JPS62129207A - Production of biological agricultural chemical - Google Patents

Abstract

PURPOSE:To obtain a biological pesticide, by selecting a specific base sequence or corresponding gene from plural crystalline toxin genes of Bacillus thuringiensis, introducing the sequence, etc., into a cell of a bacterial strain and letting the strain to produce an active biological pesticide having high insecticidal activity. CONSTITUTION:Crystalline toxin genes cryl-1-1 and cry-1-2 coding a crystalline toxin having high insecticidal power are selected from Bacillus thuringiensis. The selected genes are separately clones to E.coli vector to effect the production of crystalline toxin in the cell of E.coli. A biological pesticide having high insecticidal activity can be produced by this process. EFFECT:Reduction of production cost.

Description

Detailed Description of the Invention (a) Object of the invention "Field of industrial application" The present invention relates to a method for producing a biological pesticide that is effective against Lepidoptera pests, and is widely used in agriculture and the pesticide industry. It is something that

``Prior art'' Intracellular inclusions formed during the %L offspring formation period during the KQ life of Pacillus thuringiensis are called crystalline toxins, and are toxic to many A4-ptera insects. It is known that the production of biopesticides containing crystalline toxins as active ingredients is based on Bacillus thuringiensis cristaki mHD-
1 strain (Bacillus thuringiensis
var kurstaki f (D-1-Dipel)
It is carried out using the fermentation solution during the sporulation stage.

"Problem to be Solved by the Invention" The production of biopesticides containing crystalline toxin as an active ingredient, which is carried out using fermentation liquid during the sporulation stage, requires relatively high production costs for the reasons described below. .

In other words, since crystalline toxins are present during the spore formation stage and appear in synchronization with spore formation, it takes time to operate the -m fermenter, and the yield is low (and production efficiency is excellent). This cannot be said to be true.

Furthermore, the insecticidal power (insecticidal activity per unit of insecticide) of crystalline toxins is lower than that of chemical pesticides, and in order to obtain sufficient efficacy, it is necessary to specially prepare and manufacture filters containing high-quality toxins. I need to make one.

Therefore, reducing the manufacturing cost of this expensive biological pesticide has become a major problem for those skilled in the art to solve.

(B) Structure of the Invention ``Means for Solving the Problem'' In order to solve the above problems, the present inventors have proposed the method disclosed in Japanese Patent Application Laid-open No. 58-50 (HoR, Whiteley).
We attempted to reproduce the production of crystalline toxin by genetic manipulation disclosed in 0565, and extracted the crystalline toxin gene encoding the crystalline toxin present in Bacillus thuringiensis. The present invention was completed by discovering that there are three or more types of genes, and that the crystalline toxins produced by bacterial species incorporating each gene have different insecticidal powers.

In addition, the present inventors have identified two types of crystalline toxin genes.
The structure was determined, and cry-1-1 (Feikoken Bibori No. 8482) and cry-1-2 (Feikoku Kenkyori No. 848) were determined.
No. 6).

That is, the present invention relates to Bacillus thuringiensis (13
acillus thuringiensis), the nucleotide sequence shown in Figure 4(A) or a gene substantially homologous thereto is selected and introduced into a bacterial species, and the highly insecticidal gene is introduced into the bacterial species. The present invention relates to a method for producing a biopesticide, which is characterized by producing an active form of the biopesticide. The gene substantially homologous to the nucleotide sequence here is one that provides a gene product that exhibits the same insecticidal activity as the gene product shown in Figure 4), and is preferably the gene product shown in Figure 4 Low). Genes that give the same genetic product.

◎cry-1-2 The present inventors decided to clone the crystalline toxin gene present in the Bacillus thuringiensis crystaki variety HD-1 strain, and
, (::hem, Vol, 258. 1960-19,
! A restriction enzyme cleavage map of the obtained crystalline toxin gene was created with reference to the description disclosed in S7.1983.
The result was as shown in Figure 1), which was similar in general to the one shown by Whitley, but differed in parts. In addition, the cry-i determined by the present inventors
The nucleotide sequence of -2 is shown in Figure 4). Although this sequence was highly homologous overall to the one disclosed by Whitley, there were multiple parts with different sequences. Therefore, the crystalline toxin gene cry-1-2 cloned by the present inventors and the crystalline toxin gene cloned by Whitley are different.

