JPH0817707B2 - BT insecticidal protein gene - Google Patents

Description

TECHNICAL FIELD The present invention relates to an insecticidal protein of Bacillus thuringiensis islaerensis strain, which exhibits high insecticidal activity against dipteran larvae represented by Aedes japonicus, and the insecticidal protein. A protein gene, an expression plasmid containing the gene and expressing it in Escherichia coli, Bacillus subtilis, etc., a microorganism that retains the plasmid and produces an insecticidal protein of Bacillus thuringiensis islaerensis, and culturing the microorganism. The present invention relates to a method for producing a characteristic insecticidal protein.

2. Description of the Related Art Various strains of Bacillus thuringiensis form crystals of insecticidal proteins ranging from 1 to 2 μm during sporulation.
It is known that insects that feed on this crystal protein stop their feeding activities, cause intestinal rupture, etc., and then die, and formulations of certain strains are used as insecticides.

Bacillus thuringiensis strains are classified into 29 subspecies according to flagella antigen, esterase activity, etc., and each strain shows a specific and different insecticidal activity.
The insecticidal protein of Bacillus thuringiensis islaerensis is encoded by a gene on the plasmid of this strain, Sekar et al. (Gene, 33 (1985) p151-158), Waa.
lwijk et al. (Nucleic Acids Research 13 (1985) p8207-82
17), Ward et al. (Journal of Molecular Biology, 191 (198
6) p1-11), Bourgouin et al. (Molecular General Genetic)
s, 205 , (1986) p390-397) and Himeno et al. (Agricultu).
ral and Biological Chemistry, 49 (1985) p573-580)
, Etc.

In addition, the properties of the insecticidal protein of Bacillus thuringiensis Islaerensis are described in Thomas et al. (Journal of Cell Scinc
e, 60 (1983) p181-197) and Tyrell et al. (Journal of Bac
Teriology, 145 , (1981) p1052-1062).

Means for Solving the Problem The present inventors cloned a gene encoding a novel insecticidal protein produced by Bacillus thuringiensis Islaelensis strain showing high insecticidal activity against larvae of Aedes mosquito from a large plasmid of the strain. After that, the entire sequence of 3408 bases of the structural gene portion of the gene was determined to elucidate the entire primary structure of the insecticidal protein. Furthermore, by connecting this insecticidal protein gene to an expression vector such as Escherichia coli or Bacillus subtilis and introducing it into a microorganism such as Escherichia coli, a microorganism producing a large amount of insecticidal protein is created, and by culturing this microorganism, B. A manufacturing method for mass-producing the insecticidal protein of Lingensis islaerensis was completed. The Bacillus thuringiensis islaerensis insecticidal protein gene of the present invention is specified by the nucleotide sequence and amino acid sequence shown in FIG. The insecticidal protein according to the present invention has a molecular weight of 124 KDa and has already been clarified. It is an insecticidal protein of Bacillus thuringiensis Islaelensis having a molecular weight of 68 KDa and 28 K.
It is a novel insecticidal protein that is clearly different from Da. For the plasmid containing the insecticidal protein gene of the present invention, a gene library was prepared from the plasmid DNA of Bacillus thuringiensis islaelesis, and the anti-Israelensis insecticidal protein Ig
It can be isolated by screening by the Eliza method using G. Alternatively, it can be isolated by screening by a conventional method using a nucleotide synthesized from the nucleotide sequence shown in FIG. 2 as a probe.

As is well known, there are multiple DNA base sequences encoding many amino acids. Therefore,
The base sequence is not uniquely determined, and there are many possibilities. Also in the case of the gene encoding the amino acid sequence of the insecticidal protein of the Bacillus thuringiensis islaerensis strain revealed by the present inventors, the DNA base sequence has a number of nucleotides other than the base sequence of the natural gene. There is a possibility that the gene of the present invention is not limited to the natural DNA base sequence, but includes other DNA base sequences encoding the amino acid sequence of the insecticidal protein revealed by the present invention.

