CN101984045A - The Cry8Na1 gene of bacillus thuringiensis, expression protein and application thereof - Google Patents
The Cry8Na1 gene of bacillus thuringiensis, expression protein and application thereof Download PDFInfo
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Abstract
The invention relates to the Cry8Na1 gene of Bacillus thuringiensis (Bt), expression protein and application thereof, which belongs to the field of biological control. The preservation number of Bt strains BTQ52-7 is CGMCC No.4188. The amino acid sequence of the insecticidal protein is shown in SEQ ID No.2. Preferred nucleotide sequences of genes encoding the above insecticidal protein is shown in SEQ ID No.1. The above genes with high virulence to coleoptera insects are applicable for transforming micro-organisms and plants. Thus the transformed micro-organisms and plants are resistant to relevant insects. The development of drug resistance of insects to engineering bacteria and transgenic plants can also be overcome or delayed.
Description
Technical field
The present invention relates to the biological control technical field, particularly further, the present invention relates to that coleopteran pest is had the Bt cry8Na1 gene of high virulence and by the protein of this coded by said gene.
Background technology
Bacillus thuringiensis (Bacillus thuringiensis, be called for short Bt) is a kind of widely distributed gram positive bacterium, is a kind of strong and to the avirulent entomopathogen of natural enemy, to higher animal and people's nontoxicity to the insect virulence.It is that research is at present goed deep into the most, the most widely used microbial pesticide, and 16 order 3000 various pests are had activity.Bt can form insecticidal crystal protein (Insecticidal CrystalProteins in the gemma formation phase, ICPs), also claim delta-endotoxin (delta-endotoxin), its shape, structure and size all have substantial connection [Schnepf.E with its virulence, Crickmore.N, Van Rie.J., Lereclus.D, Baum.J, Feitelson.J, Zeigler.D.R., Dean.D.H.Bacillus thuringiensis and its pesticidal crystalproteins.Microbiol.Mol.Biol.Rev, 1998,62 (3): 775-806.].Cloned first ICPs gene of Bt from Schnepf in 1981 etc., and its DNA base sequence and the aminoacid sequence of proteins encoded thereof have been delivered in 1985, at present (in July, 2010) found 530 kinds of kind of insecticidal crystal proteins, be divided into 67 class cry albumen and 2 class cyt albumen.Now, employing sprays the chemical pesticide control means no doubt can alleviate insect causing harm to farm crop, but chemical pesticide causes environmental pollution, for a long time, spray chemical insecticide in a large number, not only can strengthen the resistance of insect, beneficial insect and other ecosystem are wrecked, and serious environment pollution, improve production cost, destroy the eubiosis.The Tribactur insecticidal crystal protein is widely used in pest control because of its good disinsection effect, safety, advantage such as efficient.As the biological pesticide, got permission to use in the U.S. in 1996 by the routine transgenic anti-insect plants that beats the world except directly for Tribactur, and the gene that it uses is from Bt cry1Ac.In ensuing several years, change the pest-resistant corn of cry1Ab gene, change the appearances apart such as pest-resistant potato of cry3Aa gene.In China, since the formal popularization of beginning in 1998 contains the Insect Resistant Cotton of cry1Ac/cry1Ab gene, generally planted.In genetically modified crops business-like first 12 years (1996-2007), owing to can obtain continual and steady income, peasant planting genetically modified crops amount increases year by year.2009,1.34 hundred million hectares genetically modified crops were planted by 25 countries, have increased by 7% than 2008.The U.S. remains maximum biotechnology crop-planting state, and cultivated area is 6,400 ten thousand hectares, China, 3,700,000 hectares.First 12 years, the genetically modified crops commercialization had all brought economy and environmental benefit for the peasant of industrialized country and developing country.Tribactur and gene thereof are excavated has become important topic in the Sustainable development agricultural.
