JPH0611229B2 - Bacillus subtilis producing anti-insect protein toxin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Purpose of the invention "Industrial field of application" The Bacillus subtilis of the present invention has an extremely excellent insecticidal effect against larvae of Lepidoptera insects (moths and butterflies) effective as pesticides. It is a very effective use in the agrochemical manufacturing industry because it produces a crystalline toxin that does not form spores, and by doing so does not form spores, it contributes significantly to the agricultural field as a safe agricultural chemical. Is.

"Prior art" Bacillus thuringiensis var.
Intracellular inclusions formed during the sporulation stage during the life cycle of us thuringiensis variety krustaki) are called crystal toxins and are toxic to many lepidopteran insect larvae. Widely used. Microbial pesticides containing crystalline toxin as an active ingredient are mainly cultured by culturing Bacillus thuringiensis var. Kurustaki HD-1 and the fermentation product, crystalline toxin, and live spores mixed therewith are not separated but directly formulated. It is done by becoming.

"Problems to be solved by the invention" The crystalline toxin produced by Bacillus thuringiensis var. Kurustaki, which is widely used in the production of microbial pesticides, shows strong toxicity not only to pests but also to silkworms. It is considered extremely dangerous to use a formulation containing viable bacteria and spores that have fertility in the field. That is, when sprayed with a preparation containing live spores that have strong resistance to live bacteria, chemicals, heat, etc., it is easily conjectured that they will germinate / proliferate and cause great damage to sericulture. It

Furthermore, it has been clarified that Bacillus thuringiensis var. Kurustaki exerts the same pathogenicity as Bacillus cereus, which is known to have pathogenicity to humans and livestock ( Takehisa Akiyama et al. Kitasato medicine 14 , 236-247, 1984, Nobutaka Osawa et al. Kitasato medicine 14 , 320-329, 1984), and the hemolytic poison produced by Bacillus thuringiensis var. It was also suggested that Bacillus thuringiensis var. Kurusthaki may produce some diarrhea-causing toxin (Takeshi Honda et al., Journal of Japanese Society of Bacteriology 4
0, 240,1985 from) spraying this bacterium formulation in the form containing the live bacteria or live spores can be easily inferred to contain high risk for humans and animals, in fact, containing live spores present There is also a report that a corneal ulcer was brought into the eyes of a farmer to whom the fungal preparation was sprayed (Samples. JR and Buettn.
er, H., Am.J.Opthalmol., 95 , 258-260, 198.
3).

As can be seen from the above, spraying pesticides formulated in the crystalline toxins produced by Bacillus thuringiensis var. Kurusthaki with live bacteria or live spores into the natural world is a public health practice from the perspective of sericulture. It is extremely dangerous from the point of view.

The present inventors have variously studied methods of preparations containing no viable bacteria or live spores in the production of the above-mentioned crystal toxin-containing pesticides, but it is a step further to produce crystal toxins without producing spores. We conducted a thorough study.

(B) Structure of the Invention "Means for Solving Problems" The present inventors have conducted various studies on methods for producing a crystalline toxin that does not cause the above problems, and have identified the crystalline toxin gene of Bacillus thuringiensis var. The present invention was completed by finding that the introduced Bacillus subtilis produces crystal toxin without forming spores.

More specifically, according to the present invention, the crystal toxin gene of Bacillus thuringiensis krustaki HD-1 strain is ligated to a Bacillus subtilis vector or an E. coli plasmid and a shuttle vector of Bacillus subtilis plasmid to prepare a crystal toxin gene expression plasmid. When it was introduced into Bacillus subtilis, a transformant stably holding the plasmid was obtained. This transformant was inoculated into an appropriate medium and the agar culture was performed under aerobic culture conditions.
When cultivated at a temperature of 0 to 37 ° C., this culture broth has a strong insecticidal activity against silkworms and is not only found to be very effective as a pesticide raw material. The present inventors have completed the present invention by discovering that spores are scarcely contained in the surprising thing and that the conventional problems can be solved at once.

That is, the present invention relates to an anti-insect protein toxin-producing Bacillus subtilis into which the crystal toxin gene of Bacillus thuringiensis var.

Bacillus subtilis It is widely known that Bacillus subtilis adopted as a bacterium to which the crystal toxin gene should be introduced in the present invention is extremely safe without parasitism and pathogenicity to humans and plants. It can be supported by the following papers.

Mitsuoka et al. (Mitsuoka T. et al. Goldschmidtin Formiert 2/7
3 23, 23-41, 1973), the Bacillus subtilis in the feces of various animals was examined, and it was found in many herbivores that were moderately present or absent in omnivores, and not in carnivores at all. It has been reported. In addition, even in the case where the presence is recognized, it is recognized that it is derived from Bacillus subtilis mixed in the feed, does not settle in the intestine, and is transient.

