JPH06192295A - Fused crystalline toxin protein and gene coding the protein - Google Patents

Abstract

PURPOSE:To relate to the crystal toxin protein having an excellent insecticidal effect and produced by Bacillus thuringiensis, and to construct the protein stable against proteases without deteriorating the characteristics of the protein. CONSTITUTION:The C side terminal of a crystal toxin protein CRY-1-2 is substituted with the C side terminal of a crystal toxin protein CRY-1-2 to provide the crystal toxin protein. The crystal toxin protein excellent in an insecticidal effect and stable against proteases can be constructed, and when a gene coding the protein is used, the stable crystal toxin protein can be produced.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a crystalline toxin protein showing an excellent insecticidal effect against larvae of Lepidoptera insects (moths and butterflies), and is effective for stable and large-scale production of the crystalline toxin protein. It relates to genes and is effectively used in the agricultural and agrochemical industries.

[0002]

BACKGROUND OF THE INVENTION Intracellular inclusions formed during sporulation during the life cycle of Bacillus thuringiensis are called crystal toxin proteins and are toxic to many lepidopteran larvae. Is widely used as a pesticide.

As a method for producing a microbial pesticide containing a crystalline toxin protein as an active ingredient, mainly Bacillus thuringiensis var.
nsis var. kurstaki HD-1) is cultured, and a crystalline toxin protein, which is a fermentation product thereof, is formulated as it is without separating it from live spores mixed with it.

On the other hand, with the recent progress of gene recombination technology, an attempt has been made to clone a gene encoding a crystal toxin protein and introduce it into a heterologous microorganism such as Escherichia coli or Bacillus subtilis to produce the crystal toxin protein. ing.

Anapurashi Shibakuma, etc. (JP-A-62-
181777, J. Bacteriol. 166 , 194, 198.
6) is Bacillus thuringiensis var. Kurustaki H
A gene encoding a crystal toxin protein of D-1-Dipel was ligated to a plasmid capable of replicating (growing) in Bacillus subtilis and introduced into Bacillus subtilis to induce bipyramid
It has been reported that it was produced as a crystal with a (dal) structure.

Furuya et al. (Japanese Patent Laid-Open No. 62-294080)
Higher than that of the parent strain Bacillus thuringiensis var. Sotto after ligating the gene encoding the crystal toxin protein of Bacillus thuringiensis var. Sotto into a vector capable of growing in Bacillus subtilis and then introduced into Bacillus subtilis to express the crystal toxin protein. It has been reported that a substance exhibiting insecticidal activity was obtained.

[0007] Furthermore, John W. Krismann et al. Introduced a gene encoding a crystal toxin protein of Bacillus thuringiensis var. Kurusutaki into a low copy plasmid pSY228 capable of replicating in Bacillus subtilis and then introduced into Bacillus subtilis. And succeeded in producing a crystalline toxin in an amount of 25% or more of the total bacterial protein under the control of spoVG promoter (Appl. Environ. Microbiol. 55 , 2302, 198).
9).

However, the insecticidal activity of the crystal toxin protein produced by Bacillus subtilis is lower than that produced by Bacillus thuringiensis, the stability of the plasmid is not satisfactory, and the transformed Bacillus subtilis When the bacterium is continuously cultured for a long time, the crystal toxin protein produced by the bacterium is also decomposed by protease.

[0009]

Bacillus subtilis is not toxic to humans, its genetic properties have been investigated in detail and various mutants have been obtained, and proteins are secreted efficiently outside the cells. Therefore, it is widely used as a host for gene recombination.

However, as described above, the substance production by the gene recombination technique using Bacillus subtilis as a host has a big problem that the product (protein) is decomposed by protease.

Decomposition by a protease means a phenomenon in which a protein produced by genetic recombination is decomposed by a protease produced by a host Bacillus subtilis after or during the culturing process. Damaged by reduced yield and activity. Usually, in order to avoid this problem, the target protein is produced using a mutant strain lacking the protease-producing ability as a host.

The present inventors have conducted various studies by speculating that a crystal toxin protein having improved stability against proteases could be obtained by changing the structure of the crystal toxin protein.