◎cry-1-1 As the above results revealed that there are multiple crystalline toxin genes in the Hasilus thuringiensis cristaki mutant FJtHD-1 strain, further cloning was performed using cry-1-1. Restriction enzyme EcoRI cuts approximately 1.8 Kb of D located on the 5-terminal side of the jilt construction gene of 1-2.
NA fragment (E-1,8) and a restriction enzyme present from the center to the downstream of the structural gene) (indl cleavage approximately 1.
Using the OKb DNA fragment (H-1,0) as a probe, crystalline toxin genes were selected from a similar phage library, and 21 recombinant phage strains were positive for both probes and DNA probe E-1, We obtained 13 phage strains that were positive only for H-8 and negative for H-1,0, and analyzed the genes of these 21 positive phage strains. ), the crystalline toxin gene cry-1-1 contained the regulatory gene region and the structural gene of crystalline toxin.

As shown in the map in Figure 1, the structure of the crystalline toxin gene cry-1-1 is relatively similar to that of cry-1-2 in the upstream region, but the downstream region is significantly different and has different restrictions. It has an enzyme cleavage site and is a crystalline toxin gene different from cry-1-2.

The base sequence of cry-1-1 was determined and shown in Figure 4(A).

This nucleotide sequence was recently published by Whitley in J. Biol.

Chem, Vol, 260.6264-6272.19
Although it is substantially homologous to the base sequence of the crystalline toxin gene shown in No. 85, it is partially different.

◎Other crystalline toxin genes D obtained when cloning the above cry-1-1
Phage 16, which is positive only for NA probes E-1 and 8 and negative for H-1 and 0, contains the regulatory gene region of the crystalline toxin gene and approximately I Kb at the 5th end of the structural gene. Based on the restriction enzyme map, it can be divided into four groups, two of which are cry-1-1 and c
cry-1-2, but the remaining two groups do not belong to them and are called cry-1-1 and cry-1-.
It was shown that there are different crystalline toxin genes.

Furthermore, the DNA completely cleaved with the restriction enzyme Hindl is developed by electrophoresis, adsorbed onto a filter, and cry-
Approximately 0.4 produced by cutting 1-2 with restriction enzyme Xba
Southern hybridization was performed using the Kb DNA fragment as a probe, and the restriction enzyme XbaI cut D
NA! Three bands, 4.7 Kb, 5.4 Kb, and 6.6 Kb, were generated due to hybridization with the fi piece.
was detected. As shown in Figure 5, this result shows that there are three different sizes of restriction enzyme 1 (indl) cleavage fragments that contain the restriction enzyme belongs to cry-1-1,
5.4Kb was determined to belong to cry-1-2,
The existence of another crystalline toxin gene with a 6.6 Kb DNA fragment was also shown here.

◎Crystalline toxin production Crystalline toxin genes cry-1-1 and cry-1-2
We were able to clone each into an E. coli vector and express the crystalline toxin in E. coli.

Expression of the crystalline toxin can be confirmed by electrophoresing the bacterial extract.

"Function" The present inventors have discovered that there are six or more crystalline toxin genes that encode crystalline toxins in Pacillus thuringiensis, but even more surprisingly, each crystalline toxin gene encodes a crystalline toxin. There are large differences in the insecticidal power of the crystalline toxins obtained from the sex toxin genes, and the crystalline toxins obtained from the Bacillus thuringiensis chrystakii strain ID-1 that were previously obtained were the average of these. It is.

Therefore, the present invention was made based on the discovery of these facts, namely, a crystalline toxin gene (cry-
The method of selecting 1-1) and producing a crystalline toxin has an excellent effect in that it can provide a crystalline toxin with much higher insecticidal power than conventional crystalline toxins.

〔Example〕

The following detailed methods were used to create recombinant plasmids and measure their insecticidal activity in the present invention.

a) Bacillus thuringiensis kurstakii [D
-I strain crystalline toxin gene + Preparation of gene library for selection of cry-1-2 Bacillus thuringiensis kurstakii variant HD-1 strain was cultured at 30°C in L-medium up to 240 Klett units, and lysozyme After digestion, total DNA was extracted and purified by SDS treatment.

