CN104845914B

CN104845914B - Bacillus thuringiensis bacterial strain, expressing protein and its application

Info

Publication number: CN104845914B
Application number: CN201510275530.1A
Authority: CN
Inventors: 张�杰; 束长龙; 王杨; 林克剑; 刘永磊; 耿丽丽; 王桂荣; 宋福平; 彭琦
Original assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Priority date: 2015-05-26
Filing date: 2015-05-26
Publication date: 2018-04-06
Anticipated expiration: 2035-05-26
Also published as: CN104845914A

Abstract

The present invention relates to " Bacillus thuringiensis bacterial strain, expressing protein and its application ", belong to technical field of biological control.Insecticidal proteins, there is such as SEQ ID NO：Amino acid sequence shown in 1, and the gene of the insecticidal proteins is encoded, the nucleotide sequence such as SEQ ID NO of preferably described gene：Shown in 2.Said gene has high virulence to planthopper insect, can apply to microbial and plant, is allowed to show the toxicity to related insect, and overcomes, delays insect to produce the resistance to the action of a drug of engineering bacteria and genetically modified plants.

Description

Bacillus thuringiensis bacterial strain, expressing protein and its application

Technical field

Particularly further the present invention relates to technical field of biological control, the present invention relates to have to planthopper agricultural pests There are the Bt bacterial strains, killing gene and the protein by the coded by said gene of high virulence.

Background technology

Planthopper category Homoptera Delphacidae.Hazard rice mainly has three kinds of brown paddy plant hopper, white backed planthopper and small brown rice planthopper.Harm Heavier is brown paddy plant hopper and white backed planthopper, and the later stage is based on brown paddy plant hopper based on white backed planthopper early stage for early rice；Middle late rice is flown with brown Based on lice.Small brown rice planthopper seldom directly causes disaster, but can propagate paddy and wheat, the virus of seeding corn and other crops.

The long wing adult of planthopper can migrate over long distances, and adult and nymph are clustered in thorn in the stalk of rice clump bottom and inhale juice Liquid, meet agitation and jump overboard face or flee from, phototaxis is strong, and likes light green；But the phototaxis of small brown rice planthopper is slightly weak.

The overwintering stage of planthopper and region of surviving the winter are because of species and different.Brown paddy plant hopper is in Guangxi and South Guangdong to Fujian LongXi Areas to the south, each worm state can all survive the winter.Winter warms up the time, and the north limit survived the winter is in 23 °~26 ° of north latitude, all winter ratooning rice and premature rice The area that seedling can survive all can safe overwintering.4~11 generations, some areas generation overlap occur every year for each province on the south the Changjiang river.Its Field full incidence period average rice earing stage.White backed planthopper is got in Guangxi to Dehua County, Fujian Province areas to the south with ovum on volunteer and trip grass In the winter, north limit of surviving the winter is in 26 ° or so of north latitude.Occurred for 3~8 generations Chinese annual, in single cropping of causing harm, late rice and late-seaon rice it is heavier. Small brown rice planthopper is survived the winter in North China with nymph under weeds clump, rice stub or fallen leaves, is survived the winter in Zhejiang with nymph on wheat weedses, in good fortune Building each worm state in south can all survive the winter.In 4~5 generations, the generation of lower Yangtze 5~6, the generation of Fujian 7~8, occur every year for North China.Field Though the phase of causing harm is slower than white backed planthopper, but still is caused most serious harm with ear period.

Planthopper is caused harm to rice, except directly thorn inhales juice, makes growth retardation, and rice clump is agglomerating withered when serious, even The complete dead stalk lodging in field is outer, and spawning can also stab plant, destroy conducting tissue, hinder nutriment transport and transmitted virus disease.

Preventing and treating for planthopper, mostly using traditional chemical prevention and control method, as chlopyrifos, imidacloprid, malathion, Pymetrozine, ethofenprox etc., easily cause environmental pollution, and drug-fast generation.For this kind of phytophagous sucking mouth parts of plant hopper Insect, not efficient insecticidal proteins are used for biological control at present and genetically modified plants are developed.

The content of the invention

For the defects of above-mentioned field, the present invention provides and screens a thuringiensis strain bacterial strain of bacillus, to planthopper There is stronger toxic action.

