7.0～7.8, and sterilization is PH6.8～7.8 afterwards.

The fermentation tank culture formula of liquid: analysis for soybean powder 2～8%, corn starch 0.3～1.5%, NaOH 0.2～0.8%, behind 121 ℃ of hydrolysis 20min, filters and removes impurity, by volume adds glucose 1.3～2.0%, KH ₂PO ₄0.08～0.22%, K ₂HPO ₄0.10～2.2%, CaCO ₃0.1～2.20%, FeSO ₄7H ₂O0.001～0.005% stirs, and pH value sterilization is preceding 8.5～11.0, sterilization afterwards 6.5～9.5.

Double effect biological insecticide Synergistic additives prescription: Tea Saponin 0.05～25%, dimethylbenzene 0.5～3%, sorbierite 1～4%, Tween 80 1～4%, glycerine 3～8%, honey 3～8%.

6. zymotic fluid requirement

Zymotic fluid contains brood cell's number alive and reaches more than 8,000,000,000 for every milliliter, puts jar during biomass less than 5%, pH value 7.0-8.0, no living contaminants.

The present invention compares with the prior biological agricultural chemicals and has the following advantages:

(1) good disinsection effect: because engineered strain has multiple Pesticidal toxins Cry1Aa respectively, Cry1Ab, Cry1Ac, Cry2Ac, HWTX-I, HWTX-II, Raventoxin-I, Raventoxin-II, atracotoxin-HV1C, delta-atracotoxin-HV1, omego-actx-Hv1a, mu-agatoxinI～VI, Alpha-Latroinsectotoxin, delta-Latroinsectotoxin, except the killing ability of self, also has significant positive cooperativity, can produce stomach toxicity and the double effects of tagging to insect, therefore desinsection is tired higher, tiring to reach 6000 international units/more than the μ g, has very strong competitiveness on using.

(2) insecticidal spectrum is wide: engineering bacteria self has multiple Pesticidal toxins, simultaneously since spider virus gene such as HWTX-I, HWTX-II and improved ICPs domain II zone with fusion protein form expression, its discernible scope is increased, simultaneously can be effective to multiclass insects such as Lepidoptera, diptera, coleopteras, therefore it is very wide to prevent and treat face in the use, and cost is relatively low.

(3) safety is higher: because the entrained toxin of engineering bacteria is natural toxin, easily for Institute of Micro-biology's degraded of occurring in nature be subject to UV sunlight and destroy, do not form residual; Simultaneously, various toxin are all harmless to people, animal.Therefore, form great advantage at aspects such as ecological environmental protection, foreign exchange earning and promotion human healths.

Embodiment:

Be specific embodiments of the invention with recipient bacterium bacillus thuringiensis,Bt 4.0718 (Bacillus thuringiensis subsp.Kurstaki) below.

1. activation medium: peptone 1%, yeast extract 0.5%, NaCl 1%, pH7.2-7.4.

The preservation bacterial classification on the solid activation medium 30 ℃ leave standstill and cultivate 24h, transfer on the liquid activation medium, in 30 ℃, 200rpm, cultivate 18～24h, centrifugal collection 3ml precipitation in 1.5ml Eppendorf pipe, with STE (50mmol/l TrisCl, 50mmol/l NaCl, 5mmol/l EDTA pH8.0) washing once, add 100 μ l solution I (10mg/ml lysozymes, 100 μ g/ml RNA enzymes) 37 ℃ of incubation 60min, add 100 μ l solution II (0.5%TritonX-100,50mol/l TrisCl, 10mmol/l EDTA, 0.1mg/ml proteinase K, pH8.0) 40 μ l 5mol/l NaCl are in 37 ℃ of incubation 90min, with twice of the saturated phenol extracting of pH8.0, chloroform extracting twice, the long-pending absolute ethyl alcohol precipitation of dliploid is spent the night, with 14000rpm with at 4 ℃ of following centrifugal 20min collecting precipitations.Precipitation is dissolved in TE (10mmol/l TrisCl, 1mmol/l EDTA pH8.0) buffer solution.With each 10U of PstI/BamHI, the buffer solution composition is 20mMTrisCl (pH8.5), 10mM MgCl2,1mmol/L Dithiothreitol, 100mM KCl, 37 ℃ of incubation 1h, the dna fragmentation about the 4kb that can obtain being fit to clone.

3. bacillus thuringiensis,Bt---shuttle vehicle pHT3101, this plasmid has the two cover dubbing systems that can duplicate in Bt and E.coli, performance erythromycin resistance in Bt shows amicillin resistance, as the carrier that the present invention relates to gene in E.coli simultaneously.

4.HWTX-I, HWTX-II, u-agatoxins, δ-Latroinsectotoxin, δ-AcTX-Hvla etc. are the polypeptide class neurotoxins of separation and purification from different spider venoms, can cause a large amount of releases of neurotransmitter, cause that insect is benumbed rapidly, death, its gene and amino acid sequence are seen 14 pages-23 pages in specification.

5. after carrier pHT3101 being used the PstI/BamHI double digestion, use phenol: chloroform extracting sample, precipitate 1h, 14000rpm, 4 ℃ of down centrifugal 25min recovery DNA with 2 times of volume of ethanol in 10 ℃, with the heavy molten DNA of TE, add 1/10 volume, 10 * calf intestinal alkaline phosphatase buffer solution (10mM ZnCl ₂, 10mM MgCl ₂, 100mMTrisCl (pH8.0)) and an amount of calf intestinal alkaline phosphatase in 37 ℃ of incubation 30min, 65 ℃ of heating 1h deactivation phosphatases under 5mmol/LEDTA solution (pH8.0) condition then.Use phenol again: chloroform extracting, the dephosphorylized DNA of purifying.Dephosphorylized carrier DNA is mixed with fragment equimolar amounts about above-mentioned 4Kb, add water to 7.5 μ l, 45 ℃ of 5min that heat unwind the cohesive end of annealing again, are cooled to 0 ℃, add 10 * T ₄DNAligase buffer solution 1 μ l, T ₄DNA ligase 0.1weiss unit, 5mM ATP 1 μ l, in 16 ℃ of incubation 1～4h, produce new plasmid pXLZ401, get 1-2 μ l sample and transform (electric-field intensity 8.75KV/cm by electricity, resistance 100 Ω～200 Ω, electric capacity 25 μ F and 2 * phosphate buffers), particle gun or at 36%PEG, 50mM CaCl ₂The method that mediation is induced by metal ion down transforms Bt does not have crystal mutant strain or plasmid-free change strain, selects transformant by erythromycin resistant panel (10 μ g/ml), and observes the transformant of selecting to produce crystal by gram stain microscopy.

6. plasmid is cultivated and extracted to the transformant that can produce crystal by the method for front, behind the pstI/BamHI double digestion by CsCl density gradient centrifugation, Glass-milk method or with total plasmid by behind the agarose gel electrophoresis, downcut the purpose band, again electrophoresis on the nucleic acid electrophoresis recovery instrument is to obtain the purpose fragment and to be purified.Behind this sequencing fragment, (as Jelly-fish) analyzes sequence with computer software, find its initiating sequence and SD sequence, and by the total length initiating sequence before the PCR method amplification initiation codon ATG, and 5 ' end, 3 ' end adds ECoRI, SalI recognition site respectively, be inserted in the multiple clone site of pHT3101 by previously described DNA method of attachment, form plasmid pXLZ402.Electricity consumption conversion, particle gun or metal ion revulsion Transformed E .Coli DH5 α are by the dull and stereotyped transformant of selecting of ampicillin (50 μ g/ml).

