CN101709270A - Bacterial strain capable of increasing insecticidal effectiveness of biocontrol fungi and construction method thereof - Google Patents
Bacterial strain capable of increasing insecticidal effectiveness of biocontrol fungi and construction method thereof Download PDFInfo
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Abstract
The invention provides a bacterial strain capable of increasing insecticidal effectiveness of biocontrol fungi. The bacterial strain is a transgenic strain containing Bacillus thuringiensis vegetative insecticidal protein gene and the preservation number of the strain is CGMCCNO.3283. The invention also provides a method for constructing the bacterial strain. The method comprises the following steps: obtaining Vip3A protein gene of Bacillus thuringiensis (Bt); amplifying and extracting fungal expression plasmid pAN52-1N; constructing Vip3A protein gene binary vector; and obtaining recombinant biocontrol fungi through transferring the constructed Vip3A protein gene binary vector to biocontrol fungi by means of genetic transformation technology. The bacterial strain and the construction method provide a novel solution scheme for preventing pests from generating resistance to Bt crystal insecticidal protein and solving the problems of the present pest-resistant transgenic plants and constructed recombinant insecticidal microorganism such as single variety of insecticidal gene and narrow insecticidal spectrum, thereby showing an enormous application prospect.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of bacterial strain and construction process thereof that the biocontrol fungi desinsection is renderd a service that strengthen.
Background technology
The hyphomycetale disinsection fungal is important insect biocontrol fungi, and under field conditions (factors), its topmost infection way is by body wall contact infection each class pest that causes death.This process comprises that spore adsorbs, sprouts, penetrates insect body wall on insect body wall, and mycelia breeding fast in host's haemocoele then causes host's death because of exhausting nutrition eventually.Unique mode of infection makes the hyphomycetale disinsection fungal become the first-selected biological and ecological methods to prevent plant disease, pests, and erosion factor of control sucking pest.The member of hyphomycetale disinsection fungal is a lot, bacterial classification as muscardine (Beauveria), green muscardine fungus (Metarhizium) and Paecilomyces varioti (Paecilomyces) and Verticillium (Verticillium) etc. belong to has been developed to the control that several formulations is used for agriculture and forestry injurious insect.Kind surplus the Hyphomycetes sterilant 60 that the present whole world is successively registered is becoming pest control new technology and rule of origin that pollution-less agriculture can rely on.
Although hyphomycetale disinsection fungal pest control long-lasting is good, desinsection speed is still difficult fully up to expectations, is the important factor that influences this type of fungus insecticide application always, also is to attempt one of technical barrier that solves by the tackling key problem research emphasis in the world.Bag muscardine as hyphomycetale disinsection fungal typical case representative usually occurs in postvaccinal 48 hours to infecting of insect body wall.Simultaneously, do not infect the host, and may be lost because of drainage, thereby cause through intestinal tract infections and lethal time-histories longer by the part that insect is taken in spore owing to beauveria bassiana has special enteron aisle virulence factor to rely on the mode that spore penetrates body wall.But making, the desinsection time that lags behind causes tremendous loss in pests with chewing mouthparts position such as damage to crops base of leaf still after microbiological contamination.How effectively to improve beauveria bassiana and the quick poisoning ability of pests with chewing mouthparts is received all the time the concern of biological and ecological methods to prevent plant disease, pests, and erosion circle.So far, it is many based on strengthening effect and the virulence of beauveria bassiana to each link in host's body wall infection processs to be intended to improve the research work of bacterial strain virulence and desinsection speed, as: strengthen the absorption of spore by form and the hydrophobic substance of regulating the sporoderm surface to host's body wall; Improve the desinsection speed that storage nutrition promotion spore is sprouted and formed germ tube and improve bacterial strain by the vigor that strengthens lytic enzyme outside the beauveria bassiana born of the same parents in the spore under the condition of no exogenous nutrition.Therefore, be necessary to utilize insect enteron aisle virulence albumen to transform disinsection fungal, it can not only effectively be infected through body wall, and the spore that enters enteron aisle can discharge accumulation and the subsequent growth process in the virulence albumen that produces directly suppress insect fast and get food, destroy the enteron aisle inner membrance, and cause pathogenic fungi to be easier to invasion and attack and to penetrate the insect enteron aisle enter hemolymph, thereby reach the purpose that strengthens the disinsection fungal virulence and improve desinsection speed.