The present invention also provides the insecticidal proteins for having poisoning planthopper effect isolated in the bacterial strain simultaneously.

Bacillus thuringiensis bacterial strain IPPBIOTSUC-C9F1, its deposit number are：CGMCC No.10782.

Application of the above-mentioned bacterial strains in planthopper insect is killed.

A kind of insecticidal proteins, its amino acid sequence such as SEQ ID NO：Shown in 1.

Encode the gene of above-mentioned insecticidal proteins.

The nucleotide sequence of the gene such as SEQ ID NO：Shown in 2.

Application of the insecticidal proteins in planthopper insect is killed.

The application is that insecticidal proteins are made into insecticide as active insecticidal components to enter planthopper action killing.

The application is that the white gene of expression desinsection lice is transferred to the host plant of planthopper, plant expression is flown rice The resistance of lice insect.

The insect is small brown rice planthopper.

This experiment, as insecticidal activity bacterial strain screening object, is chosen substantial amounts of Bt bacterial strains and screened, obtained from small brown rice planthopper Go out：Bacterial strain IPPBIOTSUC-C9F1 has stronger toxic action to small brown rice planthopper.

The positive colony of 1 gene is obtained from the bacterial strain, analysis result proves that cloned gene is new gene, TOXIN1 is named as, by purifying, detection, the protein coding gene is obtained and has 382 amino acid altogether, such as SEQ ID NO：Shown in 1, The dalton of molecular weight 43358；Encoding gene has 1149 bases altogether, and specifying information is as follows：

Composition 421 A；190 C；182 G；356 T；0OTHER

Percentage:37% A；17% C；16% G；31% T；0%OTHER

The gene order such as SEQ ID NO of the coding albumen：Shown in 2.

The gene that clone obtains is expressed, protein function verifies the death rate up to 95% (protein concentration 25ppm), table The bright albumen has stronger toxic action to planthopper.

Bacterial strain preservation information：

Bacterium classification is named：Thuringiensis (Bacillus thuringiensis)

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

Preservation date：On May 6th, 2015

Deposit number：CGMCC No.10782.

Preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.

Brief description of the drawings

Fig. 1 parts bacterial strain SDS-PAGE results,

M1, HMW Marker；M2. low molecule amount Marker.

Induced expression result in Fig. 2, Rosetta bacterial strain.

Wherein M molecular weight marker；1, blank control (comprising only pET28a empty carriers) soluble component；2, containing killing The soluble component of worm genophore (containing pET28a+Toxin1 carriers)；3, blank control insolubility component；4, containing killing The insolubility component of worm genophore；Target insecticidal proteins band of expression is shown in arrow.

Embodiment

With reference to embodiment, the present invention is described in further detail.

1st, the foundation and checking of raw survey system

Sod cultivation

With reference to the sod cultivation of the reports such as Fu, and improved.

The foundation of new life survey system improves on the basis of original raw survey system by multiple spot.First, raw survey is used instead Transparent PET plastic bottle (volume 50mL, bottle height 69mm, diameter 42mm) is carried out, and opens the hole of 6mm diameters in its body side, and Perforate is sealed in the medical adhesive tape for pasting ventilative in bottle, can prevent test worm escape and can from providing enough skies for the growth of test worm Gas, solves test worm unexpected death to the raw influence for surveying result.Secondly, bottle cap separates with body, only can be completed with bottle cap The preparation of raw survey feed.White bottle cap, with diameter 30mm hole making drills punch, by double-deck Parafilm (parafilm M, USA the lid that perforate is covered after 4 times) is stretched, adds 200 μ L man-made feeds, then artificial feeding is overlying on the Parafilm of stretching Sealed on material, be fabricated to feed capsule.The feed that need to be replaced is removed together with bottle cap, more renewed.Whole process can be with Carry out step by step, beneficial to operation, reduce test worm escape, greatly facilitate the raw progress for surveying experiment.Finally, added in bottom of bottle The Ago-Gel of 2% concentration, humidity suitable to small brown rice planthopper in bottle can be maintained, as long as the good incubator of temperature control can all enter The raw of row small brown rice planthopper surveys experiment.

Every bottle is put into test worm 20, and each processing is repeated 3 times.Body is covered with black cloth, by bottleneck towards light source, every two It changes feed.Raw survey continues 3-6d.