[0013]

The Bacillus of the present inventors
By investigating various crystal toxin proteins produced by Thuringiensis and producing a fused crystal toxin protein by replacing the C-terminal side of a specific crystal toxin protein with the C-terminal side of another crystal toxin protein, stability against protease The present invention has been completed by succeeding in obtaining a crystal toxin protein having improved properties and a gene encoding the protein.

[0014] Further, to explain in detail the examination process of the present invention, in the crystalline toxin protein of Bacillus thuringiensis variant Kristaki HD-1, there is a portion functioning as a toxin at the N-terminal side and at the C-terminal side. The present inventors have found that there is a portion that is susceptible to degradation by proteases, and in particular, the amino acid sequences 795 to 820 of the crystal toxin protein CRY-1-1 contribute to the improvement of stability to proteases. It is predicted that the C-terminal side of CRY-1-1 including that portion is approximately corresponding to CRY-1-2.
The present inventors have completed the present invention by finding that the above-mentioned problems can be solved by constructing a novel fused crystal toxin protein in which the C-terminal side is replaced.

That is, the present invention relates to C of the crystalline toxin protein of Bacillus thuringiensis var. Kristaki HD-1.
The C-terminal side of RY-1-2 is CRY- which is a crystal toxin protein of Bacillus thuringiensis var.
The present invention relates to a fused crystal toxin protein which is substituted on the C-terminal side of 1-1, and a gene encoding the protein.

The gene of the present invention can of course be applied to the conventional method for producing a crystal toxin protein, whereby the crystal toxin protein of the present invention stable to protease can be obtained. According to the method of culturing the Bacillus subtilis, which is constructed based on the method for constructing a recombinant vector separately found by the above, the gene of the present invention is incorporated into the chromosome of the recombinant vector ligated into Bacillus subtilis, The fused crystal toxin protein stable to protease can be stably and produced in large quantities even in a medium containing no antibiotic.

That is, the gene of the present invention is expressed in Escherichia coli plasmids such as pUC19, pUC9 and pBR32 which are replicable in Escherichia coli, and in Bacillus subtilis such as chloramphenicol (Cm) resistance gene and pUB110-derived kanamycin resistance gene. After ligation to the obtained DNA fragment containing the antibiotic resistance gene and the Bacillus subtilis spoVG gene promoter, it is introduced into Bacillus subtilis to obtain a transformant in which the gene is integrated into the chromosome. This gene is stably retained not only in a medium containing Cm but also in a medium containing no Cm, and the fused crystal toxin protein is stably maintained without being decomposed by protease even in long-term culture. Produced and accumulated.

Fused crystal toxin protein gene cry-1-1 (Microtechnology Research Institute No. 8482) and cry
-1-2 (Ministry of Industrial Science and Technology, No. 8483) is a plasmid prepared by Tamura et al., And the crystal toxin gene of Bacillus thuringiensis varieties Kristaki HD-1 is the EcoRI site of E. coli vector pUC9 and BamHI of pUC9, respectively.
The restriction enzyme maps are those shown in FIG. 1.

The C-terminal side of the CRY-1-2 protein is CRY-1
The gene encoding the fusion crystal toxin protein in which the C-terminal side of -1 was substituted is the sequence of amino acid sequence 795 to 820 of CRY-1-1 (Gly Glu Pro Asn Arg Cys Ala Pro Hi
s Leu Glu Trp Asn Pro AspLeu Asp Cys Ser Cys Arg A
sp Gly Glu Lys Cys) arbitrary DNA fragment encoding the latter half of the amino acid sequence, for example, the C-terminal side and 3'of the crystal toxin protein obtained by cleaving cry-1-1 with KpnI.
A 5'untranslated region obtained by cleaving a cry-1-2 corresponding to the N-terminal side of the DNA fragment containing the untranslated region with a restriction enzyme KpnI and the 1st to 725th positions of the crystalline toxin protein are encoded. It can be prepared by ligating the DNA fragment with The restriction enzyme map is shown in FIG. 1, and the amino acid sequence of the fusion crystal toxin protein and the base sequence of the gene encoding it are shown in SEQ ID NO: 1 in the sequence listing.

