JP2779440B2 - Insecticidal protein gene of Bacillus thuringiensis Aizawai IPL strain - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Industrial applications   The present invention relates to lepidopteran insects, moth moth and hasmon.
Bacillus thuringien with strong insecticidal activity against tow
Insecticidal protein gene of cis isawai IPL strain, said gene
And a plasmid transformed with the plasmid
The present invention relates to a microorganism that is converted to produce an insecticidal protein. Conventional technology   Various strains of Bacillus thuringiensis sporulate
Formed crystals of 1-2 μm consisting of insecticidal proteins
Insects who take this crystal protein stop their ingestion activity
It is known that after the rupture of the intestinal tract or the like, death occurs.
Bacillus thuringiensis strains can be
It is classified into 29 subspecies according to its activity, etc.
The strains show specific and different pesticidal activities, respectively.   The present inventors have found that among the lepidopteran insects, they are vegetable pests.
Strong insecticidal activity against Japanese long moth and cutworm
Bacillus thuringiensis Aizawai IP
Use of insecticidal proteins produced by L strains as insecticides
Research was conducted for the purpose of, and the present invention was completed. Problem-solving measures   The present inventors have found that potatoes and moths
Bacillus thuringiensis isa showing high insecticidal activity
Cloning the gene for insecticidal protein produced by YIPL strain
After that, the entire sequence of 3465 bases in the structural gene part of the gene
And elucidated the primary structure of the insecticidal protein. In addition,
Connect the insecticidal protein gene to various expression vectors,
Microbes that produce insecticidal proteins when introduced into microorganisms
By producing organisms and culturing these microorganisms,
Insecticidal protein of S. thuringiensis Aizawai IPL strain
Completed a manufacturing method for mass-producing.   Bacillus thuringiensis Isawai IP of the present invention
The insecticidal protein gene of the L strain has the nucleotide sequence and nucleotide sequence shown in FIG.
And the amino acid sequence.   The plasmid containing the insecticidal protein gene of the present invention,
Bacillus thuringiensis Aisawai IPL
Create a gene library from the plasmid DNA
Lobes, for example, the insecticidal protein inheritance of the invention shown in FIG.
Synthetic oligonucleotides based on the nucleotide sequence of
Bacillus thuringiensis.
From the known nucleotide sequence of the insecticidal protein gene of HD-1 dipel strain
Use synthetic oligonucleotides as probes
Screening this library and killing it according to the present invention.
Isolating plasmids containing worm protein genes
You can get more. In this case, the insecticidal property of the present invention
Preparation of recombinant plasmids containing protein genes
High killing from Las Thuringiensis Isawai IPL shares
This can be done advantageously by selecting and using strains that exhibit insect activity.
Can be. As is well known, many amino acids
There are multiple gene DNA sequences that encode
Exist. The nucleotide sequence is undefined and many possible
There can be sex. Bachira revealed by the present inventors
Insecticidal protein of S. thuringiensis Aizawai IPL strain
In the case of a gene encoding the amino acid sequence of
Many nucleotide sequences besides the DNA sequence of the natural gene
However, the gene DNA of the present invention may be a natural DNA salt.
The present invention is not limited to the base sequence
Encodes the amino acid sequence of a disturbed insecticidal protein
It also includes other DNA base sequences.   According to the genetic recombination technology, the basic DNA can be specified.
Changes the basic properties of what is encoded by nuclear DNA.
Without improving or improving its properties
In addition, mutation can be artificially caused. The present invention provides
Provided, a gene DNA having a natural nucleotide sequence or
Gene DNA with a base sequence different from that of the natural
In the same way, artificially inserting, deleting, or substituting
Equivalent or improved properties to more natural genes
It is possible that the present invention
Is also included.   Bacillus thuringiensis Isawai IP of the present invention
An L-strain insecticidal protein gene with an appropriate expression plasmid, e.g.
For example, the expression vector pUC18 (Fr
Pharmacia), Tac, a strong promoter for E. coli
Promoter and terminator for rrbB ribosomal RNA
Expression vector pKK223-4, Trp promoter
Expression plasmid pDR720 (Pharmacia),
Inducible expression plasmid pPL-Lambda (Pharmacia)
By connecting to E. coli etc.
Insect killing of Bacillus thuringiensis Aizawai IPL strain
Expression plasmids that produce soluble proteins
Wear.   Expression plasmid containing the insecticidal protein gene of the present invention
To E. coli (for example, E. coli JM103 strain (Pharmacia))
Insecticidal proteins in the cells by introduction into host microorganisms such as
Microorganisms that produce white can be obtained.   Appropriate culture of the transformed microorganism thus produced
Mass production of insecticidal proteins by cultivation under local conditions
It is possible to In this case, the inducing agent
Insecticide by adding propylthiogalactoside
Production of a sex protein can be advantageously performed.   Isolation of insecticidal proteins after culture can be performed, for example, by sonicating the cells.
