CN1196408C - Agricultural chemical contg. insect viruses synergist protein, and use thereof - Google Patents
Agricultural chemical contg. insect viruses synergist protein, and use thereof Download PDFInfo
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- CN1196408C CN1196408C CN 02139035 CN02139035A CN1196408C CN 1196408 C CN1196408 C CN 1196408C CN 02139035 CN02139035 CN 02139035 CN 02139035 A CN02139035 A CN 02139035A CN 1196408 C CN1196408 C CN 1196408C
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Abstract
The present invention discloses a chemical pesticide containing synergistic protein of insect viruses and an application, which is characterized in that the synergistic protein of insect viruses is mixed and compounded with a chemical pesticide; the synergistic protein of insect viruses is applied to the chemical pesticide and a composite pesticide; purified polyhedrons of insect viruses are utilized for separation and purification to obtain the synergistic protein of insect viruses, or a gene engineering method is adopted for expressing and purifying the synergistic protein of insect viruses. The present invention has the advantages of improved biologic prevention efficiency of the chemical pesticide, effective content reduction of the chemical pesticide, increased pesticide effect, improved prevention and treatment efficiency and pesticide resistance reduction or elimination of insects to the chemical pesticide; the synergistic protein of insect viruses is safe for people and livestock and is safe for crops, and the synergistic protein of insect viruses does not pollute an environment.
Description
Technical field
The present invention relates to chemical pesticide and composite insecticide, more specifically relate to a kind of chemical pesticide that contains the insect viruses synergistic protein, the invention still further relates to the application of insect viruses synergistic protein in chemical pesticide and composite insecticide.
Background technology
1956, Tanada etc. are first from America pseudatetia unipuncta granulosis virus (Pseudaletia unipunctagranulosis virus, PuGV) isolate synergistic protein, and confirm that this albumen is to America leucania separara nuclear polyhedrosis virus (Pseudaletia unipuncta nuclear polyhedrosis virus, PuNPV) has effect (the Tanada Y et al. that strengthens infection, J Invertebr Pathol, 1959,1:215-231).Hashimoto (HashimotoY et al., J Gen Virol, 1991,72:2645-2651) and Roelvink (Roelvink P W et al., J GenVirol, 1995,76:2693-2705) respectively to cabbage looper PuGV (Trichoplusia ni granulosisvirus, TnGV), PuGV, heliothis armigera granulosis virus (Heliothis armigera granulosis virus, HaGV) enhancing protein gene has carried out clone and sequence analysis, has compared their autoploidy.At present, (Lymantria dispar nuclear polyhedrosisvirus LdNPV) He in 4 kinds of poxvirus has found insect viruses synergistic protein (Hayakawa T et al., Gene at 9 kinds of PuGVs, lymantria dispar nuclear polyhedrosis virus, 1996,177:269-270).
The insect viruses synergistic protein is a kind of metalloproteinases of viral gene coding, molecular weight is 89-110kDa, has a typical metalloproteinases zinc-in conjunction with territory HEXXH, the position is very conservative, and the peritrophic degradation of midgut is subjected to inhibition (the Lepore L S et al. of metal-chelator, Invertebr Pathol, 1996,68:131-140).
The insect viruses synergistic protein can improve the susceptibility of insect, quicken progression of infection (the Tanada Y et al. of nuclear polyhedrosis virus, Insect Pathol, 1959,1:215-231), can improve simultaneously the bacillus thuringiensis,Bt parasporal crystal to the virulence of larva (Corsaro B G et al., A parasites and pathogens of insects:II.Pathogens.San Diego:Academic Press, 1993.127-145).The mechanism of action of insect viruses synergistic protein has two kinds: a kind of is that synergistic protein can combine with the specific site on the midgut epithelial cells film, and the fusion between mediation virus envelope and the cytoplasma membrane promotes that virus enters in the host cell, thereby increases the effect of virus infections.But it is not that to promote virus infections necessary that synergistic protein combines with specific site on the midgut epithelial cells film.Another kind of mechanism is that synergistic protein destroys insect larvae midgut peritrophic membrane by the intestines mucoprotein IIM that degrades, make the easier midgut epithelial cells that enters of virion, improve some simultaneously and must penetrate peritrophic membrane and usefulness (the Wang P et al. of the biological insecticides of having an effect, Natl Acad Sci USA, 1997,94:6977-6982).
