CN103396977A - Bacillus thuringiensis engineering bacterium for killing coleopteran pests as well as preparation method and application thereof - Google Patents
Bacillus thuringiensis engineering bacterium for killing coleopteran pests as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a bacillus thuringiensis engineering bacterium for killing coleopteran pests as well as a preparation method and application thereof. The preservation number is CCTCCM2013259. The preparation method comprises the following steps of: A, cloning insecticidal crystal protein gene cry3Aa8 from a Bt bacterial strain YC-03 for killing the coleopteran pests, inserting the gene into a disaggregation carrier pHT304/res and obtaining a recombined plasmid pHT3Aa8/res, converting the DNA (deoxyribonucleic acid) of the pHT3Aa8/res into a Bt aphakic mutant strain BMB171 through electric shock, screening so as to obtain transformants, shifting an auxiliary plasmid pBMB1200 into the transformant, selecting a positive transformant not growing on an Amp/Erm flat panel but growing on an LB flat panel, breeding in an LB fluid nutrient medium, and selecting a single colony growing on the LB flat panel but not growing on a tetracycline flat panel; B, taking plagiodera versicolora and the larva of phaedon brassicae baly as the test pest, carrying out toxicity bioassay, comparing the toxicity lethal dose LC50, and screening the high-toxicity engineering bacterium strain Ace-38. The method is practicable, and is simple and convenient to operate, the bacillus thuringiensis engineering bacterium has high effect on killing a plurality of coleopteran pests such as plagiodera versicolora, potato beetles, vegetable phaedon brassicae baly, aulacophora indica and the like, and the bacterium has good fermenting property, and has development and application prospects.
Description
Technical field
The present invention relates to the microbial pesticide technical field, be specifically related to a bacillus thuringiensis strain, more specifically relate to a kind of preparation method who kills the bacillus thuringiensis engineering bacteria of coleopteran pest, also relate to simultaneously a kind of purposes of bacillus thuringiensis engineering bacteria in sterilant of killing coleopteran pest.
Background technology
Coleoptera is an order maximum in Insecta, and coleopteran pest is only second to lepidoptera pest to the harm of agricultural and forestry production.Coleopteran pest be distributed widely in all over the world (Biodiversity of Coleoptera/Insect biodiversity:science and society, West Sussex:Blackwell Publishing, 2009,265-301).More common as colorado potato bug (Leptinotarsa decemlineata), corn root leaf A (Diabrotica virgifera), daikon leaf beetle (Phaedon brassicae Baly), Anomala Corpulenta (Anomala corpulenta), willow fleautiauxia armata (Plagiodera versicolora), elm fleautiauxia armata (Pyrrhalta aenescens) etc.These insects are to harm (Xinjiang Agricultural Sciences, 2012,48 (12): 2217-2222 such as potato, corn, vegetables, farm crop, forests; Plant Quarantine, 1997,11 (2): 101-103; Plant protection technology and popularization, 1995, (5): 29-30; Biological Control, 1995,5 (2): 290-296; Journal of northeast Forestry university, 2003,31 (3): 65-66).Mostly can only rely on chemical pesticide to the control of coleopteran pest at present, chemical pesticide desinsection poor specificity, cause simultaneously environmental pollution, people and animals are had harm, so the desinsection specificity is good, environmental friendliness, person poultry safety's biotic pesticide more and more are subject to people's attention.Bacillus thuringiensis is the most successful, the typical case of biological control the most widely (Microbiology and Molecular Biology Reviews to the control of lepidoptera pest, 1998,62 (3): 775-), but the Bt sterilant application of killing coleopteran pest is less, and this is with the Bt bacterial strain that kills coleopteron and that genetic resources is less, insecticidal crystal protein toxicity is lower etc. is relevant.Therefore, constantly excavate new specific b t bacterial classification and genetic resources, improve insecticidal proteins output and toxicity by engineered means, construct Coleoptera is had the Bt bacterial strain of efficient insecticide activity and uses its production novel pesticide to have realistic meaning and urgency.