Man-made feeds

(2000) report, brown paddy plant hopper Chemically defined diet formula (table 2-1) are waited using Fu Qiang.

Component of (i) concentration more than 0.05mg/mL is directly weighed with ten a ten thousandth balances in table；(ii) it is less than 0.05mg/mL component prepares 10 × mother liquor；(iii) biotin, inorganic salts are prepared 100 × mother liquor and used.Use 1mol/LNaOH The pH value of feedstuff soln is adjusted to 6.8 by solution, volumetric flask constant volume.Finally by man-made feeds with disposable Millipore micropores (0.22 μm) filter filtration sterilization, is divided in 2mL centrifuge tubes, be stored in -20 DEG C it is standby.All reagents of feed formulation are purchased from Amresco or Sigma-Aldrich.

Table 2-1. lifes, which are surveyed, uses small brown rice planthopper Chemically defined diet formula

Sod cultivation test result

The foundation of new life survey system improves on the basis of original raw survey system by multiple spot.Correlation, which is improved, to be caused first Previous existence surveys control treatment group in 20% death rate to fluctuate, is down to less than 10%.

2nd, the screening of Bt strain activities and identification

BT strain proteins extract

A. the plate of 90mm solid LB flat boards 20 is prepared, it is each to be coated with 200 μ L bacterium solutions；

B. 48h is cultivated, thalline is collected in 50mL centrifuge tubes with bacterium device is scraped.Add the milli-Q water thalline of 40mL precoolings；

C.12000g centrifugation 5min abandons supernatant；

D. 40mL lysates (50mmol/L Na are added₂CO₃, NaHCO₃, pH=10), ultrasonic 50% power output, super 3s Stop 5s, common 5min.Mixture is in ice chest 4h on shaking table after ultrasound；

E.4 a DEG C 12000g centrifugations 10min takes supernatant；

F. H is added₂CO₃Regulation pH=5.0 makes albumen isoelectric precipitation, and 4 DEG C of refrigerators are stood overnight；

G. supernatant is abandoned in centrifugation, and precipitation 5mL lysates dissolve；

H. after precipitation is dissolved, trypsase (10mg/ml, with mass ratio 1 is added：10 ratios add) 37 DEG C of digestion, 2h Albumen；

I. extract albumen and run 10%SDS-PAGE detections.

SDS-PAGE detects albumen

A. used reagent：

30%- acrylamides

Acrylamide 37g

Methylene diacrylamide 1g

Water is added to be settled to 100mL

Separation gel buffer solution (1.5mol/L Tris-HCl, pH=8.8)

Tris 9.1g

H₂O 40mL

Concentrated hydrochloric acid adjusts pH to 8.8, and ultra-pure water is settled to 50mL, 4 DEG C of preservations

Concentrate glue buffer solution (1.0mol/L Tris-HCl pH 6.8)

Tris 6.1g

H₂O 40mL

Concentrated hydrochloric acid adjusts pH to 6.8, and ultra-pure water is settled to 50mL, 4 DEG C of preservations

10% ammonium persulfate

Ammonium persulfate 1.0g

H₂O 9.0mL

It is packed as 0.5mL often to manage, -20 DEG C of preservations

10% (w/v) SDS

SDS 1.0g

H₂O 8mL

68 DEG C of heating for dissolving, concentrated hydrochloric acid adjust pH to 7.2, and ultra-pure water is settled to 10mL

5X electrophoretic buffers

Tris 15.0g

Glycine 94.0g

SDS 5.0g

Ultra-pure water is settled to 1000mL, and the used time dilutes 5 times

Prescription of its dyeing liquor

Solution 1 (dehydration) 1L:500mL ethanol, 100mL acetic acid, 400mL ultra-pure waters

Solution 2 (dyeing) 1L:50mL ethanol, 75mL acetic acid, 875mL ultra-pure waters

Coomassie brilliant blue R250：0.25g coomassie brilliant blue R250s, it is dissolved in the ethanol of 100mL 95%, filter paper filtering. B.SDS-PAGE gel formulas

Various concentrations SDS-PAGE separation gel formulas：

SDS-PAGE concentration glue (5%)

C. glue and electrophoresis

Separation gel is prepared according to above-mentioned formula, gel maker is added into and, along 1.5cm, adds ultra-pure water fluid-tight on, 20min is placed at room temperature.Concentration glue is prepared, pours into gel maker, and insert comb.After gelling collection is good, loading, electrophoresis.Upper strata Glue 60-80V voltages, when sample to separation gel, with 180V voltages.Judge to stop in good time according to pre-dyed albumen Marker bands Electrophoresis.