At present, the mode of acquisition insect viruses synergistic protein has following several: (1) separation and purification from PuGV, caryogram polyhedron and poxvirus obtains synergistic protein.(2) enhancing protein gene is binned among the wide spectrum MNPV such as AcMNPV, makes up the recombinant virus of broad-spectrum high efficacy.The reorganization AcMNPV that contains enhancing protein gene is more lower slightly than the infection ability of wild type AcMNPV, but when mixing with wild type AcMNPV proper proportion, then its infection ability is higher than wild-type virus.(3) make up the genetically modified plants that contain enhancing protein gene, improve the sensitivity of insect insect viruses.(4) in prokaryotic and eukaryotic, express synergistic protein.
World agriculture faces annual 7000 ten thousand increasing population and insufficiency that increase, and is limited and plough.Chemical pesticide has been brought into play huge effect in agricultural development, integrated pest management and agricultural sustainable development, be the essential means of production of modern agriculture.Under the situation that improves existing quality of ploughing and yield per unit area, come the harm of pest control with chemical pesticide to agricultural production, avoid loss, be unusual important measures.Advantages such as that chemical pesticide has is efficient, quick-acting, easy to use, wide adaptability.But the chemical pesticide that uses is the poisonous chemical substance of synthetic mostly at present.Since chemical pesticide for a long time in large quantities, widely, continuously, unreasonable use, caused a series of serious consequences.Cause pest resistance to insecticide to be on the rise on the one hand, dosage is increasing, forms vicious circle.On the other hand, excessively use chemical pesticide, cause a large amount of natural enemies to be killed and wounded, the beneficial organism kind reduces, and less important insect breaks out, and has destroyed the ecological balance.Particularly pesticide pollution environment, atmosphere, soil and water source are contaminated, the residual quantity of agricultural chemicals in agricultural byproducts increases, severe overweight has endangered human body health more and more bigly, and has caused very big economic loss.
At present, countries in the world use, production and exploitation to chemical pesticide on the rules proposed a series of regulations, carried out the pesticide registration resignation system.The development trend of chemical pesticide comprises: the consumption that (1) reduces chemical pesticide, improve the intrinsic activity of former medicine and service efficiency and result of use.(2) reduce the negative effect of chemical pesticide to non-target organism and ecotope.(3) improve the compatibility of chemical pesticide and environment.(4) improve the safety of chemical pesticide to people, animal and crop.Wherein, reduce the usage amount of chemical pesticide, the cry in each developed country of the world is surging day by day, and the laws and regulations on the management of strict limit production and use have all been formulated in countries in the world.
Summary of the invention
The object of the present invention is to provide a kind of chemical pesticide that contains the insect viruses synergistic protein, utilize insect viruses synergistic protein and chemical pesticide or chemical pesticide complexing agent composite, reduced the effective content of chemical pesticide, improve the chemical pesticide insecticidal activity, increase drug effect, improved the biological and ecological methods to prevent plant disease, pests, and erosion efficient of chemical pesticide.
The invention still further relates to the application of insect viruses synergistic protein in chemical pesticide.
The invention still further relates to the application of insect viruses synergistic protein in composite insecticide.
In order to achieve the above object, the present invention adopts following technical measures:
By biochemical technologies such as gel filtration and ion exchanges, utilize the insect viruses polyhedron separation and purification of purifying to obtain the insect viruses synergistic protein, perhaps utilize gene engineering to make up engineered strain, to express, purifying obtains the insect viruses synergistic protein., be mixed with chemical pesticide by a certain percentage as the synergy intermediate with the insect viruses synergistic protein.It is characterized in that insect viruses synergistic protein final concentration is 1-3 * 10
2μ g/L and chemical pesticide are mixed.Composite with insect viruses synergistic protein and chemical pesticide or chemical pesticide complexing agent, be used for the preparation of chemical pesticide or chemical pesticide complexing agent, be applied to biological control.
The present invention compared with prior art has the following advantages and effect: 1. reduced the effective ingredient consumption of chemical pesticide significantly, increased drug effect, improved the biological and ecological methods to prevent plant disease, pests, and erosion efficient of chemical pesticide; 2. reduced the residual of chemical pesticide significantly, synergistic protein is to the person poultry safety, and is free from environmental pollution to crop safety, and the residual of chemical pesticide reduced relatively significantly; 3. reduction or elimination insect are to the pesticide resistance of chemical pesticide.
