JPH074233B2 - Bacillus subtilis producing insecticidal protein and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a Bacillus subtilis transformant strain produced by a gene recombination technique, which produces a protein having no pathogenicity or natural growth ability and high insecticidal activity, and its fungus. Regarding the manufacturing method of the body.

Bacillus thuringiensi
s, hereinafter abbreviated as BT), a crystalline insecticidal protein formed in the spore sac has a strong insecticidal action against Lepidoptera insects and is harmless to animals and plants including humans, It has been widely used in Europe and America as an agricultural and horticultural insecticide.

However, an insecticide containing an insecticidal protein as an active ingredient (hereinafter referred to as BT
The agent is called), because it contains BT cells and spores, it was feared that in Japan the BT bacteria would grow after spraying the insecticide, causing serious damage to the sericulture.

In order to prevent this damage, live cells and spores in the BT agent have been sterilized by heat treatment, chemical treatment, etc., but violent sterilization conditions must be used, and insecticidal activity is reduced by denaturation of insecticidal protein. However, in order to maintain the insecticidal activity, a combination of heat treatment and chemical treatment is required, which causes an increase in manufacturing cost due to an industrially complicated treatment process.

Furthermore, BT bacterium itself is said to be closely related to Bacillus cereus, which has pathogenicity for humans (protein / nucleic acid / enzyme 29: 444, 1984).

Under these circumstances, in Japan, it is not considered preferable to use the BT bacterium itself or its preparation as an insecticidal protein.

However, in recent years, genetic recombination techniques have been developed, and it has become possible to breed microorganisms having desired and useful properties.

The present inventors have attempted to create a Bacillus subtilis strain producing an insecticidal protein by a gene recombination method in order to overcome various drawbacks of the insecticide containing the BT insecticidal protein described above, and as described below, it is high. The present invention was completed by creating a Bacillus subtilis strain having activity and insecticidal activity.

In addition, as a result of subjecting the bacterial cells obtained by culturing the transformant strain and the parent strain BT Sotohto strain to an insecticidal effect test, the transformed strain shows an excellent insecticidal effect as compared with BT bacteria, Proved the effectiveness of.

Bacillus subtilis has no pathogenicity to humans since it has been used for the production of natto since ancient times.
Since it has been used for the production of nucleic acids, enzymes, etc., it has a long experience in use as an industrial microorganism.

Bacillus subtilis has been elucidated for its genetic properties next to Escherichia coli, and various mutant strains have been constructed.

Therefore, the toxicity to silkworm and sterilization of spores, which are problems in the BT preparation, can be easily overcome by producing an insecticidal protein by using spore-deficient Bacillus subtilis or auxotrophic Bacillus subtilis. For example, spore-deficient Bacillus subtilis that produces an insecticidal protein does not need to be sterilized with high heat, and therefore does not cause a decrease in insecticidal activity.

In addition, since auxotrophic Bacillus subtilis that produces insecticidal proteins does not exist in the natural world in the form that can be utilized by Bacillus subtilis even if it is not sterilized, it can grow even if it is sprayed in the natural world. It cannot be done, and it quickly dies.

Therefore, the results of the present invention have a great significance in terms of expanding the applications of microbial insecticides in the future.

The present invention will be described in detail below.

The intracellular insecticidal protein gene of BT bacterium used in the present invention is a gene consisting of a region encoding the amino acid sequence of the intracellular insecticidal protein produced by BT bacterium and a promoter region, and the Bacillus thuringiensis sotto (American type culture A gene derived from a collection (abbreviated as ATCC) conservation number 19270) can be used.

Any vector can be used as a vector that binds to these intracellular insecticidal protein genes as long as it can be propagated in Bacillus subtilis, but the plasmid pUB110 is used because of its well-known structure and widespread use. It is convenient.

The intracellular insecticidal protein gene is ligated to the vector cleaved with an appropriate restriction enzyme using a DNA-binding enzyme. This binding method does not interfere with the usual gene recombination method.

Next, the obtained recombinant DNA molecule is introduced into Bacillus subtilis to obtain a Bacillus subtilis transformant containing the recombinant plasmid.

The recombinant DNA molecule is usually introduced into Bacillus subtilis by the protoplast method (Chang, S., and Cohen, SN ,: Mol.Gen, Genet, 168).
111 (1978)) or competent cell method (Anagnos
topoulos, C., and Spizizen, J.: J.Bactriol. 81 , 741 (196
1)).

The transformant can be easily selected by using a marker (eg, antibiotic resistance) contained in the vector.
By culturing the transformant thus obtained in an ordinary medium used for culturing microorganisms, Bacillus subtilis producing an insecticidal protein can be easily obtained.