7. adopt the synthetic respectively complementary strand of method of chemosynthesis respectively according to spider virus gene sequence, complementary strand annealing back has ECoRI, XbaI cohesive end respectively at 5 of gene ' end, 3 ' end, and 3 ' end is added with terminator TGA in addition.By utilizing existing ICPs gene data among the GeneBanK, function in conjunction with its space conformation and domain, adopt the array of computers technology to search in the domain II zone of ICPs comparatively conserved sequence, compare with the order-checking fragment, determine its domain II border according to conserved region, adopt the method for chemosynthesis or PCR to obtain domain II genetic fragment, and be added with SalI, EcoRI recognition site at 5 of fragment ' end, 3 ' end respectively, be added with initiation codon ATG (being positioned at downstream, SalI site) in addition at 5 ' end.

With above-mentioned spider virus gene fragment with after domain II fragment is connected by previously described DNA method of attachment, and make to use the same method and be inserted under the initiating sequence of plasmid pXLZ402, form plasmid pXLZ403, the method that electricity conversion, particle gun or metal ion are induced transforms Bt plasmid-free mutant strain, selects transformant by the erythromycin resistance.

9.75 the unknown antigen solution that μ l is dissolved in the carbonate buffer solution joins in 4 holes of microwell plate, 4 ℃ are spent the night or 37 ℃ of 3h; Be inverted microwell plate, the water of the inside is most, in every hole, add 200 μ l PBS-Tween-20s, after a while the PBS-Tween-20 is toppled over totally, so triplicate.Every hole adds 200 μ l confining liquids (carbonate buffer solution or 5% milk power solution that contain 2% calf serum) in microwell plate, hatches 30min for 37 ℃.The same step is equally washed microwell plate, adds the suitably spider toxin antibody of dilution of 75 μ l in every hole, hatches 30min for 37 ℃, and the same step is equally washed microwell plate, adds the suitably second antibody of dilution of 100 μ l in every hole.Second antibody is the goat-anti immunoglobulin g antibody with horseradish peroxidase-labeled, 37 ℃ of incubation 30min.The same step is equally washed microwell plate, adds 100 μ l zymolytes in every hole, covers microwell plate, and jog 15min shows certain color in the at this moment visible hole, adds 50 μ l stop buffers in every hole and stops reaction, and positive control hole color reaction is green.Behind high performance liquid chromatograph (HPLC) purifying, whether correct to C end order-checking detection polypeptide expressed amino acid sequence, indoor insecticidal test detects the biologic activity of the nerve polypeptide of expressing.

10. wild type transposons Tn5401 is removed the border with exonuclease, be used in the restriction enzyme cutting that there is recognition site at the transposase two ends, purifying is removed the fragment of transposase, with rest segment T ₄Dna ligase connects, and is inserted among the pHT3101, forms plasmid pXLZ404, increases among electric conversion, particle gun or the metal ion revulsion Transformed E .coli DH5 α, and amicillin resistance is selected transformant.

11. extracting plasmid pXLZ403, pXLZ404, and by CsCl density gradient centrifugation, the Class-milk method or cut glue after with the nucleic acid electrophoresis recovery instrument separate, complete recombinant fragment among the plasmid purification pXLZ403, be inserted on the site of plasmid pXLZ404 Central Plains transposase, and enzyme cuts except that the formation of the Bt replicon on carrier plasmid pXLZ405.Increase among electricity conversion, particle gun or the metal ion revulsion Transformed E .coli DH5 α, amicillin resistance is selected transformant.

12. change wild type transposons Tn5401 on the Bt4.0718 chromosome new bacterial strain of formation, extracting plasmid pXLZ405, electricity conversion, particle gun or metal ion are induced and are transformed the new bacterial strain that produces, then improved Tn5401 and total length recombinant fragment can be incorporated among the wild type Tn5401 that is present on the chromosome, and lose transfer ability, select transformant with the erythromycin resistance.

13. insecticidal toxicity and insecticidal spectrum by indoor insecticidal test testing engineering bacterium, choose the of the right age larva of a plurality of purposes, to join in the 9cm culture dish through the plant leaf blade that different dilution zymotic fluids soak, put into 10 diamond-back moths or cotton bollworm larvae, record worm rate alive detects its killing rate and insecticidal spectrum under 30 ℃ of temperature, 85% humidity, certain illumination condition.

14. produce 10 tons of microbial inoculums with 15 tons of fermentation tanks.With the slack tank sterilization of fermentation tank elder generation, real disappearing behind the culture fluid of packing into.High pressure steam sterilization is adopted in sterilization, the 30min that promptly under 121 ℃, pressure 0.5kg/cm2 condition, sterilizes, and seeding tank, fermentation tank culture liquid sterilization mixing speed 250rpm, pressurize is cooled to 30 ℃ then.Seeding tank, fermentation tank feed filtrated air with throughput 2Vols/vol/min, oxygen content 40% and carry out fermented and cultured after inoculating by 5% inoculum concentration.

The fermentation tank culture formula of liquid: analysis for soybean powder 6.6%, corn starch 0.825%, NaOH 0.5%, at 121 ℃, behind the hydrolysis 20min, filters and removes impurity.By volume add glucose 1.65%, KH ₂PO ₄0.165%, K ₂HPO ₄0.165%, CaCO ₃0.165%, FeSo ₄.7H ₂O0.0033% stirs, and pH value sterilization is preceding 9.8, sterilization afterwards 7.4.

Seed culture based formulas: beef extract 0.5%, peptone 1.0%, glucose 0.3%, NaCl0.2%, MgSO ₄7H ₂O 0.03%, K ₂HPO ₄0.03%, MnSO ₄0.005%.PH7.5 before the sterilization, sterilization back pH7.3.

Double effect biological insecticide Synergistic additives prescription: Tea Saponin 1～5%, dimethylbenzene 1%, sorbierite 2%, Tween 80 2%, glycerine 5%, honey 5%.