In addition, Sakaguchi (Fuji Techno System, the first practical technology for gene recombination) has not been reported to be poisoned by natto, which is made using Bacillus subtilis taxonomically known natto bacteria. Bacillus subtilis is said to be non-pathogenic to humans.

Preparation of crystal toxin gene expression plasmid (pBTK-1) Crystal toxin gene expression plasmid (pBTK-1) was used for E. coli plasmid (pUC9) and Bacillus subtilis plasmid (pUB11).
It can be prepared by ligating the crystal toxin gene of Bacillus thuringiensis krustaki HD-1 strain to the shuttle vector of 0), and its structure is shown in FIG.

The plasmids are not limited to pUC9 and pUB110, and pBR322 and the like for E. coli plasmids and pC194 and the like for Bacillus subtilis plasmids can be used. It is not necessary to use a shuttle vector, and a Bacillus subtilis vector can be used. Good.

Transformant carrying the crystal toxin gene was 5-10 μg
2 × SG medium (Leighton,
TJand Doi, RHJ Biol. Chem. 246 3189-31
95, 1971) and the like at a temperature of 30 to 37 ° C. under aerobic culture conditions.

Production of toxin protein Unlike the Bacillus thuringiensis krustaki HD-1 strain, the production of toxin protein has already been performed in cells in which the bacterial growth has entered the stationary phase by the above culture. As shown in FIG.

“Action” The present inventors succeeded in producing a crystalline toxin of Bacillus thuringiensis var. Kurustaky using Bacillus subtilis, and further, the fermentation broth contained almost no spores to be originally formed by Bacillus subtilis. I found that not.

Therefore, Bacillus subtilis introduced with the crystal toxin gene encoding the crystal toxin of Bacillus thuringiensis var. Kurustaky has an excellent effect of producing crystal toxin without producing spores, and is extremely safe without containing spores. It is possible to provide various microbial pesticides.

Detailed examples are given below, but the method according to the invention is not limited to the examples.

〔Example〕

1) Construction of Bacillus thuringiensis varieties Kurstaki crystal toxin gene expression vector cry-1-1 is a plasmid produced by Tamura et al. Vector pUC
9 ligated to the EcoRI site.

Cry-1-1 is digested with the restriction enzyme BamHI at 37 ° C for 2 hours, and then treated with alkaline phosphatase at 37 ° C for 1 hour. On the other hand, Bacillus subtilis plasmid pUB110 (Gryc
zan, T. J. J. Bacteriol. 134 318-32
9, 1978) is digested with the restriction enzyme BamHI at 37 ° C. for 2 hours. These cry-1-1 BamHI digestion product and pUB110 BamHI digestion product were digested with T4 ligase.
The reaction was carried out at 15 ° C for 15 hours to ligate and cyclize. This is Method specified by Maniatis et al. (Molecular Cloning, A La
Escherichia coli HB101 was transformed by the boratory Manual, Cold Spring Harbor Laboratory). The transformant was LB-agar medium containing 40 μg / m of ampicillin (10 g of bactotryptone, 5 g of bacto yeast extract, 5 g of sodium chloride, 15 g of agar, 1 part of distilled water).
) Selected. Plasmid DNA was isolated from the obtained transformant by the known method of alkali-SDS. These plasmids were digested with BamHI and pU
Recombinant plasmid p having a 4.5 kb DNA fragment corresponding to B110 and a 9.7 kb DNA fragment corresponding to cry-1-1
BTK-1 was obtained (Fig. 1).

2) Introduction of pBTK-1 into Bacillus subtilis Using pBTK-1 isolated from E. coli, Chang, S. and Co
hen, SN (Mol. Gen. Genet. 168 , 111-115, 1)
979) and the Bacillus subtilis PSL
1 (Ostroff, GRand Pene, JJ, J.Bacteriol. 156 ,
934-936, 1983).

Fifteen colonies were selected from the obtained transformants and plasmid D was prepared by the same alkali-SDS method as that for E. coli.
NA was separated. Then, these plasmids were subjected to agarose gel electrophoresis according to a conventional method to isolate pB isolated from E. coli.
Three samples showing the same migration distance as TK 1 were obtained. The remaining plasmid was deleted in Bacillus subtilis to miniaturize the plasmid. Furthermore, these three plasmids were digested with BamHI, and a 4.5 kb DNA fragment corresponding to pUB110 and cry were digested.
It was confirmed to have a 9.7 kb DNA fragment corresponding to 1-1. Of these three strains of Bacillus subtilis recombinants stably retaining pBTK-1, one strain was designated as IAA-1 (ATCC No. 67).
No. 136).

3) Production of Bacillus thuringiensis varieties Kurstaki crystal toxin by IAA-1 IAA-1 was inoculated into a Penassay medium (Difco, Antibiotic Medium 3) containing 5 µg / m of kanamycin and incubated at 37 ° C for 12 to 15 hours. Incubate with shaking. 5 m of this fermentation broth was inoculated into 2 x SG medium containing 5 µg / m of kanamycin, and cultured at 37 ° C with shaking.

