CN104611260A

CN104611260A - Bacillus thuringiensis LTS290 as well as insecticidal gene cry57Ab, expression protein and application of bacillus thuringiensis LTS290

Info

Publication number: CN104611260A
Application number: CN201510009827.3A
Authority: CN
Inventors: 高继国; 李海涛; 刘荣梅; 周国旺; 张�杰
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2015-01-09
Filing date: 2015-01-09
Publication date: 2015-05-13
Anticipated expiration: 2035-01-09
Also published as: CN104611260B

Abstract

The invention relates to bacillus thuringiensis LTS290 as well as an insecticidal gene cry57Ab, an expression protein and application of the bacillus thuringiensis LTS290, and belongs to the technical field of biological control. The preservation number of the bacillus thuringiensis LTS290 is CGMCC No.10232. The insecticidal protein separated from the bacterial strain LTS290 has the amino acid sequence as shown in SEQ ID NO.2 and a gene for encoding the insecticidal protein; the nucleotide sequence of the gene is preferred as SEQ ID NO.1. The gene has a certain killing force for lepidoptera pests, shows toxicity to related pests when being applied to the conversion of microorganisms and plants, and can be used for overcoming and delaying generation of drug resistance of pests to the engineering bacteria and transgenic plants.

Description

Tribactur LTS290, killing gene cry57Ab, expressing protein and application thereof

Technical field

The present invention relates to technical field of biological control, particularly the present invention relates to, to lepidopteran Agricultural pests, there is the Bt killing gene of high virulence and the protein by this coded by said gene.

Background technology

Tribactur (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 insect virulence, to higher animal and people's nontoxicity.It is that research is at present goed deep into the most, the most widely used microbial pesticide, has activity to 16 order 3000 various pests.Bt can form insecticidal crystal protein (Insecticidal CrystalProteins in the sporulation phase, ICPs), also delta-endotoxin (delta-endotoxin) is claimed, its shape, structure and size all with its virulence close relation [Schnepf.E, 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.].First ICPs gene of Bt has been cloned from Schnepf in 1981 etc., and in the aminoacid sequence of DNA base sequence and proteins encoded thereof that to have delivered it for 1985, (in April, 2014) totally 776, wherein cry gene 738 at present, cry pattern gene 289; Cyt gene 38, cyt pattern gene 11.Now, employing sprays chemical pesticide control means and 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.Tribactur insecticidal crystal protein is because of its good disinsection effect, safety, the advantage such as efficient and be widely used in pest control.Tribactur is except directly as except biological pesticide, and the routine transgenic anti-insect plants that beats the world for 1996 gets permission application in the U.S., and the gene that it uses is from Bt cry1Ac.In ensuing several years, turn the pest-resistant corn of cry1Ab gene, turn the pest-resistant potato of cry3Aa gene etc. and apart come out.In China, since starting the formal Insect Resistant Cotton promoted containing cry1Ac/cry1Ab gene from 1998, be widely 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.2013,27 national 1,800 ten thousand peasant plantings genetically modified crops of 1.752 hundred million hectares (4.33 hundred million acres), than sustainable growth in 2012 3%, namely 5,000,000 hectares (1,200 ten thousand acres).1800 ten thousand peasants benefit from genetically modified crops, and wherein 90% is the smallholder of scarcity of resources.The developing country of the first five plantation genetically modified crops is the nations of China and India in Asia, Hispanic Brazil and Argentina and African South Africa, plant the genetically modified crops of 8,270 ten thousand hectares altogether, account for 47% of global genetically modified crops cultivated area, and these five national populations account for 41% of the whole world 7,000,000,000 populations.The smallholder of China's 7,500,000 scarcity of resources has planted the Bt cotton of 4,200,000 hectares, and employing rate is 90%, the Bt cotton of average each household peasant planting 0.5 hectare.Genetically modified crops commercialization all brings economy and environment benefit to the peasant of industrialized country and developing country.Tribactur and Gene mining thereof have become important topic in Agricultural Sustainable Development.

Because the anti insect gene kind of current commercial insect-resistant transgenic crops is more single, there is insect sanctuary and reduce the risk risen with pest resistance to insecticide in spread plantation like this.Therefore that be constantly separated high virulence or the new incompatible risk avoiding pest resistance to insecticide to rise of genome is needed.Therefore, screening and separating clones Bt killing gene that is new, high virulence, killing gene resource can be enriched, for genetically modified crops and engineering strain provide new gene source, improve the insect resistant effect of Bt transgenic product, and the resistance risk of insect to Bt toxalbumin can be reduced, avoid new eco-catastrophe to come, there are important economy, society and ecological benefits.