The preservation bacterial classification on seed culture medium solid inclined-plane 30 ℃ cultivate 36h, wash with sterile water, formerly smash in Mie Jun the triangular flask that bead is housed behind the lawn by in the 5% access first class seed pot, culture fluid is a seed culture medium.Mixing speed 250rpm presses 5% amount access secondary seed jar behind the cultivation 8h down at 30 ℃.Mixing speed 250rpm, 30 ℃ cultivate that the inoculum concentration by 5% inserts in 15 tons of production fermentation tanks behind the 8h, and culture fluid is for producing fermentation culture.Mixing speed 250rpm, 30 ℃ of cultivation 42-48h, throughput 2Vols/vol/min, oxygen content 40%, online detection.When thalline degraded discharges gemma and crystal, put jar in its culture fluid during biomass less than 5%, ultrafiltration and concentration, add Synergistic additives, make preparation or make dry powder.Produce the Bt preparation with the method, its zymotic fluid crystalline content can reach 1.6g/l, spider poison protein content 50～100mg/l.Quality standard in conjunction with domestic and international microorganism insecticide, make tiring of microbial inoculum of production can reach 6000 international units/μ g, there is not residual contamination, to Lepidoptera, diptera, the killing rate of coleoptera primary pest reached 90～98% in 3 days, and the longevity of residure reaches 15～20 days in use in the land for growing field crops, brood cell's number alive of microbial inoculum reaches 80～10,000,000,000/ml, and the shelf life of concentrate and pulvis was respectively 1～2 year.The microbial inoculum of producing is used for field experiment after suitably concentrating, and after through the checking of a series of lands for growing field crops application technology, is identified for the control of crops, vegetables, fruit tree, forest and sanitary insect pest.It is residual that the agricultural product of listing do not have microbial inoculum, harmless to people, animal, and ecotope is not had destruction, promptly can be applicable to produce.

Derive from special neurotoxin gene of various insects and the amino acid sequence of spider

The Selenocosmiahuwena neurotoxin:

HWTX-I：5′GCT?TGC?AAA?GGT?GTT?TTC?GAC?GCT?TGC?ACC?CCG?GGT?AAAAAC?GAG?TGC?TGC?CCG?AAC?CGT?GTT?TGC?TCT?GAC?AAA?CAT?AAA?TGG?TGC?AAATGG?AAA?CTG?3′.

Amino acid sequence: Ala cys lys Gly Val phe Asp Ala cys Thr pro Gly lysAsn Glu cys cys pro Asn Arg Val cys ser Asp lys His lys Trp cys lysTrp lys leu.

HWTX-II (a subunit): 5 ' CTG TTC GAG TGC TCT TTC TCT TGC GAG ATAGAG AAA GAG GGT GAC AAA CCG TGC AAA AAA AAA AAA TGC AAA GGT GGT TGGAAA TGC AAA TTC AAC ATG TGC GTT AAA GTT 3 '

Amino acid sequence: leu phe Glu cys ser phe ser cys Glu Ile Glu lys GluGly ASp lys pro cys lys lys lys lys cys lys Gly Gly Trp lys cys Lysphe Asn Met cys Val lys val

HWTX II (β subunit): 5 ' CTG TTC GAG TGC TCT TTC TCT TGC GAG CAG GAGAAA GAG GGT GAC AAA CCG TGC AAA AAA AAA AAA TGC AAA GGT GGT TGG AAATGC AAA TTC AAC ATG TGC GTT AAA GTT 3 '

Amino acid sequence: leu phe Glu cys ser phe ser cys Glu Gln Glu lys GluGly Asp lys pro cys lys lys lys lys cys lys Gly Gly Trp lys cys lysphe Asn met cys Val lys Val

Lei Shi ravenizhu toxin:

Raventoxin-I：cys?gly?thr?asn?arg?ala?trp?Cys?arg?asn?ala?lysasp?his?cys?cys?cys?gly?tyr?ser?cys?val?lys?gly?ile?trp?ala?ser?lyslys?glu?asp?asp?gly?tyr?cys?trp?lys?lys?phe?gly?gly?cys

Gene order: 5 ' TGC GGT ACC AAC CGT GCT TGG TGC CGT AAC GCT AAA GACCAT TGC TGC TGC GGT TAC TCT TGC GTT AAA GGT ATC TGG GCT TCT AAA AAAGAG GAC GAC GGT TAC TGC TGG AAA AAA TTC GGT GGT TGC 3 '

Raventoxin-II：icadeggpcvagigccaglrcsgailglagscq

Gene order: 5 ' ATC TGC GCT GAG GAG GGT GGT CCG TGC GTT GCT GGT ATCGGT TGC TGC GCT GGT CTG CGT TGC TCT GGT GCT ATC CTG GGT CTG GCT GGTTCT TGC CAG 3 '

Australia Criny spider insect specific neurotoxin:

1.Atracotoxin-Hv1c:aictgadrpc aaccpccpgt sckaesngvs ycrkdep (amino acid sequence)

Gene order: 5 ' GCT ATC TGC ACC GGT GCT GAC CGT CCG TGC GCT GCT TGCTGC CCG TGC TGC CCG GGT ACC TCT TGC AAA GCT GAG TCT AAC GGT GTT TCTTAC TGC CGT AAA GAC GAG CCG 3 '

2.delta-atracotoxin-Hv1:cakkrnwcgk tedcccpmkc vyawyneqgscqstisalwk kc (amino acid sequence)

Gene order: 5 ' TGC GCT AAA AAA CGT AAC TGG TGC GGT AAA ACC GAG GACTGC TGC TGC CCG ATG AAA TGC GTT TAC GCT TGG TAC AAC GAG CAG GGT TCTTGC CAG TCT ACC ATC TCT GCT CTG TGG AAA AAA TGC 3 '

3.omega-actx-hv1a:sptcipsgqp cpynenccsq sctfkeneng ntvkrcd (amino acid sequence)

Gene order: 5 ' TCT CCG ACC TGC ATC CCG TCT GGT CAG CCG TGC CCG TACAAC GAG AAC TGC TGC TCT CAG TCT TGC ACC TTC AAA GAG AAC GAG AAC GGTAAC ACC GTT AAA CGT TGC GAC 3 '

America Criny spider insect specific neurotoxin:

1.mu-agatoxin I:ecvpenghcr dwydeccegf ycscrqppkc icrnnn (amino acid sequence)

Gene order: 5 ' GAG TGC GTT CCG GAG AAC GGT CAT TGC CGT GAC TGG TACGAC GAG TGC TGC GAG GGT TTC TAC TGC TCT TGC CGT CAG CCG CCG AAA TGCATC TGC CGT AAC AAC AAC 3 '

2.mu-agatoxin II:ecatknkrca dwagpwccdg lycscrsypg cmcrpss (amino acid sequence)

Gene order: 5 ' GAG TGC GCT ACC AAA AAC AAA CGT TGC GCT GAC TGG GCTGGT CCG TGG TGC TGC GAC GGT CTG TAC TAC TCT TGC CGT TCT TAC CCG GGTTGC ATG TGC CGT CCG TCT TCT 3 '

3.mu-agatoxin III:adcvgdgqrc adwagpyccs gyycscrsmp ycrcrsds (amino acid sequence)

Gene order: 5 ' GCT GAC TGC GTT GGT GAC GGT CAG CGT TGC GCT GAC TGGGCT GGT CCG TAC TGC TGC TCT GGT TAC TAC TGC TCT TGC CGT TCT ATG CCGTAC TGC CGT TGC CGT TCT GAC TCT 3 '

4.mu-agatoxin IV:acvgenqqca dwagphccdg yyctcryfpk cicrnnn (amino acid sequence)

Gene order: 5 ' GCT TGC GTT GGT GAG AAC CAG CAG TGC GCT GAC TGG GCTGGT CCG CAT TGC TGC GAC GGT TAC TAC TGC ACC TGC CGT TAC TTC CCG AAATGC ATC TGC CGT AAC AAC AAC 3 '

