CN1159971C - A method for preparing insecticide from recombinant broad-spectrum bacillus thuringiensis - Google Patents
A method for preparing insecticide from recombinant broad-spectrum bacillus thuringiensis Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 12
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Abstract
本发明提供了一种重组广谱苏云金芽孢杆菌(Bacillusthuringiensis LCJ-12)制备杀虫剂的方法，该方法通过制备菌种、种子发酵、生产发酵、生成悬浮剂及生成粉剂等步骤制成杀虫剂；该杀虫剂对鳞翅目害虫和鞘翅目害虫均有良好的防治效果，对人、畜及其它动植物无害，不污染环境，且可降低生产和使用成本，并提高农产品的质量。The invention provides a method for preparing insecticides from recombinant broad-spectrum Bacillus thuringiensis LCJ-12. The method prepares insecticides through the steps of preparing strains, seed fermentation, production fermentation, generation of suspending agent and generation of powder. The insecticide has a good control effect on Lepidoptera pests and Coleoptera pests, is harmless to humans, livestock and other animals and plants, does not pollute the environment, and can reduce production and use costs and improve the quality of agricultural products .
Description
The present invention relates to the preparation method of biopesticide, more specifically relate to the method that bacillus thuringiensis prepares insecticide.
The alleged recombined broad spectrum bacillus thuringiensis (Bacillusthuringiensis LCJ-12) of the present invention is by what genetic engineering means made up Lepidoptera and coleopteran pest all to be had the bacterial strain of high virulence, not only killed lepidopterous insects but also killed the characteristics of coleopteron according to this bacterium, get Lepidoptera (Lepidoptera), coleoptera (Coleoptera), its code LCJ formed in first letter of bacterium (Jun), be that the comform virulence that screens in the multiple group of engineering bacteria is the highest because of this bacterium again, and be numbered a strain of 12, so with this recombined broad spectrum bacillus thuringiensis called after Bacillus thuringiensis LCJ-12.
Lepidoptera is the maximum insect of harm during China's agriculture forest and husbandry is produced, and coleopteran pest is only second to lepidoptera pest to the harm of agriculture forest and husbandry production, colorado potato beetle invasion is found in area, Xinjiang of China Huocheng in 1991 first, and rapid spread also causes significant damage to crops.Therefore, construct coleoptera and all effective recombined broad spectrum bacillus thuringiensis of lepidoptera pest and use it to produce novel pesticide and have urgency and realistic meaning.
The insecticide of producing with this bacterium can be widely used in killing farming, woods, really, coleopteran pests such as lepidoptera pest such as the diamond-back moth on the vegetables, beet armyworm, cotton bollworm and Phaedonbrassicae, colorado potato beetle.
Prepare aspect the insecticide the recombined broad spectrum bacillus thuringiensis, document " the bacillus thuringiensis,Bt recon crystal proteins that contains binary toxin gene analyze and to the toxicity of mosquito larvae " (JOURNAL OF MICROBIOLOGY,-1999,19 (1): 1-5) the recombinant plasmid pCW2 that will contain the Bacillus sphaericus binary toxin gene by electrotransformation transforms the wild type bacillus thuringiensis subsp israelensis and obtains the recombinant bacterial strain B+CW-1 that a strain contains binary toxin gene.Recombinant bacterial strain can express binary toxin and-endotoxin and cytA toxin, and form 2 classes and be positioned at the outer parasporal crystal of gemma spore adventitia.Document " structure of disinsection prophylaxis gene engineering bacillus subtilis " (biotechnology journal,-1999,15 (2): be carrier with bacillus subtilis-bacillus coli shuttle plasmid vector pHB201 and pRP22 respectively 215-220), by the competence method for transformation, Bt HD-1 insecticidal protein gene cry1Ac is imported rice sheath blight disease biocontrol bacterial strain bacillus subtilis B916.The electrophoretic analysis of engineered strain plasmid enzyme restriction, analysis of the Southern marking and desinsection biological activity determination result have confirmed the importing of cry1Ac gene and the effective expression in B916 thereof.Bacteria inhibition assay has proved that engineering bacteria has kept the good bacteriostatic activity of former wild-type strain.Document " bacillus thuringiensis subsp israelensis 130KDa kills the expression of mosquito protein gene in bacillus subtilis " kills mosquito protein gene subclone to the pNQ122 carrier with bacillus thuringiensis subsp israelensis, by the bacillus subtilis protoplast transformation, obtain forward and reverse clone's of KMr-CMs.Protein (western-blotting) immuning hybridization proof is killed the mosquito protein gene and has been expressed in bacillus subtilis and have the immunocompetent mosquito protein that kills.The expressed mosquito albumen that kills has mosquito activity extremely in experiment.But above-mentioned various insecticide does not all possess the function of killing lepidoptera pest and coleopteran pest simultaneously.