Tribactur cry8 genoid has special insecticidal activity to multiple coleopteran pests such as Scarabaeidae, Culculionidae, Chrysomelidaes.The cry8 genoid is made up of 1160-1210 amino acid, and molecular weight is between 128-137kDa.1992, people such as Ohba filter out new bacterial strain (B.thuringiensis subsp.japonensis BuiBui) (the Ohba M. that the chafer larva is had special insecticidal activity first from the Bt bacterial strain, Iwahana H., Asano S., Sato R.and Hori H..A unique isolate of Bacillus thuringiensis serovar japonensis with a high larvicidal activity specific for scarabaeidbeetles, Letters in Applied Microbiology 1992,14:54～57), this bacterial strain is mainly to the red copper rutelian, the insect of Rutilidaes such as Japanese beetle has toxic action.1994, the toxicity of proof such as Hori bacterial strain is from the protein of the 130kDa in the bacterium, Sato etc. have therefrom cloned a kind of new killing gene cry8Ca1 (Sato R., Takeuchi K., Ogiwara K., Masayosi Minami, Kaji Y., Suzuki N., Hor H.i, Asan S.O, Ohba M.and Iwahana H..Cloning, heterologous expression, and localization of a novel crystal protein gene from Bacillus thuringiensisserovar japonensis strain Buibui toxic to Scarabaeid insects.Current Microbiology 1994,28:11～15), this gene has been used for the exploitation of biotic pesticide.Because the cry8 genoid has very big potential using value, most of genes are all applied for a patent, and isolating cry8Aa1 of U.S. Mycogen company and cry8Ba1 have tangible insecticidal activity to the various pests of Scarabaeidae; Du pont company clone's cry8Bb1, cry8Bc1 gene pairs west corn root leaf A (Westerncorn rootworm) have remarkable insecticidal effect (Abad, Andre, R., Duck Nicholas, B., Feng, Xiang, FlannaganRonald, D., Kahn, Theodore, W., Sims, Lynne, E..Genes encoding novel proteins with pesticidalactivity against Coleopterans.2002, WO 02/34774A2), and be used for the exploitation of transgenic insect-resistant corn; Clones' such as Japan Asano cry8Da1, cry8Da2 and cry8Da3 are also at Japanese publication patent (Asano S., Yamashita C., Iizuka T., Takeuchi K., Yamanaka S., Cerf D.and T.Yamamoto..A strain of Bacillus thuringiensissubsp.Galleriae containing a novel cry8 gene highly toxic to Anomala cuprea (Coleoptera:Scarabaeidae) .Biological Control 2003,28:191～196).At present; the screening successively in recent years of China Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie and Agricultural University Of Hebei obtain many strains to yellowish-brown rutelian (Anomala exoleta) and anomala corpulenta (A.corpulenta) larva have special insecticidal activity the Bt bacterial strain (Feng Shuliang etc. a strain has the new strain isolated of Bacillus thuringiensis of insecticidal activity to cockchafer subclass larva. Chinese biological is prevented and treated; 2000,16 (2): 74～78).2006; Plant Protection institute, Chinese Academy of Agricultral Sciences has found Holotrichia parallela is had the first strain Bt bacterial strain Bt185 of insecticidal activity; they have cloned respectively the cry8Ea1 that Holotrichia parallela (Holotrichia parallela) and big black gill cockchafer (Holotrichia oblita) is had special insecticidal activity subsequently; cry8Fa1 (Yu H.; Zhang J.; Huang D.; Gao J.and Song F..Characterization ofBacillus thuringiensis strain Bt185 toxic to the Asian cockchafer:Holotrichia parallela.CurrentMicrobiology 2006,53:13～17.Shu C., Yu H., Wang R., Fen S., Su X., Huang D., Zhang J., Song F..Characterization of two novel cry8 genes from Bacillus thuringiensis strain BT185.Current Microbiology 2009,58 (4): 389-92.) with cry8Ga1 gene (Shu C.L., Yan G.X., Wang R.Y., ZhangJ., Feng S.L., Huang D.F., Song F.P..Characterization of first cry8 gene specific to Melolonthidaepests:Holotrichia oblita and Holotrichia parallela.Applied Microbiology and Biotechnology.2009.3.17).At present, whole world clone's cry8 genoid is existing 33 kinds.
