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

Abstract

The invention relates to a genetically cloned Bacillus thuringiensis (abbreviated as Bt) double-effect engineering bacterium agent and a production method of the double-effect engineer bacterium biopesticide. The present invention takes Bacillus thuringiensis 4.0718 strain and Bacillus thuringiensis subspecies as hosts, and integrates the arachnoid gene with a strong promoter sequence and a fusion protein gene containing a domain II sequence into the DNA of Bt bacteria through the construction of an adjustable expression vector Stable genetics, construction of genetically engineered bacteria that produce spider venom and Bt toxin, and production of double-effect biopesticides through fermentation; The insecticidal rate of coleopteran larvae is 90-96%. The bacterial agent is a green pesticide with no residue, which is safe for humans and animals and beneficial to the protection of the ecological environment.

Description

A kind of double-effect engineering bacteria biopesticide and production method thereof

Technical Field The present invention relates to a genetically cloned Bacillus thuringiensis (Bt) double-effect engineering bacterium, and a production method of the double-effect engineering bacterium biopesticide.

Background technology The long-term use of a large number of chemical pesticides increases the resistance of pests, causing serious damage to the ecological environment and directly endangering human health; at the same time, the return of exported agricultural products due to the problem of pesticide residues directly leads to huge economic losses. Losses amounted to $7.4 billion. Since the 1980s, more and more attention has been paid to the application of Bt as an insecticide for agricultural, forestry and sanitary pests, especially because of its strong insecticidal toxicity, wide insecticidal spectrum, difficulty in producing resistance, and residual Short term, no pollution, safe for humans and animals, easy to industrialized production and other important features, so it has achieved three important social, economic and ecological benefits. However, Bt insecticides still have a series of problems: (1) the insecticidal spectrum is relatively narrow; (2) the insecticidal effect and quick-killing effect are significantly lower than those of chemical pesticides; (3) the insecticidal residual period is short, so There are certain limitations in use.

In recent years, due to the rapid development of genetic engineering technology, scientists have used genetic engineering methods to open up the field of new biopesticides. Chinese patent 86108756 discloses a recombinant virus constructed by Japan's Susumu Maeda that contains DNA fragments of two nuclear polyhedrosis viruses. Since the host insects of the two viruses are different, the insecticide produced by it can exterminate and control the two viruses. Insects, expanding the insecticidal spectrum. Notification number is CN-1044298A M E H Howden of Australia etc. applied for a patent, is to control pests by utilizing the funnel web spider toxin gene to construct engineered plants, and has achieved certain effects. Michael J. Adam of the United States expressed Bt toxin in plants in 1989 by using preferred codons in highly expressed monocotyledonous or dicotyledonous plant proteins, thus realizing a new method for direct plant resistance to insects. The patent announcement number is CN-1044298A. Although the research work on using insect baculovirus as insecticidal gene carrier and cultivating transgenic plants is relatively extensive, its application has been limited by its own characteristics. Since most baculoviruses cannot express a sufficient amount of foreign protein in a short period of time, it takes at least 2-3 days for the insects to stop feeding, and generally 4-14 days to kill the host. Food has caused significant damage to crops. In addition, the host range of baculovirus is relatively narrow, so its application is quite limited. Because people are worried about the pollution of genetically modified food, the sales and research of genetically modified agricultural products in the European market and developed countries such as Japan and Thailand have been ordered by the government departments. At present, the production and development of genetically modified crops are still under intense debate around the world, but most countries have adopted resistance. Developed countries such as the United States and Canada advocate research on genetically modified foods, but in fact they rarely sell them domestically and mainly sell them to third world countries.

Bt bacteria can produce crystal protein, which has poisonous effect on Lepidopteran, dipteran and coleopteran larvae, and the crystal protein is degraded into toxicity under the alkaline condition of larval gut The core fragment acts on the cell membrane to cause perforation to form ion channels, causing the death of pests.

Huwentoxin (HWTX for short) is a polypeptide neurotoxin isolated and purified from the venom of a rare spider species in China, Selenocosmia Huwena. Two toxins have been named, namely HWTX-I and HWTX- II. The number of amino acid residues is 33 and 74, respectively. Toxicity experiments showed that these two toxins can specifically block the nerve-muscle conduction mediated by glutamate in insects and cause paralysis and death. Raventoxin-I, Raventoxin-II are two types of polypeptides isolated and purified from the crude venom of the recently discovered and named Macrothele genus Macrothele genus Neurotoxin, the number of amino acid residues is 43 and 33 respectively, has a strong inhibitory effect on the nerve activity of insects and can cause death. Agatoxins are polypeptide neurotoxins isolated and purified from the venom glands of the American funnel web spider (Agelenopsis aperta). Among them is a group of toxins (u-agatoxins I-VI), which contains 6 kinds of toxins, which can cause irreversible nerve paralysis of insects and lead to death. Australian funnel web spider toxins (atracotoxins) are polypeptide neurotoxins isolated and purified from the spider venom of the genus Atrax in Australia, among which δ-ACTX-HVIa, Atracotoxin-HVIC, Omega-actx-hvla three types of toxins It has specific nerve inhibitory effect on insects. Insect-specific neurotoxin (α-Latroinsectotoxin, δ-Latroinsectotoxin) in the toxin family of black widow spider (Latrodectus Mactaustredisimguttatus) is a polypeptide neurotoxin purified from the venom of this species of spider, which can cause a large amount of release of insect neurotransmitters and quickly paralyze, die.