The extracted DNA was partially digested with restriction enzyme 3au3AI. Restriction enzyme reactions were performed according to the manufacturer's recommendations (Takara Shuzo Co., Ltd.). DNA digested under various conditions was subjected to sucrose density gradient centrifugation, and a 4-6 Kb fraction was collected and purified.

Next, the purified 4-6Kb fraction was treated with the restriction enzyme BamH.
It was ligated to the previously opened E. coli plasmid pUC-9 by digestion with I. Plasmid pUC-9 was isolated from PUC by the known Alka IJ-3DS method.
It was isolated from E. coli JM83 containing 9. The bond is T4D
The reaction was carried out using NA'J gauze (manufactured by Takara Shuzo Co., Ltd.) for 16 hours under conditions recommended by the manufacturer.

Next, the recombinant plasmid was added to the large intestine mJM83Mo1.
The method described in Bio153154 (1970) was used. The transformed Escherichia coli strain JIVI83 was treated with 50μ9/rnl of ampicillin and 20μg of 5-promo 4-chloro-6-indolyl β-D-galactopyranoside.
Plated on L-agar medium containing ~. Plasmid pU
C-9 was Gene 19,2 by Messing et al.
59 (19B2), ampicillin resistance: colonies with the lt gene are white if recombination has been performed in E. coli JM 83, and blue colonies if no recombination has been performed. It has the property of being formed by the action of the 1aC2 gene. Therefore, the white colonies formed on the agar medium were used as a library into which genes were integrated.

b) Selection of recombinant with crystalline toxin gene cry-1-2 Bacillus thuringiensis kurstakii variety HD-1
The genetic structure of one of the crystalline toxin genes of the strain was revealed by Whiteley et al. (J,
Biol, Chem, vol 258.1960-19
67.1985).

Based on this information, the present inventors synthesized two types of DNA globes for crystalline toxin gene selection using the triester method. The synthesized oligonucleotide is ATGGAT
AACAATCC and CTCCTGTAGGGTTTT
There are two types of base sequences. Each S' end is "p-r"
It was labeled with ATP (manufactured by Amerjam) and used in the experiment.

mK created h? Using the DNA probe, a recombinant having the crystalline toxin gene was selected from the gene library of the Sils thuringiensis kurstakii variant t-1iHD-1 strain by colony hybridization. Colony hybridization was performed according to the method described in Kodansha's Genetic Manipulation Manual. Nitrocellulose filter is 5chleicher & 5chneLl
BA85, manufactured by S.A., was used. As a result of colony hybridization, two positive recombinants were obtained from approximately 600 colonies.

A detailed restriction enzyme cleavage map was created for the positive recombinants obtained by colony hybridization. The restriction enzyme used to create the restriction map was EC0RI.)
(indl [, ACCI, Aha l.

Hinc l, pst i, pvu l, Sal l
, Sma I, Xba, KpnI (manufactured by Takara Shuzo Co., Ltd.), Ban1 (manufactured by Toyobo Co., Ltd.), and were used according to the methods recommended by the respective manufacturers.

Next, based on the created restriction enzyme cleavage map, Figure 1(B), the 11 crystalline genes were fragmented into various W fragments and recloned into plasmids pUc-9 and puc-15.Re-cloning This was mainly done based on the following description.

The crystalline toxin gene was cut with various restriction enzymes according to the method recommended by the manufacturer, and using agarose gel, the desired DNA fragment was extracted according to the method of Southern et al., J8Mo1, Biol, 98,503°1975. Recovered. Next, using T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.), a ligation reaction was performed on the 1 lasmid pUC-9 or pUC-13, which had been ring-opened with the target restriction enzyme, according to the manufacturer's recommended method. The ligated and recircularized plasmid was transformed into E. coli JM 83 as described above and selected on a grate.

Next, the primary structure of the crystalline toxin gene was determined using the constructed recombinant plasmid.

- Determination of the secondary structure was performed by Sanger et al. in 1977 proc.

Utilizing the principle described in Nat., Acad, Sci., USA 74, the method was carried out by using a plasmid directly in an enzyme reaction. The primers used were 17 Dotai Primer Meigetsu and Soyo manufactured by PL Biochemical.