In addition, according to gene recombination technology, artificial mutation is made at a specific site of basic DNA without changing the basic characteristics of the one encoded by the DNA or so as to improve the characteristics. be able to. With respect to a gene having a natural base sequence or a gene having a base sequence different from that of the natural gene provided by the present invention, the gene is equivalent to the natural gene by artificially inserting, deleting, or substituting. Improved properties are possible and the invention includes such mutated genes.

A suitable vector for the insecticidal protein gene of Bacillus thuringiensis islaerensis of the present invention, for example, the expression vector pUC18 or pUC19 (Pharmacia), which holds the 1ac promoter, and the tac promoter and rrnB ribosomal RNA, which are strong promoters of E. coli. Connect to expression vector pKK223-3 with terminator (Pharmacia), expression vector pDR720 with trp promoter (Pharmacia), inducible expression vector pPL-Lambda (Pharmacia), Bacillus subtilis vector pC194, pVB110, etc. As a result, it is possible to construct an expression vector for producing an insecticidal protein of Bacillus thuringiensis islaelensis strain by microorganisms such as Escherichia coli and Bacillus subtilis. By introducing the expression vector carrying the insecticidal protein gene of the present invention into a host microorganism such as Escherichia coli JM109 strain (Pharmacia) or Bacillus subtilis, a microorganism producing an insecticidal protein can be obtained.

The thus-produced transformed microorganism is cultured under appropriate conditions (for example, medium; 10 g of tryptone, 5 g of NaCl, 5 g of yeast extract, 1 g of glucose (pH 7.2), 1 culture of distilled water, and culture). Temperature; 37 ° C., culture time; 8-24 hours,
It is possible to mass-produce the insecticidal protein by culturing under culture conditions; shaking culture). Isolation of the insecticidal protein after culturing, for example, by crushing the cells with ultrasonic waves and centrifuging, to easily concentrate the aggregate consisting of the insecticidal protein,
It can be performed by an operation of collecting. Further, not only host-vector systems of Escherichia coli and Bacillus subtilis, but also host-vector systems of yeast, Pseudomonas or actinomycetes can be used, and mass production of insecticidal proteins utilizing the characteristics of each host-vector system is possible. You can do it. The insecticidal protein or the treated product of bacterial cells thus obtained is useful as an insecticide.

The present invention will be described in more detail with reference to the following examples.
The present invention is not limited to the following examples, and can be modified in a usual manner in the technical field of the present invention.

Example 1. Cloning of insecticidal protein gene of Bacillus thuringiensis islaerensis Step 1 Preparation of plasmid DNA Bacillus thuringiensis Islaerensis HD52
2 strains (US Department of Agriculture (USDA) deposited strain Goldberg ONR6
0) 100 ml of PY medium (tryptone (Sigma) 10 g, Na
Cl (Hanai Chemical) 5 g, yeast extract (Sigma) 5 g were added, and the mixture was cultivated at 30 ° C. overnight in a medium adjusted to 1 with distilled water and adjusted to pH 7.0), and collected by centrifugation at 3,500 rpm for 15 minutes. Birnboim et al. [Nucleic Acids Res. 7 : 1513-152
3 (1979)], prepare plasmid DNA, and use CsCl-EtB
r Purified by equilibrium density gradient centrifugation. Furthermore, the prepared plasmid DNA was added to 31,000 r in a 5-25% sucrose gradient.
Size fractionation was performed by centrifugation at pm for 2.5 hours to collect a large plasmid fraction. As a result of 0.5% agarose electrophoresis, the molecular weight of the plasmid contained in this fraction was at least 63 Md or more.