Because the anti insect gene kind of present commercial transgenic pest-resistant crop is more single, so the big area popularizing planting exists insect sanctuary to reduce the risk that rises with pest resistance to insecticide.Therefore need constantly to separate the incompatible risk of avoiding pest resistance to insecticide to rise of genome high virulence or new.Therefore, screening and separating clone Bt killing gene new, high virulence, can enrich the killing gene resource, for genetically modified crops and engineering strain provide new gene source, improve the pest-resistant effect of Bt transgenic product, and can reduce the resistance risk of insect, avoid new eco-catastrophe to come, have important economy, society and ecological benefits the Bt toxalbumin.
Summary of the invention
The invention provides a kind of to the high virulence Tribactur of Scarabeidae insect Holotrichia parallela Q52-7, and the new gene cry8Na1 of desinsection gene and its crystal insecticidal proteins, to be applied to transform microorganism and plant, make it to show toxicity, and overcome, delay the resistance generation of insect engineering bacteria and transgenic plant to relevant insect.
Bacillus thuringiensis bacterial strain BTQ52-7, its deposit number is: CGMCC No.4188.
The application of Bacillus thuringiensis bacterial strain BTQ52-7 in killing coleopteran pest.
A kind of cry8Na1 gene that obtains that separates from Bacillus thuringiensis bacterial strain BTQ52-7, its nucleotide sequence is shown in SEQ IDNO1.
The encode insecticidal proteins of this gene, its aminoacid sequence is shown in SEQ ID NO 2.
A kind of expression vector is characterized in that containing said gene.
Described expression vector is pEB 8, and its skeleton carrier is pEB, and its structure as shown in Figure 6.
A kind of microbial transformant is characterized in that containing said gene.
The application of said gene in anti-coleopteran pest.
Described application is with the albumen of this genetic expression effective constituent as biotic pesticide.
Described application is that this gene is transferred to microorganism or plant, makes it to express the toxic protein to Coleoptera.
The present invention separates the soil near the flexible sight of Liaoning numerous mountains and obtains a bacillus thuringiensis strain bacterial strain BTQ52-7, its deposit number is CGMCC No.4188, this bacterial strain biological characteristics is for producing the brood cell in growth cycle, and produce the parasporal crystal that the effect of poisoning coleopteran pest is arranged simultaneously, it has the very strong ability of killing to Holotrichia parallela; Obtain the positive colony of a new gene from this bacterial strain, promptly the pEB8 (see figure 6) is carried out sequencing analysis to it, finds to contain 3519 bases among the clone pEB 8, sees SEQ ID NO1, and 1174 amino acid of encoding are seen SEQ ID NO2.Compare with the gene of having delivered, the highest with the cry8Ca similarity, similarity is 70%, is a new gene; Extract plasmid from above-mentioned positive colony, change recipient bacterium over to, obtain explaining bacterial strain, measure the proteic activity of expression of gene, gene cry8Na1 expressed proteins gives birth to the primary dcreening operation of Holotrichia parallela that to survey the result be 100% to see Table 1 for corrected mortality, there is certain activity to see Table 2 to the holotrichia oblita larva, coleopteran pest elm fleautiauxia armata larva and anomala corpulenta larva do not had insecticidal activity see Table 3, table 4.
The cry8Na1 gene can transform microorganism, plant by the ordinary method of biotechnology, shows the toxicity to relevant coleopteran pest.
Said gene is transformed bacterial strain, and the albumen that expression obtains can be made biological pesticide and be used to kill coleopteran pest.Simultaneously, can change plant over to and make up insect-resistant transgenic plants, be used for the control of insect.