D. dyeing course：

Solution 150mL microwave stove heat 30s are added, are placed in 10~30min of shaking table

Solution 250mL+R250200 μ L heating 30s are added, are placed in shaking table dyeing.

Bt strain activities screen and qualification result

1 is shown in Table using the selection result.Division avirulent strain, less toxic strain and high strain are used as using the corrected mortality of biologicall test Foundation：(containing 75%) 75%, above person is high strain, person is avirulent strain, in 12% (containing 12%) to 75% below 12% Between person be less toxic strain.The result of biologicall test is shown：In tested Bt bacterial strains high strain have 1 plant (IPPBIOTSUC-C9F1), Less toxic strain has 30 plants, and remaining 35 plants are avirulent strain.

Bacterial strain screening result shows that bacterial strain IPPBIOTSUC-C9F1 has preferable insecticidal activity to small brown rice planthopper.

In order to confirm in the sample extracted from Bt spore crystalline substance mixtures, if be that protein component plays active function, respectively Sample is digested with Proteinase K, or the inactivation treatment of HTHP is carried out to sample, the sample after processing is carried out It is raw to survey result such as Fig. 1.

Protease K digesting：20mg/mL Proteinase Ks are prepared, 200mg Proteinase K is added to 9.5mL pH=8.0PBS In buffer solution, gently shake, until Proteinase K is completely dissolved.Should not vortex mixed.Add buffer solution constant volume to 10mL, Ran Houfen It is filled to 1.5mL centrifuge tubes and is stored in -20 DEG C.Protein sample addition Proteinase K, 1:The μ g/mL of 1000 final concentration 20.37 DEG C, it is incubated 1h。

High-temperature inactivation processing：By protein sample, it is put into autoclave and handles, 121 DEG C, 15min.

The Bt of table 1 is to the preliminary biological activity determination result of small brown rice planthopper

Biological characteristis shows that IPPBIOTSUC-C9F1 protein concentrations are that result is surveyed in primary dcreening operation lifes of the 70 μ g/ml to small brown rice planthopper It is 88% for the death rate.By the bacterial strain preservation, its deposit number is：CGMCC No.10782.

3rd, bacterial strain IPPBIOTSUC-C9F1 genomes determine

Genome extracts

Extraction Methods of Genome：

A. by IPPBIOTSUC-C9F1 streak inoculations on LB culture mediums, 30 DEG C of overnight incubations；

B. thalline is collected in 1.5mL centrifuge tubes, is fully suspended with 150 μ L S1；

C. 100mg quartz sands are added, 1min is crushed on historrhexis's instrument；

D. 200 μ L S2 are added fully to mix；

E. 400 μ L S3 are added, are fully mixed, 12000g centrifugations 10min；

F. upper strata supernatant is transferred to 1.5mL centrifuge tubes, adds isometric isopropanol, mixed, be placed in -20 DEG C of standings 20min；

G.12000g centrifugation 10min abandons supernatant；

H.70% washes of absolute alcohol is precipitated, and room temperature is dried；

I. 100 μ L ultra-pure waters water dissolving precipitation is added.

Each solution formula：

S1：10mmol/L Tris-HCl, 1.0mmol/L EDTA, 1mol/L sucrose pH=7.0

S2：4%SDS

S3：5mol/L guanidinium isothiocyanates, 1mol/L NaAc

Gene order-checking

Genome is built library, is sequenced using Illumina microarray datasets through interrupting at random.Utilize SOAPdenovo Reads sequencing datas are assembled, obtain Scaffold sequences.Gene order-checking data use after splicing GeneMark.hmm PROKARYOTIC (Version 3.2) are using Bacillus thuringiensiskonkukian as reference Genome, enable RBS model.Protein coding gene is predicted.The protein coding base obtained in draft genome Because annotation is using Blastx and NCBI non-redundant databases (Nr) progress.

