CN1184890C

CN1184890C - Thuricide engineering bacterium double-effect pesticide containing amatoxin gene

Info

Publication number: CN1184890C
Application number: CNB011286652A
Authority: CN
Inventors: 夏立秋; 丁学知; 莫湘涛; 陈作红; 陈宇; 谢伟岸; 张何
Original assignee: Hunan Normal University
Current assignee: Hunan Normal University
Priority date: 2001-10-09
Filing date: 2001-10-09
Publication date: 2005-01-19
Anticipated expiration: 2021-10-09
Also published as: CN1341362A

Abstract

The present invention relates to a bacterial agent of Bacillus thuringiensis engineering bacteria (Bt), which is cloned by genes. The bacterial agent uses a Bacillus thuringiensis 4.0718 strain and the subspecies of Bacillus thuringiensis, etc. as parasitifer, amanitin toxin protein genes of fusion protein genes with a strong initiating sequence and a domain I 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 amanitin toxin protein and Bt toxin protein, and double-effect insecticide is produced by fermentation. The titer of the bacterial agent is more than 8000 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

The Thuricide engineering bacterium double-effect pesticide that contains amatoxin gene

Technical field the present invention relates to a kind of Biotrol BTV, especially through the Biotrol BTV of gene clone.

Background technology Bt 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, causes insect death.And the spider endotoxin has special inhibitory action to the nervous activity of insect, causes that the insect paralysis is dead.Lepidoptera and dipteral insect all there is toxic action.What Chinese patent application number 01114592.7 proposed is a kind of Bt of generation toxalbumin and the bacillus thuringiensis,Bt double engineering bacterium preparation that produces spider poison protein after gene clone, has also proposed to comprise the method that makes up Thuricide engineering bacterium and produce the Thuricide engineering bacterium agent simultaneously.Problem that double engineering bacterium preparation solution Bt insecticide exists and gene pesticide plant be an open question still.But how faster kill insects, and expansion insecticidal spectrum and raising safety are to need the constantly problem of solution.

Summary of the invention: the present invention is intended to the further speed that improves the Thuricide engineering bacterium agent that contains amatoxin gene and kills effect, enlarges insecticidal spectrum and improve safety.

The technical scheme that solves the object of the invention is: the Thuricide engineering bacterium double-effect pesticide that contains amatoxin gene is except that producing the Bt toxalbumin, after gene clone, can also produce goose cream peptide toxoid albumen, the living spores content of the engineering microbial inoculum of this insecticide is at 8,000,000,000-10,000,000,000/ml, tiring in that 8000 international units/more than the μ g, preparation reaches 90-96% to the larva killing rate of Lepidoptera, diptera and coleoptera of preparation.Have cry1Aa, cry1Ab, cry1Ac, cry1Ax, cry1Cb and the cry2Ac except that carrying the Bt toxoprotein gene on the engineering bacteria plasmid, carry gene α-amanitin, phalloin, the viroidin of goose cream peptide toxoid albumen in addition.

Be described in further detail the present invention below in conjunction with accompanying drawing:

Fig. 1 plasmid pHT3101 structure chart;

Fig. 2 goose cream peptide toxoid gene and amino acid sequence and amino acid molecular diagram;

ICPs gene strong promoter and SD sequence are obtained schematic diagram on Fig. 3 bacterial strain 4.0718 plasmids;

Fig. 4 Amanita fuliginea peptide toxoid expression vector makes up schematic diagram;

Fig. 5 fusion is incorporated into schematic diagram on the Bt bacterium chromosome;

Fig. 6 economic benefits and social benefits disinsection engineering bacteria fermentation manufacturing technique schematic flow sheet;

Recipient bacterium of the present invention is the new bacterial strain 4.0718 of bacillus thuringiensis,Bt efficient insecticide (the Bacillus thuringiensis subsp.Kurstaki through seed selection, country's culture presevation number: (" life science " 2001 CCTCC NO:M200016), 5 (5): 242-245 " researchs of bacillus thuringiensis,Bt 4.0718 bacterial strain insecticidal crystal character " and " life science " 2001,5 (5): 278-280 " the method research of high efficiente callback bacillus thuringiensis,Bt plasmid DNA ") and Su Yun gold subspecies (Bacillusthuringiensis subsp.thuringiensis), Kustak subspecies (Bacillusthuringiensis subsp.kurstaki), galleria mellonella waxmoth subspecies (Bacillus thuringiensissubsp.galleriae), Wuhan subspecies (Bacillus thuringiensis subsp.wuhanensis), China's subspecies (Bacillus thuringiensis subsp.chinensis).