Aspect the screening of killing Coleoptera Bt bacterial strain and genetic resources excavation, document " Bacillus thuringiensis var.tenebrionis; a new pathotype effective against larvae of Coleoptera " has reported that the first strain has Bt bacterial strain (the Zeitschrift Fur Angewandte Entomologie-Journal of Applied Entomology of special insecticidal activity to coleopteran pest, 1983,96 (5): 500-508).Document " A new strain of Bacillus thuringiensis with activity against coleopteran insects " and " Nucleotide sequence and deduced amino acid sequence of a coleopteran-active delta-endotoxin gene from Bacillus thuringiensis subsp.san diego " have reported the second strain to the virose Bt bacterial strain of coleopteron and have cloned its insecticidal crystal protein encoding gene cry3Aa (Bio-Technology, 1986,4 (4): 305-308; Gene, 1987,57 (1): 37-46).Above two kinds of Bt bacterial strains all have efficient insecticidal activity to endangering serious colorado potato bug.document " A unique isolate of Bacillus thuringiensis serovar japonensis with a high larvicidal activity specific for scarabaeid beetles " reaches " Insecticidal spectrum of a novel isolate of Bacillus thuringiensis serovar japonensis " and has reported the activated Bt bacterial strain of chafer larva grub, this bacterial strain has efficiently poisoning active to multiple chafer larva, but to lepidopteran, Diptera, Orthoptera and Coleoptera (golden Dascillidae) insect non-activity, showed specificity (the Letters in Applied Microbiology of height, 1992, 14 (2): 54-57, Biological Control, 1992,2 (2): 138-142), after this, document " Characterization of larvicidal toxin protein from Bacillus thuringiensis serovar japonensis strain Buibui specific for scarabaeid beetles " and " Cloning, heterologous expression, and localization of a novel crystal protein gene from Bacillus thuringiensis serovar japonensis strain Buibui toxic to scarabaeid insects " reported that the insecticide active substance of this bacterial strain is the Cry albumen of 130KD, corresponding gene is cry8Ca1 (Journal of Applied Bacteriology, 1994, 76 (4): 307-313, Current Microbiology, 1994,28 (1): 15-19).Over 30 years, have successively different to the activated Bt bacterial strain of coleopteran pest with gene is separated and the clone.at present, the known Bt that coleopteran pest is had an insecticidal activity mainly contains and intends ground beetle subspecies (Bt subsp.tenebrionis), San Diego subspecies (Bt subsp.sandiego), subspecies morrixoni (Bt subsp.morrisoni), many nests subspecies (Bt subsp.tolworthi), Kumamoto subspecies (Bt subsp.kumanotoensis), Ku Sita subspecies (Bt subsp.kurstaki), galleria mellonella waxmoth subspecies (Bt subsp.galleriae), Su Yun gold subspecies (Bt subsp.thuringiensis), Japan's subspecies (Bt subsp.japonensis) and Chinese subspecies (Bt subsp.chinensis) etc.Document " several to the virose bacillus thuringiensis strains of coleopteron " has been studied Bt bacterial strain C-001～006 and ES-01 toxicity (the insect knowledge to anomala corpulenta (Anomala corpilenta Motschulsky), tenebrio molitor (Tenebrio molitor), 1991, (3): 151-152); " kill the research of the new bacterial strain of Coleoptera bacillus thuringiensis and sterilant " and " kill coleoptera larvae Bt bacterial strain YM-03 delta-endotoxin genes location " reported that Bt bacterial strain YM-03 is to the insecticidal activity of the coleopterons such as colorado potato bug and willow fleautiauxia armata and location (the microorganism journal of killing gene, 1999,39 (6): 515-520; JOURNAL OF MICROBIOLOGY, 2000,20 (1): 18-19), the insecticidal protein gene in YM-03 has been cloned and by definite designation, has been cry3Aa8 (accession number:AY572010)." strain has the new strain isolated of Bacillus thuringiensis of insecticidal activity to cockchafer subclass larva " reported Bt bacterial strain HBF-1 (the Chinese biological control that anomala corpulenta (Anomala corpilenta Motschulsky) and Anomala exoleta (Anomala exoleta Faldermann) larva is had cytotoxicity, 2000,16 (2): 74-77)." foundation of bacillus thuringiensis cry gene PCR-RFLP identification system ", " research of bacillus thuringiensis cry1Ie1 and cry8 gene " reach " Improving toxicity of Bacillus thuringiensis strain contains the cry8Ca gene specific to Anomala corpulenta larvae " perfect the PCR-RFLP identification system, and utilize this technology be cloned into from Bt bacterial strain HBF-1 one with the cry8Ca1 insecticidal protein gene cry8Ca2 (Scientia Agricultura Sinica of a codon (amino acid) difference only, 1998, 31 (3): 11-14, Biological Technology institute, Chinese Academy of Agricultural Sciences's post-doctoral research work report, 2003,23-46, Current Microbiology, 2007,55 (6): 492-496).Document " research overview of Bt insecticidal crystal