Summary of the invention

The invention provides a kind of to lepidoptera pest small cabbage moth, beet armyworm, the Tribactur LTS290 of bollworm isoreactivity, and desinsection new gene gene cry57Ab1 and its crystal insecticidal proteins, to be applied to microbial and plant, make it to show the toxicity to relevant insect, and overcome, delay insect the resistance of engineering bacteria and transgenic plant is produced.

Bacillus thuringiensis bacterial strain LTS290, its deposit number is: CGMCC No.10232.

Bacillus thuringiensis bacterial strain LTS290 is killing lepidopteran small cabbage moth, beet armyworm, bollworm Agricultural pests and the application suppressed in Fusariumsp.

Insecticidal proteins Cry57Ab, its aminoacid sequence is as shown in SEQ ID NO 2.

Killing gene cry57Ab, encoding insecticidal proteins Cry57Ab.

Killing gene cry57Ab, its nucleotide sequence is as shown in SEQ ID NO 1.

A kind of expression vector, is characterized in that containing Cry57Ab1 gene.

Described expression vector is pEB-cry57Ab, and its skeleton carrier is pEB, and its structure as shown in Figure 6.

A kind of microbial transformant, is characterized in that containing cry57Ab gene.

Killing gene cry57Ab is killing the application in lepidopteran Agricultural pests.

Described application is transformed in plant by killing gene cry57Ab, makes expression of plants to the resistance of Agricultural pests, or the albumen that killing gene cry57Ab expresses is killed Agricultural pests as the effective constituent of biotic pesticide.

The present invention is separated and obtains a thuringiensis strain bacterial strain of bacillus LTS290 from soil near La Lin town, Wuchang City of Heilongjiang Province, its deposit number is CGMCC No.10232, this Biological Characteristics of Strain for can produce brood cell in growth cycle, and produce simultaneously and have poisoning lepidoptera pest small cabbage moth, beet armyworm, Pyrausta nubilalis (Hubern)., the parasporal crystal of the effect of bollworm and the effect of suppression Fusariumsp; The positive colony of a new gene is obtained from this bacterial strain, i.e. pEB-cry57Ab (see Fig. 6), sequencing analysis is carried out to it, BLAST is applied in NCBI website and applies the biosoftwares such as DNAMAN and analyze, analytical results proves that the gene of cloning is cry57Ab1 gene, this genes encoding frame is 2064bp by size, to encode 687 amino-acid residues, be 90.2% with the amino acid similarity degree of cry57Aa1, so this gene should belong to the new gene of the tertiary gradient, be cry57Ab1 through the definite designation of international delta-endotoxin NK.

Cry57Ab1 gene can, by the ordinary method microbial of biotechnology, plant, show relevant lepidopteran small cabbage moth, beet armyworm, Pyrausta nubilalis (Hubern)., the toxicity of bollworm insect.

Said gene is transformed bacterial strain, expresses the albumen obtained and can make biological pesticide for killing lepidoptera pest.Meanwhile, plant can be proceeded to and build insect-resistant transgenic plants, for the control of insect.

The Bt cry57Ab1 gene order of separating clone of the present invention and gene expression product thereof can to lepidopteran Agricultural pests beet armyworms, bollworm is without toxic action, and having certain poisoning power to small cabbage moth, Ostrinia furnacalis, cry57Ab1 can expand the insecticidal spectrum to lepidoptera pest.By being applied to microbial and plant, making 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 is named: Tribactur (Bacillus thuringiensis)

Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center

Preservation date: on December 23rd, 2014

Deposit number: CGMCC No.10232

Accompanying drawing explanation

The form of Fig. 1 optical microphotograph Microscopic observation bacterial strain LTS290 thalline,

Wherein: A is observations under opticmicroscope, B is bacterial strain LTS290 thalli morphology observations under Electronic Speculum,

Fig. 2 Bt LTS290 without the bacteriostatic action of fermented liquid to dry rot of potato 6 kinds of pathogenic Fusariumsps,

Fig. 3 Bt bacterial strain LTS290 to the restraining effect of oat Fusariumsp mycelia,

Wherein A:Bt bacterial strain LTS290 is to the restraining effect of oat Fusariumsp mycelia thalline; The hypha form of normal growth under B opticmicroscope; Repressed hypha form under C opticmicroscope

Fig. 4 cry57Ab full length gene PCR result,

Fig. 5 cry57Ab gene at the SDS-PAGE of expression in escherichia coli albumen,

Wherein: M: high-molecular-weight protein Marker 1:pEB empty carrier component; 2:Cry57 protein ingredient,

The structure iron of Fig. 6 recombinant vectors pEB-cry57Ab.