SUMMARY OF THE INVENTION The present invention aims to utilize the bred Bacillus thuringiensis subsp. Bacillus thuringiensis subsp.kurstaki, Bacillus thuringiensis subsp.galleriae, Bacillus thuringiensis subsp.wuhanensis, and Bacillus thuringiensis subsp.chinensis were used as recipient bacteria. The insect-specific neurotoxin genes of a variety of spiders were cloned with suitable vectors and achieved high expression, and a new strain for the production of new biological insecticides was constructed, and a super-effective, broad-spectrum, and safe insecticide was developed to overcome Bt Problems with pesticides and unresolved problems with current genetically engineered insecticidal plants.

The double-effect engineered bacteria biopesticide is a Bacillus thuringiensis engineered bacteria preparation that produces Bt poisonous protein and spider venom protein after gene cloning. ml, the titer of the preparation is above 6000 international units/μg, and the insecticidal rate of the preparation to larvae of Lepidoptera, Diptera and Coleoptera reaches 90-96%.

The preparation method of the double-effect engineering bacteria biopesticide consists of two parts: the construction of the Bacillus thuringiensis engineering bacteria and the production of the Bacillus thuringiensis engineering bacteria agent:

(1) Construction of Bacillus thuringiensis double-effect engineering bacteria

① Construction of regulatable expression vector

——The recipient bacteria were Bacillus thuringiensis 4.0718 and subsp. thuringiensis, subspecies Kustak, subsp.

——Plasmid pXLZ401 was constructed, and the total plasmid of strain 4.0718 was extracted by Triton X-100 gentle method. After PstI/BamHI double digestion, a gene fragment of about 4Kb was obtained, which was separated and purified by agarose gel electrophoresis, and then PstI/BamHI Digest the shuttle vector pHT3101 with double enzymes to obtain chain DNA with two different cohesive ends, dephosphorylate it with alkaline phosphatase, mix the isolated and purified gene fragment of about 4Kb with the dephosphorylated carrier pHT3101, and use T ₄ DNA ligase ligation, resulting in plasmid pXLZ401.

——ICPs gene strong promoter and SD sequence acquisition, the plasmid pXLZ401 was electrotransformed into a crystal-free mutant strain, and the transformant was screened by erythromycin plate, and the crystal-producing transformant was selected by Gram staining microscope, and the LB Activate and culture the transformant on the medium, extract the plasmid with TritonX-100, separate and purify the plasmid pXLZ401 by agarose gel electrophoresis, and digest it with pstI/BamHI double enzymes, after the double enzyme digestion, pass CsCl density gradient centrifugation, and Glass-milk method to obtain For fragments with complete open reading frames of ICPs, sequence the fragments and analyze the sequences with computer software to determine their starting sequence and SD sequence.

——The construction of plasmid pXLZ402, the above fragment was amplified by PCR to amplify the full-length promoter sequence before the start codon ATG, and ECoRI and SalI sites were added to the 5' end and 3' end respectively, after purification, ECoRI/SalI was used Double enzyme digestion, the vector pHT3101 was also double enzyme digested with ECoRI/SalI, dephosphorylated with alkaline phosphatase, the two were mixed, and the two were connected with T ₄ DNA ligase in the presence of ATP to form plasmid pXLZ402, Transformed into E.coli DH _5α and amplified.

②Chemical synthesis of spider-specific insect neurotoxin gene and acquisition of domain II sequence

——Chemical synthesis of spider venom gene, based on the sequence of the spider venom gene, the full-length gene of the toxin polypeptide is synthesized by chemical synthesis method, with ECoRI and XbaI sites at the 5' end and 3' end of the gene respectively, and at the same time at the 3' end Add the stop codon TGA.

——Acquisition of the domain II sequence of ICPs acting on insect midgut cell membrane receptors, search for the domain II sequence of the existing ICPs in Gene Bank, use the sequence analysis software, compare the conserved regions with the array, and compare with the sequenced fragments, according to The conserved region determines its domain II boundary, and the domain II gene fragment is obtained by chemical synthesis and PCR, with SalI and ECoRI restriction sites at the 5' end and 3' end respectively, and an additional start codon ATG at the 5' end , the domain II segment can be obtained by PCR.