The enzyme reaction was carried out using Takara M13 Sequencing Kit manufactured by Takara Shuzo Co., Ltd. under conditions recommended by the manufacturer. For DNA labeling, 32p-α manufactured by Amerjam
-dCTP was used. The labeled DNA sample was electrophoresed using a 40 cm x 20 c+n polyacrylamide gel with a thickness of 0.2 mm. Polyacrylamide gels of 6 and 8 degrees A are used, and 200
Electrophoresis was performed using a constant voltage of 0V. 'The gel after electrophoresis was adhered to Whatman 5MMF paper, dried, and then subjected to autoradiography for 14 hours using an X-ray film manufactured by Fuji Photo Industries, Ltd.

d) Bacillus thuringiensis kurstakii var. HD
- Characterization of the crystalline toxin gene present in one strain It is clear that the crystalline toxin gene that the inventors cloned and revealed the structure of is different from the crystalline toxin gene shown by Whiteley et al. It became. From this result, Bacillus thuringiensis kurstakii variety HD-
It was suggested that at least two or more crystalline toxin genes were present in one strain. Based on the above background, the present inventors characterized the crystalline toxin in Bacillus thuringiensis kurstakii variety HD-1 strain by Sasanplot analysis.

Biochem was developed by Godson et al. from Bacillus thuringiensis kurstakii var.

Total DNA was extracted and purified according to the method described in Biophys, Acta 299.516.1973. Extracted D
NA was completely cleaved using a restriction enzyme (indl (Takara Shuzo Co., Ltd.) under the conditions recommended by the manufacturer. The cleaved DNA fragments were developed by 0.8% agarose gel electrophoresis, and then et al. J, Mol.

The DNA analysis method described in Biol, 98, 503.1975 was applied. Southern plotting was performed according to the method of Kodansha's Genetic Manipulation Manual. The DNA globe for hybridization was prepared by cutting the crystalline toxin gene cry-1-2, which the inventors had cloned, with the restriction enzyme Using the ration kit, 32
One labeled with P-α-dCTP (manufactured by Amarjam) was used.

e) Bacillus thuringiensis kurstakii var. HD
- Preparation of gene library for selection of strain 1 crystalline toxin gene cry-1-1 Bacillus thuringiensis kurstakii variety HD-1
Since it became clear that there are multiple crystalline toxin genes in the strain, the present inventors used a method different from the previous section to
We created a gene library again and attempted cloning.

Basil Sutlingiensis Yukurustaki var. HD-1
Total DNA extracted from the stock using Motohi's method was treated with restriction enzyme Ec.
It was partially cut using oRI (manufactured by Takara Shuzo Co., Ltd.). The cut DNA fragments were fractionated by ultracentrifugation under a sucrose density gradient, and fragments of 11 Kb to 21 Kb that could be incorporated into phage heads were collected.

On the other hand, the input phage Charon 4A was treated with the restriction enzyme EC0RI (
(manufactured by Takarahozo Co., Ltd.) to prepare fragments with cohesive ends. The Pacillus thuringiensis kurstakii modified mHD-1 DNA fragment and phage DNA fragment prepared as above were combined with DNA binding enzyme T4DNAIJ gauze (
(manufactured by Takara Shuzo Co., Ltd.). Binding reactions were performed according to the manufacturer's recommended methods.

Next, the ligated DNA fragments were prepared using the method of Stambarb et al.
ene 1.255 (1977)). This operation was carried out using Takara Shuzo Co., Ltd.'s in vitro packaging kit according to the method recommended by the manufacturer. The phages that had been packaged were plated on a T-agar medium using Escherichia coli LE392 as an indicator bacteria, and cultured at 37°C for 12 hours. The appearing plaques were scraped off and used as a Bacillus thuringiensis subspecies HD-1 strain gene phage library.

f) Bacillus thuringiensis kurstakii var. HD
-1 strain crystallizing toxin gene cry-1-1 selection from a phage library Item M Selection of phage carrying the crystallizing toxin gene from the gene library of the described B. thuringiensis kurstakii variant HD-1 strain using a plaque. This was done by hybridization method.