protein " has been summed up the insecticidal crystalline gene that kills coleopteran pest has cry1B, cry1I, cry3A, cry3B, cry3C, cry7A, cry8A, cry8B, cry18A etc. (the Exploitation of Agriculture in Heilongjiang science, 2004,5:37-39).document " Insecticidal activity of Bacillus thuringiensis crystal proteins " has been summed up the insecticidal activity of desinsection Bt crystallin, wherein 30 kinds of Cry albumen (secondary classification) have insecticidal activity to coleopteran pest, except Cry8 proteinoid (7 kinds), all the other kind numbers are less (≤3 kinds) all, with regard to insecticidal spectrum, that has found at present has activity to the activated Cry albumen of coleopteran pest to multiple coleopteran pest, wherein Cry3Aa proteinoid insecticidal spectrum is the widest, even Hemiptera (Hemiptera) and Hymenoptera (Hymenoptera) insect there is activity (Journal of Invertebrate Pathology, 2009, 101 (1): 1-16).Document " to the Screening and Identification of the high virulence bacillus thuringiensis of coleopteron " has been summed up different coleopteran-toxic Cry albumen and insecticidal spectrum (Heilongjiang University's Life Science College, 2012) thereof." the high virulence bacillus thuringiensis research of grub " (modern commerce and trade industry, 2010,9:353-355) and " bacillus thuringiensis kills the line activity to pine wood nematode " (Chinese biological control, 2010,26 (1): 85-89) philosophy has screened the efficient specific strains to insects such as lasioderma serricorne, fall webworms, beet armyworm, grub, nematodes from numerous bacillus thuringiensis bacterial strains.
aspect killing gene clone and engineering bacteria structure, the gene that 71 nearly more than 600 bacillus thuringiensiss of class have been cloned in the present whole world, document " progress of bacillus thuringiensis and insecticidal crystalline gene resource thereof " (Shanghai Agricultural journal, 2010, 26(4): 135-139), " the clone of bacillus thuringiensis cry9Aa gene, express and activation analysis " (plant protection journal, 2010, 37(6): 541-546), " clone and the prokaryotic expression of bacillus thuringiensis cry1Ac28 gene " (Northeast Agricultural University's journal, 2010, 41(10): 61-67), " strain is identified the killing gene of the high virulence bacillus thuringiensis of fall webworms " (Chinese agronomy circular, 2010, 26(3): 242-244), " Tribactur cry1Ia17 gene clone, the research of expressing " (Northeast Agricultural University's journal, 2011, 42(4): 93-97), " clonal expression and the insecticidal activity thereof of Bacillus thuringiensis cry7Ab8 gene " (North China agronomy newspaper, 2011, 26 (2): 35-38), and " strain contains the research of the bacillus thuringiensis strains of the new gene of cry " (North China agronomy newspaper, 2011, 26 (1): 54-57) philosophy separates, identify, expressed a series of new active gene.Document " Construction of novel Bacillus thuringiensis strains with different insecticidal activities by transduction and transformation " successfully utilizes phage CP-54Ber to change the cry1A gene over to Bt Ku Sita subspecies (Bt subsp.kurstaki) 407 without (Applied and Environmental Microbiology in crystal mutant strain 407Cry-, 1992,58 (3): 840-849)." Transgenic Bacillus thuringiensis ", " Genetic improvement of Bt strains and development of novel biopesticides; Sansinenea E., Bacillus thuringiensis biotechnology " have reported respectively structure and the commercialization preparation of Bt engineering bacteria EG2348 and EG2424
With
Effective constituent (Phytoprotection, 1998,79:127-130; Heidelberg:Springer Science+Business media, 2012,215-228) document " Engineered Bacillus thuringiensis GO33A with broad insecticidal activity against lepidopteran and coleopteran pests " utilizes shuttle vectors pSTK to carry cry3Aa7 importing recipient bacterium G03 and has built engineering bacteria G033(Applied Microbiology and Biotechnology, 2006,72 (5): 924-930)." structure of Tribactur transposon integrative vector ", " utilizing integrative vector to build pesticidal engineering bacteria of Bacillus thuringinsis " utilize transposon Tn4430 to build engineering bacteria pBMB-F7, pBMB-R14, the pBMB-F7E(Journal of Agricultural Biotechnology, 2000,8 (4): 321-325; Acta Genetica Sinica, 2003,30 (8): 737-742)." Construction of new insecticidal Bacillus thuringiensis recombinant strains by using the sporulation non-dependent expression system of cryIIIA and a site specific recombination vector ", the carrier that dissociates of cry1Ac10 gene " utilize Tribactur transposon Tn4430 to build contain " philosophy utilization has been dissociated vector construction and has been contained Bt engineering bacteria (the Journal of biotechnology of cry1C and cry1Ac, 1996,48 (1-2): 81-96; The microorganism journal, 2000,40 (3): 264-269).