Embodiment
1, the preparation of insect viruses gene engineering synergistic protein
Picking contains the engineered strain of insect baculovirus enhancing protein gene, as E.coliM15 (pQE-TnGVEn Cp96), E.coli M15 (pQE-TnGVNp85), E.coliM15 (pQE-TnGVCp80), E.coli M15 single bacterium colonies such as (pQE-TnGV-Enp108) in the 25ml LB medium that contains 100 μ g/ml ampicillins and 25 μ g/ml kanamycin, 37 ℃, the 200rpm incubated overnight.Activation bacterium liquid contains in the 500ml LB culture fluid of 100 μ g ampicillins and 25 μ g/ml kanamycin with inoculation in 1: 20, continues to cultivate, as its concentration OD
600=0.3 o'clock, add isopropyl-to final concentration 1mmol/L, induce 5h for 37 ℃, results bacterium liquid, the centrifugal 10min of 4000rpm collects bacterial sediment.
The ultrasonic treatment fluid (50mmol/L Tris-HCl pH8.0-0.1mol/L NaCl-2mg/ml lysozyme) that in bacterial precipitation, adds 2-5 times of volume, multigelation 3 times, 37 ℃, insulation 1h places ice to use the ultrasonic disruption bacterium then.Place the light microscope high power objective to observe, a large amount of crystal are arranged.30-60% saccharose gradient on the treatment fluid, the centrifugal 40min of 4000rpm takes out the inclusion body band, washes 3 times the synergistic protein crystal of results purifying, 4 ℃ of preservations with the sterilization distilled water.
2, the evaluation of synergistic protein
Adopt the SDS-PAGE electrophoretic analysis.
(1) goes up sample and electrophoresis
With reference to Sambrook " molecular cloning handbook ", prepare 10% separation gel and concentrated glue.In sample, add equal-volume 2 * sds gel sample loading buffer, in 100 ℃ of heating 3-5min sex change.Sample on the sample.Enter separation gel with 100V electrophoresis to bromjophenol blue, improve voltage, finish electrophoresis when bromjophenol blue arrives precontract 1cm place, bottom to 150V.
(2) fix, dye and decolouring
Fix, dye and spend the night with 5 times of volume dyeing liquors behind the stripping glue, the receipts gel is soaked in and shakes gently in the destainer more than the 4h, changes destainer 4-5 time therebetween.
(3) interpretation of result
Can observe after the decolouring fully and take pictures, and determine the purity and the content of synergistic protein with laser scanning.
3, cabbage looper PuGV synergistic protein P108 is to the synergistic effect of stuck gram
The active ingredient of stuck gram (Cascade) is fulfenoxuron, is a kind of insect growth regulator, IGR, kills the mite desinsection by stoping larval molting, can influence adult spawning rate and egg hatching rate simultaneously.Stuck gram has tags and the anelasticity insecticidal action, to various pests, as cabbage caterpillar, diamond-back moth, beet armyworm, two spotted spider mite, cotton bollworm etc. preventive and therapeutic effect is arranged all.
But in 5% stuck gram dispersant, add cabbage looper PuGV synergistic protein P108, be made into mixture by 200 μ g/L.With coubling dilution above-mentioned mixture and the single agent of 5% stuck gram are made into some concentration gradients, promptly extension rate is 1,000-64,000 times.Getting at the beginning of 3 ages under the beet exigua larvae room temperature single head raises in 24 hole Costar Tissue Culture Dishs.Infect liquid with each dilution factor respectively and make the infection feed, each dilution factor sample infects for 96 of examination beet armyworm third-instar larvaes, and establishes single water contrast of steaming.Every hole adds an amount of artificial feed, and adds the above-mentioned various treatment fluids of 30 μ l in the artificial feed surface, and it is fully absorbed.Dead and the survival borer population of 48h statistics adopts DPS to carry out data and handles.The result shows that cabbage looper PuGV synergistic protein P108 has improved the virulence of stuck gram to beet armyworm, and coefficient of synergism is 178.
4, insect viruses synergistic protein P108 is to the synergistic effect of " coming the good fortune spirit " synthetic pyrethroid insecticide
Synthetic pyrethroid insecticide (5% missible oil) is the synthetic pyrethroid insecticide that the height desinsection is renderd a service that has of SUMITOMO CHEMICAL chemical industry Co., Ltd. research and development " to come the good fortune spirit ".To the insect on the various crop, as cotton bollworm, cabbage caterpillar, peach fruit borer, aphid etc. have strong insecticidal effect and also the residual effect time long.