The Bt cry8Na1 gene order and the gene expression product thereof of separating clone of the present invention can produce strong virus force to coleopteran pest, particularly Holotrichia parallela are had high reactivity, are good Biocidal genes, and very application prospects is arranged.By the combination of gene expression products such as this cry8Na1 gene and cry1Ab, cry1Ba, cry2Ab, can enlarge insecticidal spectrum to lepidopteran, coleopteran pest.By being applied to transform microorganism and plant, make them show toxicity to relevant insect, can overcome or delay insect to engineering bacteria and the drug-fast generation of transgenic plant.
Bacterial strain preservation information:
Bacterium classification name: bacillus thuringiensis (Bacillus thuringiensis)
Preservation mechanism: China Committee for Culture Collection of Microorganisms common micro-organisms center
Preservation date: on 09 20th, 2010
Deposit number: CGMCC No.4188
Description of drawings
Fig. 1 opticmicroscope is observed the form of bacterial strain BTQ52-7 thalline down,
Bacterial strain BTQ52-7 thalli morphology under Fig. 2 Electronic Speculum
Fig. 3 contains the genome PCR evaluation of goal gene,
Wherein 1: negative control 2: bacterial strain BTQ52-7 S5un8/S3un8 pcr amplification product
M：Marker(100、200、300、400、500、600、700、800、900、1000、1500bp
Fig. 4 cry8Na1 full length gene PCR result
1: negative control M:Marker (300,500,800,1000,1500,2000,3000,4000,5000,6000,8000,10000bp) 2:cry8 genoid total length amplified production
Fig. 5 cry8Na1 gene is at the proteic SDS-PAGE of expression in escherichia coli
M: (200,116,97,66,44kDa) the crystallin 2:pEB8 of 1:BTQ52-7 bacterial strain is at the albumen 3:pEB of expression in escherichia coli empty carrier for albumen high molecular marker
Fig. 6 pEB8 structural representation,
Fig. 7 homology analysis figure.
Embodiment
Near the applicant's laboratory worker isolating bacillus thuringiensis strain that obtains the soil flexible sight of Liaoning numerous mountains, the gemma ecto-entad of bacillus thuringiensis is followed successively by exine, gemma clothing, cortex, gemma inwall, plasmalemma and protoplastis.The main component of its mediopellis is a peptidoglycan, the saccharan teichoic acid that does not contain vegetative cell, it is keeping the dewatering state and the thermotolerance of gemma, on the other hand, in the gemma forming process, can produce a large amount of DPA-Ca huge legendary turtle compounds, make the biomacromolecule in the gemma form heat-resisting gel, at 80 ℃ of following thermal treatment 20min, the bacillus thuringiensis gemma can death yet and the gemma of dormancy under 75 ℃ inferior fatal temperature, handle 15min, activation effect is best, not only urgees its fast-germination, also can improve the surviving rate (explaining sub-ox 1990) of gemma.According to this characteristic, can implement temperature screening (Knowles B H, EllarD J.Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillusthuringiensis d-endotoxins with different specificity[J] .Biochimica et biophysicaacta, 1987,924:509-518.; Dai Lianyun, Wang Xue engage etc. the distribution [J] of Su Yun gold gemma bar in eight wilderness area forest soil of China. and microorganism journal, 1994,30 (2) 117-121).
1, the separation of 1 bacterial strain
1) soil sample of getting packing joins in the big centrifuge tube of 50ml, to tapered tube taper place.
2) add aqua sterilisa to the 15ml place, put into 5～10 of granulated glass spherees.
3) with vibrator soil sample is smashed.
4) put into 80 ℃ of water-baths, 20 minutes.
5) EP that gets 1.5ml manages, and adds the 1ml aqua sterilisa in each pipe, gets 10 microlitre bacterium liquid again and join mixing in the EP pipe from the 50ml pipe.
6) from the EP pipe, get 100 microlitres and be sprayed onto in the 1/2LB substratum, smoothen.
7) put in 30 ℃ of incubators and cultivated 2～3 days.
8) microscopy is observed.