Composed of insecticidal proteins by crushing and centrifugation
This can be done by easily concentrating and collecting the aggregates.
Wear.   In addition to E. coli host-vector systems,
Hosts such as fungi, yeasts, Pseudomonas or actinomycetes
Vector systems are also available, each host-vector
-Mass production of insecticidal proteins taking advantage of the characteristics of the system.   Hereinafter, the present invention will be described in more detail by way of examples. Book
The invention is not limited to only the following examples,
Normal changes in the technical field of the present invention can be made.
You. Example 1 (Isolation of insecticidal protein gene)   High against lepidopteran moths and cutworm
Bacillus thuringiensis Aizawa showing insecticidal activity
Selection of IPL No. 7 strain Step 1: Bacillus thuringiensis Isawai IP
Purification of L strain   Isawai IPL shares [US Department of
  Agriculture Research Service (U.S.Depart
ment of Agriculture Research Service)
Plate medium (10 g of tryptone (Sigma), NaCl
G) 5 g, yeast extract (Sigma) 5 g,
And agar (Sigma) 12g to make 11
Medium), and purify each of the 32 purified strains.
Sumid analysis was performed. 10 ml of PY liquid medium for each strain
After culturing for 24 hours in (PY plate medium without agar),
Bacteria are collected by centrifugation (20,000 xg, 30 minutes), and Birn
boim and Doly (Nucleic Acids Res. 7,1513-1523.)
Prepare plasmid DNA according to the method and add 0.8% agarose
Plasmid analysis was performed by gel electrophoresis. The result
As a result, the 32 purified strains analyzed had the same plasmid pattern.
At least 9 different plasmid patterns
4.OMd, 4.8Md, 5.4Md, 8.5Md, 12Md, 15Md, 17M
d, 21Md, 37Md, 40Md, 50Md, 52Md
Strain named Izawai IPL No. 7
Was.   The Izawai IPL No. 7 strain is
Bacillus thuringiensis subsp.aizawai
 Deposited as IPL No. 7, Deposit number FERM P-8654
I have. Currently, the deposit has been changed under Budapest conditions.
And deposited under accession number FERM BR-1150. Step 2: Bacillus thuringiensis by insecticide test
・ Selection of Izawai IPL No.7 strain   Each of the nine purified Isawai IPL strains
Incubate on 30 pieces of PY plate medium (15 cm diameter) at 30 ° C for 7 days.
After confirming the formation and crystal formation with a phase contrast microscope,
After three freeze-thaw cycles, suspend in 2 ml of distilled water.
The suspension is crystallized by turbidity and sonication.
Prepared. Stock solution of the resulting preparation, 10-fold dilution, 100-fold
1 ml of each diluted solution was soaked in artificial feed
After that, feed four 10-year-old cutworms to cutworm and feed 3 days later
The number of insects was observed. Using the third-instar larvae for Japanese moth,
A similar test was performed. Purification of strain Aizawai IPL No. 7 strain
Crystal suspension (2.2 × 10⁷1ml Kona (including spores)
When fed to moths, 10 out of 10 died, and
Liquid (2.6 × 10⁸(Including spores)
When fed, 10 out of 10 died.   Therefore, the Izawai IPL No. 7 strain is high against both pests
It was revealed that it shows insecticidal activity. (Cloni of the insecticidal protein gene of Izawai IPL No. 7 strain
Ng) Step 1: preparation of synthetic DNA probe   Bachiras Thuringiensis Kristaki HD-1
The nucleotide sequence of the insecticidal protein gene of Dipel strain (Whiteley et al.,
J. Biol. Chem. 260, 6264-72 (1985))
Preparation of probe (5'-CACAAATCCAGCACCGGG-3 ')
Was. Applied Biosystems DNA Synthesizer Model 3
After synthesizing the DNA oligomer using 80A,
Add 1 ml of 27% ammonium hydroxide to the mixture and place at 55 ° C for 4 hours
Warmed for a while and concentrated to dryness in a concentrator. After drying, 100μl
Dissolved in 0.01M triethylamine-acetic acid (TEAA) (pH 7.2)
And applied to a reverse phase HPLC column C18, acetonitrile-0.1
Elution was performed with a solvent system of MTEAA (pH 7.2). 260nm absorption
Separate the peak fraction and dry to 100 μl of acetonitrile.