The DNA probe used for selection of recombinants was E, which is located approximately 270 bases from the 5' end of the structural gene region of the crystalline toxin gene cry-1-2 cloned in the previous section.
From the restriction enzyme cleavage site of C0RI, about 1 point toward the 3' side.
The region up to EC0RI located 700 bases downstream (hereinafter referred to as E
-1,7)) and the region up to indll (located about i ooo bases toward the 6' side from the restriction enzyme cleavage site of Hindl, which is also located about 1690 bases downstream from the 5' end of the structural gene) 2 (hereinafter abbreviated as H-1,0)
One was used. E-1,8 and H-1,0 prepared by restriction enzyme ECORI and Hindl digestion were manufactured by Amerjam.

%”P-α-dCT using the translacein kit
P, (manufactured by Amarjam).

Darak hybridization was performed as described in the Kodansha ENTIFIC Genetic Manipulation Manual.

g) Banrus thuringiensis kurstakii variant mHD
-1 Structural analysis of crystalline toxin gene cry-1-1 Structural analysis was performed in the same manner as for cry-1-2 described in the previous section.

h) Cloned crystalline toxin gene cry-1-1
Identification of the products of cry-1-2 and measurement of insecticidal power The crystal toxin gene cry-1- Cry-1 and cry-1-2 were each cloned into Escherichia coli plasmid pUC9, and their base sequences were revealed. As a result, the gene product of cry-1-1 is composed of 1176 amino acids, and the molecular weight is 1.
33020 Furthermore, the gene product of cry-1-2 is 115
It consisted of 5 amino acids and had a molecular weight of 150,625.

This result is in good agreement with the molecular weight of crystalline toxin, which has been previously shown to be approximately 130,000.

Next, the cloned crystalline toxin gene was transferred to E. coli JM8.
It was introduced into 5 strains, expressed, and the protein in the bacterial cell extract was analyzed using the method described below.

E. coli strain JM83 having each gene was cultured in L-medium,
After centrifuging the culture solution 150- and washing the bacterial cells with water,
30ff1Mto! JS hydrochloric acid buffer (pH 8,0)-10
ff1M ethylenediaminetetraacetic acid - 50mM chloride)
IJum liquid 1ONIC! After the mixture became cloudy, lysozyme (manufactured by Seikagaku Kogyo Co., Ltd.) was added to the mixture to give a concentration of 15 μm/h, and the mixture was allowed to react at 37° C. for 1 hour. Next, 301Le of DNase I (manufactured by Takara Shuzo Co., Ltd.) was added, and freezing and thawing at -80°C and room temperature were repeated four times to destroy the cells. 5DS using this sample
- Polyacrylamide gel electrophoresis and insecticidal activity against silkworm larvae were tested.

For electrophoresis, a 2° sample was placed on a 2 mm thick 5% polyacrylamide gel prepared in a size of 15 cL x 15 cL, and a constant current of 100 mA was applied for 5 hours.

Oriental Yeast Co., Ltd. RMW as a molecular weight marker
-Marker (HPLC) was run simultaneously. After electrophoresis, the gel was stained with 25% Coomassie Brilliant Blue R-250 and decolorized with a 10-thiacetic acid-10% methyl alcohol bath. .

cry-1-1 and cry-1 transferred into E. coli
The insecticidal power of the gene product No.-2 was measured by the method described below. Samples prepared from recombinant strains and Bacillus
0.5 d of a solution of crystalline toxin isolated from the culture solution of T. thuringiensis kurstaki strain HD-1 was mixed with artificial feed manufactured by Kyodo Feed Co., Ltd., and then spread in a 9 ctnl petri dish. Ten silkworm larvae reared to 4th instar were transferred to one Petri dish, and after soaking at 57° C. for 72 hours, the number of dead insects in the Petri dish was measured. For the silkworm larvae, silkworm eggs were purchased from Kanebo Silk Elegance Co., Ltd., and those raised to 6 instars were used for the test. The description of insecticidal power is based on Howard Dalmage et al.
The method described in Pathology 18.240.1971 was followed.

The crystalline toxin content in the sample was determined using the following formula by subjecting the aforementioned 5DS-polyacrylamide gel electrophoresis image to a densitometer.

Crystalline toxin gene cry-1-1 tested by the above method,
Translated gene products of cry-1-2 Furthermore, the results of the insecticidal activity of crystalline Yangzi produced by Bacillus thuringiensis kurstakii variety HD-1 strain are shown in Table-1.