With regard to the coleopteran pest prevention and control, due to bacterial strain and genetic resources less, build engineering bacteria and obtain even more important with high yield, high virulence, the expansion of insecticidal spectrum and the prolong of long-lasting that obtains insecticidal proteins.document " spbA locus ensures the segregational stability of pTH1030, a novel type of Gram-positive replicon " utilize integrative vector pHT315ts to eliminate integrative vector after the cry3A gene integration is to the interior living plasmid of Ku Sita subspecies (Bt subsp.kurstaki) HD73, obtain lepidoptera pest small cabbage moth (plutella xylostella), prodenia litura (Spodoptera littoralis), coleopteran pest colorado potato bug (Leptinotarsa decemlineata), horseradish daikon leaf beetle (Phaedon cochleariae) has Bt engineering bacteria HD73-II (the Molecular Microbiology of insecticidal activity, 1992, 6 (1): 35-46), " Engineering Bacillus thuringiensis bioinsecticides with an indigenous site-specific recombination system " utilizes the carrier system that dissociates build engineering bacteria EG7673 and be applied to commercially produce, this engineering bacteria contains two coleopteran-toxic cry 3A genes and cry3B, and lepidopteran virulent gene cry1A, it is the Bt engineering bacteria of a high-efficiency broad spectrum, be also an at present unique employing dissociate system constructing to the activated engineering strain of coleopteran pest (Applied and Environmental Microbiology, 1996, 62 (12): 4367-4373)." Construction of a Bacillus thuringiensis engineered strain with high toxicity and broad pesticidal spectrum against coleopteran insects " built the activated engineering bacteria BIOT185 of coleopteran pest, this engineering bacteria contains insecticidal protein gene cry8Ca2 and cry8Ea1 (the Applied Microbiology and Biotechnology that anomala corpulenta and Holotrichia parallela is had virulence, 2010,87 (1): 243-249).
Aspect the activated Bt preparation of coleopteran pest, 8 kinds that have commercially produced are at present killed in Coleoptera Bt preparation, 4 kinds is engineering bacteria, shown the importance of engineering bacteria in killing Coleoptera Bt preparation, simultaneously, in killing the development from now on of Coleoptera Bt preparation, build the engineering bacteria that is easy to the genetic manipulation improvement and remain an important directions of its development.
Summary of the invention
The objective of the invention is to be to provide a kind of bacillus thuringiensis (Bacillus thuringiensis) engineering bacteria that kills coleopteran pest, called after Ace-38(preserving number of the present invention: CCTCC M2013259), this bacterium is delivered to Chinese Typical Representative culture collection center on June 14th, 2013 and carries out preservation, deposit number: CCTCC NO:M2013259, Classification And Nomenclature: bacillus thuringiensis Ace-38Bacillus thuringiensis Ace-38, address: Wuhan, China Wuhan University.This bacterium take Bt without crystal mutant strain BMB171 as recipient bacterium, utilize genetic engineering technique to build, containing this gene of insect-killing protein encoding gene cry3Aa8(numbers at GenBank: AY572010), without external source antibiotic marker gene, the various pests such as the willow herb to Coleoptera is chrysomelid, colorado potato bug, vegetables daikon leaf beetle, aulacophora femoralis have the efficiently poisoning effect, and to be a strain efficient to coleopteran pest and the bacillus thuringiensis engineering bacteria of wide spectrum.This bacterium leavening property is good, is that a strain has bacillus thuringiensis engineering bacteria exploitation, application prospect, environmentally safe.
Another object of the present invention is to be to provide a kind of preparation method who kills the bacillus thuringiensis engineering bacteria of coleopteran pest, easy to implement the method, easy and simple to handle, utilize genetic engineering technique from killing the bacillus thuringiensis YC-03(patent No. ZL01114288.X of Coleoptera) clone's insecticidal crystal protein encoding gene, the system of dissociating by improvement, structure is without the Bt engineering bacteria of external source antibiotic resistance gene, take two age the willow fleautiauxia armata and daikon leaf beetle as the examination worm, carry out virulence bioassay, through comparing virulence median lethal dosage LC
50, screen high virulence Bt engineering bacterial strain Ace-38.