Preparation of a fusion crystal toxin gene-integrated expression plasmid (pVGCRY2 / 1) A plasmid (pVGCRY2 //) for expressing the gene encoding the fusion crystal toxin protein in the chromosome of Bacillus subtilis
1) is a fusion crystal toxin protein gene, E. coli plasmid p
S from UC19, Cm resistance gene, and pZL207
It is composed of a DNA fragment containing the poVG promoter, and its structure is shown in FIG.

○ Stability of fused crystal toxin protein to protease Inoculate an appropriate medium with transformed Bacillus subtilis in which the gene is integrated in the chromosome, and culture. This can be confirmed by the presence or absence of disappearance of a band having a molecular weight of about 130,000 corresponding to the fused crystal toxin protein by subjecting the extract of the bacterial cells sampled from this culture solution to SDS-polyacrylamide gel electrophoresis over time.

Stability of the gene integrated into the Bacillus subtilis chromosome The stability of the gene encoding the fused crystal toxin protein integrated into the Bacillus subtilis chromosome is determined by subculturing the transformed Bacillus subtilis in a medium containing no Cm. It can be confirmed by examining by SDS-polyacrylamide gel electrophoresis whether or not the expression level of the toxin protein is reduced by repeated subculture.

[0023]

The present inventors succeeded in constructing a gene encoding a fused crystal toxin protein in which the C-terminal side of the crystal toxin protein CRY-1-2 was replaced with the C-terminal side of another crystal toxin protein CRY-1-1. Therefore, the crystal toxin protein can be stably produced without being decomposed by the protease, which is based on the unique action of the gene.

Although detailed examples are shown below, the present invention is not limited to the examples shown below.

[0025]

【Example】

1) Preparation of cry-1-2 and cry-1-1 fusion gene cry-1-1 (Microtechnology Research Institute, No. 8482) and cry-1-
No. 2 (Microtechnological Research Institute No. 8483) is a plasmid prepared by Tamura et al., Which is a crystal toxin gene of Bacillus thuringiensis variant Kristaki HD-1 ligated into E. coli vector pUC9. The enzyme map is shown in FIG.

After cry-1-2 was completely digested with KpnI, it was subjected to agarose gel electrophoresis, a gel containing a DNA fragment of about 7.9 kb was cut out, and DNA DREP (Asahi Glass) was used to perform D
NA was recovered.

Then, 55 with alkaline phosphatase
It was treated at ℃ for 1 hour. On the other hand, after cry-1-1 was completely digested with KpnI, it was electrophoresed on agarose gel to give a DNA of about 2 kb.
The gel containing the A fragment was cut out and the DNA was recovered using DNA PREP.

This approximately 2 kb DNA fragment and the above-mentioned approximately 7.9
The kb DNA fragment was ligated and circularized using a DNA ligation kit (Takara Shuzo). The method specified by Maniatis et al. (T. Maniatis et al., Molecular Cloning
, A Laboratory Manual, Cold Spring Harbor Laborat
ory) was used to transform E. coli HB101. A plasmid was isolated from the obtained transformant by the alkali-SDS method. These plasmids were completely digested with KpnI, and plasmids producing DNA fragments of about 7.9 kb and 2 kb were selected. These plasmids were then digested to completion with AhaIII to yield approximately 3.6 kb, 2.6 kb, 2.3 kb and 0.7 kb.
Recombinant plasmids producing the DNA fragment of E. coli were selected, and one of them was named pCRY2 / 1K.

2) Preparation of integrative expression vector SalI-SmaI adapter (5'-TCGACCCGG
G-3 ', Takara Shuzo and pSmaI linker (5'-CCC)
GGG-3 ', Takara Shuzo) are mixed in equimolar amounts, and the temperature is 70 ° C.
It is annealed by heating for 0 minutes and then slowly cooling. This double-stranded oligonucleotide and the E. coli vector pUC19 that had been completely cleaved with the restriction enzyme SmaI in advance.
Were ligated using a DNA ligation kit (Takara Shuzo), and a SalI protruding end was newly introduced into the SmaI site of pUC19.