80% acetic acid prepared in the above was added and left for 15 minutes. 15 minutes
After a while, if it becomes pale orange, it is dried to dryness and 0.01M TEAA
(PH 7.2) 100 µl was added, and ethyl acetate was further added to 100 µl
Was added and mixed. Discard the ethyl acetate phase and add diethyl ether
Add 100 μl of ter, repeat the same operation twice, and dry
Was. Dissolve in 0.01M TEAA (pH 7.2) and re-
Fractions of the nm absorption peak fraction were collected and dried, and then 10 mM Tris-HCl
(PH 7.5) + 1 mM EDTA (TE) solution. Next,
Prepared synthetic DNA probe³²P labeling was performed. 5 μl
Escherichia coli polynuclease on synthetic DNA probe (about 100 pmole)
15 units of Ochidokines (Takara Shuzo), 100μCi
[Γ-³²P] ATP (Amersham Japan) and 10 times
Concentration reaction mixture (0.5M Tris-HCl (pH7.6), 0.1MMgCl_Two
0.1 M2-mercaptoethanol)
Add distilled water to make the total volume 100 µl, and react at 37 ° C for 1 hour
Then, after adding chloroform: phenol (1: 1) and mixing,
The supernatant was collected. The collected supernatant is used for TE buffer (pH 7.5)
DE-52 column (Whatman, 0.5 ml
And 3 ml of TE buffer (pH 7.5)
After washing, elution was performed with 0.7 M NaCl-TE buffer (pH 7.5).
Separate the radioactive fraction,³²Preparation of P-labeled synthetic DNA probe
did. Step 2: Plasmid DNA license of Ausawai IPL No. 7 strain
Making a library   Izawai IPL No. 7 strain cultured in 200 ml PY liquid medium
Cells in 30 ml of G buffer (10% glycerol, 1 mM EDT)
A, Wash with 50 mM Tris-HCl (pH 8.0)
(5 mg / ml) and reacted at 30 ° C. for 2 hours. Next
And an alkaline solution (0.2N NaOH, 1% sodium dodecyl sulfate)
(SDS)), mix and leave at room temperature for 10 minutes
After that, add neutralization solution (3M sodium acetate (pH4.8))
2 ml was added and left at 4 ° C. for 1.5 hours. Centrifugation operation (14,000rp
m, 20 minutes), and add 70 ml of cold ethanol.
And left at -20 ° C for 1 hour.
A was recovered. After drying, suspend in 5 mL of 0.1 M sodium acetate
And phenol equilibrated with TE buffer (pH 7.5)
After the treatment was performed twice, the supernatant was separated, and 2 volumes of cold ethanol
And ethanol precipitated again.   After drying, the suspension was suspended in 6 ml of TE buffer (pH 7.5).
In accordance with a conventional method, cesium chloride
Rasmid DNA was purified.   Next, restrict 5 μg of plasmid DNA to 10 units.
Cleavage with enzyme BamH I, equal volume of phenol: chloroform
(1: 1) was added and mixed. The supernatant was collected and ethanol precipitated.
The DNA is recovered by precipitation and after drying, 20 μl of TE buffer (pH
7.5). In addition, 2 μg of the pUC8 plasmid (
(Pharmacia) with 5 units of restriction enzyme BamHI
Thereafter, the DNA was recovered by the same operation, and 20 μl of TE buffer (pH
After suspending in 7.5), 5 μl of E. coli alkaline phosphater
Ze (Takara Shuzo, 2.0 units), 50 μl of 0.1 M Tris-salt
After adding an acid buffer (pH 8.0) and 15 μl of distilled water,
Incubate for 1 hour with alkaline phosphatase
Was done. After the reaction, perform phenol-chloroform treatment twice
After that, the supernatant was separated and the DNA was precipitated by ethanol precipitation.