——The construction of plasmid pXLZ403, the plasmid pXLZ402 was extracted by alkaline lysis, digested with SalI/XbaI, dephosphorylated with alkaline phosphatase, and the obtained domain II sequence was digested with ECoRI, and chemically synthesized The toxin gene with the ECoRI site at the 5' end was mixed and ligated with T ₄ DNA ligase. After the ligated fragment was purified, it was digested with XbaI and mixed with the dephosphorylated chain plasmid pXLZ402, ligated with T ₄ DNA Enzyme ligation to form plasmid pXLZ403, transform the mutant without plasmid, and select transformants by erythromycin plate.

③Integrate the fusion protein gene with the promoter sequence into the DNA of Bt bacteria for stable inheritance

——Construction of plasmid pXLZ404, using exonuclease at both ends of the wild-type transposon Tn5401 to remove the border recognized by the transposase, deleting the transposase by enzyme cutting, purifying the remaining fragments, connecting with T4DNA ligase, and inserting into pHT3101 to form plasmid pXLZ404, which was transferred into E.coli DH _5α for amplification.

——Construction of plasmid pXLZ405, using TritonX-100 gentle method to extract plasmids pXLZ403 and pXLZ404 respectively, isolate and purify the recombinant fragment of pXLZ403, insert it into pXLZ404 to delete the transposase site, and remove Bt in plasmid pXLZ404 by enzyme digestion The replicon was used to generate plasmid pXLZ405, which was amplified in E.coli DH _5α by electroporation, gene gun or metal ion induction method, and the transformants were selected on the ampicillin plate.

——The plasmid pXLZ405 was integrated into the chromosome of strain 4.0718, and the wild-type transposon Tn5401 was transferred into the chromosome of Bacillus thuringiensis 4.0718 to generate a new strain, and the plasmid pXLZ405 was electrotransformed into the new strain, and the transformed Tn5401 and full-length fusion gene were integrated Into the wild-type transposon Tn5401 already present on the chromosome, transformants were selected with erythromycin plates.

(2) Production of Bacillus thuringiensis engineering bacteria agent

①The strains are activated. After the preserved engineering strains are cultured on a slant, they are transferred to the culture medium of a flat bottle and cultured for 30 to 48 hours at 28 to 35°C. Inoculate in seed pots.

②Seed tank fermentation, the spore liquid is connected to the sterilized first-level seed tank equipped with culture medium, and sterile air is introduced to cultivate to obtain the first-level seed tank fermentation liquid, and the inoculation amount is 5% to 8%. The primary seed fermentation liquid is connected to the secondary seed tank culture liquid, fed with sterile air and stirred for cultivation, to obtain the secondary seed tank fermentation bacterial liquid.

③Fermentation tank culture, according to the inoculum amount of 5% to 8%, put the fermentation liquid of the secondary seed tank into the fermenter tank, and under the temperature condition of 25°C to 30°C, feed sterile air for 42-48h,

④ Concentration. After the fermentation in the fermenter is completed, the bacterial liquid is concentrated by ultrafiltration, supplemented with synergistic additives, and bottled.

Below in conjunction with accompanying drawing, describe the present invention in detail.

Fig. 1 Structural diagram of plasmid pHT3101;

Figure 2 Schematic diagram of obtaining strong ICPs gene promoter and SD sequence on the plasmid of strain 4.0718;

Fig. 3 is a schematic diagram of constructing a spider toxin gene expression vector;

Fig. 4 is a schematic diagram of the integration of the fusion protein into the chromosome of Bt bacteria;

Fig. 5 schematic diagram of the fermentation production process of double-effect insecticidal engineering bacteria;

(1) Construction of regulated expression vector

1. Recipient strain

The acceptor strain of the present invention is the bacillus thuringiensis subsp. kurstaki (Bacillus thuringiensis subsp. kurstaki) efficient insecticidal new strain Hu4.0718 (National strain preservation number: CCTCC NO: M200016), with a total of 6 plasmids of different sizes , carrying cry1Aa, cry1Ab, cry1Ac, cry1Ax, cry1Cb, cry2Ac and other genes on its No. 1~3 plasmids, which have strong toxicity to Lepidoptera and Diptera insects. The recipient bacteria also include Bacillus thuringiensis subsp.thuringiensis, Bacillus thuringiensis subsp.kurstaki, Bacillus thuringiensis subsp.galleriae, Bacillus thuringiensis subsp. wuhanensis), Chinese subspecies (Bacillusthuringiensis subsp.chinensis).