The objective of the invention is to, a kind of method for preparing insecticide with recombined broad spectrum bacillus thuringiensis (Bacillus thuringiensis LCJ-12) is provided, insecticide with this method preparation all has good control efficiency to lepidoptera pest and coleopteran pest, and it is harmless to people, animal and other animals and plants, free from environmental pollution, and can reduce and produce and use cost, and improve the quality of agricultural product.
In order to achieve the above object, the present invention adopts the method that is prepared as follows:
1 material and method
1.1 the preparation method of recombined broad spectrum bacillus thuringiensis:
A, employing preserving number are the recipient bacterium of the bacterial strain of CCTCC NO:M 201003 as bacillus thuringiensis, and this bacterial strain contains the cry3A gene; Adopt the pHT304/1Ac10 recombinant plasmid, this recombinant plasmid contains the cry1Ac gene of lepidopterous insects extremely;
B, containing the LB inoculation of medium Escherichia coli of ampicillin, 35-37 ℃ is acutely rocked and cultivated 8-12 hour, and bacterial precipitation is resuspended in the ice-cold solution I of 100 μ l, and concuss adds 200 μ l solution II; Add the ice-cold solution III of 150 μ l,,, in-20 ℃ of deposit D NA, obtain Escherichia coli recombinant plasmid pHT304/1Ac10 with the ethanol of 2 times of volume supernatants with equivalent phenol-chloroform-isoamyl alcohol extracting supernatant in centrifugal 10 minutes of 4 ℃ of following 12000g;
C, recipient bacterium is seeded in the LB medium, 28-30 ℃ after wave and culture 8-10 hour, ice bath 20-30 minute, centrifugal 10 minutes of 4 ℃ of following 12000g, earlier wash twice, wash once with the electric shock buffer solution again, be suspended at last in the electric shock buffer solution with the electric shock eluent, obtain recipient bacterium electric shock competent cell, put-20 ℃ of preservations;
D, pHT304/1Ac10 plasmid DNA and recipient bacterium competent cell suspension fully are mixed, get 200 μ l and inject electric shock conversion cup, shock by electricity once under 2.5KV with the Escherichia coli conversion instrument (E.coliGene Pulser) that shocks by electricity, the liquid that will shock by electricity changes in the 4mlLB medium, 28-30 ℃ of vibration cultivated after 2 hours, coat on the LB flat board that contains erythromycin 20 μ g/ml, cultivated 3 days down for 28-30 ℃;
E, on erythromycin (20 μ g/ml) resistant panel, select single bacterium colony, smear for microscopic examination, choosing the bacterium colony with bacillus thuringiensis feature goes down to posterity on the erythromycin flat board, to detect its stability, go down to posterity and virulence bioassay and PCR detection through the heredity in 20 generations, obtain virulence recombined broad spectrum bacillus thuringiensis the highest and that contain cry1Ac and cry3A gene, be a strain of recombined broad spectrum bacillus thuringiensis LCJ-12 of the present invention.
Described LB medium is: 0.5% sodium chloride (NaCl), and 0.5% dusty yeast, 1.0% tryptone the rest is water, pH 7.2-7.4.
Described antibiotic adopts: the ampicillin working concentration is 100 μ g/ml, and the erythromycin working concentration is 20 μ g/ml.
Described electric shock eluent is: two (2-ethoxy) piperazine-N '-2-(ethyl sulfonic acid) of 1mmol/L (pH7.0) N-, be called for short HEPES.
Described electric shock buffer solution is: 1mmol/L (pH7.0) HEPES, 10% glycerine.
Described solution I is: 50mmol/L glucose, 25mmol/L trishydroxymethylaminomethane (pH8.0), 10mmol/L ethylenediamine tetra-acetic acid (pH8.0).
Described solution II is: 0.2mol/mL sodium hydroxide, 1% dodecyl sodium sulfate.
Described solution III is: 5mol/L potassium acetate, 2.5mol/L glacial acetic acid.
The used examination worm of described virulence bioassay is: just incubate cotton bollworm (Helithisarmigera) larva and 2 willow fleautiauxia armata in age (Playiodera versicolora) larvas.