Was collected and suspended in 20 μl of TE buffer (pH 7.5). One
15 μl of BamHI digested plasmid DNA solution and 15 μl
After mixing the BamHI digested pUC8 plasmid DNA solution of
T4 DNA ligase (Takara Shuzo, 0.1 unit), 7.5 μl
0.1 M dithiothreitol (Hansui Chemicals), 7.5 μl of 10 mM
Adenosine triphosphate (Hansui Chemicals), 25μl, 3 times concentration
Reaction mixture (200 mM Tris-HCl (pH 7.6) 20 mM MgCl_Two) And
And 5 μl of distilled water to make a total volume of 75 μl, at 14 ° C. for 6 hours
For a while. 10 μl of the obtained ligase reactor
The method of Scott et al. (Cell engineering,2, 616-626, 198
100 μl of the E. coli DH1 strain (ATCC33849) prepared in 3)
Incubate at 0 ° C for 15 minutes
And heat-treated at 42 ° C for 40 seconds, then 0.9 ml L broth solution
Body medium (tryptone 10g, yeast extract 5g, N
Add 5 g of aCl and 1 g of glucose (Hansui Chemicals), and add
And incubate at 37 ° C for 1 hour,
L-broth plate containing 50 μg / ml final concentration of ampicillin
Ground (agar added to L broth liquid medium to 1.2%
). Step 3: insecticidal properties by colony hybridization
Isolation of protein gene clones   Spread the colonies on the plate with two nitrocellulose
Replicate to the filter and add ampicillin (50μg /
ml) and chloramphenicol (600 μg / ml)
Transferred to plate and incubated overnight. One sheet
Add 2.5 ml of 0.5N NaOH per filter and treat for 5 minutes
After air-drying, add an equal volume of 1 M Tris-HCl (pH 7.5),
Left for 5 minutes. After air drying, another 2.5 ml of 1 M Tris-salt
Treated with acid (pH 7.5) -1.5M NaCl for 5 minutes, air-dried, and 80 ° C
For 3 hours under vacuum. 10 per 4 filters
ml of 0.1% SDS-4 × SSC (SSC is 0.15M NaCl-0.015M
Sodium enoate (pH 7.5)), and add 60 ° C for 15 minutes
After the treatment, wipe the colonies on the filter and
Rehybrid solution (4 × SSC, 10 × Denhardt, 100 μg / ml
Thermomontistis single-stranded DNA (Sigma);
× Den Heart is 0.2% Ficoll, 0.2%, polyvinyl
Pyrrolidone, a solution containing 0.2% BSA).
For 3 hours. Next, the above solution was prepared in step 1.
Made³²Hybridization solution with P-labeled synthetic DNA probe
The filter was placed inside and incubated at 56 ° C. overnight.
After hybridization, 4 × SSC-0.1% SDS at 56 ° C.
Wash with the solution four times for 15 minutes, dry the filter,
The film was sandwiched and autoradiographed.
Master play colonies that give a positive signal
And then again, colony hybridization
To isolate the positive clone E. coli DH1 / pAB6.
Was. (Bacillus thuringiensis Aisawai IPL No.7 strain
Analysis of insecticidal protein genes in rice)   Birn from the isolated positive clone E. coli DH1 / pAB6
Isolate plasmid pAB6 according to the method of boim and Doly et al.
Was. Plasmid pAB6 is replaced with restriction enzymes BamHI, HindIII, Pat
 I, Bgl II, Pvu I, EcoR I, Aha III, Kpn I, Cla I
And analyzed by 0.7% agarose gel electrophoresis.
Creates a restriction map of 22.4 kb of insert DNA
(Upper part of FIG. 3). In addition, the synthetic DNA probe described above
The Southern hybridization method used (J. Mol. Biol. 9
8,503-517 (1975))
Identifies the sex protein gene region and detailed restriction enzymes for that region
A map was created (FIG. 3, bottom). Then, with various restriction enzymes
Subcloning cut DNA fragment into plasmid pUC8
After that, include the DNA fragment by the method of Birnboim and Doly et al.
Plasmid DNA was prepared. 18 μl of TE obtained
(PH 7.5), add 2 μl of 2N NaOH, and add
For 3 minutes, add 3 μl of 7.5 M ammonium acetate, add 10
0 μl of cold ethanol was added to perform ethanol precipitation.
Was.   Centrifuge at 12,000 rpm for 5 minutes, collect the precipitate, dry it,
It was dissolved in distilled water to 0.5 pmol / 5 ml. 5 μl
Add 1.5 μl of 10 times volume to the prepared plasmid DNA (5 pmol)
Concentration Kleno-buffer (Takara Shuzo), 1 μl of primer DN
A (PL Biochemical), add 4.5 μl of distilled water
Make the volume 12 μl, warm at 60 ° C for 15 minutes, then release to room temperature for 20 minutes
Was placed. Add 2 μl of [α-³²P] ATP (400 Ci /
mmol, Amersham Japan) and 1 μl Klenow digestion
One side enzyme (Takara Shuzo, 2 units) was added and mixed. this
3.2 μl of the mixture was mixed with 4 types of 2 μl of dNTP + ddNTP.