5.mu-agatoxin V:acvgenkqca dwagphccdg yyctcryfpk cicrnnn (amino acid sequence)

Gene order: 5 ' GCT TGC GTT GGT GAG AAC AAA CAG TGC GCT GAC TGG GCTGGT CCG CAT TGC TGC GAC GGT TAC TAC TGC ACC TGC CGT TAC TTC CCG AAATGC ATC TGC CGT AAC AAC AAC 3 '

6.mu-agatoxin VI:dcvgesqqca dwagphccdg yyctcryfpk cicvnnn (amino acid sequence)

Gene order: 5 ' GAC TGC GTT GGT GAG TCT CAG CAG TGC GCT GAC TGG GCTGGT CCG CAT TGC TGC GAC GGT TAC TAC TGC ACC TGC CGT TAC TTC CCG AAATGC ATC TGC GTT AAC AAC AAC 3 '

Latrodectus mactans's insect specific neurotoxin:

1.Alpha-latroinsectotoxin： acsspevsif?hffvyagsfv?knfkkmkgssaiskremsra?dqckllayta?vgyetvgnva?adiasiegan?lvaapvaagg?hlgkgltdaamiamdcssip?feeikeilnk?efkemgrkld?kntealehvs?klvsktlstv?ekirvemregfklvietien?iatkeivfdi?nkivqyfnne?reninsrqke?efvaklqepa?pgnfllylrnsrtsesgtly?sllfriidqe?laipnnagdn?naiqalyalf?ygtetfisim?fylvkqysylaeyhyqkgnl?eefntnfdhm?kivfqdfkfs?liginqntkp?lvdevlnvln?nvknksfirnvqnklfydlm?kqtesllelk?keianmelpi?idetprlsis?isfkersddk?pvdtpllkwdkgkevkyaiq?feqdgkfski?sswskpvtvq?hlacpfisvd?kdrrnrlifr?qfgdqipelvgtlrgsqvef?rdihrdlyna?aqvpyareal?sisrtliqng?anvsetfelg?rgaihaaasagnydvgelll?nkdinlleka?dkngytplhi?aadsnkndfv?mflignnadv?nvrtksdlftplhlaarrdl?tdvtqtlidi?teidlnaqdk?sgftplhlsi?sstsetaail?irntnavinikskvgltplh?latlqnnlsv?skllagkgay?lndgdangmt?plhyaamtgn?lemvdfllnqqyininaatk?ekkwtplhla?ilfkkndvae?rllsdenlni?rletngginp?lhlasatgnkqlviellakn?advtrltskg?fsalhlgiig?kneeipfflv?ekganvndkt?nsgvtplhfaaglgkanifr?lllsrgadik?aedinsqmpi?heavsnghle?ivriliekdp?slmnvknirneypfylavek?rykdifdyfv?skdanvnevd?hngntllhlf?sstgelevvq?flmqnganfrlknnerktff?dlaiengrln?ivafaveknk?vnlqaahrgk?tilyhaicds?akydkieivkyfieklnese?cnplheaaay?ahldlvkyfv?qerginpaef?neenqaspfc?itihgapcgysldcdtpdrl?evveylsdki?pdingkcdvq?entpitvaif?ankvsilnyl?vgigadpnqqvdgdpplyia?arqgrfeivr?clievhkvdi?ntrnkerfta?lhaaarndfm?dvvkylvrqgadvnakgidd?lrpidiagek?akaylqssrf?lrsghsfqsn?eidsfgntih?gismsartndkltqqisskg?trsdsnsteg?kmhsenvhvr?sidvngalll?ldfmirvfas?kktnfapygsriktrsaaea?qaealimter?fenllsglig?dpipdsidfs?nvhskiykai?msgrrsvisemlcsfaeeys?klnhesikql?lsefetlttt?kaseihiees?vpyapfeice?lkvnsnvsqik