2. Acquisition of ICPs gene strong promoter and SD sequence on the strain 4.0718 plasmid

Fig. 2 is a schematic diagram of searching for the strong promoter and SD sequence of the ICPs gene from the plasmid of bacterial strain 4.0718. The total plasmid of strain 4.0718 was extracted by TritonX-100 gentle method, and agarose gel electrophoresis was performed after PstI/BamHI double digestion, and the cloned gene fragment of about 4Kb was separated and purified. pHT3101 (see Figure 1 for the structure) is a shuttle vector that can replicate in both Bt and E.coli. Show ampicillin resistance. Use PstI/BamHI double enzyme digestion to obtain a chain-like DNA with two different sticky ends, dephosphorylate it with alkaline phosphatase to prevent its own circularization and increase the difficulty of work, and dephosphorylate the above 4kb fragment with The resulting vectors were mixed and ligated with T4 DNA ligase to generate plasmid pXLZ401. A crystal-free mutant strain was electroporated, and the transformants were screened by erythromycin plate, and the transformants with complete open reading frames of ICPs that could produce prismatic crystals were detected by microscope observation. Transformants were activated on the LB medium slant for 24 hours, inserted into LB liquid medium, cultured at 30° C. and 200 rpm for 18-24 hours, and the plasmid was extracted by the TritonX-100 method. After agarose gel electrophoresis, isolate and purify the target plasmid. The fragments with complete open reading frames of ICPs can be obtained by double-digesting the plasmid with PstI/BamHI. After the fragments were sequenced, the sequence was analyzed with computer software to determine its initiation sequence and SD sequence.

3. Construction of plasmid pXLZ402

Design primers based on the fragment sequence, add monocloning sites at both ends of the primers, so that the upstream of the full-length promoter sequence before the ATG is amplified by PCR with an ECoRI site, and the downstream has a SalI site. After the PCR product is purified, use ECoRI/ SalI double enzyme digestion, the vector pHT3101 was also digested with ECoRI/SalI double enzymes, and dephosphorylated with alkaline phosphatase. Mix the two, and use T ₄ DNA ligase to connect the two in the presence of ATP to form plasmid pXLZ402, which is transformed into E.coli DH _5α and amplified.

(2) Chemical synthesis of spider-specific insect neurotoxin gene and acquisition of domain II sequence

1. The gene sequences of a variety of spider insect-specific neurotoxins are known. According to the spider venom gene sequence (see page 14-23 of the description), the full-length gene of the toxin polypeptide is synthesized by chemical synthesis, and the 5' end has a EcoRI site, XbaI site at the 3' end, and a stop codon TGA at the 3' end.

2. Acquisition of domain II sequences of ICPs acting on insect midgut cell membrane receptors

Search the domain II sequences of existing ICPs in the Gene Bank, compare the conserved regions with arrays of sequence analysis software (such as Jellyfish, etc.), compare them with the sequenced fragments, and determine the domain II boundaries based on the conserved regions. Design primers according to the border sequence, in the case of avoiding frameshift mutation after toxin fragment insertion, the amplified domain II gene has ATG at the 5' end, and SalI and ECoRI at the 5' end and 3' end respectively Restriction sites. The domain II segment can be obtained by PCR.

3. Construction of plasmid pXLZ403

Figure 3 is a schematic diagram of the construction of the spider toxin expression vector. The plasmid pXLZ402 was extracted by alkaline lysis, dephosphorylated with alkaline phosphatase after SalI/XbaI double digestion; the domainII sequence generated by PCR was digested with ECoRI, and digested with Chemically synthesized toxin genes with ECoRI sites at the 5′ end were mixed, ligated with T ₄ DNA ligase, digested with XbaI after purification of the ligated fragments and mixed with dephosphorylated chain-like plasmid pXLZ402, ligated with T ₄ DNA Ligase was used to form plasmid pXLZ403, and the plasmid-free mutant was transformed, and transformants were selected by erythromycin plate.

(3) Verification of insecticidal function

1. ELISA detection of expression products of toxin genes

The purified target spider toxin was immunized into rabbits, and after the antibody was obtained, it was linked with horseradish peroxidase to form an enzyme-linked antibody; after the cultured bacteria were treated with ultrasonic waves, the total protein was electrophoresed, transferred to a membrane, hybridized with the antibody, and eluted , Color development, to determine whether there is toxin expression.

2. Verify the correctness of toxin expression

The fusion protein is purified by HPIC, and the C-terminal sequencing can confirm whether the expression is correct.

3. Indoor insecticide test

(4) Integrating the fusion protein gene with the promoter sequence into the DNA of Bt bacteria for stable inheritance

1. Construction of plasmid pXLZ404.

Fig. 4 is a schematic diagram of integrating the entire fusion protein gene into the chromosome. The two ends of the wild-type transacne Tn5401 were treated with exonuclease to remove the border recognized by the transposase, and the transposase was deleted by enzyme cutting, and then inserted into pHT3101 to generate plasmid pXLZ404, which was transferred into E.coli DH _5α Amplify.