In 5% " coming the good fortune spirit " pyrethroids missible oil, add cabbage looper PuGV synergistic protein P108 by 180 μ g/L, be made into mixture.Come the single agent of good fortune spirit to be made into some concentration gradients above-mentioned mixture and 5% with coubling dilution, promptly extension rate is 2500-64000 times.Getting at the beginning of 3 ages under the beet exigua larvae room temperature single head raises in 24 hole Costar Tissue Culture Dishs.Infect liquid with each dilution factor respectively and make the infection feed, each dilution factor sample infects for 96 of examination beet armyworm third-instar larvaes, and establishes single water contrast of steaming.Every hole adds an amount of artificial feed, and adds the above-mentioned various treatment fluids of 30 μ l in the artificial feed surface, and it is fully absorbed.Dead and the survival borer population of 48h statistics adopts DPS to carry out data and handles.The result shows that cabbage looper PuGV synergistic protein P108 has improved the virulence of 5% " coming the good fortune spirit " pyrethroids missible oil to beet armyworm, and coefficient of synergism is 250.
5, insect viruses synergistic protein P108 is to pressing down the synergistic effect of hooligan's insecticide
But press down in hooligan's dispersant 5%, add cabbage looper PuGV synergistic protein P108, be made into mixture by 160 μ g/L.With coubling dilution above-mentioned mixture and 5% is pressed down the single agent of hooligan and be made into some concentration gradients, promptly extension rate is 1,000-64,000 times.Getting at the beginning of 3 ages under the beet exigua larvae room temperature single head raises in 24 hole Costar Tissue Culture Dishs.Infect liquid with each dilution factor respectively and make the infection feed, each dilution factor sample infects for 96 of examination beet armyworm third-instar larvaes, and establishes single water contrast of steaming.Every hole adds an amount of artificial feed, and adds the above-mentioned various treatment fluids of 30 μ l in the artificial feed surface, and it is fully absorbed.Dead and the survival borer population of 48h statistics adopts DPS to carry out data and handles.The result shows that cabbage looper PuGV synergistic protein P108 has improved and presses down the virulence of hooligan to beet armyworm that coefficient of synergism is 671.
6, insect viruses synergistic protein P108 is to the synergistic effect of the full insecticide of rice
But in 20% meter full dispersant, 240 μ g/L add cabbage looper PuGV synergistic protein P108, are made into mixture.With coubling dilution above-mentioned mixture and 20% meter full single agent are made into some concentration gradients, promptly extension rate is 2,000-64,000 times.Getting at the beginning of 3 ages under the beet exigua larvae room temperature single head raises in 24 hole Costar Tissue Culture Dishs.Infect liquid with each dilution factor respectively and make the infection feed, each dilution factor sample infects for 96 of examination beet armyworm third-instar larvaes, and establishes single water contrast of steaming.Every hole adds an amount of artificial feed, and adds the above-mentioned various treatment fluids of 30 μ l in the artificial feed surface, and it is fully absorbed.Dead and the survival borer population of 48h statistics adopts DPS to carry out data and handles.The result shows that cabbage looper PuGV synergistic protein P108 has improved the full virulence to beet armyworm of rice, and coefficient of synergism is 472.
Claims (7)
1. a composition pesticide is characterized in that described composition pesticide contains chemical pesticide and final concentration is 1-3 * 10
2The insect viruses synergistic protein of μ g/L.
2. composition pesticide according to claim 1 is characterized in that described chemical pesticide is selected from the group of being made up of organic phosphor, chrysanthemum ester class, carbamate, organic nitrogen and insect growth auximone class.
3. composition pesticide according to claim 1 is characterized in that, described composition pesticide contains the cabbage looper PuGV synergistic protein P108 of 200 μ g/L, and 5% stuck gram dispersible agent.
4. composition pesticide according to claim 1 is characterized in that, the cabbage looper PuGV synergistic protein P108 and 5% that described composition pesticide contains 180 μ g/L comes the good fortune missible oil.
5. composition pesticide according to claim 1 is characterized in that, the cabbage looper PuGV synergistic protein P108 and 5% that described composition pesticide contains 160 μ g/L presses down hooligan's missible oil.
6. composition pesticide according to claim 1 is characterized in that, it is full that described composition pesticide contains the cabbage looper PuGV synergistic protein P108 of 240 μ g/L and 20% meter.
7. composition pesticide according to claim 1, it is characterized in that described insect viruses synergistic protein is by utilizing the insect viruses polyhedron of purifying, separation and purification obtains, or utilize the gene engineering to make up engineered strain, express, purifying obtained.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 02139035 CN1196408C (en) | 2002-09-12 | 2002-09-12 | Agricultural chemical contg. insect viruses synergist protein, and use thereof |
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| CN 02139035 CN1196408C (en) | 2002-09-12 | 2002-09-12 | Agricultural chemical contg. insect viruses synergist protein, and use thereof |
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| CN1401246A CN1401246A (en) | 2003-03-12 |
| CN1196408C true CN1196408C (en) | 2005-04-13 |
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