A further object of the invention is to be to provide the application of a kind of bacillus thuringiensis engineering bacteria in preparation Bt sterilant, and this application of fermentation condition is reasonable, simple, fermentation period is short, the fermented liquid virulence is high.Utilize its production novel B t sterilant not only can the multiple coleopteran pest of high-efficiency prevention and control, guarantee the farm crop high and stable yields, and to ecological environment security, therefore have important practical significance and urgent.
In order to realize above-mentioned purpose, the present invention adopts following technical measures:
Bacillus thuringiensis (Bacillus thuringiensis, being called for short Bt) Ace-38 a kind ofly utilizes genetic engineering technique to build by this laboratory, and chrysomelid as for the examination insect take the coleopteran pest willow herb, through what virulence bioassay filtered out, coleopteran pest there is is the bacterial strain of high virulence.Effective object according to this bacterial strain, the first letter of getting anti-(Anti-), Coleoptera (coleopteran), engineering bacteria (engineering strain) forms its code Ace, again because of this bacterial strain be screen from the numerous engineering bacterias that build and be numbered a strain of 38, therefore with this bacillus thuringiensis engineering bacteria called after Ace-38.
A kind of preparation method of bacillus thuringiensis engineering bacteria, the steps include:
1, duplicate field ori1030 the dissociate structure of carrier: Bt-E.coil shuttle vectors pHT304(professor Sun Ming of Hua Zhong Agriculture University is so kind as to give) replaces carrier pBMB1205R (plasmid pBMB1205R is so kind as to give by professor Sun Ming of Hua Zhong Agriculture University) duplicate field ori44, builds the carrier pHT304/res that dissociates.Concrete operations are: pHT304 after Hind III enzyme cuts back to close, then carries out incomplete digestion and reclaims the 3657bp fragment with Sca I.Simultaneously pBMB1205R cuts and with the Klenow enzyme, fills also recovery of cohesive terminus afterwards with Nco I enzyme, then with Hind III enzyme, cuts back to close the 3688bp fragment.The 3657bp fragment is connected with the 3688bp fragment obtains the novel carrier pHT304/res that dissociates, size is 7345bp (seeing Fig. 1).
2, take cry3A-F (5 ' GGA TCC TTT GAA AAT ATA ACT ACC3 ') and cry3A-R (5 ' GGA TCC AAG CTT ACA GAG AAA TAC) as primer, utilize round pcr (polymerase chain reaction), from killing the bacillus thuringiensis bacterial strain YC-03(patent No. ZL01114288.X of Coleoptera) genomic dna amplification insecticidal crystal protein plasmagene cry3Aa8, its method is: 94 ℃ of sex change 5 minutes, then by follow procedure, carry out the amplification of 35 circulations: 94 ℃ 30 seconds, 52 ℃ 1 minute 30 seconds, 72 ℃ 2 minutes, extended 7 minutes at 72 ℃ finally.The PCR product is connected (Promega) with carrier pGEM-T-Easy after sepharose test kit (QIAGEN) reclaims, and conversion e. coli tg1 (available from clean overabundance of yang bio tech ltd), utilize blue, hickie selection positive colony, extract DNA from positive colony, cut DNA with restriction enzyme BamHI enzyme, sepharose reclaims test kit and reclaims the gene fragment of 2811bp.
3, the carrier pHT304/res that will dissociate cuts with the BamHI enzyme, the sepharose test kit reclaims the carrier DNA fragment that enzyme is cut, after dephosphorylation, be connected with the gene fragment of above-mentioned 2811bp, obtain recombinant plasmid pHT3Aa8/res(Fig. 2), recombinant plasmid pHT3Aa8/res transforms e. coli tg1, utilizes blue, hickie selection positive colony.Extract plasmid pHT3Aa8/res DNA from the TG1 transformant, and transform Bt without crystal mutant strain BMB171 (being derived from Bt subsp.kurstuki), with two anti-Screening of Medias clone of Amp/Erm and carry out PCR checking and the observation by light microscope of cry3Aa8 gene, after verified recombinant plasmid transformed success, with engineering bacteria called after BMB3Aa8/res.
4, the neat BMB3Aa8/res transformant of observation by light microscope picking growth, the electricity for preparing BMB3Aa8/res with SG solution turns competence, and helper plasmid pBMB1200(professor Sun Ming of Hua Zhong Agriculture University that will contain resolvase gene (tnp I) is so kind as to give) transform BMB3Aa8/res, with tetracyclin resistance plate screening positive transformant.Respectively dibbling is dull and stereotyped in the LB of the two anti-flat boards of Amp/Erm and nonreactive with positive transformant, and picking is not grown and the transformant of growing on the LB of nonreactive flat board on the two anti-flat boards of Amp/Erm, extract plasmid and carry out enzyme with Hind III and BamH I and cut checking.The errorless transformant of empirical tests is the upper amp of recombinant plasmid pHT3Aa8/res
rAnd erm
rGene is dissociated the transformant of elimination, called after BMB3Aa8nr.