As everyone knows, Bt (Bacillus thuringiensis) produces insecticidal crystal protein (the Insecticidal Crystal Proteins that is called delta-endotoxin (delta-endotoxin) in the gemma forming process, ICPs), these albumen have very high enteron aisle toxicity.In the past few decades, Bt and insecticidal crystal protein thereof have been developed to several formulations and have been widely used in control agricultural, forest and sanitary insect pest.Moreover, the important farm crop that change the Bt insecticidal crystalline gene constantly occur and application, have obtained remarkable economic efficiency and social benefit.However, also there are problems such as insecticidal spectrum is narrow, virulence is not high in the Bt preparation.Especially, it is found that since the eighties in last century various pests has shown the resistance of different levels to insecticidal crystal protein and genetically modified crops thereof, needs means of prevention safely and effectively badly.Yet, since the last century the nineties, be that new era has been started in the research that is found to be Bt and the application of the Bt Vegetative Insecticidal Proteins of representative with Vip3A.Vip3 is that the novel insecticidal proteins family of a class is different with ICPs.At first, Vip3 is at Bt vegetative phase excretory, has the albumen of signal peptide.It begins secretion mid-term from logarithmic growth, reaches the climax in stable early stage.Secondly, the aminoacid sequence of Vip3 and known crystallin do not have any homology.Most important difference is that Vip3 has unique insecticidal mechanism.Vip3 albumen is lower than 7.5 just solubilized at pH, and its carbon teminal need not excision, directly combines with sensitive insect midgut epithelial cell (mainly being the mast cell), brings out the insect cell apoptosis, and karyolysis causes insect death.Vip3 has insecticidal activity than wide spectrum to lepidopterous insects, or even some have the insect of very strong resistance to the Bt insecticidal crystal protein, and is higher 260 times than CrylA to the toxicity of black cutworm (Agrotis ipsilon) as Vip3A.This shows, Vip3 albumen produces resistance to Bt crystal insecticidal proteins and overcomes present insect-resistant transgenic plants and make up difficult problems such as the killing gene kind that the reorganization insecticidal microorganism exists is single, insecticidal spectrum is narrow new solution is provided for delaying insect, has showed very wide prospect.So far, Vip albumen successfully is applied to the structure of transgenosis pesticide plant, obtains efficient pest-resistant multivalence transgenic corns.In addition, make up Vip fusion protein and utilize it and the synergy of insecticidal crystal protein increase desinsection speed.In like manner, Vip albumen is expected to become the efficiency factor that strengthens hyphomycetale disinsection fungal enteron aisle virulence.
Summary of the invention
Technical problem to be solved by this invention provides a kind of bacterial strain that the biocontrol fungi desinsection is renderd a service that strengthens, and it is the biocontrol fungi bacterial strain that utilizes genetically engineered to render a service with the raising desinsection in conjunction with the bacillus thuringiensis Vegetative Insecticidal Proteins gene with unique insecticidal mechanism and high insecticidal activity.The present invention by the following technical solutions for this reason: it is the transgenosis bacterial strain (its Latin formal name used at school is Beauveria bassiana) that contains bacillus thuringiensis Vegetative Insecticidal Proteins gene, its deposit number is CGMCC NO.3283, its preservation time is on September 16th, 2009, depositary institution is China Committee for Culture Collection of Microorganisms common micro-organisms center, the address is the Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
The present invention also provides a kind of method that makes up the bacterial strain that strengthens biocontrol fungi desinsection effectiveness, it comprises from bacillus thuringiensis (Bacillus thuringiensis, amplification and the extraction of the acquisition of Vip3A protein gene Bt) and expressed in fungi plasmid pAN52-1N, it also may further comprise the steps successively on the basis of above-mentioned steps: the structure of Vip3A protein gene binary vector; Utilize the genetic transformation technology to change the Vip3A protein gene binary expression vector that makes up over to biocontrol fungi, obtain the reorganization biocontrol fungi.