Described PCR detects and is meant with Auele Specific Primer detection cry1Ac and cry3A gene;
The specific primer sequence that detects the cry1Ac gene is:
TYIUNI2?5’ATCACTGAGTCGCTTCGCATGTTTGACTTTATC3’
TYIIAC?5’TCACTTCCCATCGACATCTACC3’
The specific primer sequence that detects the cry3A gene is:
CoL1A?5’GTCCGCTGTATATTCAGGTG3’
CoL2A?5’AGGTGCCAACTAACCATGTT3’
1.2 factory formula
The main component of factory formula has: 2.5% starch; 0.4% peptone; 2.6% soybean cake powder; 0.1% dusty yeast; 2.0% corn steep liquor; 0.2% fish meal; 0.2% potassium dihydrogen phosphate (KH
2PO
4); 0.04% epsom salt (MgSO
47H
2O); 0.1% calcium carbonate (CaCO
4); α-Dian Fenmei (account for content of starch 1/400).Total solid content 6.4%.
1.3 detect insect
Just incubate cotton bollworm (Helithis armigera) and 2 willow herbs in age chrysomelid (Playioderaversicolora).
1.4 production process
1.4.1 seed fermentation
The 500ml triangular flask, dress medium 50ml, 30 ± 1 ℃, shaking speed is 200r/min, the termination in 10-12 hour of fermenting.
1.4.2 produce fermentation
40 tons of fermentation tanks are feeded 25 tons, two triangular flasks of every jar of inoculation, and inoculation temperature 35-37 ℃, fermentation temperature 28-31 ℃, constant speed stirs 250r/min, throughput 1: 0.5-1: 1.5, fermenting stops when the gemma crystal forms and the brilliant separation of 40-50% spore is arranged.Defoamer is bubble enemy BAPE, an amount of adding.
Fermentation situation in table 1 fermentation tank
Jar time thalline developmental state is put a jar pH production cycle and is tired
20 hours 38 hours (hour) (IU/ μ L)
1 neat spore crystalline substance synchronously comes off 20% 8.0 38 2458
The 2 30% spore crystalline substances that physically well develop come off 8.0 38 2628
1.5 zymotic fluid post processing
1.5.1 suspending agent production
Zymotic fluid is only removed about 40% supernatant behind GF-105 disc-type supercentrifuge circular centrifugal.In the zymotic fluid that this kind low power concentrates, add preservative and emulsifier etc. on request and make suspending agent.
1.5.2 pulvis production
The low power concentrated broth is directly or after adding 10% precipitated calcium carbonate, carries out drying, 195-200 ℃ of spray tower inlet temperature, outlet temperature 85-90 ℃ with the high pressure draught spray dryer of BLSA specification.Make recombined broad spectrum thuringiensis cladosporioides bacillus insecticide wetting powder.
Table 2 aftertreatment technology is produced the result of microbial inoculum
Lepidoptera toxicity evaluation coleoptera toxicity evaluation
Formulation water content (%)
IU/μl IU/mg CU/μl CU/mg
Suspending agent 4600-5300--
Pulvis (directly spraying)-28600-32,000 5.12
Pulvis (adding calcium carbonate)-16000-21,500 2.75
1.6 the toxicity evaluation of insecticide is measured
1.6.1 kill the Lepidoptera titration
Select for use and just incubate cotton bollworm, carry out according to the regulation among the agricultural industry criteria NY293-95 of the People's Republic of China (PRC) " sporeine preparation ".The toxicity evaluation that records this recombined broad spectrum thuringiensis cladosporioides bacillus insecticide suspension emulsion is 4600IU/ μ l; The toxicity evaluation of pulvis is 28600IU/mg; The pulvis toxicity evaluation that adds 10% precipitated calcium carbonate is 16000IU/mg.
1.6.2 kill the coleoptera titration
Selecting the chrysomelid larva of 2 willow herbs in age for use, is standard preparation (toxicity evaluation is decided to be 20000CU/mg) with the self-control pulvis, measures with reference to the industry standard method.The toxicity evaluation that records this recombined broad spectrum bacillus thuringiensis suspension emulsion is 5300CU/ μ l; The toxicity evaluation of pulvis is 32000CU/mg; The pulvis toxicity evaluation that adds 10% precipitated calcium carbonate is 21500CU/mg.