Crystal is observed
Opticmicroscope:
Born of the same parents' crystalline substance is mixed drop on slide glass, smear evenly, oven dry is fixing, carbolfuchsin dye liquor dyeing 3min, flushing with clean water, 100x oil mirror carries out microscopy, the carbolfuchsin dye liquor preparation method referring to document (Baroy F, Lecadet M M, Deleluse A.Cloning and sequencing of three new putative toxin genes from Clostridiumbifermentans[J] .Gene, 1998,211:293-295).See shown in Figure 1.Form single bacterium colony after cultivating 48h on the 1/2LB substratum, observing bacterial strain BTQ52-7 thalline under the opticmicroscope is elongated rod shape, and gemma is oval bar-shaped, and crystal is spherical.
The Electronic Speculum microscopic examination:
The scanning electron microscope sample preparation: the brilliant drop that mixes of spore is on sheet glass, and drying is fixed through osmic acid, and after the dehydration of alcohol gradient, critical point drying, ion sputtering metal spraying (2nm is thick), New Bio-TEM H-7500 scanning electron microscopic observation is taken pictures.As shown in Figure 2.
Biology is measured and to be shown, the primary dcreening operation of Holotrichia parallela is given birth to survey the result for corrected mortality is 100% to see Table 1, has certain biological activity to see Table 2 to the holotrichia oblita larva.Coleopteran pest elm fleautiauxia armata, anomala corpulenta there is not insecticidal activity.
2.1 utilize cry8 genoid universal primer to detect bacterial strain BTQ52-7, primer is as follows
Amplification cycles: 94 ℃ of sex change 1 minute, 56 ℃ of annealing 1 minute, 72 ℃ were extended 4 minutes, 25 circulations, last 72 ℃ were extended 10 minutes.
The result carries out nucleic acid, amino acid after the order-checking and relatively finds it is new gene as shown in Figure 3.
2, the clone of cry8 genoid in the 1BTQ52-7 bacterial strain
Adopt rapid clon method that the new cry gene in this bacterial strain is carried out separating clone.
With reference to 5 ' end and 3 ' terminal sequence of the gene coding region of the cry8 class of announcing among the GenBank, designed a pair of primer of the total length cry8 genoid that increases, primer sequence is as follows:
cry8N5：5′-3′：TTACTCTTCTTCTAACACGAGTTCTACAC
cry8N3：5′-3′：ATGAGTCCGAATAATCAAAACGAAT
Plasmid DNA with bacterial strain BTQ52-7 is a template, total length primer with above-mentioned design carries out the amplification of PCR total length, and having amplified total length is 3.5kb cry8 full-length gene, (see figure 4), different with the collection of illustrative plates of known cry8 genoid, show and may contain new cry8 killing gene in this bacterial strain.
Recovery is connected with expression vector pEB through DNA, obtains recombinant plasmid called after pEB8, and its structure iron is shown in 6.
Use the pfuDNA polysaccharase, carry out pcr amplification with following system.
10×PCR?buffer 5μL
dNTP(10mM) 1μL
Primer is to (10mM) 1 μ L/
Template 1uL
PfuDNA polysaccharase (5U/ μ L) 0.5 μ L
Ultrapure water is mended to 50 μ L, and mixing is centrifugal.
Amplification cycles: 94 ℃ of sex change 1 minute, 54 ℃ of annealing 1 minute, 72 ℃ were extended 4 minutes, 25 circulations, last 72 ℃ were extended 10 minutes.
The purifying fragment is connected transformed into escherichia coli JM109 with carrier pEB, obtains positive transformant.Transformant is carried out enzyme cut, the result obtains about 3.5kb band, shows the successfully insertion of the new gene order of cry8 class.Carry out sequencing analysis to inserting segment, obtain sequence SEQ ID NO 1, sequence total length 3522bp, the albumen that 1173 amino acid of encoding are formed.After measured, its aminoacid sequence is shown in the SEQ ID NO 2.
2.2 connectivity scenario
Carrier 0.1-0.2 μ g
Purpose sheet segment DNA 0.5-1.0 μ g
5×Ligation?Buffer 2μL
T4?DNA?Ligase 1μL
Supply volume to 10 μ L with ultrapure water, abundant mixing, 16 ℃ of connection 4h or 4 ℃ of connections are spent the night.