On the other hand, pZL207 (American
S obtained from Type Culture Collection)
After complete digestion with alI, perform agarose gel electrophoresis and
A gel of about 3.1 kb consisting of a Bacillus subtilis chromosomal DNA fragment containing a resistance gene and spoVG gene promoter was cut out and the DNA was recovered using DNA PREP (Asahi Glass).

This DNA fragment was ligated to pUC19 into which the SalI protruding end was introduced by using a DNA ligation kit, and circularized. The method defined by Maniatis et al. (T. Maniatis et al., Molecular Cloning, A Labor
Escherichia coli HB101 was transformed with the Laboratory Manual, Cold Spring Harbor Laboratory). 50 transformants
LB-agar medium containing 10 μg / ml of ampicillin (10 g of bactotryptone, 5 g of yeast extract, 5 g of sodium chloride, 15 g of agar, 1 liter of distilled water) was used for selection. Plasmid DNA was isolated from the obtained transformant by the known method of alkali-SDS. These plasmids were digested with SalI to obtain a DNA fragment of about 2.7 kb corresponding to pUC19, Cm resistance gene and spoVG.
A plasmid having a DNA fragment of about 3.1 kb containing a promoter was selected. Then, those plasmids are
Recombinant plasmids which were completely digested with II and produced DNA fragments of about 0.2 kb and about 5.6 kb were selected, and one of them was selected.
It was named pVG1.

3) Cloning of the gene encoding the fusion crystal toxin protein into the integrated expression vector pVG1 pCRY2 / 1 was completely digested with AhaIII and subjected to agarose gel electrophoresis. After electrophoresis, the gel containing the DNA fragment of about 3.6 kb was cut out, and DNA PREP was used for DN.
A was collected.

On the other hand, pVG1 prepared in 2) above is Bam
After completely digested with HI, it was blunted using a DNA blunting kit (Takara Shuzo).

This BamHI site-blunted pVG1 and the above-mentioned about 3.6 kb AhaIII DNA fragment of pCRY2 / 1 were ligated using a DNA ligation kit to transform Escherichia coli HB101. A plasmid was isolated from the obtained transformant by the alkali-SDS method, and pVG
1 was subjected to agarose gel electrophoresis. as a result,
Select a plasmid with a higher molecular weight than pVG1
The direction of insertion of the cloned fused crystal toxin protein gene was determined by complete digestion with RI. As a result, a recombinant plasmid having the gene inserted in the forward direction of the spoVG gene promoter was obtained, and pVGC was obtained.
It was named RY2 / 1. In addition, this plasmid was introduced into Escherichia coli HB101 to prepare HB101 (pVGCRY2 / 1).
Has been deposited with the Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology (Microtechnology Research Institute, No. 12053).

4) Introduction of pVGCRY2 / 1 into Bacillus subtilis and multicopy on the chromosome Using pVGCRY2 / 1 isolated from E. coli, it was described in Biology Experiment Course 7 Prokaryotic Biology (Maruzen Co., Ltd.). Bacillus subtilis 1A96 (this strain was obtained from Bacillus Genetic Stock Center of Ohio State University, USA) was transformed according to the method described in the above. The transformant has a Cm of 5 μg / ml.
LB-agar medium containing 10 g (bactotryptone 10 g, yeast extract 5 g, sodium chloride 5 g, agar 1
5 g, 1 l of distilled water) were selected. From the obtained transformants, the method of Saito and Miura (Saito H. and Miura
K. Biochim. Biophys. Acta 72 , 612, 196
Chromosomal DNA was separated according to 3), digested completely with EcoRI, and subjected to agarose gel electrophoresis. Then, the DNA digested with EcoRI is transferred to a nitrocellulose membrane, and [α
Hybridized with pVGCRY2 / 1 labeled with -32P] dCTP.

As a result of hybridization, pVGC
The same band as the band generated when RY2 / 1 was completely digested with EcoRI was used as a transformant.
It was named CRY2 / 1-5.