2. Construction of plasmid pXLZ405

The plasmid pXLZ403 was extracted by the gentle method of TritonX-100, the recombinant fragment was separated, and after purification, it was inserted into the transposase-deleted site in the plasmid pXLZ404, and the Bt replicon in the plasmid pXLZ404 was removed by enzyme digestion to generate the plasmid pXLZ405.

3. Integration of plasmid pXLZ405 into the chromosome of strain 4.0718

The wild-type transposon was transferred to the chromosome of the Bt4.0718 strain, and the plasmid pXLZ405 was electrotransformed into the strain 4.0718. The transformed Tn5401 and the full-length fusion gene would be integrated into the wild-type transposon Tn5401 already present on the chromosome, and Lose transfer ability, use erythromycin plate to select transformants.

4. Functional verification

Through southern hybridization and ELISA, it was confirmed that the cloned spider venom gene existed on the chromosome and could be expressed, and the insecticidal test was used to determine the insecticidal potency of the engineered strain.

(5) Fermentation production

Figure 5 is a schematic diagram of the fermentation process of double-effect insecticidal engineering bacteria, which mainly includes the activation of strains, the fermentation and cultivation of primary and secondary seed tanks, and the cultivation of fermentors for production.

1. Activation of strains

Streak inoculation of the preserved high-efficiency broad-spectrum insecticidal engineering bacteria on the slant of the solid seed medium, sterilize the flat bottle containing the medium at 121°C for 30 minutes before inoculation, and inoculate it at 28-35°C after inoculation Cultivate for 30-48 hours, make spore liquid, and use 5% inoculum for seed tank inoculation.

2. Seed tank fermentation

Sterilize the primary seed tank first, put it into the culture medium and then sterilize it, cool it to 30°C, put the spore liquid into the culture medium, and pass it into sterile air for cultivation to obtain the fermentation liquid of the primary seed tank; Sterilize the secondary seed tank at 121°C for 30 minutes, put it into the medium and then sterilize it, cool to 30°C, put the fermentation liquid of the primary seed tank into the secondary seed tank according to the inoculation amount of 5% to 8%, and pass it into the Cultivate with sterile air and stirring to obtain the secondary seed tank fermentation liquid.

3. Production fermenter culture

Sterilize the fermenter first, put it into the culture medium and then sterilize it, keep the pressure and cool down to 25°C-30°C, put the fermentation liquid of the secondary seed tank into the fermenter at a rate of 5%-8% according to the seed amount, and cultivate it. by sterile air.

4. Concentrate

After the fermentation in the fermenter is completed, the bacterial liquid is concentrated by ultrafiltration, supplemented with synergistic additives, and then bottled before being put on the market.

5. Sterilization and culture conditions

The above-mentioned sterilization adopts high-pressure steam sterilization, that is, it is sterilized at 121°C and the pressure is 0.3-0.5kg/cm2 for 30 minutes. After the fermenter is inoculated, it is cultivated at 28-35°C for 36-48 hours, and the ventilation rate is 1-2Vols /vol/min, the oxygen content is 30-40%. Seed pot culture solution formula: beef extract 0.3-0.8%, peptone 0.7-1.2%, glucose 0.1-0.6%, NaCl 0.1-0.6%, MgSO ₄ 7H ₂ O 0.01-0.06%, K ₂ HPO ₄ 0.01%- 0.06%, MnSO ₄ 0.02% ~ 0.08%, pH value before disinfection 7.0 ~ 7.8, after disinfection pH 6.8 ~ 7.8.

Fermentation tank culture solution formula: soybean powder 2-8%, corn starch 0.3-1.5%, NaOH 0.2-0.8%, after hydrolysis at 121°C for 20 minutes, filter to remove impurities, add glucose 1.3-2.0% by volume, KH ₂ PO ₄ 0.08～0.22%, K ₂ HPO ₄ 0.10～2.2%, CaCO ₃ 0.1～2.20%, FeSO ₄ 7H ₂ O 0.001～0.005%, stir evenly, pH value before disinfection is 8.5～11.0, after disinfection 6.5～9.5 .

Double-effect biological insecticide synergistic additive formula: tea saponin 0.05~25%, xylene 0.5~3%, sorbitol 1~4%, Tween 80 1~4%, glycerin 3~8%, honey 3~ 8%.

6. Fermentation broth requirements

The number of viable spores in the fermentation broth is more than 8 billion per milliliter, and when the amount of bacteria is less than 5%, put it into the tank, the pH value is 7.0-8.0, and there is no contamination by bacteria.