The confirmation that the crystal toxin gene was expressed in Bacillus subtilis was confirmed by SDS-polyacrylamide gel electrophoresis of the cultured cells and microscopic observation. That is, 6,
8, 10, 12, 24, 30, 48 hours culture medium 10m each
The cells were collected by centrifugation and 2 m of 30 mM Tris-HCl (pH 8.
0) -10 mM ethylenediaminetetraacetic acid-50 mM suspension in sodium chloride solution, lysozyme was added to 10 mg / m, and the mixture was allowed to stand at 37 ° C for 2 hours. Next, -80 ℃
After repeating freeze-thawing at room temperature four times, ultrasonic treatment was performed for 30 seconds 5 times with an ultrasonic breaker model UR-20P manufactured by Tommy Seiko. Lyophilize this sonicated product,
500μ 0.01M phosphate buffer, 1% SDS, 5%
It was suspended in 2-mercaptoethanol and used as an electrophoresis sample. Electrophoresis was carried out on a 5% polyacrylamide gel.
A sample corresponding to 1 m was placed and carried out. At the same time, the crystalline toxin protein isolated from Bacillus thuringiensis krustaki HD-1 strain was electrophoresed.

Microscopic observation was performed at a magnification of 2000 times for the 24-hour culture solution.

As is clear from FIG. 2, the crystal toxin gene-introduced Bacillus subtilis IAA-1 produces a protein having a molecular weight of about 130,000, which is the same as Bacillus thuringiensis krustaki HD-1 strain, ie, a crystal toxin protein. Was there. This protein was not found in Bacillus subtilis harboring only pUB110. In addition, unlike the Bacillus thuringiensis krustaki HD-1 strain, the crystal toxin protein was already produced in the cells (TO) immediately after the growth of the bacteria entered the stationary phase in Bacillus subtilis, but after 24 to 48 after culturing. Maximum amount in time.

Further, as is clear from FIG. 3, the same bipyramidal crystalline protein particles as in Bacillus thuringiensis krustaki HD-1 were formed in Bacillus subtilis.

4) Biological test 0.5 m of the stock solution and the diluted solution (diluted with distilled water) of the 48-hour culture solution of IAA-1 obtained by the above method were mixed with artificial feed manufactured by Kyodo Feed Company and spread on a petri dish of 9 cmφ. . Transfer 10 silkworm larvae raised up to 4th in one dish,
After standing still at 37 ° C. for 72 hours, the number of dead insects in the petri dish was measured. The description of insecticidal power is given by Howard Dalmage et al.
The method described in of Invertebrate Pathology 18 , 240, 1971 was followed.

Crystal toxin produced by IAA-1 and Bacillus
The insecticidal activity of the crystal toxin produced by Thuringiensis krustaki HD-1 is shown in Table 1.

5) Measurement of the number of spores The number of spores was measured by utilizing the fact that the spores have heat resistance. That is, an appropriate dilution of the above culture solution was added at 80 ° C.
Let stand in the warm bath for 30 minutes. After treatment, cool and determine the number of viable cells on nutrient agar.

The results of spore number measurement are shown in Table 2.

(C) Effect of the Invention According to the present invention, it is possible to produce a crystalline toxin that does not contain spores using Bacillus subtilis, and as a result, it is extremely safe for humans and animals, and has an effect on silkworms. It can provide a small amount of microbial pesticides, and its effect on the pesticide industry and general farmers is immeasurable.

[Brief description of drawings]

Figure 1 shows Bacillus thuringiensis
1 shows a method for constructing a plasmid for producing a crystal toxin of Kurusthaki HD-1 strain. In the restriction enzyme action site in the figure, B represents BamHI and E represents EcoRI. In addition, the thin line indicates pUC9, the white thick line indicates pUB110, and the black thick line indicates the DNA fragment containing the crystal toxin gene derived from Bacillus thuringiensis krustaki HD-1 strain. FIG. 2 shows the SDS-polyacrylamide gel electrophoresis image of the IAA-1 cultured bacterial cell extract. The numbers in the figure are
1 to 7 are IAA-1, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 30 hours and 48 hours, respectively, electrophoretic images of the cultured bacterial cell extract, and 8 is Bacillus thuringiensis krustaki HD-1 1 shows a migration image of a crystalline toxin protein isolated from a strain. The arrow indicates a crystalline toxin protein having a molecular weight of 130,000. FIG. 3 represents a micrograph of the crystal toxin produced by IAA-1. The arrow in the figure indicates crystal toxin.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1: 125) (C12N 15/32 C12R 1:07)

Claims (2)

[Claims] 1. An anti-insect protein toxin-producing Bacillus subtilis into which a crystal toxin gene of Bacillus thuringiensis variety krustaki has been introduced. 2. The anti-insect protein toxin-producing Bacillus subtilis according to claim 1, wherein the Bacillus subtilis is Bacillus subtilis PSL1.