Amanita peptides toxin protein is the peptide toxoid of separation and purification from the multiple fungi of Amanita (amanita), mostly is dicyclo or monocycle polypeptide that 7-8 amino acid is formed, mainly comprises amanita hemolysin, Phallus phallotoxins and amanita phalloides phallotoxins three classes.Wherein the amanita hemolysin activity that can suppress rna plymerase ii causes eukaryotic cell nucleus leafing and death, Phallus phallotoxins and amanita phalloides phallotoxins can destroy that thereby the dynamic equilibrium of filamentous actin and globular actin causes the cell function atrophy in the eukaryotic, cause cell death.(α-amanitin) is a kind of dicyclo octapeptide to amanita hemolysin, and molecular weight is 918.99; Phallus phallotoxins (phalloin) is an a kind of pair of ring seven peptide, and molecular weight is 772.9; Amanita phalloides phallotoxins (viroidin) is a kind of single ring seven peptide, and molecular weight is 864.89; These three kinds of phallotoxins are the kill insects cell at short notice, causes the forfeiture of insect body function and death.

The carrier that the present invention advances the engineering bacteria plasmid with the gene clone of Amanita fuliginea peptide toxoid is a kind of shuttle vector PHT3101 (structure is seen Fig. 1) that can both duplicate at Bt and E.coli.

The gene order and the amino acid sequence of Amanita peptides toxin protein see Table 2.

The structure of the Thuricide engineering bacterium of Amanita peptides toxin protein is as follows:

(1) structure of controllable express carrier

With the gentle method extracting of Triton X-100 bacterial strain 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, produce plasmid pXLZ601, electricity transforms the no crystal mutant strain of plasmid pXLZ601, 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/BamHI double digestion or 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, determine its initiating sequence and SD sequence

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 ECoR I/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 pXLZ602, Transformed E .coli DH _{5 α}Middle amplification;

(2) chemosynthesis of Amanita fuliginea peptide toxoid gene and domain II sequence obtains

Adopt chemical synthesis process according to Amanita fuliginea peptide toxoid gene order, 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,

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 domain II section by PCR

With alkaline lysis method of extracting plasmid pXLZ602, behind Sal I/Xba I double digestion, use the alkaline phosphatase dephosphorylation, after in addition acquired domain II sequence being cut with EcoR worker's 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 pXLZ602 with Xba I enzyme, uses T ₄Dna ligase connects, and forms plasmid pXLZ603, transforms the plasmid-free mutant strain, by the dull and stereotyped transformant of selecting of erythromycin;

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

Wild type is changeed Cuo Tn5401 two ends remove the border that transposase is discerned with exonuclease, 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 pXLZ604, changes E.coli DH over to _{5 α}Middle amplification,

Extract plasmid pXLZ603, pXLZ604 respectively with the gentle method of Triton X-100, the recombinant fragment of separation and purification pXLZ603, be inserted on the site of having deleted transposase among the pXLZ604, cut except that the Bt replicon among the plasmid pXLZ604, produce plasmid pXLZ605 with enzyme.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,

Wild type transposons Tn5401 is changed on the chromosome of bacillus thuringiensis,Bt 4.0718, produce new bacterial strain, plasmid pXLZ605 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;

The production zymotechnique flow process of bacterium agent of engineering bacterium is seen Fig. 5, mainly comprises actication of culture, first class seed pot fermented and cultured, secondary seed jar fermented and cultured and production fermentation tank culture.The living spores content of engineering microbial inoculum is at 8,000,000,000-10,000,000,000/ml, the tiring in 8000 international units/more than the μ g of preparation.

The present invention compares with existing biological pesticide and has the following advantages:

(1) speed is extremely effective: since engineered strain respectively with multiple Pesticidal toxins Cry1Aa, Cry1Ab, Cry1Ac, Cry2Ac, α-amanitin, phalloin, viroidin are except the desinsection energy of self Power also has significant positive cooperativity outward, can produce stomach toxicity and the double effects of tagging to insect, therefore Desinsection is tired higher, rapidly kill pests.

(2) insecticidal spectrum is wide: engineering bacteria self is with multiple Pesticidal toxins, simultaneously because α-amanitin, The Amanita fuliginea such as phalloin, viroidin Peptide toxin gene and improved ICPs domain II district The territory increases its discernible scope with fusion protein form expression, simultaneously can be to Lepidoptera, dipteron The multiclass such as order, coleoptera insect is effective, and it is very wide therefore to prevent and treat in the use face, 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.

Claims

1, a kind of Thuricide engineering bacterium double-effect pesticide that contains amatoxin gene, it is characterized in that engineering bacteria except that carrying gene cry1Aa, cry1 Ab, cry1Ac, cry1Ax, cry1Cb and the cry2Ac that produces the Bt toxalbumin, also carry gene α-amanitin, phalloin, the viroidin of goose cream peptide toxoid albumen.