Multi-copying of the integrated fused crystal toxin protein gene was performed by the following method. 1A96 / pVGCRY
1-5 was inoculated into an LB liquid medium containing 5 µg / ml of Cm and shake-cultured at 37 ° C for 10 to 12 hours. Of this culture, 50 μl was added to 5 ml of L containing 10 μg / ml Cm.
B liquid medium is inoculated and shake-cultured at 37 ° C. for 10 to 12 hours. Similarly, Cm is 20 μg / ml, 40 μg / m
l and 60 μg / ml of medium were successively subcultured, and finally 60 μg / ml of Cm-resistant strain was obtained.
It was named A96 / pVGCRY2 / 1-60 and deposited at the Institute for Microbial Technology, National Institute of Advanced Industrial Science and Technology
54).

5) Stability of fused crystal toxin protein to protease 1A96 / pVGCRY2-60 (1A96 / pVGCRY2-60, which produces the crystal toxin protein CRY-1-2 prepared by Akashi et al. No. 12
No. 050) was inoculated into LB-liquid medium (10 g of bactotryptone, 5 g of yeast extract, 5 g of sodium chloride, 1 l of distilled water) each containing 60 μg / ml of Cm,
Incubate with shaking at 37 ° C overnight. 5 ml of 1 ml of these cultures
0 ml of 2 × SG medium (Leighton TJ and Doi R. et al.
H. J. Biol. Chem. 246 , 3189, 1971) and shake-cultured at 37 ° C. After the start of culture 4,
6, 7, 8, 9, 10, 11, 12, 24, 36, 48
At 1 hour and 72 hours, 1 ml of the culture solution was collected, collected by centrifugation, and
The cells were suspended in an aqueous solution of mM ethylenediaminetetraacetic acid and 2 mM phenylmethylsulfonyl fluoride and subjected to ultrasonic treatment to disrupt the cells. 2x equal amount of this ultrasonic breaker
Sample buffer (125 mM Tris-HCl (pH 6.
8), 20% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.02% bromophenol blue), boiled for 5 minutes, cooled and centrifuged. This supernatant was used for the Laemmli method (UK Laemmli Nature, 22
7 , 680, 1970), and SDS-polyacrylamide gel electrophoresis was performed. After the electrophoresis, the gel was stained with Coomassie Brilliant Blue to remove excess stain solution, and then the protein band was observed.

The CRY-1-2 protein was stably produced and accumulated until 12 hours after the start of the culture, but when the culture was continued for a longer time, the molecular weight of 130,000 corresponding to the crystal toxin protein gradually increased due to the action of protease. The number of bands has decreased. on the other hand,
The C-terminal side of the CRY-1-2 protein is the C-terminal of the CRY-1-1 protein.
The fusion toxin protein exchanged with the end side was hardly decomposed by protease, and was stably accumulated in the culture for 72 hours.

When the production amount of the fused crystal toxin protein was measured by a densitometer, the production amount was about 20% of the total bacterial protein.

6) Stability of the fused crystal toxin protein gene integrated into the chromosome The stability of the gene integrated into the Bacillus subtilis chromosome was determined by transforming Bacillus subtilis 1A96 / pVGCRY2 / 1-60 into LB-liquid medium containing no Cm. Whether the amount of the fused crystal toxin protein produced by subculturing in
It was confirmed by examining by S-polyacrylamide gel electrophoresis.

As a result, there was almost no difference in the production amount of the fusion toxin protein even after repeated subculture 5 times in the Cm-free medium, suggesting that the gene integrated in the Bacillus subtilis chromosome is stably retained. Was done.

7) Insecticidal test 1A96 / pVGCRY2-60 and 1A9 described above
After culturing 6 / pVGCRY2 / 1-60 in LB liquid medium containing 60 μg / ml of Cm overnight, 1 ml of it was added to 50 ml.
LB liquid medium was inoculated and cultured at 30 ° C. for 24 hours.
The bacterial cells collected by centrifuging the culture solution were suspended in 1 mM ethylenediaminetetraacetic acid, 2 mM phenylmethylsulfonyl fluoride, 100 μg / ml lysozyme solution, and 3
After keeping the temperature at 7 ° C. for 10 minutes, ultrasonic treatment was performed. next,
The solution was centrifuged, and the precipitate was collected and suspended in 1 mM ethylenediaminetetraacetic acid and 2 mM phenylmethylsulfonyl fluoride solution.