：5′ GCTTGCTCTT CACCAGAAGT AAGTATTTTT CATTTCTTTGTTTATGCAGG TAGCTTTGTG AAGAATTTTA AAAAAATGAA GGGAAGTAGC GCGATTTCTAAGAGAGAAAT GTCAAGAGCA GATCAGTGTA AGCTTCTTGC GTATACTGCT GTAGGATACGAAACGGTGGG CAACGTTGCA GCCGATATCG CTTCCATTGA GGGAGCGAAT CTGGTAGCCGCACCGGTTGC AGCTGGCGGT CACCTTGGCA AAGGTCTGAC TGACGCCGCA ATGATAGCCATGGATTGCTC TAGCATACCA TTTGAGGAAA TAAAAGAAAT CTTAAATAAA GAATTTAAAGAAATGGGTAG AAAACTCGAT AAGAACACCG AAGCCTTAGA ACACGTTTCT AAATTAGTCTCTAAAACATT GTCCACCGTC GAAAAGATTA GAGTTGAGAT GAGGGAAGGG TTTAAACTTGTTATCGAAAC AATTGAGAAC ATTGCAACAA AAGAAATAGT TTTTGATATT AATAAAATTGTTCAATACTT TAATAACGAA AGAGAAAATA TAAATTCTCG ACAAAAAGAA GAATTTGTTGCTAAACTGCA AGAACCAGCT CCTGGAAATT TTTTGCTCTA TTTAAGAAAT TCTAGAACTTCTGAAAGTGG CACTCTTTAT TCTTTACTAT TTAGAATTAT CGACCAAGAG TTGGCAATTCCAAATAATGC TGGGGATAAT AATGCAATTC AGGCTCTTTA CGCTCTGTTC TATGGTACTGAGACGTTCAT TTCTATCATG TTTTATTTAG TCAAGCAATA CTCGTATCTC GCAGAGTATCACTACCAAAA AGGTAATCTG GAGGAATTCA ATACAAATTT CGATCATATG AAAATAGTATTCCAAGATTT CAAATTCTCT CTTATTGGTA TTAACCAAAA CACGAAGCCT TTGGTGGATGAAGTCCTTAA TGTTTTAAAC AATGTAAAAA ATAAAAGCTT TATTAGAAAT GTCCAAAATAAATTGTTTTA TGACCTAATG AAACAAACTG AATCTTTGCT GGAACTCAAG AAAGAAATTGCGAATATGGA ACTGCCTATT ATAGATGAAA CTCCCAGATT ATCCATTTCT ATTTCTTTTAAAGAGAGAAG TGATGATAAG CCTGTTGATA CACCACTTTT AAAGTGGGAT AAGGGAAAGGAAGTAAAATA TGCTATACAA TTCGAGCAAG ATGGTAAGTT CTCTAAAATT AGCAGTTGGTCGAAACCGGT AACAGTGCAG CACTTAGCAT GCCCATTTAT TTCGGTAGAT AAAGATAGGAGAAACAGATT AATTTTTCGG CAATTTGGAG ACCAAATACC AGAGCTAGTG GGCACTCTGAGAGGTTCTCA GGTTGAATTT CGGGATATCC ATCGTGATCT GTACAATGCA GCGCAAGTTCCTTATGCGAG AGAAGCACTG AGTATCAGCC GAACGCTCAT ACAAAACGGT GCCAATGTGAGTGAAACATT CGAATTGGGC AGAGGAGCTA TTCACGCAGC TGCATCAGCT GGAAATTATGATGTTGGGGA ATTGCTTCTA AATAAAGACA TTAATTTGCT CGAAAAGGCT GATAAAAACGGTTACACTCC ACTTCACATA GCTGCTGATT CAAATAAGAA TGATTTCGTT ATGTTTCTAATCGGAAATAA TGCAGATGTT AATGTTCGAA CTAAATCAGA TTTATTTACT CCTTTACATTTAGCTGCACG GCGGGATTTA ACAGATGTTA CTCAAACATT GATTGATATC ACTGAAATAGACCTTAATGC GCAAGACAAA TCTGGATTCA CTCCATTGCA TCTCTCTATC TCTAGTACTTCTGAAACTGC TGCGATTCTC ATACGAAATA CAAACGCAGT AATAAACATA AAATCTAAGGTCGGATTAAC TCCTTTACAT CTGGCCACGC TTCAAAATAA CTTAAGCGTT TCCAAGTTATTAGCTGGTAA AGGAGCTTAT TTGAATGACG GCGATGCTAA TGGGATGACT CCTCTGCATTATGCAGCGAT GACGGGGAAT TTAGAAATGG TTGATTTTCT TCTTAACCAA CAGTACATAAATATTAATGC AGCTACGAAG GAGAAAAAAT GGACACCTTT GCATTTAGCC ATTCTGTTTAAAAAGAATGA TGTTGCGGAA AGGTTACTAA GTGATGAAAA TCTTAATATA CGCTTGGAAACCAATGGAGG TATTAATCCT TTGCATTTAG CTTCCGCAAC TGGAAATAAG CAATTAGTAATTGAATTATT AGCAAAGAAT GCGGATGTGA CCAGATTAAC ATCCAAAGGT TTTTCTGCCCTTCATTTGGG GATAATAGGT AAAAACGAGG AAATTCCATT CTTTCTGGTT GAAAAAGGAGCAAATGTTAA CGATAAAACT AACAGTGGAG TGACACCTTT ACATTTTGCA GCTGGGTTGGGAAAAGCCAA TATTTTCAGG CTACTGCTCA GCCGAGGAGC AGATATTAAA GCTGAAGATATAAATTCTCA AATGCCTATT CATGAAGCTG TATCGAATGG ACATCTAGAA ATTGTTAGAATACTCATTGA GAAAGATCCC TCCCTGATGA ACGTAAAAAA TATCAGGAAT GAATATCCCTTTTACCTAGC AGTGGAAAAA CGCTATAAGG ATATATTCGA TTACTTTGTA AGCAAAGATGCTAACGTAAA TGAGGTTGAT CATAACGGGA ACACACTTTT GCACTTATTT TCCAGTACAGGAGAACTTGA AGTTGTGCAG TTCTTAATGC AAAACGGCGC TAACTTTCGC CTTAAAAATAATGAAAGGAA GACCTTCTTC GATCTTGCTA TTGAAAATGG ACGCTTAAAC ATTGTGGCCTTTGCTGTAGA GAAAAACAAG GTGAATCTCC AAGCAGCCCA CAGAGGAAAA ACGATTCTGTATCATGCAAT TTGTGATTCT GCAAAATACG ACAAGATTGA AATTGTAAAA TATTTCATTGAAAAACTTAA TGAAAGTGAA TGCAATCCAT TGCATGAAGC CGCGGCTTAC GCGCATTTAGATTTAGTGAA ATACTTCGTT CAGGAAAGAG GAATAAATCC GGCAGAATTT AATGAGGAAAATCAAGCGTC TCCTTTCTGT ATCACTATAC ACGGGGCGCC ATGCGGATAT TCACTTGACTGTGATACGCC TGACCGGTTA GAAGTGGTTG AGTATCTCTC AGACAAAATA CCCGATATTAACGGAAAGTG TGATGTCCAG GAAAACACTC CAATAACCGT AGCCATATTT GCAAATAAAGTCAGCATTTT AAATTATTTA GTAGGGATCG GAGCTGATCC CAACCAACAA GTTGATGGAGATCCTCCTTT ATACATTGCG GCAAGGCAAG GACGTTTCGA AATTGTAAGA TGTTTGATAGAAGTGCATAA GGTCGACATA AATACTAGAA ATAAAGAGCG GTTTACGGCA CTACACGCTGCAGCCCGGAA CGATTTTATG GATGTAGTGA AATATCTCGT AAGGCAAGGG GCCGATGTCAATGCTAAAGG TATAGATGAT CTTAGGCCCA TCGACATTGC TGGAGAAAAA GCAAAAGCATACCTGCAATC GTCTCGTTTC CTTCGAAGCG GTCATTCTTT TCAATCAAAC GAAATCGATAGTTTTGGTAA TACGATACAC GGTATTTCTA TGTCAGCAAG GACAAATGAT AAATTAACTCAACAAATATC TTCTAAAGGA ACCAGATCGG ATTCTAATTC AACGGAAGGC AAAATGCATTCAGAAAACGT CCATGTCCGC AGCATTGACG TTAACGGAGC ACTTCTGCTA TTGGATTTTATGATTAGAGT TTTTGCGAGT AAGAAAACGA ATTTCGCCCC CTACGGCTCC AGAATAAAGACGCGCTCAGC TGCAGAAGCG CAGGCTGAAG CACTGATAAT GACAGAACGA TTCGAAAATCTTTTGAGTGG TTTGATTGGC GACCCGATTC CCGATTCTAT AGACTTTTCC AACGTTCATTCGAAGATATA CAAAGCCATT ATGAGTGGGA GACGAAGTGTAATATCAGAA ATGCTATGTT CCTTTGCAGA AGAGTATTCT AAACTGAATC ATGAAAGTATAAAACAGCTT CTATCAGAAT TCGAAACGCT CACTACTACT AAAGCATCCG AAATTCATATTGAAGAAAGT GTTCCTTATG CTCCTTTTGA AATATGTGAA TTAAAGGTTA ATTCGAATGTCTCACAAATC AAGTAATTAA AGAAAATGAA ATATATATGG ATTATCTTAT GAAGTAAGTATTGCATTTTA CCTATTAAGT TCACTCGAAA TTATTTATTT TATTTTGTTT AACTCATACGCTTAACGCAA TAATATAAAA TAGCTATTAA TTTATTATAA TACATAGTAA GTTTTATTTATCATTAAATG AAAAGGGGCA TTTTTCATTT AATGATAAAA TAATAATTCC ATCTCCATAGTGGTTTTTTT TTTTTTACCT AATTTAAATA GTTTGTAACA AGAGATTTAA TTTTCTAATGCATAATTAGA AACATAGGTT ATTGATTTAT TTCTTAATAA ATTAAAATTA AAATTTGCAACAAATTATTT TAATTGTTTA TTTTAAAGCT TTTTTTTTAT GCATTCCAAA TTCTCTGAAATTTTCGATGT TTGAAATTCT AAGTAAAAGC ACATTCAATA TAAATGTATT TAATTAAAATTTTGTTTTAA TACTTTTATT AAAATTCTGA ACTTGACCTA TGAATTGAAA TCGCAATTATATAACACATC ATATAAAAAA AATTATAAAT ATTATGATAT AAATATCTAA CCAAATTGCAAAGAGTGTAG TTCTTTAAAT AGAAAGAAAC AACTAAATTT ACTTTAAAGT GGAACAATTGCATTTATTTA AATAAAAAAA TGATTCCATA TGTAATTCCA GCTAACATTT GAAAACTTCTTAGTTTTAAT AAATTTTTTG CGTTTTCTTA AATTTCTAAA AGAATTGATT TTGTTAGAATTATCCTTTTA TTTTTGAGAA ATTTTTTTCA TTTTAACTTC TATTCTTTAA AATAGTACTGCAATGTATTT AATTTAATAT ATTTCATTTT TAACACAGTA CTTTTCTTAT TTCAAAACCTATCGATTTCT TAAGGCACTA AATGAAATTA TTGCTTAAAT CGATCATATG CATAGTTATATACTATGTTC TTCCTCCAAG TCTTT 3′