The acquisition of Vip3 protein gene:, obtain the Vip3 protein gene with the PCR method amplification according to the primer of gene order design band restriction enzyme site Nco I and BamH I.
Amplification and extraction expressed in fungi plasmid pAN52-1N in intestinal bacteria.
The structure of Vip3 protein gene binary vector: cut Vip3 protein gene PCR product and plasmid pAN52-1N respectively with restriction endonuclease Nco I and BamH I, the Vip3 gene is connected with plasmid.Thereby anti contravariance related gene is placed respectively between the terminator TtrpC of the promotor Pgpd of Aspergillus nidulans (Aspergillius nidulans) glyceraldehyde 3-phosphate dehydro-genase gene and tryptophane gene, be connected into the expressed in fungi carrier with biocontrol fungi selection markers grass fourth phosphine resistant gene Bar Expression element respectively then.
The acquisition of reorganization biocontrol fungi: utilize the genetic transformation technology, change the binary expression vector that makes up over to biocontrol fungi, screening obtains the desinsection of constitutive expression Vip3 protein gene and renders a service enhanced reorganization biocontrol fungi bacterial strain.
The present invention is with the Vip3 protein gene cloning among the Bt, and be placed under the fungi constitutive promoter and make up the expressed in fungi plasmid, and utilize genetic conversion system to change the expressed in fungi plasmid over to biocontrol fungi, obtain the recombinant bacterial strain of constitutive expression Vip3 protein gene, by the enteron aisle toxicity action of Vip3 albumen unique efficient, the desinsection of insect is renderd a service thereby strengthen biocontrol fungi.The present invention provides new solution for delaying insect Bt crystal insecticidal proteins is produced resistance and overcoming present insect-resistant transgenic plants and make up difficult problems such as the killing gene kind that the reorganization insecticidal microorganism exists is single, insecticidal spectrum is narrow, has showed very wide application prospect.
Embodiment
Below in conjunction with experiment content of the present invention is further described.
Embodiment 1: make up desinsection and render a service enhanced beauveria bassiana recombinant bacterial strain.
The Vip3A gene generally is directed to Bt among the present invention, and it is the class new type disinsection albumen that bacillus thuringiensis produces in the mid-log phase secretion of nourishing and growing.Vip3A extensively is present among the Bt, and (Insecticidal CrystalProteins ICPs) without any homology, does not have diversity, and is more conservative in heredity with crystal desinsection egg.
(1) acquisition of Vip3 protein gene.
Synthesize the vip3Aa gene voluntarily according to the sequence that GenBank DQ539887.1 provides.Vip3A albumen (GenBank accession number:AAC37036.1) gene at Bt adds restriction enzyme site Nco I and BamH I from beginning to end at it, restriction enzyme site following (the wherein restriction enzyme site sequence of the base of italic for introducing):
Vip3aF：5′-CTGCCATGGACAAGAACAACACC-3′(NcoI)，
vip3aR：5′-GCTGGATCCCTACTTGATGCTCACGTCGT-3′(BamHI)。
(2) amplification and extraction expressed in fungi plasmid pAN52-1N in intestinal bacteria.
Cut vip3Aa gene product and plasmid pAN52-1N respectively with restriction endonuclease Nco I and BamH I.