For Bacillus subtilis into which a recombinant DNA molecule is introduced, various mutant strains of Bacillus subtilis can be selected depending on the purpose of use. For example, in order to produce an insecticide consisting of an insecticidal protein derived from Bacillus subtilis that cannot proliferate in nature and rapidly dies, an auxotrophic mutant of Bacillus subtilis (ATCC33608), a spore-deficient strain (BGSC 1S1), or two properties are required. A mutant strain (ATCC35148) having the same is used.

By using these Bacillus subtilis mutant strains, it is possible to obtain a microbial insecticide having high activity and safety because the bacterial cells containing the insecticidal protein do not need to be heat-treated or treated with chemicals, and the cost is low. .

The transformant strain containing the recombinant DNA molecule produces insecticidal protein, as described below, by an immune double-diffusion test method using an antibody against the intracellular insecticidal protein derived from the BT bacterium, and also in larvae of Lepidoptera insects. It is clearly shown by the insecticidal activity test used.

Particularly noteworthy is that Bacillus subtilis producing the insecticidal protein produced by the gene recombination method showed stronger insecticidal activity than the parent strain BT bacterium from which the insecticidal protein gene was derived (first to (Table 3).

This shows that Bacillus subtilis producing the insecticidal protein obtained by the present invention is extremely useful as an active ingredient of insecticides.

Examples and test examples are shown below.

Example 1 (Preparation of recombinant DNA molecule containing intracellular insecticidal protein gene of Bacillus thuringiensis sotto) Bacillus thuringiensis sotto (ATCC19270)
3) using Pen Assay Broth (Difco) 20
After culturing at 0 ° C for 15 hours, the cells were harvested and the method of Hansen (J. Bac
The plasmid was prepared according to teriol, 135 , 227 (1978).

50 μg of this plasmid was added to the restriction enzyme Pst I (Takara Shuzo) 50
Cleavage was performed by incubating the unit for 2 hours at 37 ° C. The composition of the reaction system is 10 mM Tris-HCl buffer (pH
7.5), 10 mM MgCl ₂ , 50 mM NaCl, 1 mM dithiothreitol.

The DNA fragment cleaved by this reaction was purified by phenol extraction and ether extraction according to a conventional method, and recovered by ethanol precipitation.

The resulting Pst I-cleaved DNA fragment (donor DNA fragment) was then completely cleaved with the restriction enzyme Pst I and then ligated to the plasmid pBR322 (ATCC37017) in which the terminal phosphate was hydrolyzed with Escherichia coli alkaline phosphatase.

The reaction using of T ₄ ligase to bond donating DNA fragment 20μ
g, pBR322 10 μg was used. The composition of the reaction system is 10 units of T ₄ ligase (Takara Shuzo), 6.6 mM Tris-HCl buffer (p
H7.6), 6.6 mM MgCl ₂ , 10 mM dithiothreitol, 1 mM A
It is TP. The reaction was carried out at 4 ° C for 15 hours.

After the reaction, an amount corresponding to 0.1 μg DNA was extracted from the reaction solution and subjected to 0.7% agarose electrophoresis. As a result, it was confirmed that the vector bBR322 and the donor DNA fragment were bound to form a recombinant DNA molecule. It was

The recombinant DNA molecule thus obtained was used to transform E. coli HB 101.
Strain (ATCC33694) with calcium treatment competent cell method (Mandel, M., and Higa, A ,: J. Mol. Biol. 53 159 (1970))
Introduced according to.

All resulting transformants were, radioimmunoprecipitation assays using antibodies of the insecticidal protein of Bacillus Churingenshisu Sotto (Ehrlich et al.:Cell 13, 681 ( 1978)) of the sorted strain was positive by Select one strain (# 801) and use the method described in the document ("Molecular Cloning" p86.Cold Spri
ng Harbor Laboratory) to prepare a plasmid.

After cutting the obtained plasmid with the restriction enzyme Pst I, 0.7
As a result of examination by% agarose gel electrophoresis, it was found that the plasmid (designated pSP801) was a recombinant DNA molecule in which a donor DNA fragment of about 6.6 Kb was inserted into the Pst I cleavage site of vector pBR322. did.

The plasmid pSP801 has two Hpa I cleavage sites. Therefore, after obtaining pSP801 Hpa I-degraded fraction, this was re-ligated by the method using T ₄ DNA ligase to construct a smaller recombinant plasmid containing the insecticidal protein gene.

The plasmid prepared by recombination was introduced into Escherichia coli HB101 strain by the above-mentioned calcium treatment competent cell method, and the plasmid was prepared from the obtained transformant by the above-mentioned conventional method. A plasmid pHP1 into which was introduced was obtained.

100 μg of this plasmid pHP1 was digested with the restriction enzyme Pst I under the above reaction conditions, and then electrophoresed at 0.7% agarose gel electrophoresis for 40 V for 5 hours.