Recombined broad spectrum thuringiensis cladosporioides bacillus insecticide preventive effect to lepidoptera pest in field trial and application is well stablized, and has produced 30 tons of suspension emulsions, 625 kilograms in efficient pulvis, and production technology is mature on the whole, and has reached suitability for industrialized production demonstration scale.Because the desinsection scope has comprised Lepidoptera and coleopteran pest, the multiple insecticidal properties of a kind of insecticide tool need not to produce multiple insecticide simultaneously, has improved use value, has saved production cost and use cost.And domestic still do not have extremely coleoptera bacillus thuringiensis commodity preparation at present, thereby economic worth improves, estimate that therefore production cost per ton can reduce by 30%, price can improve 20%, the outstanding agent of breast is 4000 yuan/ton routinely, 50000 yuan of/ton meters of one-level pulvis, overall economic efficiency can increase by 500 yuan/ton (emulsions) and 4000 yuan/ton (pulvis).According to 210,000 mu of incomplete statistics of using data, use the recombined broad spectrum insecticide, control efficiency to coleopteran pest obviously is better than commercially available sporeine preparation, has reduced the pesticide control expense, has increased crop yield, nearly 1,367 ten thousand yuan of overall economic efficiency.The experiment proved that its safety and wild thuringiensis cladosporioides bacillus insecticide are as good as, harmless, free from environmental pollution to people, animal and other animal and plant, and can improve agricultural product quality, good society and ecological benefits are arranged.Thereby, there is aspect such as demand to consider that the recombined broad spectrum thuringiensis cladosporioides bacillus insecticide has good popularizing application prospect from insecticidal properties, production and the use cost reduction of superiority, suitability for industrialized production technology maturation, to people and animals and plants safety, free from environmental pollution and market.
The pilot scale of recombined broad spectrum Bt insecticide and industrialization demonstration large-scale production are carried out in Hubei Province Kang Xin biopesticide company and starch chemical plant, Jiangdu, Jiangsu Province.Fermentation lab scale and pilot scale, microbial inoculum production, field trial, demonstration and land for growing field crops application, quality standardization and safety testing have been carried out.Produce 30 tons of the outstanding agent of breast, 625 kilograms in efficient pulvis.Outstanding agent toxicity evaluation 4600IU/ μ L of breast (killing Lepidoptera) and 5200-5500CU/ μ L (killing coleoptera).Wetting powder toxicity evaluation 28600IU/mg (not filled), 16000IU/mg (filled) and 18000-32000CU/mg.Finish field plot trial and Demonstration Application to lepidoptera pests such as cotton bollworm, rice leaf roller and beet armyworm and coleopteran pests such as ape is chrysomelid, colorado potato beetle.In the small plot experiment, preventive effect to above-mentioned lepidoptera pest is respectively 78.8-92.0%, 69.8-85.7%, 58.5-61.9% and 58.1-63.7%, above-mentioned coleopteran pest preventive effect is respectively 97.0-100% and 38.9-44.5%, and domestic 3 kinds of commodity preparations and U.S.'s product Dipel pulvis are all invalid to coleopteran pest.In the demonstration test, lepidoptera pest preventive effects such as cotton bollworm, diamond-back moth, oriental tobacco budworm are reached 66.5-92.0%; The Phaedonbrassicae preventive effect is reached 81.0-94.0%, is 47.3-48.1% to the colorado potato beetle preventive effect.21.05 ten thousand mu of areas are used in the land for growing field crops, control efficiency good (table 3).Tentatively set up the detection technique system of the quality standardization of recombined broad spectrum Bt insecticide.Acute toxicity experience and ecological safety Journal of Sex Research have been carried out.Get permission Ministry of Agriculture's genetic engineering body pilot scale level security evaluation and declared the evaluation of open-air release level security.