Genome extraction, sequencing and killing gene the selection result

Genome splicing result

Genome splices statistical form 2

Parameter type	Numerical value
		Genome Size	6981828
Contig quantity	7909
		Average Contig length	882

Maximum Contig length

17694

Killing gene Blastx and NCBI non-redundant database (Nr) comparison result

Compared by gene, find a possible killing gene, the killing gene coded sequence is as follows, is named as Toxin1, sequence are shown in SEQ ID NO：2, its amino acid sequence encoded is SEQ ID NO：1.

Following data shows, the gene and bacillus thuringiensis [Bacillus thuringiensis] killing gene gb | EEM92619.1 | there is certain similitude；With Bacillus weihenstephanensis [Bacillus weihenstephanensis] killing gene Gb | KEZ80012.1 | there is certain similitude.Concrete outcome is as follows.

>gb|KEZ80012.1|insecticidal toxin[Bacillus weihenstephanensis]

Length=375

Score=365 bits (938), Expect=2e-120, Method:Compositional matrix adjust.

Identities=204/376 (54%), Positives=261/376 (69%), Gaps=19/376 (5%)

Frame=+1

5th, gene cloning and expression

Using the method for gene chemical synthesis by gene cloning between pET28a BamHI and HindIII sites, and ensure base Because being properly inserted ORFs, gene is synthesized by Shanghai life work.The plasmid for carrying synthetic killing gene is transformed into greatly In enterobacteria Rosetta (DE3), for further protein expression and insecticidal activity assay.

IPTG induced expressions

A.PCR detects correct positive colony, is stayed overnight with 5mLLB bacterium solutions, activates；

B. 300mLLB culture mediums are added afterwards, and 300 μ L ammonia benzyl antibiotic, 37 DEG C, 220rpm shakes bacterium；

C. bacterium 2h or so is shaken, detects OD to OD₆₀₀Between 0.5-0.8, the μ L of 1mol/LIPTG 500 are added；

D. 16-30 DEG C of temperature is adjusted, rotating speed 180rpm, continues to cultivate 10-12h；

E. bacterium solution is poured into centrifuge tube, trim, and 8000g, 10min, 4 DEG C are centrifuged with console mode high speed freezing centrifuge；

F. supernatant is abandoned, adds 10mL 20mmol/LpH=8.0Tris-HCl buffer solutions that precipitation is resuspended, is transferred to 50mL centrifugations Pipe, sonicated cells wall (power 50%, 5min, super 3s, stop 5s)；

G. centrifuge again, 13000g, 15min, 4 DEG C；

H. separated after centrifuging, retain soluble constituent and insoluble component respectively.

Protein expression situation analysis

Induced expression is carried out to the bacterial strain containing Toxin1 genes with IPTG, soluble constituent and insoluble component are entered respectively Row electrophoretic analysis, as shown in Fig. 2

Insecticidal activity assay

Further insecticidal activity assay is carried out with extraction albumen, protein concentration 25ppm, test result is as follows.As a result table Bright, Toxin1 has preferably pesticidal to small brown rice planthopper.

Biological activity determination result of the brand-new Toxin1 Primary structures product of table 3 to small brown rice planthopper

Beneficial effects of the present invention：The TOXIN1 gene orders and its gene expression product of present invention separation clone can be right Planthopper produces virulence, can be by applied to microbial and plant, making them show to phase especially for small brown rice planthopper The toxicity of insect is closed, can overcome or delay insect to engineering bacteria and the drug-fast generation of genetically modified plants.

Claims

1. a kind of insecticidal proteins, its amino acid sequence such as SEQ ID NO：Shown in 1.

2. encode the gene of the insecticidal proteins described in claim 1.

3. gene according to claim 2, its nucleotide sequence such as SEQ ID NO：Shown in 2.

4. application of the insecticidal proteins in planthopper insect is killed described in claim 1.

5. application according to claim 4, to make insecticide to planthopper using insecticidal proteins as active insecticidal components Enter action to kill.

6. application according to claim 4, the application is that the white gene of expression desinsection lice is transferred to the host of planthopper Plant, make resistance of the plant expression to planthopper insect.

7. application according to claim 4, the insect is small brown rice planthopper.