delta-latroinsectotoxin： mhskelqtis?aavarkavpn?tmvirlkrdeedgemtleer?qaqckaieys?nsvfgmiadv?andigsipvi?gevvgivtap?iaivshitsagldiastald?cddipfdeik?eileerfnei?drkldkntaa?leevsklvsk?tfvtvektrnemnenfklvl?etieskeiks?ivfkindfkk?ffekerqrik?glpkdryvak?lleqkgilgslkevrepsgn?slssalnell?dknnnyaipkvvddnkafqa?lyalfygtqt?yaavmfflleqhsyladyyy?qkgddvnfna?efnnvaiifd?dfkssltggd?dglidnviev?lntvkalpfiknadsklyre?lvtrtkalet?lknqikttdl?pliddipetl?sqvnfpnden?qlptpignwvdgvevryavq?yeskgmyskf?sewsepftvq?gnacptikvr?vdpkkrnrli?frkfnsgkpqfagtmthsqtnfkdihrdlydaalninklkavdeattliekgadieakfdndrsamhavayrgnnkialrfllknqsidielkdkngftp?lhiaaeagqa?gfvkllinhg?advnaktskt?nltplhlatrsgfsktvrnl?lespnikvnekeddgftplh?tavmstymvv?dallnhpdid?knaqstsgltpfhlaiines?qevaeslves?nadlniqdvn?hmapihfaas?mgsikmlryl?isikdkvsinsvtennnwtp?lhfaiyfkkedaakellkqd?dinltivadg?nltvlhlavs?tgqiniikellkrgsnieek?tgegytslhi?aamrkepeia?vvliengadi?earsadnltp?lhsaakigrkstvlyllekg?adigaktadgstalhlavsg?rkmktvetll?nkganlkeyd?nnkylpihkaiinddldmvr?lflekdpslk?ddeteegrts?imlivqklll?elynyfinny?aetldeealfnrldeqgkle layifhnkegdakeavkpti lvtiklmeyclkklreesgapegsfdspsskqcistfsed emfrrtlpei vketnsrylp?lkgfsrslnkflpslkfaes?knsyrsenfv?snidsngall?lldvfirkft?nekynltgke?avpyleakasslriaskfee?lltevkgipa?gelinmaevs?snihkaiasg?kpvskvlcsy?ldtfselnsqqmeelvntyl?stkpsvitsa?sadyqklpnl?ltatcleper?maqlidvhqk?mflr