The effect of table 3 recombined broad spectrum thuringiensis cladosporioides bacillus insecticide control in field insect
Application site pest control crop preventive effect (%) area (ten thousand mu)
7 districts and cities cotton bollworm cotton 63.0-90.0 11.6 such as Dingzhou, Hebei
The chrysomelid vegetables 70.0-92.0 1.5 of 3 districts and cities apes such as Jiashan, Zhejiang
Huocheng, Xinjiang colorado potato beetle potato 67.8-85.7 2.95
Vegetables base, Shenzhen diamond-back moth vegetables 70.5-85.6 5.0
Add up to 7 kinds 5 kinds 21.05
Claims (1)
1, a kind of recombined broad spectrum bacillus thuringiensis prepares the method for insecticide, it is characterized in that, this preparation method follows these steps to order and carries out:
(1) at first prepare recombined broad spectrum bacillus thuringiensis Bacillusthuringiensis LCJ-12, method is as follows:
A, employing preserving number are the recipient bacterium of the bacterial strain of CCTCC NO:M 201003 as bacillus thuringiensis, and this bacterial strain contains the cry3A gene; Adopt the pHT304/1Ac10 recombinant plasmid, this recombinant plasmid contains the cry1Ac gene of lepidopterous insects extremely;
B, containing the LB inoculation of medium Escherichia coli of ampicillin, 35-37 ℃ is acutely rocked and cultivated 8-12 hour, and bacterial precipitation is resuspended in the ice-cold solution I of 100 μ l, and concuss adds 200 μ l solution II; Add the ice-cold solution III of 150 μ l,,, in-20 ℃ of deposit D NA, obtain Escherichia coli recombinant plasmid pHT304/1Ac10 with the ethanol of 2 times of volume supernatants with equivalent phenol-chloroform-isoamyl alcohol extracting supernatant in centrifugal 10 minutes of 4 ℃ of following 12000g;
C, recipient bacterium is seeded in the LB medium, 28-30 ℃ after wave and culture 8-10 hour, ice bath 20-30 minute, centrifugal 10 minutes of 4 ℃ of following 12000g, earlier wash twice, wash once with the electric shock buffer solution again, be suspended at last in the electric shock buffer solution with the electric shock eluent, obtain recipient bacterium electric shock competent cell, put-20 ℃ of preservations;
D, pHT304/1Ac10 plasmid DNA and recipient bacterium competent cell suspension fully are mixed, get 200 μ l and inject electric shock conversion cup, shock by electricity once under 2.5KV with E.coli Gene Pulser Escherichia coli electric shock conversion instrument, the liquid that will shock by electricity changes in the 4mlLB medium, 28-30 ℃ of vibration cultivated after 2 hours, coat on the LB flat board that contains erythromycin 20 μ g/ml, cultivated 3 days down for 28-30 ℃;
E, on the erythromycin resistant panel of 20 μ g/ml, select single bacterium colony, smear for microscopic examination, choosing the bacterium colony with bacillus thuringiensis feature goes down to posterity on the erythromycin flat board, to detect its stability, go down to posterity and virulence bioassay and PCR detection through the heredity in 20 generations, obtain virulence recombined broad spectrum bacillus thuringiensis the highest and that contain cry1Ac and cry3A gene, be a strain of recombined broad spectrum bacillus thuringiensis LCJ-12 of the present invention;
(2) composition of factory formula is: 2.5% starch; 0.4% peptone; 2.6% soybean cake powder; 0.1% dusty yeast; 2.0% corn steep liquor; 0.2% fish meal; 0.2% potassium dihydrogen phosphate; 0.04% epsom salt; 0.1% calcium carbonate; Account for the α-Dian Fenmei of content of starch 1/400, the rest is water, total solid content 6.4%;
(3) detect insect adopt just incubate cotton bollworm and 2 age willow herb chrysomelid;
(4) seed fermentation 500ml triangular flask, dress medium 50ml, 30 ± 1 ℃, shaking speed is 200r/min, ferments 10-12 hour;
(5) production is fermented with 40 tons of fermentation tanks, feed 25 tons, two triangular flasks of every jar of inoculation, inoculation temperature 35-37 ℃, fermentation temperature 28-31 ℃, constant speed stirs 250r/min, throughput 1: 0.5-1: 1.5, fermenting stops when the gemma crystal forms and the brilliant separation of 40-50% spore is arranged, and defoamer is bubble enemy BAPE;
(6) zymotic fluid is only removed about 40% supernatant behind GF-105 disc-type supercentrifuge circular centrifugal, adds preservative and emulsifier and makes suspending agent;
(7) the low power concentrated broth is directly or after adding 10% precipitated calcium carbonate, high pressure draught spray dryer with the BLSA specification carries out drying, 195-200 ℃ of spray tower inlet temperature, makes recombined broad spectrum thuringiensis cladosporioides bacillus insecticide wetting powder by outlet temperature 85-90 ℃.
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| CN103396977B (en) * | 2013-07-25 | 2014-10-15 | 中国科学院武汉病毒研究所 | Bacillus thuringiensis engineering bacterium for killing coleopteran pests as well as preparation method and application thereof |
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