The mixture was added to the mixture (Takara Shuzo) and left at 42 ° C for 20 minutes. To each
Add 1 μl of chase solution (Takara Shuzo), and further add 42 ° C
For 20 minutes. Finally, 6 μl of 95% formamide
Dye (0.1% bromophenol blue and 0.1% xylene
(Including cyanol). 6% acrylic according to the usual law
Make a lamide / urea gel and apply 2 μl of the above reaction solution.
And electrophoresed at 1700 V for 6 hours. Dry gel
After drying, sandwich the X-ray film, read the sensitivity, and read the base sequence.
I took it.   The elucidated nucleotide sequence is shown below and in FIG. Bachiras
Insecticidal protein of Thuringiensis Aisawai IPL No. 7 strain
The gene starts with the start codon ATG and the stop codon T
It has a coding region of 3465 bases ending in AA and 1155
Coded for amino acids. Example 2 (Bacillus thuringiensis Isawai
Expression of an insecticidal protein gene from an IPL strain for expression in Escherichia coli
Construction of current plasmids pAH8 and pAH7) Step 1: preparation of Aha III DNA fragment   About 10 μg of recombinant plasmid containing the insecticidal protein gene
Add about 30 units of the restriction enzyme Aha III to pAB6 and add
μl of Aha III reaction solution [10 mM Tris-HCl (pH 7.5), 60 mM
M NaCl, 7mM MgCl_Two, 10 mM EDTA, 1 mM dithiothreito
After reacting for 1 hour at 37 ° C, the reaction mixture was brominated at 0.1 μg / ml.
0.8% low melting point agarose gel containing ethidium
Suda Research Laboratory Co., Ltd.)
Electrophoresis was performed. 3.5 kb under UV lamp after electrophoresis
Cut out the gel portion corresponding to the Aha III DNA fragment of
Placed in test tubes and heated at 65 ° C. for 5 minutes. 2 for melted gel
Double volume of TE buffer [10 mM Tris-HCl (pH 8.0), 0.5 mM E
DTA], and add phenol saturated with TE buffer.
Then, phenol extraction was performed. Centrifuge at 10,000rpm for 5 minutes
After separating the upper layer, 1/40 volume of 4M NaCl and 2 volumes
Of ethanol and leave at -80 ° C for 10 minutes.
After ethanol precipitation from DNA, centrifuge at 10,000 rpm for 10 minutes.
The DNA was collected and suspended in 10 μl of distilled water. Step 2: plasmid preparation   1 μg of expression plasmid pUC18 (Pharmacia)
And 1 unit of restriction enzyme Hinc II (Takara Shuzo)
0 μl of Hinc II reaction solution [10 mM Tris-HCl (pH 7.4), 10
0mM NaCl, 7mM MgCl_Two, 10 mM dithiothreitol)
The reaction was carried out at a temperature of 1 hour. After the reaction, add an equal amount of phenol
-Add a chloroform (1: 1) mixture, mix, and 10,000 rpm
After centrifugation at for 5 minutes, the supernatant was collected. Next, double the amount of cold
Add ethanol and leave at -80 ° C for 15 minutes.
Centrifuge at 00 rpm for 10 minutes, collect DNA, and place in 10 μl of distilled water.
Suspended. Step 3: Construction of expression plasmids pAH7 and pAH8   Aha III DNA prepared in Step 1 and Step 2
1 μg each of the fragment and the expression plasmid pUC18
Mix and add 7.2 units of T4 DNA ligase (Takara Shuzo).
Of a ligase reaction solution [66 mM Tris-HCl (pH 7.
6), 6.6mM MgCl_Two, 10mM dithiothreitol, 1.0mM ATP)
At 16 ° C for 2 hours. Then, follow the method of Cohen et al.
Therefore, the reaction solution was transformed into E. coli JM103 (Pharmacia).
Converted. Culture of emerging ampicillin-resistant colonies
The plasmid DNA was prepared according to the method of Birnboim et al.
Was. About 1 μg of plasmid DNA and 3 units of restriction enzyme
Hydrogen Hind III was added, and a Hind III reaction solution (10 mM Tris-hydrochloric acid) was added.
(PH7.5), 60mM NaCl, 10mM MgCl_Two, 1 mM 2-mercapto
37 ℃ in ethanol, 100μg / ml bovine serum albumin)
The reaction was carried out for an hour and analyzed by agarose electrophoresis. Agarlow
From the results of electrophoresis, the Lac promoter
Expression plus the insecticidal protein gene connected in the forward direction
The plasmid was pAH8 and the expression plasmid connected in the reverse direction was pAH8.