After electrophoresis, cut out the 5.6 Kb gel, purify by phenol extraction and ether extraction, and precipitate by ethanol.
The DNA was recovered.

The recovered DNA was treated with TE buffer (10 mM Tris-HCl buffer (pH 8.
0, 1 mM EDTA) 100 μ, and then 0.1 μg of DNA was extracted from the solution, and 0.7% agarose electrophoresis confirmed that the recovered DNA was purified as a fragment having a size of about 5.6 Kb. The following experiments were carried out.

Example 2 (obtaining auxotrophic transformant by ligation of vector plasmid and donor DNA fragment and transformation into Bacillus subtilis) 50 μl of the donor DNA fragment obtained in Example 1 was used as Klenow fragment 10 of E. coli DNA polymerase I. Using the unit, 30 ℃
A blunt end was obtained by the reaction for 30 minutes.

The composition of the reaction system is 0.1 mM dNTPs, nick translation buffer (50 mM Tris-HCl buffer (pH 7.2), 10 mM Mg).
SO ₄ , 0.1 mM dithiothreitol, 5 μg / ml bovine serum albumin).

The product of the above reaction was purified by phenol extraction and ether extraction, and recovered by ethanol precipitation.

The recovered DNA fragment was then completely cleaved with the restriction enzyme Pvu II, and the resulting plasmid pUB110 (ATCC37015) obtained by hydrolyzing the terminal phosphate ester with Escherichia coli alkaline phosphatase was used.
₄ Ligated with DNA ligase. In the reaction, 20 μg of recovered DNA (donor DNA fragment) and 10 μg of pUB110 were used.

Transformation with the obtained recombinant DNA molecule is performed by the protoplast method of Chang et al. (Chang, S., and Cohen, SN: Mol.Gen.
Genet. 168 111 (1978)). The protoplast regeneration medium contains kanamycin at a final concentration of 150 μg / ml.
Was added. The auxotrophic Bacillus subtilis 1A 510 strain (BGSC preserved strain) was used as the host bacterium for cloning.

Plasmids were prepared from all kanamycin resistant strains obtained by transformation.

The plasmid was prepared by culturing the kanamycin resistant strain in 20 ml of Pen assay medium (Difco) at 37 ° C. for 15 hours,
The cells were collected and the collected cells were treated with 50 mM Tris-HCl buffer (pH 7.
5), washed with 5 mM EDTA and 50 mM NaCl, and then subjected to an alkaline method (Birnboim, HC, and Poly, J.: Nucleic Acid Res. 7 151).
3 (1979)).

The obtained plasmid was used for restriction enzymes Bcl I, Cla I, EcoR V,
It was cleaved with Hind III, Kpn I, Pvu II, Sac I and Xba I, and the presence or absence of a donor DNA fragment was examined.

Next, the presence or absence of production of the intracellular insecticidal protein in the strain having the plasmid in which the donor DNA fragment was confirmed to be inserted was examined by the immunodouble diffusion test method (Ouchterlony, Oe., And Nilsson,
LA “Hand book of Experimental Immunology (ed.Wei
r, PM) p1 (1973)).

As a control, the host strain 1A510 having pUB110 and the Bacillus thuringiensis Sotto that is a DNA donor strain were used.

For culturing, Bacillus subtilis host strain and transformant were sporulation medium (0.8% nutrient broth (manufactured by Difco), 1 mM)
MgSO ₄ , 13.4 mM KCl, 0.01 mM MnCl ₂ , 10 ^-6 M FeSO ₄ , 10 ^-3
Bacillus thuringiensis Sotto was prepared using M Ca (NO ₃ ) ₂ ) in GYS medium (0.2% (NH ₄ ) ₂ SO ₄ , 0.2% Yeast).
extract, 0.05% K ₂ HPO ₄ , 0.1% Glucose, 0.03% MgS
O ₄ , 0.008% CaCl ₂ , 0.005% MnSO ₄ , pH 7.3)
Shaking culture was carried out at 0 ° C. for 3 days.

At this time, kanamycin was added to the sporulation medium of Bacillus subtilis at a final concentration of 5 μg / ml. After culturing, after confirming the sporulation of each strain, the cells were collected by centrifugation, and the collected cells were lysed with lysozyme and washed with 1 M NaCl, and the precipitate was collected by centrifugation. The precipitate obtained was 15 mM NaO.
After suspending in H and incubating at 25 ° C. for 10 hours, it was centrifuged and the spore-free supernatant was recovered.

The obtained supernatant was used as a test sample, and an immune double diffusion test was carried out by the method described above using an antiserum against the intracellular insecticidal protein of Bacillus thuringiensis Sotto.