Table 1 As is clear from the results, the insecticidal power was different depending on the person. That is, the insecticidal activity was a product of cry-1-1 (product of cry-1-1), a product of crystal toxin near cry-1-2 of the HD-1 strain.

From the above studies, it is clear that there are multiple crystalline toxins with different insecticidal powers in the biopesticides produced from Bacillus thuringiensis kurstaki var. It is clear that the insecticidal power of multiple crystalline toxin proteins is averaged. In fact, the insecticidal power (337,000 IU/Mg) of the crystalline toxin obtained from the fermentation liquid of Bacillus thuringiensis kurstaki'fllHD-1 strain is higher than the activity of the protein produced by cry-1-2 expression (821,240 IU/Mg).
m and 5 < 5520 IU/■).

Using the crystalline toxin gene cry-1-1, a bacterial species is made to produce a crystalline toxin, and the obtained crystalline toxin is a crystalline toxin obtained by the current manufacturing method using Bacillus thuringiensis kurstakii variety HD-1. Compared to sex toxins, 264
It is clear that the insecticidal power is twice as strong.

Therefore, Bacillus thuringiensis kurstakii
Currently, crystalline toxins are produced using bacterial species using the crystalline toxin gene cry-1-1 of the HD-1 strain or a substantially homologous gene encoding the same protein as cry-1-1. It has become possible to produce a crystalline toxin with insecticidal power 2.4 times greater than that of the conventional method, which has great economic significance.

(C) Effects of the Invention According to the present invention, it is possible to produce a crystalline toxin with high insecticidal power, and as a result, the cost associated with the production of biopesticides can be reduced and the market competitiveness over existing products can be improved. It is something that is given.

[Brief explanation of drawings]

Figure 81 shows two crystalline toxins cloned from Bacillus thuringiensis kurstaki variety HD-1 and a restriction enzyme map of their vicinity, with arrows indicating the position and direction of the crystalline toxin genes. In the diagram, the restriction enzyme action site is 13anl for B and Ec for E.
o RI, HO2 ri''-) (inc[, l (d is 1 (
ind l[, K is Kpn I, ps is pst I,
pv represents pvu l and X represents XbaI. Figure 2 shows DNA isolated from Bacillus thuringiensis kurstakii var.
-2 obtained by complete digestion with the restriction enzyme XbaI.
Southern hybridization was performed using a 4 Kb DNA fragment as a probe. MM is DNA obtained by completely digesting the DNA of the input phage with the restriction enzyme Hindl.
It shows the migration position of the A fragment and corresponds to a molecular weight marker. Figure 6 shows cry ligated with E. coli vector pUC9.
5 is a 5DS 15% polyacrylamide gel electrophoresis image of extracts of cultured E. coli cells transformed with -i-1 and cry-1-2. Staining was done with Coomassie brilliant blue. Numbers 1 in the diagram are cr
y-1-1, 2 is a crystalline toxin isolated from Bacillus thuringiensis, and 6 is cry-1-2.
and the host E. coli JM83.
represents. The arrow Fendotoxin indicates the migration position of crystalline toxin. In Figure 4, (A)-1, (A)-2, and (A)-3 are cry.
-1-1's CB>-1, CB)-2,'s)-3 is cry
-1-2 base sequences are shown, respectively. The underlined bases of cry-1-1 indicate bases lacking homology in comparison with cry-1-2. The lacrimal arrows BtI and Btl [represent the starting point of transfer in Bacillus thuringiensis, and the wavy arrow EC represents the starting point of transfer in E. coli. Figure 5 shows Southern hybridization 4,
This is a schematic representation of the meaning of the 7, 5.4 and 6.6 Kb DNA fragments, and the arrow indicates the position of the structural gene of the crystalline toxin. Abbreviations of restriction enzymes are the same as in Figure 1.

Claims (1)

[Claims] 1. Bacillus thuringiensis
thuringiensis), the base sequence shown in Figure 4 (A) or a gene substantially homologous thereto is selected and introduced into a bacterial species, and a highly insecticidal organism is introduced into the bacterial species. A method for producing a biological pesticide, characterized by producing an active form of the pesticide.