：5′ GGTCAATTGA AACTTTATGA TAGGATTCAC TTTCTTATATAGAAATGCAT TCCAAAGAAT TACAAACTAT TTCAGCAGCG GTAGCACGAA AAGCAGTACCCAATACTATG GTTATTCGGT TGAAAAGAGA TGAAGAAGAT GGAGAAATGA CTCTAGAAGAAAGACAAGCA CAATGCAAAG CAATAGAGTA CAGCAATTCA GTTTTTGGGA TGATCGCTGATGTAGCTAAC GACATCGGTT CCATTCCTGT AATTGGCGAA GTAGTTGGCA TTGTAACTGCCCCAATTGCC ATCGTAAGTC ACATTACTAG CGCAGGCTTG GATATAGCTT CTACGGCATTAGATTGTGAT GATATACCTT TTGATGAGAT TAAGGAAATA TTAGAAGAAA GATTCAATGAAATAGATAGA AAGTTGGACA AGAACACAGC TGCTTTGGAA GAGGTCTCTA AACTGGTAAGTAAAACTTTT GTTACGGTGG AAAAAACAAG GAATGAAATG AACGAAAATT TTAAGCTTGTTTTGGAAACT ATAGAAAGCA AAGAAATAAA ATCAATTGTA TTCAAAATAA ATGATTTTAAAAAGTTTTTT GAAAAAGAAC GACAAAGAAT TAAAGGTTTG CCTAAAGATA GGTATGTTGCTAAGCTTCTA GAACAAAAAG GTATTTTAGG TTCTTTAAAA GAAGTAAGAG AACCATCTGGAAACAGTCTG AGCTCCGCGT TAAATGAACT CTTAGACAAA AACAACAACT ATGCCATCCCAAAAGTGGTT GATGATAATA AGGCCTTTCA GGCGCTGTAT GCTTTATTTT ATGGAACTGAGACTTATGCA GCCGTTATGT TTTTCTTACT CGAACAACAT TCTTATCTGG CTGATTATTATTACCAAAAA GGTGATGATG TAAATTTTAA TGCAGAATTT AATAATGTAG CAATTATTTTTGATGACTTT AAATCATCAC TAACAGGAGG AGATGACGGA TTAATAGATA ATGTCATTGAGGTTCTTAAC ACCGTGAAAG GATTACCATT TATAAAGAAC GCCGACAGTA AACTATACAGAGAATTAGTA ACTAGAACAA AAGCTTTAGA GACTCTTAAA AATCAAATCA AAACGACTGATTTGCCTCTT ATAGATGATA TACCCGAAAC TTTGTCTCAA GTGAAGTTTC CGAATGAGGAAAATCAATTG CCTACACCAA TAGGAAATTG GGTTGATGGC GTAGAAGTTA GGTACGCAGTACAGTATGAA AGTAAGGGCA TGTATTCGAA ATTCAGTGAA TGGTCTGAAC CATTTACTGTCCAAGGTAAC GCTTGTCCGA CTATAAAAGT TCGTGTTGAT CCGAAAAAGA GAAATAGACTTATCTTTAGG AAGTTCAACT CAGGAAAACC TCAGTTTGCT GGAACCATGA CTCATTCACAAACAAATTTT AAAGATATTC ATCGTGATCT ATACGATGCA GCCTTAAATA TTAATAAGTTGAAAGCAGTG GATGAAGCTA CAACTTTGAT TGAAAAGGGT GCAGACATAG AAGCAAAATTTGACAATGAC AGAAGTGCAA TGCACGCAGT TGCATATCGA GGAAATAACA AAATAGCCTTAAGATTTCTT TTGAAAAATC AATCCATTGA CATCGAGTTA AAAGATAAAA ACGGCTTTACTCCTCTACAC ATCGCAGCTG AAGCAGGTCA GGCAGGATTT GTTAAGTTAC TAATAAATCATGGAGCTGAT GTGAATGCAA AAACAAGTAA GACAAATTTG ACACCATTAC ATCTTGCAACACGTAGTGGA TTTTCAAAAA CTGTAAGAAA TTTACTAGAA AGCCCAAATA TTAAGGTAAATGAAAAGGAG GATGACGGAT TTACACCTTT GCATACTGCA GTAATGAGTA CTTATATGGTTGTCGATGCT TTGCTAAATC ATCCAGACAT TGATAAAAAT GCGCAGTCTA CGTCAGGATTGACTCCTTTC CATTTAGCAA TTATTAATGA AAGTCAAGAA GTTGCAGAAT CTTTAGTGGAAAGTAATGCT GATCTAAATA TTCAGGATGT TAACCATATG GCTCCTATTC ATTTTGCAGCTTCAATGGGT AGTATTAAAA TGCTTAGATA TCTCATTTCC ATAAAAGATA AAGTTAGTATTAATTCTGTG ACTGAGAATA ATAACTGGAC ACCTTTACAT TTTGCTATAT ATTTTAAAAAAGAAGATGCT GCAAAAGAAT TGTTGAAACA AGATGACATA AATTTAACAA TTGTTGCAGATGGTAATCTT ACCGTTTTAC ATCTTGCTGT TTCGACAGGA CAAATAAATA TAATTAAAGAATTATTGAAG AGAGGCTCCA ATATAGAAGA AAAAACTGGA GAAGGATATA CATCTCTCCACATCGCTGCG ATGCGAAAGG AGCCAGAGAT AGCTGTTGTT TTGATTGAAA ACGGTGCTGACATAGAAGCT CGATCAGCTG ATAATTTAAC ACCTTTACAT TCTGCCGCAA AAATAGGAAGGAAATCTACA GTACTTTACT TATTAGAAAA AGGAGCTGAC ATTGGAGCTA AAACAGCAGACGGTTCTACT GCCTTGCATT TAGCTGTATC TGGTCGTAAA ATGAAAACTG TTGAAACTCTATTAAATAAA GGAGCAAATT TAAAAGAATA CGATAACAAT AAATATTTGC CAATACATAAAGCTATTATT AATGATGACC TTGACATGGT ACGTTTGTTT CTTGAAAAAG ATCCCAGTCTCAAAGATGAT GAAACAGAAG AGGGTAGAAC TTCAATTATG TTAATTGTTC AGAAATTGCTTCTTGAATTA TATAACTATT TTATAAATAA TTATGCTGAA ACTTTGGATG AAGAAGCTTTATTCAACCGC TTAGATGAAC AAGGGAAATT AGAGCTTGCA TATATCTTCC ATAATAAAGAAGGTGATGCA AAAGAGGCTG TTAAGCCAAC TATCCTTGTT ACAATTAAAC TTATGGAATACTGCTTAAAA AAACTTCGCG AAGAGTCTGG AGCTCCTGAA GGTAGTTTCG ATTCTCCATCTTCAAAGCAA TGTATTTCTA CCTTTTCAGA GGATGAAATG TTTCGTCGTA CTTTACCGGAAATTGTAAAA GAAACGAACA GCAGATATTT ACCACTAAAG GGCTTTTCTC GCAGCCTAAATAAGTTTCTC CCTTCTCTAA AATTTGCCGA AAGTAAGAAT AGCTACAGAT CTGAAAATTTTGTTAGCAAT ATTGATTCCA ACGGAGCATT ACTTTTACTC GATGTATTTA TCAGAAAGTTTACTAATGAG AAATACAATT TGACTGGAAA AGAAGCTGTA CCCTATCTGG AAGCAAAGGCTTCATCATTA CGTATCGCTT CTAAATTTGA AGAACTTCTA ACTGAAGTTA AAGGTATTCCGGCTGGAGAG CTAATTAATA TGGCCGAAGT GAGTTCCAAC ATACATAAGG CAATTGCAAGTGGTAAGCCT GTATCAAAAG TCTTATGTTC GTATTTGGAT ACCTTTTCTG AATTAAATTCTCAACAAATG GAAGAATTAG TTAACACATA CTTATCCACC AAACCTTCTG TAATTACGTCAGCATCTGCA GATTACCAGA AACTTCCTAA TTTGTTAACT GCAACTTGCT TAGAACCAGAAAGAATGGCT CAACTTATAG ATGTGCATCA AAAGATGTTT TTACGTTAAA ATACCATTCCTTCTGTGTCA TCCATTGAGT ATATGGATTG GCTCTTTCTT TTATGCAAAT ATTTTTTCCACTTTAATGAT TTATTTCGGA TTGTGATTTT CTCACTTCTT TCTTGTTTAT TTCGTTGTTCTGTGCTTTTG AGTGAAATTA TCTGATAATC ATTTCTGAGT TAGTTTCTCT TCTAAGGAGTTTTAGGTATT TGGAATTACA TTAGTTTTGG TTTCGGGTTT TACTTCTATA TTTTGTCCAAAGTAATTTGT AGGAGGGAAT TTCTAAAAAG GAATGGAATC GTTGATAAAT AGTTTTGTATTATTGTACCT TTATAATAAT AAGACTTTAA AGTCAATTTT TTAAAACCAA GCATAAAGTATAGAAGGACT CGTATGTTAT ATAATGAAAT AATAAGTTGC CAAATGTCAT GGATTCAATCAAACAAATTT AAATAATTAA TAATGCAAAA ATTTTTTAAC TTTTTTAATG TTCTTAGTATATTAAGTCAA TAATTTAATA AATACTTTCT AGTTTAATTT TGCTATCTGC TATTACTCTAATTATCAAAC TAGTTATAAA TTCTAAATAT TCTCGTAACA TTATTTTAAA AGTGTAAATAGAGAGACTAT ATTTTTTAAT TATATCCATT TTTATATTGT TAATTTAATT CTTCAAAATTACATTCCTTT TCCTGCATTA TTATTTACAA TTCATTAAAT GCCTATATTT TAAAGACAATACTCAAGTTT CAGATTATTA TAAACAGTCT TATATTGATT CAACGTAAAA TTTTATTGACAATTGCAAAG AAATAATAAT TTAAAATTGT ATTTAAGTAT TTTGAAACAG TTTGATTTTTATTGCAATAT GAATCTAAAT TATTTTCAAA CAATGATAGA A 3′

Claims (3)

1. double engineering bacterium biological pesticide, it is characterized in that these biological insecticides are a kind of bacillus thuringiensis,Bt (Bacillusthuringiensis) engineering bacteria preparations that contain the bacillus thuringiensis,Bt toxalbumin and contain spider poison protein after gene clone, the living spores content of the Thuricide engineering bacterium of said preparation is at 80-100 hundred million/ml, tiring in that 6000 international units/more than the μ g, preparation reaches 90-96% to the larva killing rate of Lepidoptera, diptera and coleoptera of preparation.