5, the neat BMB3Aa8nr transformant of observation by light microscope picking growth, cultivate concussion in its liquid nutrient medium of LB in nonreactive 8 hours and go down to posterity 2 times, 8 hours, each generation time interval.Get a certain amount of final nutrient solution and dilute 10
4Doubly, the LB that gets 100ul coating nonreactive is dull and stereotyped, and dibbling is dull and stereotyped in the LB of tsiklomitsin flat board and nonreactive respectively to choose bacterium with toothpick after growing single bacterium colony.Cultivate that picking after 12 hours is grown on the LB flat board and the bacterium colony of not growing extracts genomic dna and also carries out the detection of tetracycline gene on the tsiklomitsin flat board.The transformant that PCR does not detect tetracycline gene is the engineering bacteria of eliminating all resistant genes.Filter out the engineering bacterial strain Ace-38 that growth is neat, the Cry3Aa8 expressing quantity is high, insecticidal activity is high after engineering bacteria SDS-PAGE and virulence bioassay.
6, willow fleautiauxia armata and daikon leaf beetle larva, for the examination worm, carry out virulence bioassay, through comparing virulence median lethal dosage LC
50, screen supper toxic strain Ace-38.
7, the main characteristic of Ace-38 is: the cellular form of Ace-38 is shaft-like, produces oval gemma.Cultivated 24 hours for 28 ℃ in extractum carnis-peptone Agar Plating, form the oyster white small colonies, edge-smoothing, cultivated 72 hours, forms discoid bacterium colony, and edge is irregular.The insecticidal crystal protein plasmagene of Ace-38 is cry3Aa8, and the insecticidal crystal protein plasmagene be expressed as non-gemma dependent form, i.e. the formation of insecticidal crystal protein matter does not rely on the formation of gemma; Produce square parasporal crystal, its insect-killing protein is Cry3Aa8, molecular weight is 74kD, and the nutrient solution in broth culture (substratum forms: 1% peptone, 0.3% beef extract, 0.5%NaCl, accent pH to 7.0) is to two willow fleautiauxia armatas in the age median lethal concentration(LC﹠-{50}) (LC of 72 hours
50) be 3.09 μ l/ml.
The application of a kind of bacillus thuringiensis engineering bacteria in preparation Bt sterilant, the steps include:
A, shake flask fermentation:
The purpose of shake flask fermentation is to select best fermentating formula, and optimization of fermentation conditions adopts different solid contents and different C/N ratios to carry out orthogonal test.Cultivate flat board more than 3 days and scrape culture and be dissolved in sterilized water and make spore suspension, process for 80 ℃ and be used for inoculation in 30 minutes.20/250ml loading amount, 30 ℃, 200 rev/mins shake-flask culture 72 hours, obtain the Optimal compositions of fermentation medium formula of strains A ce-38 in Table 1.
Table 1 fermention medium forms
The Optimal compositions of fermentation medium formula that B, employing screen, filling 20 milliliters of substratum in 250 milliliters of triangular flasks ferments, the substratum front pH8.1 that disappears, the rear pH that disappears is 7.0, inoculation is through 80 ℃ of slant strains of processing 30 minutes, 30 ℃, 200 rev/mins shake-flask culture 72 hours, 90% bacillus exfoliation, pH8.5, insect-killing protein concentration reaches 2.0985 ± 0.25138 mg/ml.
C, adopt the aforesaid method preparation the Ace-38 fermentation culture to two age the willow fleautiauxia armata insecticidal effect in Table 2.
Table 2 engineering bacteria Ace-38 is to willow fleautiauxia armata insecticidal activity assay (72 hours)
Compared with prior art, the present invention has the following advantages and effect:
1. bacillus thuringiensis engineering bacteria Ace-38 has the advantages that to kill coleopteran pest.Killing coleopteran pest Bt bacterial strain with wild-type compares, the growth of Ace-38 is synchronous, insecticidal crystal protein matter output is high, Cry3Aa8 protein yield in fermention medium reaches 2.0985 mg/ml, be not less than and commercially produce bacterial strain, good disinsection effect, can meet the requirement of commercially producing and applying.Domestic there is no at present killed Coleoptera bacillus thuringiensis commodity preparation, thereby can utilize Ace-38 to produce novel thuringiensis cladosporioides bacillus insecticide, make product diversification and the seriation of thuringiensis cladosporioides bacillus insecticide, enlarge the use range of thuringiensis cladosporioides bacillus insecticide, reduce the pesticide control expense, reduce the pollution of chemical pesticide to environment, increase crop yield.