H7 (see FIG. 1). Example 3 (Bacillus turigensis Isawai IPL
Of a pesticidal protein gene of a strain for expression in Escherichia coli
Construction of plasmid pTB1) Step 1: Pst I-BamHI fragment containing insecticidal protein gene
Preparation of   About 5 μg of the expression plasmid pA containing the insecticidal protein gene
Add about 20 units of restriction enzyme Pst I and about 20 units to H8.
Of the Pst I reaction solution (10 mM
Tris-hydrochloric acid (pH 7.5), 50 mM NaCl, 10 mM MgCl_Two, 1mM2
-Mercaptoethanol, 100 μg / ml bovine serum albumin)
Reaction at 37 ° C for 1 hour in a buffer saturated with 30 µl of TE buffer.
The phenol was extracted by adding phenol. 10,000rp
After centrifugation at 5 m for 5 minutes and collecting the upper layer, 1/40 volume of 4 mM NaCl
Add 2 volumes of ethanol and leave at -80 ° C for 10 minutes
did. After centrifugation at 10,000 rpm for 10 minutes, collect the DNA and dry it.
After that, the cells were suspended in 20 μl of distilled water. Step 2: plasmid preparation   About 5 μg of the expression plasmid pKK223-3 (Pharmacia)
) With 0.1 unit of the restriction enzyme BamHI (Takara Shuzo)
In addition, a BamHI reaction solution [10 mM Tris-HCl (pH 8.0), 7 mM
 MgCl_Two, 100 mM NaCl, 2 mM 2-mercaptoethanol, 0.01
% Bovine serum albumin] at 37 ° C for 15 minutes. reaction
The solution was diluted to 0.8% low melting point with 0.1 μg / ml ethidium bromide.
The sample was subjected to a gelose gel and subjected to electrophoresis. After electrophoresis, UV
Under the line ramp, two BamHI recognitions carried by pKK223-3
DNA molecule presumed to have been cut only one of the sites
(4.6 kb), cut into test tubes, and heat at 65 ° C for 5 minutes.
Heated. After melting the gel and extracting with phenol, ethanol
Precipitate, recover DNA, suspend in 20 μl distilled water
Was. To the prepared DNA solution, add 4 kinds of deoxylysine at a final concentration of 3 mM.
Polynucleotide and 5 units of E. coli DNA polymerase
Add the large fragment of HindIII
Reaction solution (10 mM Tris-HCl (pH 7.5), 60 mM NaCl, 10 mM Mg
Cl_Two, 1 mM 2-mercaptoethanol, 100 μg / ml bovine serum
Bumin], at 25 ° C for 2 hours, and then extracted with phenol.
Perform ethanol precipitation, recover the DNA, and add 20 μl of distilled water.
Suspended in water. About 3 μg of T4D
Add NA ligase (Takara Shuzo) and add 45μl of ligase reaction solution
[66mM Tris-HCl (pH7.6), 6.6mM MgCl_Two, 10mM
Osleitol, 1.0 mM ATP] at 16 ° C for 2 hours.
Then, according to the method of Cohen et al., The reaction solution was used for E. coli JM10.
Three strains were transformed. Emerging ampicillin-resistant colonies
And cultivate the plasmid DNA according to the method of Birnboim et al.
Was prepared. 3 units per 1 μg of plasmid DNA
Add the restriction enzyme BamHI in the BamHI reaction solution at 37 ° C for 1 hour.
And analyzed by agarose gel electrophoresis. Analyzed program
Among the plasmids, the pKK223-3 plasmid
The BamH I site of the BamH
 A plasmid in which the I site was deleted was selected, and pKK223-4 was selected.
And Next, about 5 μg of pKK223-4 DNA was added to 10 units.
Restriction enzyme Pst I and 10 units restriction enzyme BamH I
And add 50 μl of the Pst I reaction solution [10 mM Tris-HCl (pH
7.5), 50mM NaCl, 10mM MgCl_Two, 1mM2-mercaptoethano
In 100 μg / ml bovine serum albumin) at 37 ° C for 1 hour.
After phenol extraction and ethanol precipitation,
N was recovered and suspended in 20 μl of distilled water. Step 3: Construction of expression plasmid pTB1   Pst I-BamH prepared in steps 1 and 2
I digested DNA fragment and plasmid pKK223-4
μg each, and add 7.2 units of T4 DNA ligase (Takara Shu)
Ligase reaction solution [66 mM Tris-salt]
Acid (pH7.6), 6.6mM MgCl_Two, 10mM dithiothreitol, 10m
MATP] at 16 ° C for 2 hours. Cohen's method
, The reaction solution was transformed into E. coli IM103 strain. Emergence
Cultured ampicillin-resistant colonies
A was prepared. 3 units per 1 μg of plasmid DNA
Of the restriction enzyme BamHI, and 37 μl of the BamHI reaction solution.