Compared with existing biological pesticides, the present invention has the following advantages:

(1) Good insecticidal effect: because the engineering strains contain various insecticidal toxins Cry1Aa, 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, in addition to their own insecticidal ability, also have a significant positive synergistic effect, which can produce double effects of stomach poisoning and contact killing on pests, so The insecticidal potency is higher, and the potency can reach more than 6000 international units/μg, which has strong competitiveness in application.

(2) Wide insecticidal spectrum: Engineering bacteria have a variety of insecticidal toxins. At the same time, because HWTX-I, HWTX-II and other spider venom genes are expressed in the form of fusion proteins with the modified ICPs domain II region, they can be identified The range of pests increases, and at the same time, it can be effective against many types of pests such as Lepidoptera, Diptera, Coleoptera, etc. Therefore, it has a wide range of control in use and relatively low cost.

(3) Higher security: Since the toxins carried by engineering bacteria are all natural toxins, they are easily degraded by microorganisms in nature and are easily damaged by sunlight and ultraviolet rays without forming residues; at the same time, various toxins are harmless to humans and animals. Harmful. Therefore, it has great advantages in terms of ecological environment protection, export earnings and promotion of human health.

Detailed ways:

Take the recipient bacterium Bacillus thuringiensis subsp.Kurstaki 4.0718 (Bacillus thuringiensis subsp.Kurstaki) as the specific embodiment of the present invention below.

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

2. The preserved strains were cultured statically on solid activated medium at 30°C for 24 hours, transferred to liquid activated medium, cultured at 30°C and 200 rpm for 18-24 hours, centrifuged to collect 3ml of precipitate in a 1.5ml Eppendorf tube, and used Wash once with STE (50mmol/l Tris Cl, 50mmol/l NaCl, 5mmol/l EDTA pH8.0), add 100μl solution I (10mg/ml lysozyme, 100μg/ml RNase) and incubate at 37°C for 60min, add 100μl Solution II (0.5% TritonX-100, 50mol/l Tris Cl, 10mmol/l EDTA, 0.1mg/ml proteinase K, pH8.0) 40μl 5mol/l NaCl was incubated at 37°C for 90min, and saturated phenol with pH8.0 Extract twice, extract twice with chloroform, precipitate with double volume of absolute ethanol overnight, and collect the precipitate by centrifugation at 14000rpm and 4°C for 20min. The precipitate was dissolved in TE (10mmol/l Tris Cl, 1mmol/l EDTA pH8.0) buffer. Use PstI/BamHI 10U each, the buffer composition is 20mM Tris Cl (pH8.5), 10mM MgCl2, 1mmol/L Dithiothreitol, 100mM KCl, incubate at 37°C for 1h, and a DNA fragment of about 4kb suitable for cloning can be obtained.

3. Bacillus thuringiensis-Escherichia coli shuttle vector pHT3101, this plasmid has two sets of replication systems that can replicate in Bt and E.coli, and at the same time express erythromycin resistance in Bt and ampicillin in E.coli Resistance, as the carrier of the gene involved in the present invention.

4. HWTX-I, HWTX-II, u-agatoxins, δ-Latroinsectotoxin, δ-AcTX-Hvla, etc. are polypeptide neurotoxins isolated and purified from different spider venoms, which can cause a large release of neurotransmitters, causing insects to Rapidly paralyzed and died, its gene and amino acid sequence are shown on pages 14-23 of the manual.

5. Digest the carrier pHT3101 with PstI/BamHI, extract the sample with phenol: chloroform, precipitate with 2 times the volume of ethanol at 10°C for 1 hour, centrifuge at 14,000 rpm and 4°C for 25 minutes to recover the DNA, and re-dissolve the DNA with TE. Add 1/10 volume of 10× calf small intestine alkaline phosphatase buffer (10mM ZnCl ₂ , 10mM MgCl ₂ , 100mM Tris·Cl (pH8.0)) and appropriate amount of calf small intestine alkaline phosphatase and incubate at 37°C for 30min, then in The phosphatase was inactivated by heating at 65° C. for 1 h under the condition of 5 mmol/LEDTA solution (pH 8.0). The dephosphorylated DNA was purified by extraction with phenol:chloroform. Mix the dephosphorylated carrier DNA with the above 4Kb fragments in equimolar amounts, add water to 7.5 μl, heat at 45°C for 5 minutes to dissolve the re-annealed sticky ends, cool to 0°C, add 10×T ₄ DNAligase buffer 1μl, T ₄ DNA ligase 0.1weiss unit, 5mM ATP 1μl, incubate at 16°C for 1-4h to produce a new plasmid pXLZ401, take 1-2μl sample by electrotransformation (electric field strength 8.75KV/cm, resistance 100Ω～200Ω, Capacitance 25μF and 2× phosphate buffer), gene gun or 36% PEG, 50mM CaCl ₂ mediated by metal ion induction method to transform Bt amorphic mutant strain or no plasmid mutant strain, through erythromycin resistance plate (10 μg/ml) to select transformants, and observe and select transformants capable of producing crystals by Gram staining microscope.