Step 2 Preparation of gene bank To about 1 μg of the prepared giant plasmid DNA, 10 units of restriction enzyme Hind III (Takara Shuzo) was added, and 20 μl of Hind III reaction solution [10 mM Tris-acetic acid (pH 7.5), 10 mM MgCl ₂ , 50 mM Na ₂
Cl] and reacted at 37 ° C. for 2 hours. Then, an equal volume of TE buffer saturated phenol [TE is 10 mM Tris-hydrochloric acid (pH 8.0) -1 m
M EDTA] is added, phenol extraction is performed, and 12,000 rpm / 5
Centrifugation was performed and the upper layer fraction was collected. Repeat the treatment with an equal volume of water-saturated diethyl ether twice, add 2 volumes of cold ethanol and 3M CH ₃ COONa (pH 7.0) to a final concentration of 0.3M CH ₃ COONa (pH 7.0), It was left at 20 ° C for 30 minutes. The precipitate was collected by centrifugation at 12,000 rpm for 5 minutes, washed with 70% ethanol, dried to dryness, and 5 μl.
Suspended in TE.

In addition, all the ethanol precipitations performed below followed this method. Next, the method of Birnboim and DoLY (Nucleic
Acids Res. 7 (1979) 1513-1523) by E. coli HB101 / pB
1 μg of E. coli cloning vector pBR322 prepared from R322 and purified by CsCl-EtBr equilibrium density gradient centrifugation
On the other hand, 10 units of restriction enzyme Hind III was added, and similarly reacted in 20 μl of Hind III reaction solution at 37 ° C. for 5 hours, phenol extraction, diethyl ether treatment, and ethanol precipitation were performed to recover DNA. Suspended in. 1
10 μl of calf intestinal alkaline phosphatase (equivalent to 4 units, Boehringer) was added to 0 μl of Hind III-cut pBR322, and a final volume of 20 μl of alkaline phosphatase reaction solution (50 mM Tris-HCl buffer pH 9.0, 1 mM MgCl ₂ , 0.1 mM Zn Zn) was added.
Cl ₂ and 1 mM spermidine) at 37 ° C. for 1 hour, phenol extraction was performed twice, and ethanol precipitation was performed.
A was collected and suspended in 20 μl of TE. Hind III derived from the plasmid of Islaelensis strain prepared in this manner
The DNA was cleaved with 5 μl and treated with alkaline phosphatase. pBR
After mixing 2 μl of 322 Hind III DNA fragments, 1 μl of T4 DNA ligase (Takara Shuzo, 10 units) was added to give a final volume of 20 μl.
T4 DNA ligase reaction solution (66 mM Tris-HCl buffer (pH
7.5), 6.6 mM MgCl ₂ , 10 mM DTT, 1 mM ATP) at 16 ° C
And incubated for 12 hours. After reaction, ligase reaction solution
For 20 μl, the method of Cohen et al. (Proc. Natl. Acad, Sc
200 μl prepared by i.USA, 69 , (1972) 2110-2114)
Calcium-treated Escherichia coli HB101 was added, left at 0 ° C. for 30 minutes, and then heat-treated at 42 ° C. for 60 seconds. Then, 0.3 ml of double concentration L medium was added and incubated at 37 ° C. for 1.5 hours. After the incubation, 0.1 ml was plated on an L plate medium containing ampicillin at a final concentration of 50 μg / ml (a plate medium solidified by adding 1.2% agar to the L medium) and cultured at 37 ° C. overnight.

Step 3 Screening by the Eisa method Among the resulting ampicillin-resistant colonies, a plate containing tetracycline-sensitive colonies was treated with chloroform, and the colonies were transferred to Biodyne A filter (Japan Peel Co., Ltd.) [Analytical Bioche].
mistry, 97 , p153-157 (1979)], the colonies were fixed and blocked. However, at the time of blocking, bovine serum albumin (Sigma) was used instead of normal rabbit serum. Next, the soluble fraction of all-crystal single protein obtained from the Islaelensis 4Q1 strain (Gorldberg ONR60A, Stock Center Depository strain, Ohio State University Bachuras Genetics) was used as an antigen to inject rabbits, and then anti-Islaelensis from serum was injected. Crystal protein IgG was prepared. The IgG was bound to peroxidase by the maleimide method (J. Applied Biochemistry 4 p41-57 (1982)] 0.5% BSA
PBS containing 50 ng / ml peroxysidase-conjugated IgG
Buffer solution (0.125M NaCl, 0.02M dipotassium phosphate (pH 7.
The treated filter was immersed in 2)) overnight at 4 ° C., washed with PBS buffer and air-dried, and the filter was PBS buffer containing 0.5 mg / ml 3,3′-diaminobenzidine and 0.05% H ₂ O _2. The positive colonies were screened by immersing them in water and allowing them to develop color. As a result of screening about 1,000 ampicillin-resistant tetracycline-sensitive colonies,
The positive clone HB101 / pBGH3 was isolated. When a restriction map was constructed, pBGH3 retained the 7.7 Kb Hind III fragment.