C. for 1 hour and analyzed by agarose gel electrophoresis. A
From the results of the galose electrophoresis, the Tac
Downstream of the promoter, the insecticidal protein gene
A plasmid was selected and used as an expression plasmid pTB1 (Fig.
1). Example 4 (Production of insecticidal protein in Escherichia coli)   Coat each constructed expression plasmid pAH7, pAH8 and pTB1 with Coh
Introduced into E. coli JM103 according to the method of en et al.   The obtained E. coli recombinants JM103 / pAH7, JM103 / pAH8 and
And JM103 / pTB1 produce Bacillus thuringiensis
The identification and analysis of insecticidal proteins of the Izawai IPL strain are as follows.
I went.   Each E. coli was cultured in L broth liquid medium [10 g tryptone (de
Ifco, 5g NaCl, 5g yeast extract
(Difco) in 1 l of distilled water]
Culture overnight. 0.1 ml of the final broth is added to 10 ml of L broth
Transfer to body medium and incubate at 37 ° C until OD660nm reaches 0.1
At a final concentration of 2 mM isopropylthiogalactoside.
Added. Continue culturing at 37 ° C, and after 20 hours,
0.3 ml of nutrient solution is collected and centrifuged (3,000 rpm, 15 minutes)
Collect the bacteria and add 50 μl of sample buffer [62.5 mM Tris-salt
Acid (pH 8.0), 2% (w / w) sodium dodecyl sulfate, 5%
(V / v) 2-mercaptoethanol, 10% (w / v) glycer
Roll, 0.01% bromophenol blue)
After heat treatment at 0 ° C. for 5 minutes, the method of Laemmli et al. (Nature, 227, 6
80-685), SDS-polyacrylamide electrophoresis
To After electrophoresis, run the gel on a Coomassie brilliant gel.
After dyeing with a roux, degassing and drying, it was fixed on paper. The result
As a result, the E. coli JM103 strain containing the expression plasmid has a molecular weight of 1
A 25K insecticidal protein band was detected and this protein band
Shows specific cross-reactivity with anti-insecticidal protein antibody (IgG)
Was. Measure each protein band on the gel with a densitometer.
E. coli JM103 / pAH7, JM103 / pAH8 and JM103 / p
In TB1, the prominent peaks of each 125K insecticidal protein band were
Peak was detected, and the peak of JM103 / pTB1-like was the highest.
(See FIG. 4). Therefore, these E. coli recombinants are effective.
Bacillus Thuringiensis Isawai IPL Share Killing
It was confirmed that insect proteins were produced. Example 5 (Preparation of insecticidal protein produced in Escherichia coli)   0.1 ml of E. coli cultured overnight in L broth liquid medium
Transfer the final solution to 10 ml of L broth medium and culture at 37 ℃
When the OD 660 nm value reaches 0.1, the final concentration of 2 mM
Propylthiogalactoside was added. In addition, culture
Subsequently, 5 ml of the culture solution was collected, centrifuged at 3,000 rpm for 15 minutes, and the bacteria were collected.
Were collected and frozen at -80 ° C. Next, melt at room temperature,
After repeating this operation three times, 2 ml of TE buffer (10 mM
Suspension in squirrel-hydrochloric acid (pH 7.5), 1 mM DDTA), 3 times in 30 seconds
Was subjected to ultrasonic treatment. Next, this crude extract was subjected to 7,000 r
The precipitate was collected by centrifugation at pm for 10 minutes. This precipitated fraction is
Suspend in sample buffer for electrophoresis and analyze by SDS-PAGE.
As a result, about 80% of the protein contained in the sediment fraction
Sex protein. Therefore, by using the above preparation method,
Can easily and efficiently prepare insecticidal proteins.
It became clear that. In addition, this preparation method is
It has been confirmed that the culture solution is effective.   "E.coli JM103 / pTB1 strain was added to 10 ml of L broth liquid medium.
Culture overnight, add this to 1 L of L-broth medium, and add
The culture was continued for a while. Centrifuge the culture at 6,000 rpm for 5 minutes to remove the cells.
After being collected, washed with water, and frozen at -80 ° C. Then melt at room temperature
And the freeze-thaw operation was repeated one more time
Thereafter, the cells were suspended in 50 ml of water and sonicated (5 min for 2 minutes).
Twice) to lyse the cells, and centrifuge this extract at 6,000 rpm for 5 minutes.