2.Ace-38 do not carry external source antibiotic marker gene, its security and other wild bacillus thuringiensis are as good as, and be harmless to people, animal and animals and plants, free from environmental pollution, and good economy and ecological benefits are arranged.Thereby low from superior and unique insecticidal properties, production and use cost, the aspect such as safe, the free from environmental pollution and market requirement is considered to people and animals and plants, bacillus thuringiensis Ace has good popularizing application prospect.
3. utilize the thuringiensis cladosporioides bacillus insecticide of Ace-38 preparation to have very high virulence and obvious prevention effect to coleopteran pests such as the daikon leaf beetle of danger to vegetables, forest, potato etc., aulacophora femoralis, willow fleautiauxia armata, colorado potato bugs.The Ace-38 engineering bacteria of cultivating with fermention medium is to two willow fleautiauxia armatas in the age median lethal concentration(LC﹠-{50}) LC of 72 hours
50Be 1.13 microlitre/milliliters, i.e. fermented liquid dilution approximately still can make the mortality ratio of tested worm reach 50% after 887 times.
4. utilize fermentation condition provided by the invention and method to produce bacillus thuringiensis Ace-38 sterilant and have with short production cycle, fermented liquid virulence advantages of higher.
Description of drawings
Fig. 1 is the structure schematic diagram of a kind of carrier pHT304/res that dissociates.
Fig. 2 is the structure schematic diagram of a kind of recombinant plasmid pHT3Aa8/res.
Embodiment
Embodiment 1:
A kind of preparation method of bacillus thuringiensis engineering bacteria, the steps include:
1, the dissociate structure of carrier: the duplicate field ori1030 of Bt-E.coil shuttle vectors pHT304 is replaced carrier pBMB1205R duplicate field ori44, build the carrier pHT304/res that dissociates.Concrete operations are: pHT304 after Hind III enzyme cuts back to close, then carries out incomplete digestion and reclaims the 3657bp fragment with Sca I.Simultaneously pBMB1205R cuts and with the Klenow enzyme, fills also recovery of cohesive terminus afterwards with Nco I enzyme, then with Hind III enzyme, cuts back to close the 3688bp fragment.The 3657bp fragment is connected with the 3688bp fragment obtains the novel carrier pHT304/res that dissociates, size is 7345bp (seeing Fig. 1).
2, take cry3A-F (5 ' GGA TCC TTT GAA AAT ATA ACT ACC3 ') and cry3A-R (5 ' GGA TCC AAG CTT ACA GAG AAA TAC) as primer, utilize round pcr (polymerase chain reaction), from killing the bacillus thuringiensis bacterial strain YC-03(patent No. ZL01114288.X of Coleoptera) genomic dna amplification insecticidal crystal protein plasmagene cry3Aa8, its method is: 94 ℃ of sex change 5 minutes, then by follow procedure, carry out the amplification of 35 circulations: 94 ℃ 30 seconds, 52 ℃ 1 minute 30 seconds, 72 ℃ 2 minutes, extended 7 minutes at 72 ℃ finally.The PCR product is connected (Promega) with carrier pGEM-T-Easy after sepharose test kit (QIAGEN) reclaims, and conversion e. coli tg1 (available from clean overabundance of yang bio tech ltd), utilize blue, hickie selection positive colony, extract DNA from positive colony, cut DNA with restriction enzyme BamHI enzyme, sepharose reclaims test kit and reclaims the gene fragment of 2811bp.
3, the carrier pHT304/res that will dissociate cuts with the BamHI enzyme, the sepharose test kit reclaims the carrier DNA fragment that enzyme is cut, after dephosphorylation, be connected with the gene fragment of above-mentioned 2811bp, obtain recombinant plasmid pHT3Aa8/res(Fig. 2), recombinant plasmid pHT3Aa8/res transforms e. coli tg1, utilizes blue, hickie selection positive colony.Extract the DNA of plasmid pHT3Aa8/res from the TG1 transformant, and transform Bt without crystal mutant strain BMB171 (being derived from Bt subsp.kurstuki), with two anti-Screening of Medias clone of Amp/Erm and carry out PCR checking and the observation by light microscope of cry3Aa8 gene, after verified recombinant plasmid transformed success, with engineering bacteria called after BMB3Aa8/res.