2, by the described double engineering bacterium biological pesticide of claim 1, it is characterized in that containing the bacillus thuringiensis,Bt toxoprotein gene that the Thuricide engineering bacterium of bacillus thuringiensis,Bt toxalbumin and spider poison protein carries cry1Aa is arranged, cry1Ab, cry1Ac, cry1Ax, the spider poison protein gene that cry1Cb and cry2Ac, Thuricide engineering bacterium carry has HWTX-I, α subunit among the HWTX-II, β subunit among the HWTX-II, Raventoxin-I, Raventoxin-II, Atracotoxin-Hvlc, delta-atracotoxin-Hvl, omega-actx-hvla, mu-agatoxin I～VI, Alpha-latroinsectotoxin and delta-latroinsectotoxin.

3, a kind of preparation method of double engineering bacterium biological pesticide as claimed in claim 1 is characterized in that the preparation of biological insecticides is made up of the structure of Thuricide engineering bacterium and production two parts of Thuricide engineering bacterium agent:

(1) structure of bacillus thuringiensis,Bt double engineering bacterium

1. make up the controllable express carrier

---recipient bacterium is the golden subspecies of bacillus thuringiensis,Bt 4.0718 bacterial strains and Su Yun, Kustak Asia

Kind, galleria mellonella waxmoth subspecies, Wuhan subspecies and Chinese subspecies,

---plasmid pXLZ401 makes up, with the gentle method extracting of Triton X-100 bacillus thuringiensis,Bt 4.0718 total plasmids, after Pst I/BamH I double digestion, obtain the genetic fragment about 4Kb, use the agarose gel electrophoresis separation and purification, with Pst I/BamH I double digestion shuttle vector pHT3101, obtain having the chain dna of two different cohesive ends in addition.Use the alkaline phosphatase dephosphorylation, the genetic fragment about the 4Kb of separation and purification is mixed with dephosphorylized carrier pHT3101, use T ₄Dna ligase connects, and produces plasmid pXLZ401,

---obtaining of ICPs gene strong promoter and SD sequence, electricity transforms the no crystal mutant strain of plasmid pXLZ401, by erythromycin plate screening transformant, select to produce the transformant of crystal by gram stain microscopy, on the LB medium, activate, cultivate transformant, extract plasmid with TritonX-100, through the agarose gel electrophoresis separation and purification, with passing through CsCl density gradient centrifugation behind pst I/BamH I double digestion or the double digestion, the Glass-milk method obtains to have the fragment of complete ICPS open reading frame, to with computer software sequence be analyzed behind the sequencing fragment, determine its initiating sequence and SD sequence

---the structure of plasmid pXLZ402, above-mentioned fragment is passed through the total length initiating sequence before the pcr amplification initiation codon ATG, and adds ECoR I, Sal I site respectively at 5 ' end and 3 ' end, and is purified, with ECoR I/Sal I double digestion.Carrier pHT3101 uses ECoRI/Sal I double digestion equally, uses the alkaline phosphatase dephosphorylation, and both mix, and use T ₄Dna ligase couples together both under the condition that ATP exists, and forms plasmid pXLZ402, Transformed E .coli DH _{5 α}Middle amplification;

2. the chemosynthesis of the special insect specific neurotoxin gene of spider and domain II sequence obtains

---the chemosynthesis of spider virus gene, adopt chemical synthesis process according to spider virus gene sequence, synthesize the toxin polypeptide full-length gene, hold at the 5 ' end and 3 ' of gene to have ECoR I, Xba I site respectively, add terminator TGA in addition at 3 ' end simultaneously,

---act on the obtaining of domain II sequence of insect enterocyte membrane receptor among the ICPs, in Gene Bank, search existing ICPs domain II sequence.By sequence analysis software, array is conserved region relatively, and compares with the order-checking fragment.Determine its domain II border according to conserved region, obtain domain II genetic fragment with chemosynthesis and PCR method, have Sal I, ECoR I restriction enzyme site at 5 ' end and 3 ' end respectively, add initiation codon ATG in addition at 5 ' end, can obtain the domainII section by PCR

---the structure of plasmid pXLZ403, with alkaline lysis method of extracting plasmid pXLZ402, behind Sal I/XbaI double digestion, use the alkaline phosphatase dephosphorylation, after in addition acquired domain II sequence being cut with EcoR I enzyme, with chemosynthesis 5 ' toxin gene that end has an EcoR I site mixes, and uses T ₄Dna ligase connects, and behind the junction fragment purifying, cuts and mixes with dephosphorylized chain plasmid pXLZ402 with Xba I enzyme, uses T ₄Dna ligase connects, and forms plasmid pXLZ403, transforms the plasmid-free mutant strain, by the dull and stereotyped transformant of selecting of erythromycin;

The antigen-4 fusion protein gene that 3. will have initiating sequence is incorporated into bacillus thuringiensis,Bt DNA and goes up genetic stability

---the structure of plasmid pXLZ404, the border that transposase is discerned is removed with exonuclease in wild type transposons Tn5401 two ends, with the method deletion transposase that enzyme is cut, the purifying rest segment is used T ₄Dna ligase connects, and is inserted among the pHT3101, forms plasmid pXLZ404, changes E.coli DH over to _{5 α}Middle amplification,

---the structure of plasmid pXLZ405, extract plasmid pXLZ403, pXLZ404 respectively with the gentle method of Triton X-100, the recombinant fragment of separation and purification pXLZ403, be inserted on the site of having deleted transposase among the pXLZ404, cut except that the bacillus thuringiensis,Bt replicon among the plasmid pXLZ404 with enzyme, produce plasmid pXLZ405.Electricity consumption conversion, particle gun or metal ion revulsion Transformed E .coli DH _{5 α}Middle amplification, the dull and stereotyped transformant of selecting of ampicillin,

---plasmid pXLZ405 is incorporated on the chromosome of bacillus thuringiensis,Bt 4.0718, wild type transposons Tn5401 is changed on the chromosome of bacillus thuringiensis,Bt 4.0718, produce new bacterial strain, plasmid pXLZ405 electricity is transformed new bacterial strain, improved Tn5401 and total length fusion are incorporated among the wild type transposons Tn5401 that is present on the chromosome, with the dull and stereotyped transformant of selecting of erythromycin;

(2) production of Thuricide engineering bacterium agent

1. actication of culture, the engineering bacteria bacterial classification of preservation are transferred in flat bottle medium behind slant culture, under 28～35 ℃ of conditions, cultivate 30～48h, are made into spore liquid, and the inoculum concentration with 5% is used for the seeding tank inoculation,

2. seeding tank fermentation, spore liquid is inserted through sterilizing and being equipped with in the first class seed pot of culture fluid, the feeding filtrated air is cultivated, obtain the first class seed pot zymocyte liquid, by 5%～8% inoculum concentration the first order seed zymotic fluid is changed in the secondary seed jar culture fluid, feed filtrated air and stir culture, obtain secondary seed jar zymocyte liquid

3. fermentation tank culture, the inoculum concentration by 5%～8% changes secondary seed jar zymotic fluid in the fermentation tank over to, and under 25 ℃～30 ℃ temperature condition, feed filtrated air and cultivate 42～48h,

4. concentrate, ferment tank through ultrafiltration and concentration, is added Synergistic additives with bacterium liquid after finishing, bottling.

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