The amplification of pAN52-1N plasmid and extraction: the activation of on LB (Luria-Bertani) flat board, ruling of the intestinal bacteria that contain plasmid pAN52-1N (E.coli) the DH5 α bacterial classification that will in-70 ℃ of 25% glycerine, preserve, cultivate 12h for 37 ℃ and grow to mono-clonal.Mono-clonal after the picking activation 37 ℃ of shaking table 200rpm in 2ml LB liquid nutrient medium cultivate 10h to logarithmic phase.Get 1.5ml bacterium liquid in clean 1.5mleppendorf centrifuge tube, (Doraville, GA, a small amount of plasmid extraction test kit USA) pressed standard step extracting plasmid pAN52-1N in the test kit specification sheets with Omega after the centrifugal 1min of room temperature 10000g collected thalline.Use 50 μ l DEPC water dissolution plasmids at last.
(3) structure of Vip3 protein gene binary vector.
At first, with Vip3aF and Vip3aR primer, be masterplate with synthetic vip3Aa sequence voluntarily, with PCR method NcoI and BamHI restriction enzyme site are introduced 5 ' and 3 ' of this gene respectively and hold.By standard consumption 50ul system, the PCR specific procedure: 94 ℃ of preheating 2min, then by the step cycle of 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 2.5min 35 times, last 72 ℃ are extended 4 ℃ of ∞ behind the 7min.Then, the PCR product is reclaimed with test kit, reclaim product and behind NcoI and BamHI (NEB) double digestion, be connected with plasmid pAN52-1N, the vip3Aa gene is introduced obtaining plasmid pAN52-Vip3A between the terminator TtrpC of the promotor Pgpd of Aspergillus nidulans (Aspergillius nidulans) the glyceraldehyde 3-phosphate dehydro-genase gene among the pAN52-1N and tryptophane gene and through the affirmation of checking order through same double digestion with the T4 ligase enzyme.Plasmid pET29b-bar (the Ying﹠amp that will contain then, the bar gene expression element; Feng 2006) digest with enzyme XbaI, separate enzyme through the DNA agarose gel electrophoresis and cut product and reclaim test kit with gel and reclaim and obtain bar gene expression element fragment and it is linked to each other with the plasmid pAN52-Vip3A that digested through XbaI equally, obtain containing the vip3Aa gene with identical transcriptional orientation and the binary vector pAN52-Vip3A-bar of bar gene expression element.Above-mentioned each endonuclease reaction is the various endonuclease reaction temperature to provide on NEB (Beijing) website (http://www.neb.com) all, cut reaction system and step is carried out by standard enzyme.
(4) acquisition of reorganization biocontrol fungi..
The plasmid pAN52-vip3A-Bar that step (3) is obtained changes in the beauveria bassiana genomic dna through the mediation of blastospore transformation system.Filter out the transformant BBV28 (preserving number: CGMCC NO.3283) of Vip3A albumen constitutive expression.
With beauveria bassiana Bb2860 bacterial strain is example, it is as follows to specify the blastospore method for transformation: the blastospore competence of getting the beauveria bassiana Bb2860 bacterial strain that a control gets ready, melt the back on ice in 4 ℃ of centrifugal 5min of 5000 * g, after abandoning supernatant, add following material successively: 240 μ l 50%PEG 4000,36 μ l 1M LiAc, the salmon sperm dna of 25 μ l 4g/l sex change, linearizing plasmid pAN52-Vip3A-bar of 10 μ l HindIII (0.1 μ g/ μ l) and 35 μ l 1M dithiothreitol (DTT) (dithiothreitol, DTT).With said mixture with the abundant mixing of vibrator after ice bath 30min, again in 42 ℃ of water-bath heat shock 20min, place on ice behind the 5min in 4 ℃ of centrifugal 5min collecting cells of 5000 * g.After the cell of collecting suspended with 500 μ l sterilized waters, get 100 μ l cell suspensions and evenly coat and contain 200 μ g/ml glufosinatess (phosphinothricin is on Cha Shi PPT) (CPZ) culture medium flat plate, in 25 ± 1 ℃ of cultivation 6d.The clone who grows is chosen into the enterprising row filter of CPZ substratum that contains 400 μ g/ml PPT again, pick out growing way better conversion son and identify.