2. Construction of Expression Plasmid pUCH3 Step 1 Preparation of 5.2 Kb Hind III Fragment Birnbiom and Dol from isolated positive clone HB101 / pBGH3
According to the method of y, plasmid DNA was prepared and purified by CsCl-EtBr equilibrium density gradient centrifugation. About 10 μg of the plasmid pBGH3 DNA was added with 10 units of the restriction enzyme Hind III and reacted in 50 μl of the Hind III reaction solution at 37 ° C. for 1 hour. The reaction solution was applied to 0.7% agarose gel (Agarose Type VI, Sigma) containing 1 μg / ml ethidium bromide,
Agarose electrophoresis was performed. After the electrophoresis, the portion corresponding to the 5.2 Kb Hind III DNA fragment was cut out under an ultraviolet lamp, the band was recovered by electroelution, suspended in TE, and TE saturated phenol was added to perform phenol extraction. 12,000
After centrifugation at rpm for 5 minutes to separate the upper layer, the DNA recovered by ethanol precipitation was suspended in 20 μl of TE.

Step 2 Construction of E. coli expression plasmid pUCH According to the method of Birnboim and Doly, E. coli expression vector pUCH
C13 DNA was prepared and purified by CsCl-EtBr equilibrium density gradient centrifugation. 10 to about 1 μg of plasmid pUC13 DNA
After adding the unit restriction enzyme Hind III and reacting in 20 μl of Hind III reaction solution at 37 ° C for 5 hours, phenol extraction, ether extraction, and ethanol precipitation were performed to recover DNA and
It was suspended in TE. This DNA solution was reacted with calf intestinal alkaline phosphatase (4 units, Boehringer) in 20 μl of alkaline phosphatase reaction solution at 37 ° C. for 1 hour to perform alkaline phosphatase treatment. After the reaction, phenol-chloroform treatment was performed twice, and the supernatant was collected.
The DNA was recovered by ethanol precipitation and suspended in 20 μl of TE. Next, 5 μl of the Hind III fragment prepared in step 1 and Hind III cleaved pUC13 2 μl treated with alkaline phosphatase
1 μl of T4 DNA ligase (Takara Shuzo, 10 units) was added, and the final volume of 20 μl of T4 DNA ligase reaction solution (6
6 mM Tris-HCl buffer (pH 7.5), 6.6 mM MgCl ₂ , 10 mM
Incubated for 12 hours at 16 ° C in DTT, 1 mM ATP).
20 μl of the obtained ligase reaction solution was added to the method of Hanahan et al.
nahan, DJ Mol. Biol., 166 (1983) 557-580). Add 100 μl of E. coli JM109 strain competent cell, incubate at 0 ° C. for 30 minutes, heat-treat at 42 ° C. for 90 seconds, add 0.8 ml of L broth liquid medium, 37 ° C.
After incubating for 1.5 hours, the plate was plated on L plate medium containing ampicillin at a final concentration of 50 μg / ml, X-gel.0.004%, 0.1 mM IPTG. Plasmid DNA was prepared from the obtained ampicillin-resistant white colonies by the method of Birnboim and Doly, and cleaved with restriction enzyme Hind III.
A clone carrying the 5.2 Kb Hind III fragment was isolated and cloned into pUCH
I named it 3.