Embodiment

Embodiment 1, separation obtain Bacillus thuringiensis bacterial strain LTS290

What the laboratory worker of the applicant was separated from the soil of La Lin town, Wuchang City of Heilongjiang Province obtains a thuringiensis strain bacillus, brood cell's ecto-entad of Tribactur is followed successively by Exosporium, Ya Baoyi, cortex, brood cell's inwall, plasmalemma and protoplastis.The main component of its mediopellis is peptidoglycan, not containing the saccharan teichoic acid of vegetative cell, which holds dewatering state and the thermotolerance of brood cell, on the other hand, in sporulation process, a large amount of DPA-Ca huge legendary turtle compound can be produced, the biomacromolecule in brood cell is made to form heat-resisting gel, thermal treatment 20min at 80 DEG C, Tribactur brood cell also can not be dead and the brood cell of dormancy processes 15min under the sub-fatal temperature of 75 DEG C, activation effect is best, and not only its fast-germination short, also can improve the surviving rate (explaining sub-ox 1990) of brood cell.According to this characteristic, temperature screening (Knowles B H can be implemented, 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. the distribution [J] of Su Yun gold brood cell bar in China's eight wilderness area forest soil. and microorganism journal, 1994,30 (2) 117-121).

1, the separation of 1 bacterial strain

1) soil sample of getting packing joins in the large centrifuge tube of 50ml, to tapered tube taper place.

2) add aqua sterilisa to 15ml place, put into granulated glass sphere 5 ~ 10.

3) with vibrator, soil sample is smashed.

4) water-bath 80 DEG C is put into, 20 minutes.

5) get the EP pipe of 1.5ml, in each pipe, add 1ml aqua sterilisa, then from 50ml pipe, get 10 microlitre bacterium liquid join in EP pipe and mix.

6) from EP pipe, get 100 microlitres to be sprayed onto in 1/2LB substratum, smoothen.

7) put in 30 DEG C of incubators and cultivate 2 ~ 3 days.

8) microscopy is observed.

Crystal Observation

Opticmicroscope:

By born of the same parents' crystalline substance mixing drop on slide glass, smear evenly, dry fixing, carbolfuchsin dye liquor dyeing 3min, clear water rinses, 100x oil mirror carries out microscopy, carbolfuchsin dye liquor preparation method is see 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 1A.Form single bacterium colony after 1/2LB substratum cultivates 48h, observing bacterial strain QZL38 thalline under opticmicroscope is elongated rod shape, and brood cell is oval bar-shaped, and crystal is biconical.

Electronic Speculum microscopic examination:

SEM sample preparation: spore crystalline substance mixing drop is on sheet glass, dry, fixes through osmic acid, and after through alcohol serial dehydration, 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 1B.

Biological characteristis shows, to small cabbage moth, beet armyworm, Pyrausta nubilalis (Hubern)., the primary dcreening operation raw survey result of bollworm is corrected mortality is 65%, 46%, 40%, 20%.

By this bacterial strain preservation, its deposit number CGMCC No.10232.

The crystal habit that Bt bacterial strain LTS290 produces, as shown in arrow under opticmicroscope in Figure 1A, is from left to right followed successively by sphaerocrystal and brood cell; Arrow indication in electron microscopic picture (Figure 1B), is from left to right followed successively by sphaerocrystal and brood cell.Bt LTS-290 fermented liquid to the growth-inhibiting of 6 kinds of sickle-like bacteria mycelia as shown in Figure 2, by calculation result display fermented liquid, 91.38% and 91.76% are respectively to oat Fusariumsp and Williams Elder Twig Fusariumsp mycelial growth inhibition rate, and to other mycelial growth inhibition rates for examination Fusariumsp generally between 73% ~ 87%.As shown in Figure 3.Inhibiting rate (R) value of Bt bacterial strain LTS290 to oat Fusariumsp is approximately 0.3, (Fig. 3) as calculated.By observing oat Fusariumsp mycelia microstructure, as shown in Figure 3 B, the mycelia of normal growth is long, and linearly type growth, and repressed mycelia is short, and limpen, mycelia suppressed Be very effective Fig. 3 C.