2.3 conversion scheme
1. picking list bacterium colony is in 5ml LB concussion overnight incubation;
2. be inoculated in the LB liquid nutrient medium by 1% inoculum size, 37 ℃, 230rpm cultivates 2-2.5hr, (OD
600=0.5-0.6);
3.4 ℃, 4, the centrifugal 10min of 000rpm;
4. abandon supernatant, add the 0.1M CaCl of precooling
2The 50ml suspension cell places on ice more than the 30min;
5.4 ℃, 4, the centrifugal 10min of 000rpm reclaims cell;
6. ice the 0.1M CaCl of precooling with 2-4ml
2Re-suspended cell is distributed in the 200 μ l/0.5mL centrifuge tubes, in 4 ℃ of preservations (can preserve a week).
7. get 200 μ l competent cells and be connected the abundant mixing of product, ice bath 30min with 5 μ L.
8.42 ℃ heat shock 1.5min, ice bath 3min.
9. add 800 μ l LB substratum and cultivate 45min for 37 ℃.
10. get 200 μ l coated plates, add corresponding microbiotic, and IPTG, X-gal, 37 ℃ of cultivations.
2.4 homology analysis
SEQ ID NO2 and cry8 genoid genetic distance and homology analysis: show that SEQ genetic distance and cry8Ca are 0.297 recently, amino acid identity is 70.3%, data are as follows, according to bacillus thuringiensis criteria for classification (Crickmore N, D RZeigler, J Feitelson, et al.1998.Revision of the nomenclature for the Bacillus thuringiensispesticidal crystal proteins.Microbiol.Mol Biol Rev.62:807～813) be new gene, by the called after cry8Na1 of Bt insecticidal crystal protein NK gene.Property of protein is as follows:
Protein length Protein Length=1173,
Protein molecular weight is MW=132697.5,
Iso-electric point pI=4.74.
cry8Aa.txt 0
cry8Ab.txt 0.233?0
cry8Ba.txt 0.334?0.345?0
cry8Bb.txt 0.311?0.336?0.175?0
cry8Bc.txt 0.313?0.332?0.175?0.089?0
cry8Ca.txt 0.475?0.485?0.476?0.464?0.464?0
cry8Da.txt 0.356?0.393?0.456?0.441?0.442?0.353?0
cry8Ea.txt 0.340?0.323?0.281?0.260?0.266?0.459?0.432?0
cry8Fa.txt 0.349?0.338?0.354?0.349?0.353?0.481?0.452?0.307?0
cry8Ga.txt 0.435?0.454?0.436?0.448?0.452?0.346?0.373?0.447?0.436?0
cry8Ka.txt 0.372?0.374?0.376?0.363?0.363?0.446?0.461?0.371?0.369?0.388?0
cry8Ma.txt 0.464?0.477?0.507?0.482?0.490?0.513?0.490?0.491?0.486?0.514?0.498?0
SEQ.txt 0.453?0.456?0.467?0.449?0.450?0.297?0.371?0.453?0.449?0.320?0.425?0.509?0
Homology?matrix?of?13?sequences
cry8Aa.txt 100％
cry8Ab.txt 76.7％?100％
cry8Ba.txt 66.6％?65.5％?100％
cry8Bb.txt 68.9％?66.4％?82.5％?100％
cry8Bc.txt 68.7％?66.8％?82.5％?91.1％?100％
cry8Ca.txt 52.5％?51.5％?52.4％?53.6％?53.6％?100％
cry8Da.txt 64.4％?60.7％?54.4％?55.9％?55.8％?64.7％?100％
cry8Ea.txt 66.0％?67.7％?71.9％?74.0％?73.4％?54.1％?56.8％?100％
cry8Fa.txt 65.1％?66.2％?64.6％?65.1％?64.7％?51.9％?54.8％?69.3％?100％
cry8Ga.txt 56.5％?54.6％?56.4％?55.2％?54.8％?65.4％?62.7％?55.3％?56.4％?100％
cry8Ka.txt 62.8％?62.6％?62.4％?63.7％?63.7％?55.4％?53.9％?62.9％?63.1％?61.2％?100％
cry8Ma.txt 53.6％?52.3％?49.3％?51.8％?51.0％?48.7％?51.0％?50.9％?51.4％?48.6％?50.2％?100％
SEQ.txt 54.7％?54.4％?53.3％?55.1％?55.0％?70.3％?62.9％?54.7％?55.1％?68.0％?57.5?49.1％?100％
3.1 extract plasmid DNA above-mentioned clone, change among the recipient bacterium Rosetta (DE3), obtain expression strain.