3. Expression of insecticidal protein gene in Escherichia coli 50 μg / ml ampicillin, 1 mM IPTG (isopropyl-β-D-galactoside) of E. coli recombinant JM109 / pUCH3 strain
Containing 200 ml of L broth medium (1 g of distilled water in 10 g of tryptone (Sigma), 5 g of yeast extract, 5 g of Na.
28 in Cl and 1 g glucose, pH 7.2 medium)
After culturing overnight at ℃, after centrifugation, suspended in 10 ml of 0.1 M 2-mercaptoethanol solution and sonicated for 5-10 minutes. 10 to the solution
Alkali lysis was performed by adding ml of 0.2 M glycine-NaOH (pH 10.5) and incubating at 37 ° C. for 3 hours.
Next, adjust the pH to 7.5 with 1N hydrochloric acid, centrifuge, and then collect the supernatant. Add ammonium sulfate to 50% saturation, place in ice for 1 hour, and centrifuge to wet About 1.
6 g of protein precipitate was obtained. Was suspended in a solution 4ml of precipitate 0.05M2- mercaptoethanol -0.1M glycine -NaOH (pH10.5), 136mM NaCl, 2.7mM KCl, 81mM Na 2 HPO 4, 1.5mM KH 2
Dialyze against a buffer solution of PO ₄ , collect the centrifugal supernatant,
The crude cell extract was used. 200 μg protein was electrophoresed on 12.5% TSDS-polyacrylamide gel (Laemmli method, Natur
e (London) 227 680-685 (1970)] and then T
owbin et al. [Proc. Nat. Acad. Sci. USA 76 4350-54 (197
Western blotting was performed according to 9)]. The anti-Israelensis insecticidal protein IgG used was prepared using the total insecticidal protein crystal fraction obtained from the Islaelensis 4Q1 strain as an antigen. As a result, the molecular weight is 135 to 145.
It was confirmed that the KDa protein was produced in the JM109 / pUCH3 strain.

After mixing 10 μl of 1% latex beads (Sigma: 0.8 μm) with 1 ml of 0.1 M Tris-hydrochloric acid (pH 7.4) containing bacterial protein of each protein concentration, the mixture was allowed to stand at room temperature for 1 hour to Were adsorbed on the beads. Culex of this solution
Insecticidal activity was measured by adding to 20 ml distilled water containing 20 pipiens larvae. As a result, JM109 / pUC1 used as a control
In 240 μg / ml of bacterial protein solution of 3 strains, dead insects were not observed even after 24 hours and 48 hours, but in JM109 / pUCH3 strain, 3
Dead insects were observed at around 0 μg / ml.

The LC ₅₀ value was measured and found to be 39 μg / ml after 24 hours and 32 μg / ml after 48 hours. From the above, it was revealed that the JM109 / pUCH3 strain produced a 135 to 145 KDa protein having insecticidal activity on C. pipiens .