As a result, it was revealed that this Bacillus subtilis transformant strain produces a Bacillus thuringiensis sottotype insecticidal protein.

The presence of crystalline protein was confirmed by observing this transformant with a phase-contrast electron microscope.

From the above results, it was confirmed that the auxotrophic Bacillus subtilis transformant strain produces the same insecticidal protein as Bacillus thuringiensis Sotto. Therefore, this transformant was designated as HBT1.
I named it.

Example 3 (Acquisition of Bacillus subtilis auxotrophic spore deficient transformant producing insecticidal protein) A plasmid (designated as pHBT1) was purified from the HBT 1 strain obtained in Example 2 and Bacillus subtilis auxotrophic spore deficient strain MT- SP31 shares (F
ERM BP-1034) was introduced.

Transformation of Bacillus subtilis MT-SP31 strain with 50 μg of plasmid pHBT1 was carried out by the competent method (Anagnostopoulos, C., and S.
pizizen, J .; J. Bacteriol. 81 , 741 (1961).

The culture solution (1 ml) after the plasmid was taken up was plated on TBAB medium containing 5 μg / ml kanamycin (manufactured by Dafco) in 100 μm increments.

Plasmids were prepared from all of the obtained kanamycin resistant strains and the restriction enzymes Bcl I, Cla I, Ecor V, Hind III, Kpn
Cleavage was performed using I, Pvu II, Sac I, and Xba I, electrophoresis was performed, and the digestion pattern was compared with that of the similarly digested plasmid pHBT I.

As a result, a strain having a plasmid having a cleavage pattern matching that of pHBT1 was obtained.

This transformant strain was subjected to an immuno-diffusion test using an antiserum of an intracellular insecticidal protein of Bacillus thuringiensis Sotto according to the method described above, and the positive strain was treated with SPBT 1
I named it.

This strain was cultured in the aforementioned sporulation medium, and the obtained bacterial cells were assayed by the method of Example 2 and Test Example 1, and as a result, a protein having insecticidal activity was produced, but the sporulation ability was defective. I confirmed that. Test Example 1 (Insecticidal activity of Bacillus subtilis transformant cells obtained in Example 2) As in Example 2, Bacillus subtilis was used in a sporulation medium and Bacillus thuringiensis Sutto was in a GYS medium. Subtilis HBT1, Bacillus thuringiensis Sotto, Bacillus subtilis 1A510 (pUB110) 30
After culturing for 3 days at 30 ° C and collecting the cells by centrifugation, the obtained cells were suspended in the same amount of physiological saline as the medium, washed, and then centrifuged again, and the precipitated fraction was lyophilized.

Using this freeze-dried sample as a test sample, diamondback moth larvae,
The insecticidal effect on the caterpillar larva and the larvae of the larvae, Tanagingin vulgaris, was tested. The test method was according to Tsuchiyama's method (Journal of the Japanese Society of Applied Entomology, Vol. 22, No. 4, pp. 234-237 (1978)).
That is, 200 mg of the test sample was suspended in 100 ml of deionized water, homogenized, and then diluted with deionized water to prepare a bacterial solution having a predetermined concentration. After 0.4 ml of this bacterial solution was uniformly mixed with 4 g of an artificial sample, the larva of the test insect was fed and the mortality rate was examined.

From the above results, it is clear that the insecticidal protein-producing Bacillus subtilis obtained by the gene recombination method showed a higher insecticidal activity than the parent strain Bacillus thuringiensis Sotto.

Test Example 2 (Insecticidal activity of Bacillus subtilis transformant cells obtained in Example 3) GYS medium was used for Bacillus subtilis SPBT1, Bacillus subtilis MTSP31 (pUB110) and Bacillus thuringiensis soot obtained in Example 3. After culturing, each strain was tested for insecticidal effect in the same manner as in Test Example 1. As a result, as shown in Tables 4, 5, and 6, the insecticidal protein-producing Bacillus subtilis of the present invention showed higher insecticidal activity against Lepidoptera insects than the parent strain Bacillus thuringiensis Sotto.

[Brief description of drawings]

The drawings illustrate the procedure for obtaining the Bacillus subtilis transformants shown in the examples.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12R 1:07) (C12N 15/32 C12R 1:07) (C12P 21/02 C12R 1:07) (56) References JP-A-61-5098 (JP, A)

Claims (2)

[Claims] 1. A bacillus Bacillus thuringiensis sotto protein represented by the following DNA sequence is linked to a vector capable of growing in Bacillus subtilis, and then introduced into a Bacillus subtilis spore-deficient strain. A transformant of Bacillus subtilis that produces an insecticidal protein having an insecticidal action against Lepidoptera insects. 2. The Bacillus subtilis transformant according to claim 1, wherein the Bacillus subtilis spore deficient strain is an auxotrophic spore deficient strain.