Embodiment 2. obtains new gene

By genome sequencing, find cry gene high with cry57Aa1 similarity containing on bacterial strain BtLTS290 genome, design total length primer 57F (5'CG gGATCCand 57R (5'CCC GATGGGGACATGGTGGCCT3') aACGTTaTTTGATAAATAATTAAATAAAGTATCAG3'), the 5' end of primer adds restriction enzyme site BamH I and Sal I respectively, marks with underscore.

Rapid clon method is adopted to carry out separating clone to the new sip1A gene in this bacterial strain.

Use pfuDNA polysaccharase, carry out pcr amplification by following system.

Ultrapure water is mended to 50 μ L, mixes centrifugal.

Amplification cycles: 94 DEG C of sex change 1 minute, 54 DEG C of annealing 1 minute, 72 DEG C extend 1 minute, 25 circulations, last 72 DEG C of extensions 10 minutes.As shown in Figure 4.

2.2 connection scheme

Supply volume to 10 μ L with ultrapure water, fully mix, 16 DEG C connect a 4h or 4 DEG C connection and spend the night.

The total length primer of design cry57Ab genoid, amplification obtains full-length gene, by itself and carrier pEB (open carrier, there is preservation in this institute laboratory, can external disclosure granting) carrier connects, be transformed in competence JM109, through resistance screening, PCR identification and analysis, filter out the positive recombinant plasmid containing cry57Ab gene.Fig. 4 PCR qualification result.By purified fragments and carrier pEB, carrier pET21b connects transformation of E. coli JM109, obtains positive transformant.Carry out sequencing analysis to insertion segment, obtain sequence SEQ ID NO 1, its aminoacid sequence is for shown in SEQ ID NO 2.Obtain the correct object band of size by pcr amplification, object band is carried out purifying and checks order.Sequencing result shows, and the cry57 gene size in BtLTS290 bacterial strain is 2064bp, and 678 amino-acid residues of encoding are 90.2% with the amino acid similarity degree of cry57Aa1, is cry57Ab1 by the definite designation of the Bt delta-endotoxin genes Intemational Nomenclature council.

2.3 Transformation Protocol

2.3.1 intestinal bacteria transform

1. picking list bacterium colony shakes overnight incubation in 5ml LB;

2. be inoculated in LB liquid nutrient medium by 1% inoculum size, 37 DEG C, 230rpm cultivates 2-2.5hr, (OD ₆₀₀=0.5-0.6);

3.4 DEG C, the centrifugal 10min of 4,000rpm;

4. abandon supernatant, add the 0.1M CaCl of precooling ₂50ml suspension cell, is placed in more than 30min on ice;

5.4 DEG C, the centrifugal 10min of 4,000rpm, reclaims cell;

6. with the 0.1M CaCl of 2-4ml ice precooling ₂re-suspended cell, is distributed in 200 μ l/0.5mL centrifuge tubes, in 4 DEG C of preservations (can preserve a week).

7. get 200 μ l competent cells to be connected product with 5 μ L and fully to mix, ice bath 30min.

8.42 DEG C of heat shock 1.5min, ice bath 3min.

9. add 800 μ l LB substratum 37 DEG C and cultivate 45min.

10. get 200 μ l coated plates, add corresponding microbiotic, and IPTG, X-gal, 37 DEG C of cultivations.

Embodiment 3, genetic expression and determination of activity

3.1.1 extract plasmid DNA above-mentioned clone, proceed in recipient bacterium Rosetta (DE3), obtain expression strain.

After IPTG abduction delivering, carry out the detection of SDS-PAGE protein electrophoresis.

Abduction delivering process is as follows:

1) activated spawn (37 DEG C, 12hr);

2) 10% (37 DEG C, 2hr) in LB substratum is inoculated in;

3) inductor IPTG is added, 150rpm, 18-22 DEG C of low temperature induction 4-20h;

4) collected by centrifugation thalline, adds 10mM TrisCl (pH 8.0) and suspends;

5) broken thalline (ultrasonic disruption is complete);

Centrifugal 12,000rpm 10min 4 DEG C;

Collect supernatant and precipitate each 10-15 μ L, respectively electrophoresis detection.

Polyacrylamide gel configuration is as follows.

Loading: loading 10-15 μ l, electrophoresis: 130-150V constant voltage.