4. Determination of the nucleotide sequence of the Islaelensis insecticidal protein gene To confirm the structure of the Islaelensis 135-145KDa insecticidal protein gene, various plasmids from plasmids pBGH3 and pUCH3 were used.
Prepare the cloning vector M13mp18 by adjusting the restriction enzyme fragments.
And recloned into M13mp19. pUCH3 and various M1
For the three subclones, the deletion method by Y. Perron et al. Exonuclease III and VII (Y. Perron et.al. Gene,
33 (1985) 103-119), after obtaining various deletion clones,
The nucleotide sequence was determined according to the M13 Sequencing Kit (Takara Shuzo). That is, the obtained subclone plasmid
Suspend DNA in 18 μl TE (pH8.0) and then 2 μl 2N NaOH
Was added, and the mixture was allowed to stand at room temperature for 5 minutes, 8 μl of 5.0 M ammonium acetate was added, and 100 μl of cold ethanol was added to carry out ethanol precipitation. Centrifuge at 12,000 rpm for 5 minutes, collect the precipitate, wash with 70% ethanol, dry to 0.5 pmole / 5
It was dissolved in distilled water so as to have a volume of μl or more. 5 μl of prepared alkaline-denatured plasmid DNA or phage single-stranded DNA
(5 pmol or more), 1.5 μl of 10 times concentrated Klenow buffer (Takara Shuzo), 1 μl of primer DNA (Takara Shuzo), 4.5 μl of distilled water were added to bring the total volume to 12 μl. Let stand for a minute. 2 μl of [α-
³² p] dCTP (400Ci / mmol, Amarsh Japan
am, Japan) and 1 μl of Klenow fragment enzyme (Takara Shuzo, 2 units) were added and mixed. Added portion 3.5μl of the mixture into four 2μl of dNTPs + ddNTPs mixture (Takara Shuzo), and left for 15 minutes at 39 ° C., add more 1 mM dNTPs _s of 1 [mu] l for 15 minutes, allowed to stand at 39 ° C.. Finally, 6 μl of 95% formamide dye (0.1% bromphenol blue and 0.1%
(Including xylene cyanol) was added according to the conventional method.
% Acrylamide-urea gel to prepare the above reaction solution 2
4 μl was applied and electrophoresed at 3,000 V for 7 to 24 hours. After the gel was dried, it was sandwiched between X-ray films and exposed to light, and then the base sequence was read. The determined nucleotide sequence is shown in FIG. The structural gene portion of the insecticidal protein gene of the Bacillus thuringiensis islaerensis strain HD522 of the present invention is a nucleotide ATG from the 461st position to the 463rd position in the base sequence of FIG.
It had a coding region of 3408 bases starting at (start codon) and ending at stop codon TGA, and encoded 1136 amino acids. As a result, the molecular weight of the encoded insecticidal protein is estimated to be 124 KDa.

[Brief description of drawings]

FIG. 1 shows a restriction enzyme map of the 124 KDa insecticidal protein gene of Bacillus thuringiensis islaerensis HD522. White arrows indicate coding regions for insecticidal protein genes. Black arrows indicate the dideoxy strategy for nucleotide sequencing. C is Cla I, X is Xba I, E is Eco RI, Pv is Pvu II, P is Pst I, H is Hin
d III and ISRH3 represent the Islaelensis 124 KDa insecticidal protein gene. FIG. 2 shows the entire nucleotide sequence of the gene for the insecticidal protein of the Bacillus thuringiensis islaerensis HD522 strain of the present invention. The structural gene portion of the insecticidal protein gene has a coding region of 3408 bases starting from the 461st to 463th bases ATG (start codon) of the base sequence and ending at the stop codon TGA, and encoding 1136 amino acids. . The upper row shows the nucleotide sequence and the lower row shows the amino acid sequence deduced therefrom. FIG. 3 shows the construction method of the expression plasmid pUCH3. Black box contains 5.2 Kb Hin containing insecticidal protein gene
dIII fragment, the white box represents an unknown cloned DNA fragment flanking the insecticidal protein gene. The solid line indicates the vector. Ap ^r represents the ampicillin resistance gene.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12P 21/02 C 9282-4B (C12P 21/02 C12R 1:19)

Claims (9)

[Claims] 1. An insecticidal protein gene of Bacillus thuringiensis islaelensis strain specified by the amino acid sequence shown in FIG. 2 or a DNA containing the same. 2. The gene according to claim 1, characterized in that the gene is specified by the DNA sequence shown in FIG. 2, or a DNA containing the gene. 3. A plasmid containing an insecticidal protein gene of Bacillus thuringiensis islaerensis strain specified by the amino acid sequence shown in FIG. 4. The plasmid according to claim 3, wherein the gene is specified by the DNA sequence shown in FIG. 5. The plasmid according to claim 3, which is named pBGH3 or pUCH3. 6. A bacterium into which a plasmid containing the insecticidal protein gene of Bacillus thuringiensis islaelensis strain specified by the amino acid sequence shown in FIG. 2 has been introduced. 7. The bacterium according to claim 6, wherein the gene is specified by the DNA sequence shown in FIG. 8. The bacterium according to claim 6, wherein a plasmid designated as pBGH3 or pUCH3 has been introduced. 9. The bacterium according to claim 6, which is named Escherichia coli HB101 / pBGH3 or JM109 / pUCH3.