6. For the transformant capable of producing crystals, culture and extract the plasmid according to the previous method, after pstI/BamHI double enzyme digestion, pass CsCl density gradient centrifugation, Glass-milk method or after the total plasmid is passed through agarose gel electrophoresis, cut out The target band is electrophoresed on the nucleic acid electrophoresis recovery instrument to obtain the target fragment and purify it. After sequencing the fragment, use computer software (such as Jelly-fish) to analyze the sequence, find its promoter sequence and SD sequence, and amplify the full-length promoter sequence before the initiation codon ATG by PCR method, and insert ECoRI and SalI recognition sites were added to the end and 3′ end respectively, and inserted into the multiple cloning site of pHT3101 according to the DNA ligation method described above to form plasmid pXLZ402. Transform E. Coli DH5α by electroporation, gene gun or metal ion induction method, and select transformants by ampicillin (50 μg/ml) plate.

7. According to the sequence of the spider venom gene, the complementary single strands are synthesized by chemical synthesis. After the complementary single strands are annealed, there are ECoRI and XbaI cohesive ends at the 5' end and 3' end of the gene respectively, and a termination at the 3' end Sub TGA. By using the existing ICPs gene data in GeneBanK, combined with its spatial conformation and domain functions, computer array technology was used to find the relatively conserved sequences in the domain II region of ICPs, compared with the sequenced fragments, and the domain II boundary was determined according to the conserved region. The domain II gene fragment was obtained by chemical synthesis or PCR, and SalI and EcoRI recognition sites were added to the 5' end and 3' end of the fragment respectively, and an initiation codon ATG (located at the SalI position) was added to the 5' end. point downstream).

8. Ligate the above-mentioned spider venom gene fragment and domain II fragment according to the DNA ligation method described above, and use the same method to insert it under the promoter sequence of plasmid pXLZ402 to form plasmid pXLZ403, which can be induced by electrotransformation, gene gun or metal ions Bt plasmid-free mutants were transformed using the method described above, and transformants were selected by erythromycin resistance.

Add 9.75 μl of unknown antigen solution dissolved in carbonate buffer to 4 wells of the microwell plate, overnight at 4°C or 3h at 37°C; invert the microwell plate, pour out all the water inside, and add 200μl PBS-Tween-20, and then pour the PBS-Tween-20 clean, repeat this three times. Add 200 μl of blocking solution (carbonate buffer solution or 5% milk powder solution containing 2% calf serum) to each well of the microplate, and incubate at 37° C. for 30 minutes. Wash the microplate as in the previous step, add 75 μl of appropriately diluted spider toxin antibody to each well, incubate at 37°C for 30 min, wash the microplate as in the previous step, and add 100 μl of appropriately diluted secondary antibody to each well. The second antibody was goat anti-immunoglobulin G antibody labeled with horseradish peroxidase, and incubated at 37°C for 30min. Wash the microwell plate as in the previous step, add 100 μl enzyme substrate to each well, cover the microwell plate, shake gently for 15 minutes, then a certain color can be seen in the well, add 50 μl stop solution to each well to stop the reaction, positive control well The color reaction is green. After being purified by high-performance liquid chromatography (HPLC), the amino acid sequence of the expressed polypeptide is checked by sequencing the C-terminal, and the biological activity of the expressed neuropeptide is checked by an indoor insecticidal test.

10. Use exonuclease to remove the border of the wild-type transposon Tn5401, cut it with a restriction endonuclease that has recognition sites at both ends of the transposase, purify the fragment that removes the transposase, and use _T4 DNA ligase was connected and inserted into pHT3101 to form plasmid pXLZ404, which was amplified in E. coli DH5α by electroporation, gene gun or metal ion induction method, and ampicillin resistance was selected for transformants.

11. Extract the plasmids pXLZ403 and pXLZ404, and use CsCl density gradient centrifugation, Class-milk method or gel cutting to separate and purify the complete recombinant fragment in the plasmid pXLZ403, and insert it into the original transposase in the plasmid pXLZ404 site, and digested to remove the Bt replicon on the vector to form plasmid pXLZ405. Electroporation, gene gun or metal ion induction method was used to transform E.coli DH5α, amplified, and ampicillin resistance to select transformants.

12. Transfer the wild-type transposon Tn5401 into the chromosome of Bt4.0718 to form a new strain, extract the plasmid pXLZ405, and transform the new strain produced by electroporation, gene gun or metal ion induction, then the transformed Tn5401 and the full-length recombinant fragment Will integrate into the wild-type Tn5401 present on the chromosome and lose the ability to transfer, and transformants will be selected for erythromycin resistance.