Dyeing and decolouring: take out gel after electrophoresis, after distilled water flushing, put into staining fluid, 60rpm vibration dyeing about 1hr, decolour in destainer about 2hr, and decolour to gel background transparent, in clear water, rinsing is clear to protein band.

By recombinant plasmid pEB-cry57Ab, (see Fig. 6) is transformed in E.coli Rosetta (DE3), IPTG abduction delivering, SDS-PAGE (12%) gel electrophoresis.Result shows, cry57Ab gene can be approximately 90kDa albumen by expression vector pEB at E. coli, and does not have special object band to produce (Fig. 5) through the pEB empty carrier proceeding to Rosetta (DE3) of IPTG induction.

The insecticidal activity assay of 3.2Bt bacterial strain LTS290 and cry57Ab gene coded protein

By Bt bacterial strain LTS290 and cry57Ab genetic expression albumen, be diluted with water to different concns, to the insecticidal activity of lepidoptera pest, concrete grammar is as follows, adopts feed hybrid system to carry out insecticidal bioactivity mensuration.To the expressing protein sample of different concns gradient be prepared and be sub-packed in the culture dish of sterilization, respectively with feed stirring and evenly mixing, selecting active newly hatched larvae is connected on feed, each process repetition 3 times, small cabbage moth, bollworm, Ostrinia furnacalis, each repetition of beet armyworm Agricultural pests are 30 examination worms.Negative control is that 10mmol/L Tris-Cl solution is done.The rearing conditions of examination worm is relative humidity is 70%-80%, temperature is cultivate in the illumination box of 27 DEG C, raises 48h " Invest, Then Investigate " borer population amount dead, alive, calculates mortality ratio.

This experiment has carried out preliminary insecticidal activity assay to the Cry57Ab albumen of expressing as shown in Table 1,100 μ g/mL are decided to be to the concentration of beet armyworm, bollworm and Ostrinia furnacalis, 50 μ g/mL are decided to be to small cabbage moth concentration, each process repetition 3 times, and with 10mmol/LTris-Cl solution as negative control.Primary dcreening operation result is as shown in table 1, Cry57Ab albumen to beet armyworm and bollworm insecticidal activity more weak, and have certain insecticidal activity to Ostrinia furnacalis and small cabbage moth.And existing disclosed Cry57Aa1 albumen is there are no the report of poisoning Agricultural pests effect.

Table 1Cry57Ab albumen is to the primary dcreening operation desinsection result of a few Species of Lepidopterous Insect Pests

The wherein average mortality of CK process: Tris-Cl beet armyworm 4.4%; Tris-Cl is to bollworm 5.6%; Tris-Cl is to Ostrinia furnacalis 13.3%; Tris-Cl is to small cabbage moth 15.6%.

Beneficial effect of the present invention: the Bt cry57Ab gene order of separating clone of the present invention and gene expression product thereof can produce virulence to lepidopteran, certain toxic action is had especially for small cabbage moth, Ostrinia furnacalis, the insecticidal spectrum to lepidoptera pest can be expanded, 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 transgenic plant.

Claims

1. Bacillus thuringiensis bacterial strain LTS290, its deposit number is: CGMCC No.10232.

2. Bacillus thuringiensis bacterial strain LTS290 is killing lepidopteran small cabbage moth, bollworm, Ostrinia furnacalis, beet armyworm Agricultural pests and is suppressing the application in Fusariumsp.

3. insecticidal proteins Cry57Ab, its aminoacid sequence is as shown in SEQ ID NO 2.

4. killing gene cry57Ab, encode insecticidal proteins Cry57Ab according to claim 3.

5. killing gene cry57Ab according to claim 4, its nucleotide sequence is as shown in SEQ ID NO 1.

6. an expression vector, is characterized in that containing Cry57Ab1 gene according to claim 5.

7. expression vector according to claim 6, be pEB-cry57Ab, its skeleton carrier is pEB, and its structure as shown in Figure 6.

8. a microbial transformant, is characterized in that containing cry57Ab gene according to claim 5.

9. the killing gene cry57Ab described in claim 4 or 5 is killing the application in lepidopteran Agricultural pests.

10. application according to claim 9 is transformed in plant by killing gene cry57Ab, makes expression of plants to the resistance of Agricultural pests, or the albumen that killing gene cry57Ab expresses is killed Agricultural pests as the effective constituent of biotic pesticide.