The present invention can also utilize other genetic transformation technology (as: protoplast transformation, agrobacterium mediation converted, blastospore conversion, electric shock conversion etc.), change the binary expression vector that makes up over to biocontrol fungi, screening obtains the insecticidal activity enhanced reorganization fungi of constitutive expression Vip3A protein gene.
Embodiment 2: the beauveria bassiana spore is to the mensuration of the different worm instar larvae of prodenia litura insecticidal activity.
Concrete test procedure is as follows: will be suspended in evenly that to make concentration in the 0.02%Tween-80 aqueous solution be 1 * 10 for trying dry spore powder
8The spore suspension of individual/ml.Fresh, clean cabbage leaves is cut in the disk immersion spore suspension that diameter is 8cm, dries surperficial excessive moisture at room air then.Subsequently, the diameter that places sterilising treatment to cross in blade is the 15cm glass culture dish, choose respectively into each 35 of healthy and strong prodenia litura second instar larvaes, after sealing with parafilm, place 25 ± 1 ℃, L: D=12h: raise 7d in the 12h constant temperature illumination box, every day is the dead borer population of observed and recorded regularly, and dead worm is in time shifted out.Be blank with aseptic 0.02%tween-80 solution-treated simultaneously; Wild strain Bb2860 spore powder is treated to parallel control.Every processing repeats for 3 times.
Institute obtains the living data of surveying and analyzes with DPS data handling system software (Tang Qiyi and Feng Mingguang, 2002).Utilize the analysis of scalar type data unit value to draw the median lethal time LT of biocontrol fungi high density spore inoculating to each instar larvae of prodenia litura
50, with this or 7d cumulative mortality as the virulence index of biocontrol strain to each instar larvae.
Through biological assay, (preserving number: CGMCC NO.3283) virulence to an age, second instar larvae has improved 45.3%, 48.3% and 56.0% respectively than wild strain to the recombinant bacterial strain spore.
Embodiment 3: various dose biocontrol fungi spore is to the mensuration of prodenia litura second instar larvae virulence.
Concrete test procedure is as follows: will evenly be suspended in the 0.02%Tween-80 aqueous solution for the dry spore powder of examination and make 1 * 10
8Individual/ml, 2 * 10
7Individual/ml and 4 * 10
6The spore suspension of individual/ml series concentration.With full-automatic spray tower (Potter Spray Tower, Burkard Scientific Ltd., Middx, UK) spore suspension of inoculating each concentration respectively is in two sides clean, fresh cabbage leaves, every inoculation 1ml.(20mm * 20mm) collect spore, cotton blue dyeing back is at microscopically microscopy (0.2165mm with lying against the other cover glass of blade for actual dosage of inoculation
2/ the visual field) spore count is determined.Insert in the glass dish that clean diameter is 15cm after the blade room air of inoculating dried, choose healthy and strong larva at the beginning of two ages of prodenia litura on it, 40 in every ware.Each is handled 3 times and repeats.Culture dish places 25 ± 1 ℃ after sealing with the PE preservative film, raises the dead borer population of time recording every day in the constant temperature illumination box of L: D=12h: 12h.With the aseptic 0.02%Tween-80 aqueous solution as blank.Wild strain Bb2860 spore powder is a parallel control.
Institute obtains the living data of surveying and carries out model analysis with time-dosage-mortality ratio model.Utilize DPS data handling system software carry out about this model Basic of Biology, modeling procedure, parameter fitting and check, time and dosage effect (as LT
50And LC
50) estimate to wait and simulate and computing.
Through biological assay, the recombinant bacterial strain spore is at (preserving number: be 1373/mm to second instar larvae at spore dosage CGMCCNO.3283)
2The time LT
50Shortened 21.5% than wild strain, LC the 4th day, the 5th day and the 6th day
50Reduced by 91.2%, 61.3% and 57.9% than wild strain respectively.