Behind the IPTG abduction delivering, carry out the SDS-PAGE protein electrophoresis and detect.
The abduction delivering process is as follows:
1) activated spawn (37 ℃, 12hr);
2) 10% be inoculated in (37 ℃, 2hr) in the LB substratum;
3) add inductor IPTG, 150rpm, 18-22 ℃ of low temperature induction 4-20h;
4) centrifugal collection thalline adds 10mM TrisCl (pH 8.0) and suspends;
5) broken thalline (ultrasonic disruption is complete);
Centrifugal 12,4 ℃ of 000rpm 10min;
Collect supernatant and precipitate each 10-15 μ L, respectively electrophoresis detection.
The polyacrylamide gel configuration is as follows.
Separation gel 8% (ml) spacer gel 5% (ml)
Distilled?water 4.6 2.7
30％Degassed?Acrylamide 2.7 0.67
1.5M?Tris(pH8.8) 2.5
1.0M?Tris(pH6.8) 0.5
10％SDS 0.1 0.04
10％APS 0.1 0.04
TEMED 0.006 0.004
Last sample: go up sample 10-15 μ l, electrophoresis: 130-150V constant voltage.
Dyeing and decolouring: take out gel behind the electrophoresis, behind distilled water flushing, put into staining fluid, about 60rpm vibration dyeing 1hr, about decolouring 2hr, decolour to the gel background transparent in the destainer, rinsing is clear to protein band in the clear water.
Detected result shows that the expressed proteins molecular weight is about 130kD, the results are shown in Figure 5.
3.2 the insecticidal activity assay of gene coded protein
With cry8Na1 genetic expression albumen, be diluted with water to different concns, measure insecticidal activity to coleopteran pest.
The biological activity determination of Holotrichia parallela (H.parallela), black greatly gill cockchafer (H.oblita) anomala corpulenta (A.corpulenta):
According to the contained protein content of Bt dry powder, be suspended in the sterilized water with a certain amount of, above-mentioned suspension is diluted according to 2 times of differential gradient concentrations of geometric ratio, join mixing in the sterilization fine earth of even thickness potato silk, as for examination worm kind, each processing connects 30 of worms with 5-7 age in days Holotrichia parallela and big black gill cockchafer larva, triplicate,, infect to raise and checked dead borer population, calculation correction mortality ratio in 7 days, 14 days as blank with the processing that adds clear water.
To the indoor insecticidal activity assay of elm fleautiauxia armata (P.aenescens):
The elm blade that clip is suitable cleans, and dries, and the Bt bacterium liquid good with dilution in advance soaks into 10s, takes out, and dries naturally, puts into culture dish, is used to preserve moisture at the shop, bottom of culture dish one deck wetted filter paper in advance.Handle in contrast with clear water.Insert 30 of the elm fleautiauxia armata larvas (chocolate) in 2～3 ages in each culture dish respectively, each handles triplicate, builds with culture dish, and room temperature is placed (25～28 ℃), 96h investigation larva death condition.