And separated into a precipitate and a supernatant. This precipitate fraction is used for electrophoresis.
When suspended in sample buffer and analyzed by SDS-PAGE,
Approximately 90% of the proteins contained in the sediment fraction are insecticidal proteins.
Was. Also, the protein content of the precipitate fraction and the supernatant fraction was determined by Lowry.
Quantification was performed by the method (J. Biol. Chem., 193, 265-275, 1951). So
As a result, the amount of protein in the precipitate fraction was 360 mg, and the amount of protein in the supernatant fraction was
It was 580 mg. The total amount of protein in both fractions is the total protein
Therefore, E. coli JM103 / pTB1 strain accounts for about
Producing up to 35% of insecticidal proteins
Was. Furthermore, by using the above preparation method,
It is clear that insecticidal proteins can be efficiently prepared
It was ok. "

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a method for constructing expression plasmids pAH7, pAH8, and pTB1. Each box containing black, vertical, horizontal, white, and dots indicates the insecticidal protein gene, La
The c promoter, ribosomal RNA terminator, Tac promoter and lacZ gene are shown. ATG and T
AA is the start codon and stop codon of the insecticidal protein gene, respectively. Ah, Kp, pv, Bm, Hc, Ps are restriction enzymes Aha III, Kp
nI, Pvu II, BamH I, Hinc II, and Pst I are shown. FIG. 2 shows the entire base sequence consisting of 3465 bases in the structural gene portion of the insecticidal protein gene. The upper row shows the base sequence and the lower row shows the amino acid sequence deduced therefrom. In the base sequence, the region from base number 1 to base 3465 is the region encoding the structural gene of the insecticidal protein gene. The base number
From 843 th to 848 th "T G CTCT" is "A G GCTC"
" C " at base number 2568 is a " G " error, " A " at base number 2934 is a " C " error, and " T " at base number 3107 is a " G " error. It is. As the corresponding amino acid, amino acid number 281 to 283
The second "Arg Ala Leu " is a mistake of "Arg Gly Ser "
" Gln " at amino acid number 978 is an error of " His ",
“ Phe ” at amino acid number 1036 is an error of “ Cys ”. FIG. 3 shows a restriction map of the insert DNA (22.4 kb) of the plasmid pAB6. The gene region of the insecticidal protein of the Izawai IPL strain is indicated by a thick frame. The lower part below shows the detailed restriction enzyme sites of the insecticidal protein gene region. FIG. 4 shows the results of quantification of the expressed insecticidal protein using a densitometer. (1), (2) and (3) are the results obtained by measuring the insecticidal proteins in the crude cell extracts of JM103 / pAH7, JM103 / pAH8 and JM103 / pTB1, respectively. The peak of the arrow corresponds to the insecticidal protein band.

Continuation of the front page (51) Int.Cl. ⁶ Identification code FI (C12N 15/09 ZNA C12R 1:07) (C12P 21/02 C12R 1:19) (72) Inventor Keiko Nakamura 4-2-2 Takashi Takarazuka No. 1 Sumitomo Chemical Co., Ltd. (72) Inventor Hideo Okawa 4-2-1 Takashi Takarazuka-shi Sumitomo Chemical Co., Ltd. (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 15 / 32 C12N 1/21 C12P 21/02

Claims (1)

(57) [Claims] An insecticidal protein gene characterized by the following amino acid sequence: 2. The insecticidal protein gene according to claim 1, wherein the gene is identified by the following nucleotide sequence. 3. A recombinant plasmid comprising an insecticidal protein gene specified by the following amino acid sequence: 4. 4. The recombinant plasmid according to claim 3, comprising an insecticidal protein gene specified by the following nucleotide sequence. 5. The following restriction map (Kp, Pv, Bm, Ps is the restriction enzyme Kpn
I, Pvu II, BamH I and Pst I are shown. PAH7,
4. The recombinant plasmid according to claim 3, designated as pAH8 or pTB1. 6. A microorganism which is transformed with a recombinant plasmid containing an insecticidal protein gene specified by the following amino acid sequence to produce an insecticidal protein. 7. The microorganism according to claim 6, wherein the microorganism is transformed with a recombinant plasmid containing an insecticidal protein gene specified by the following nucleotide sequence to produce an insecticidal protein. 8. The recombinant plasmid is shown in the following restriction map (Kp, Pv, B
m and Ps indicate restriction enzymes KpnI, PvuII, BamHI and PstI. ), Which is a recombinant plasmid designated as pAH7, pAH8 or pTB1.
The microorganism according to Item.