13. Test the insecticidal virulence and insecticidal spectrum of the engineering bacteria through the indoor insecticidal test, select multiple purpose-age larvae, add the plant leaves soaked in the fermentation broth of different dilutions to a 9cm petri dish, and put 10 Plutella xylostella or cotton bollworm larvae were recorded at 30°C temperature, 85% humidity, and certain light conditions to record the rate of live insects, and to detect their insecticidal rate and insecticidal spectrum.

14. Use 15 tons of fermentation tanks to produce 10 tons of bacterial agents. The fermenter is first sterilized when it is empty, and then it is sterilized after it is filled with culture medium. High-pressure steam sterilization is used for sterilization, that is, sterilization is carried out at 121°C and a pressure of 0.5kg/cm2 for 30 minutes, the culture solution of seed tanks and fermenters is sterilized and the stirring speed is 250rpm, and then the pressure is maintained and the temperature is lowered to 30°C. After seed tanks and fermenters are inoculated with an inoculum amount of 5%, they are fed with sterile air at an aeration rate of 2 Vols/vol/min and an oxygen content of 40% for fermentation.

Fermentation tank culture solution formula: 6.6% soybean flour, 0.825% corn starch, 0.5% NaOH, hydrolyze at 121°C for 20 minutes, and filter to remove impurities. Add glucose 1.65%, KH ₂ PO ₄ 0.165%, K ₂ HPO ₄ 0.165%, CaCO ₃ 0.165%, FeSo ₄ .7H ₂ O 0.0033% by volume, stir evenly, pH value before disinfection is 9.8, and after disinfection is 7.4.

Seed medium formula: beef extract 0.5%, peptone 1.0%, glucose 0.3%, NaCl 0.2%, MgSO ₄ ·7H ₂ O 0.03%, K ₂ HPO ₄ 0.03%, MnSO ₄ 0.005%. pH7.5 before disinfection, pH7.3 after disinfection.

Double-effect biological insecticide synergistic additive formula: tea saponin 1-5%, xylene 1%, sorbitol 2%, Tween 80 2%, glycerin 5%, honey 5%.

The preserved strains were cultivated on the solid slope of the seed medium at 30°C for 36 hours, washed with sterile water, crushed the bacterial lawn in a sterilized triangular flask equipped with glass beads, and then put it into the first-level seed tank at a rate of 5%. The culture medium is the seed medium. Stir at 250 rpm and culture at 30° C. for 8 hours, then add 5% to the secondary seed tank. Stirring speed is 250rpm, 30 DEG C cultivates 8 hours, transfers into 15 tons of production fermentation tanks according to the inoculum amount of 5%, and culture liquid is production fermentation culture liquid. Stirring speed is 250rpm, culture is 42-48h at 30°C, ventilation rate is 2Vols/vol/min, oxygen content is 40%, and it is detected online. When the bacterium is degraded to release spores and crystals, when the amount of bacterium in the culture solution is less than 5%, it is placed in a tank, concentrated by ultrafiltration, supplemented with synergistic additives, and made into a preparation or a dry powder. By using this method to produce Bt preparations, the fermented liquid crystal content can reach 1.6g/l, and the spider venom content can reach 50-100mg/l. Combined with the quality standards of domestic and foreign microbial pesticides, the potency of the production of bacterial agents can reach 6000 international units/μg, without residual pollution, and the insecticidal rate of the main pests of Lepidoptera, Diptera and Coleoptera is within 3 days It reaches 90-98%, the residual effect period reaches 15-20 days in field application, the number of viable spores of the bacterial agent reaches 8-10 billion/ml, and the shelf life of the concentrate and the powder is 1-2 years respectively. The produced bacterial agent is properly concentrated and used for field tests. After a series of field application technology verification, it is determined to be used for the control of crops, vegetables, fruit trees, forests and sanitary pests. The agricultural products on the market have aseptic residues, are harmless to humans and animals, and have no damage to the ecological environment, so they can be used in production.

Various insect-specific neurotoxin genes and amino acid sequences derived from spiders

Tiger tarantula 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 (subunit a): 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 Vallys 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

Raye's 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 sequence: 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 3 GT AAA GT AAA TTC T′

Raventoxin-II: icadeggpcvagigccaglrcsgailglagscq

Gene sequence: 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′

Australian Funnel Web Spider Insect Neurotoxins:

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

Gene sequence: 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 sequence: 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 TCTG TGG AGCAA 3 AA'

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

Gene sequence: 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′

American Funnel Web Spider Insect Neurotoxins:

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

Gene sequence: 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 sequence: 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 sequence: 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 sequence: 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 sequence: 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 sequence: 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′

Black Widow Spider Insect 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

基因序列：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

基因序列：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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