Table 1Cry8Na1 albumen is surveyed the result to Holotrichia parallela
Table 2Cry8Na1 albumen is surveyed the result to big black gill cockchafer
Table 3Cry8Na1 albumen is surveyed the result to the elm fleautiauxia armata
Table 4Cry8Na1 albumen is surveyed the result to anomala corpulenta
Beneficial effect of the present invention: Bt cry8Na1 gene order and gene expression product thereof that the present invention separates the clone can produce strong virus force to coleopteran pest, by the combination of the gene expression products such as cry8Na1 gene and cry8Na1, cry8Na1, cry8Na1, can enlarge the insecticidal spectrum to Lepidoptera, coleopteran pest. By being applied to microbial and plant, make them show toxicity to relevant insect, can overcome or delay insect to engineering bacteria and the drug-fast generation of genetically modified plants.
Sequence table
Claims (10)
1. Bacillus thuringiensis bacterial strain BTQ52-7, its deposit number is CGMCC No.4188.
2. the application of the described Bacillus thuringiensis bacterial strain BTQ52-7 of claim 1 in killing coleopteran pest.
3. an Accessory Right requires to separate the cry8Na1 gene that obtains among the 1 described Bacillus thuringiensis bacterial strain BTQ52-7, and its nucleotide sequence is shown in SEQ ID NO1.
4. the insecticidal proteins of the described cry8Na1 genes encoding of claim 3, its aminoacid sequence is shown in SEQ ID NO 2.
5. an expression vector is characterized in that containing the described cry8 genoid of claim 3.
6. the described expression vector of claim 5 is pEB 8, and its skeleton carrier is pEB.
7. a microbial transformant is characterized in that containing the described cry8Na1 gene of claim 3.
8. the application of the described cry8Na1 gene of claim 3 in anti-coleopteran pest.
9. the described application of claim 8 is with the albumen of the described cry8Na1 genetic expression of claim 3 effective constituent as biotic pesticide.
10. the described application of claim 8 is that the described cry8Na1 gene of claim 3 is transferred to microorganism or plant, makes it to express the toxic protein to Coleoptera.
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| CN102363630A (en) * | 2011-11-09 | 2012-02-29 | 四川农业大学 | Pesticide Bt protein Cry8Pa1 and coding gene and use thereof |
| CN103570811A (en) * | 2013-11-19 | 2014-02-12 | 中国农业科学院植物保护研究所 | Bacillus thuringiensis gene cry1Ah3 and application thereof |
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| CN101503666A (en) * | 2009-03-05 | 2009-08-12 | 四川农业大学 | Novel strain of Bacillus thuringiensis bacterial strain and use thereof |
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| CN102363630A (en) * | 2011-11-09 | 2012-02-29 | 四川农业大学 | Pesticide Bt protein Cry8Pa1 and coding gene and use thereof |
| CN103570811A (en) * | 2013-11-19 | 2014-02-12 | 中国农业科学院植物保护研究所 | Bacillus thuringiensis gene cry1Ah3 and application thereof |
| CN103570811B (en) * | 2013-11-19 | 2015-06-03 | 中国农业科学院植物保护研究所 | Bacillus thuringiensis gene cry1Ah3 and application thereof |
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| CN103667123B (en) * | 2013-11-28 | 2016-09-07 | 湖南省植物保护研究所 | Highly active bacillus thuringiensis bacterial strain HNCS-93 and application thereof |
| CN104388349A (en) * | 2014-11-21 | 2015-03-04 | 东北农业大学 | Insecticidal gene sip1A secreted by bacillus thuringiensis as well as expression protein and application thereof |
| CN104388349B (en) * | 2014-11-21 | 2017-12-29 | 东北农业大学 | Thuringiensis secretes killing gene sip1A, expressing protein and its application |
| CN104673706A (en) * | 2014-12-20 | 2015-06-03 | 东北农业大学 | Bacillus thuringiensis FH21, insecticide gene, expression protein and application thereof |
| CN117229377A (en) * | 2023-11-16 | 2023-12-15 | 中国农业大学 | Insecticidal protein and application thereof in gill-prevention and control of scarab beetles |
| CN117229377B (en) * | 2023-11-16 | 2024-02-27 | 中国农业大学 | Insecticidal protein and application thereof in gill-prevention and control of scarab beetles |
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