Green muscardine fungus, Paecilomyces varioti and muscardine are the biocontrol fungis that belongs to hyphomycetes together, has similar biological, genetic transforming method is similar, thereby the expressed in fungi plasmid pAN52-vip3A-Bar aimed strains such as green muscardine fungus, Paecilomyces varioti of also can recombinating, and in bacterial strain, reach the purpose that same raising desinsection is renderd a service, be used for the biological control of Agricultural pests.
Through evidence, the insecticidal toxicity of reorganization beauveria bassiana bacterial strain spore insect of the present invention is compared wild strain and is greatly increased, and the time of deadly insect is compared wild strain to be had and significantly reduces, but thereby alleviated pests with chewing mouthparts that existing biocontrol fungi brought because of the desinsection time lag problem at position such as damage to crops base of leaf still after microbiological contamination, can reduce massive losses that agricultural, forestry etc. are caused, have important economy and social effect.
Claims (4)
1. one kind strengthens the bacterial strain that the biocontrol fungi desinsection is renderd a service, and it is characterized in that it is the transgenosis bacterial strain that contains bacillus thuringiensis Vegetative Insecticidal Proteins gene, and its deposit number is CGMCC NO.3283.
2. a structure strengthens the method for the bacterial strain that the biocontrol fungi desinsection renders a service according to claim 1, it is characterized in that it comprises from bacillus thuringiensis (Bacillus thuringiensis, amplification and the extraction of the acquisition of Vip3A protein gene Bt) and expressed in fungi plasmid pAN52-1N, it also may further comprise the steps successively on the basis of above-mentioned steps: the structure of Vip3A protein gene binary vector; Utilize the genetic transformation technology to change the Vip3A protein gene binary expression vector that makes up over to biocontrol fungi, obtain the reorganization biocontrol fungi.
3. a kind of method that strengthens the bacterial strain of biocontrol fungi desinsection effectiveness as claimed in claim 2 is characterized in that described Vip3A protein gene binary vector changes in the biocontrol fungi genome through the mediation of blastospore transformation system.
4. a kind of method that strengthens the bacterial strain of biocontrol fungi desinsection effectiveness as claimed in claim 2, the resistant gene that it is characterized in that described Vip3A protein gene binary vector is careless fourth phosphine resistant gene Bar.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154127A (en) * | 2011-01-12 | 2011-08-17 | 浙江大学 | Genetically modified biocontrol fungus with high intestinal-toxin insecticidal activity and structuring method and application thereof |
| CN104726351A (en) * | 2015-04-14 | 2015-06-24 | 南方医科大学 | Bivalent recombination beauveria bassiana capable of killing mosquito larvae and preparation method of bivalent recombining beauveria bassiana |
| CN114214262A (en) * | 2021-12-27 | 2022-03-22 | 浙江大学 | Biocontrol fungal spore culture method for enhancing insecticidal efficacy and abiotic stress resistance |
-
2009
- 2009-11-12 CN CN200910222794A patent/CN101709270A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154127A (en) * | 2011-01-12 | 2011-08-17 | 浙江大学 | Genetically modified biocontrol fungus with high intestinal-toxin insecticidal activity and structuring method and application thereof |
| CN102154127B (en) * | 2011-01-12 | 2014-04-16 | 浙江大学 | Genetically modified biocontrol fungus with high intestinal-toxin insecticidal activity and structuring method and application thereof |
| CN104726351A (en) * | 2015-04-14 | 2015-06-24 | 南方医科大学 | Bivalent recombination beauveria bassiana capable of killing mosquito larvae and preparation method of bivalent recombining beauveria bassiana |
| CN104726351B (en) * | 2015-04-14 | 2018-06-01 | 南方医科大学 | A kind of bivalent Recombinant Strain of Beauveria bassiana for killing mosquito larvae and preparation method thereof |
| CN114214262A (en) * | 2021-12-27 | 2022-03-22 | 浙江大学 | Biocontrol fungal spore culture method for enhancing insecticidal efficacy and abiotic stress resistance |
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