4, the neat BMB3Aa8/res transformant of observation by light microscope picking growth, the electricity for preparing BMB3Aa8/res with SG solution turns competence, and helper plasmid pBMB1200(professor Sun Ming of Hua Zhong Agriculture University that will contain resolvase gene (tnp I) is so kind as to give) transform BMB3Aa8/res, with tetracyclin resistance plate screening positive transformant.The positive transformant difference dibbling that screens is dull and stereotyped in the LB of the two anti-flat boards of Amp/Erm and nonreactive, picking is not grown and the transformant of growing on the LB of nonreactive flat board on the two anti-flat boards of Amp/Erm, extract plasmid and carry out enzyme with Hind III and BamH I and cut checking.The errorless transformant of empirical tests is the upper amp of recombinant plasmid pHT3Aa8/res
rAnd erm
rGene is dissociated the transformant of elimination, called after BMB3Aa8nr.
5, the neat BMB3Aa8nr transformant of observation by light microscope picking growth, cultivate concussion in its liquid nutrient medium of LB in nonreactive 8 hours and go down to posterity 2 times, 8 hours, each generation time interval.Get a certain amount of final nutrient solution and dilute 10
4Doubly, the LB that gets 100ul coating nonreactive is dull and stereotyped, and dibbling is dull and stereotyped in the LB of tsiklomitsin flat board and nonreactive respectively to choose bacterium with toothpick after growing single bacterium colony.Cultivate that picking after 12 hours is grown on nonreactive LB flat board and the bacterium colony of not growing extracts genomic dna and also carries out the detection of tetracycline gene on the tsiklomitsin flat board.The transformant that PCR does not detect tetracycline gene is the engineering bacteria of eliminating all resistant genes.Filter out the engineering bacterial strain Ace-38 that growth is neat, the Cry3Aa8 expressing quantity is high, insecticidal activity is high after engineering bacteria SDS-PAGE and virulence bioassay.
6, willow fleautiauxia armata and daikon leaf beetle larva, for the examination worm, carry out virulence bioassay, through comparing virulence median lethal dosage LC
50, screen supper toxic strain Ace-38.
7, the main characteristic of Ace-38 is: the insecticidal crystal protein plasmagene is cry3Aa8, and the insecticidal crystal protein plasmagene be expressed as non-gemma dependent form, i.e. the formation of insecticidal crystal protein matter does not rely on the formation of gemma; Produce square parasporal crystal, its insect-killing protein is Cry3Aa8, molecular weight is 74kD, and the nutrient solution in broth culture (substratum forms: 1% peptone, 0.3% beef extract, 0.5%NaCl, accent pH to 7.0) is to two willow fleautiauxia armatas in the age median lethal concentration(LC﹠-{50}) (LC of 72 hours
50) be 3.09 μ l/ml.
Embodiment 2:
The application of a kind of bacillus thuringiensis engineering bacteria in preparation Bt sterilant, the steps include:
A, shake flask fermentation:
The purpose of shake flask fermentation is to select best fermentating formula, and optimization of fermentation conditions adopts different solid contents and different C/N ratios to carry out orthogonal test.Cultivate flat board more than 3 days and scrape culture and be dissolved in sterilized water and make spore suspension, process for 80 ℃ and be used for inoculation in 30 minutes.20/250ml loading amount, 30 ℃, 200 rev/mins shake-flask culture 72 hours, obtain the Optimal compositions of fermentation medium formula of strains A ce-38 in Table 1.
The Optimal compositions of fermentation medium formula that B, employing screen, filling 20 milliliters of substratum in 250 milliliters of triangular flasks ferments, the substratum front pH8.1 that disappears, the rear pH that disappears is 7.0, inoculation is through 80 ℃ of slant strains of processing 30 minutes, 30 ℃, 200 rev/mins shake-flask culture 72 hours, 90% bacillus exfoliation, pH8.5, insect-killing protein concentration reaches 2.0985 ± 0.25138 mg/ml.
C, adopt the aforesaid method preparation the Ace-38 fermentation culture to two age the willow fleautiauxia armata insecticidal effect in Table 2.
Claims (2)
1. a bacillus thuringiensis engineering bacteria that kills coleopteran pest, is characterized in that: bacillus thuringiensis engineering bacteria Bacillus thuringiensis, Ace-38, CCTCC M 2013259.
2. the application of a kind of bacillus thuringiensis engineering bacteria claimed in claim 1 in preparation Bt sterilant.
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