The insecticidal test is carried out by appropriately diluting this suspension,
It was carried out by immersing the cabbage leaves, air-drying them, feeding them on 30 third-instar larvae of diamondback moth, and counting the dead insects after 3 days.

As a result, the fused crystal toxin protein has a strong insecticidal activity against diamondback moth, and CRY-1-
It has been revealed that the insecticidal activity is equivalent to that of the two proteins.

[0046]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to stably produce a crystalline toxin using Bacillus subtilis in a medium containing no antibiotics without concern about degradation of the product by protease. As a result, a pesticide that is harmless to humans and livestock can be provided stably and at a low cost, which will greatly contribute to the agriculture and pesticide industries.

[0047]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1177 Sequence type: Nucleic acid and amino acid Origin organism name: Bacillus thuringiensis var. Kurstaki Strain name: HD-1 ATG GAT AAC AAT CCG AAC ATC AAT GAA TGC ATT CCT TAT AAT TGT 45 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys 5 10 15 TTA AGT AAC CCT GAA GTA GAA GTA TTA GGT GAA GAA AGA ATA GAA 90 Leu Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu 20 25 30 ACT GGT TAT ACC CCA ATC GAT ATT TCC TTG TCG CTA ACG CAA TTT 135 Thr Gly Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe 35 40 45 CTT TTG AGT GAA TTT GTT CCC GGT GCT GGA TTT GTG TTA GGA CTA 180 Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu 50 55 60 GTT GAT ATA ATA TGG GGA ATT TTT GGT CCC TCT CAA TGG GAC GCA 225 Val Asp Ile Ile Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala 65 70 75 TTT CTT GTA CAA ATT GAA CAG TTA ATT AAC CAA AGA ATA GAA GAA 270 Phe Leu Val Gln Ile Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu 80 85 90 TTC GCT AGG AAC CAA GCC ATT TCT AGA TTA GAA GGA CTA AGC AAT 315 Phe Ala Arg Asn Gln Ala Ile Ser Arg Leu Glu Gly Leu Ser Asn 95 100 105 CTT TAT CAA ATT TAC GCA GAA TCT TTT AGA GAG TGG GAA GCA GAT 360 Leu Tyr Gln Ile Tyr Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp 110 115 120 CCT ACT AAT CCA GCA TTA AGA GAA GAG ATG CGT ATT CAA TTC AAT 405 Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe Asn 125 130 135 GAC ATG AAC AGT GCC CTT ACA ACC GCT ATT CCT CTT TTT GCA GTT 450 Asp Met Asn Ser Ala Leu Thr Thr Ala Ile Pro Leu Phe Ala Val 140 145 150 CAA AAT TAT CAA GTT CCT CTT TTA TCA GTA TAT GTT CAA GCT GCA 495 Gln Asn Tyr Gln Val Pro Leu Leu Ser Val Tyr Val Gln Ala Ala 155 160 165 AAT TTA CAT TTA TCA GTT TTG AGA GAT GTT TCA GTG TTT GGA CAA 540 Asn Leu His Leu Ser Val Leu Arg Asp Val Ser Val Phe Gly Gln 170 175 180 AGG TGG GGA TTT GAT GCC GCG ACT ATC AAT AGT CGT TAT AAT GAT 585 Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg Tyr Asn Asp 185 190 195 TTA ACT AGG CTT ATT GGC AAC TAT ACA GAT CAT GCT GTA CGC TGG 630 Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val Arg Trp 200 205 210 TAC AAT ACG GGA TTA GAG CGT GTA TGG GGA CCG GAT TCT AGA GAT 675 Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg Asp 215 220 225 TGG ATA AGA TAT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val 230 235 240 TTA GAT ATC GTT TCT CTA TTT CCG AAC TAT GAT AGT AGA ACG TAT 765 Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr 245 250 255 CCA ATT CGA ACA GTT TCC CAA TTA ACA AGA GAA ATT TAT ACA AAC 810 Pro Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn 260 265 270 CCA GTA TTA GAA AAT TTT GAT GGT AGT TTT CGA GGC TCG GCT CAG 855 Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln 275 280 285 GGC ATA GAA GGA AGT ATT AGG AGT CCA CAT TTG ATG GAT ATA CTT 900 Gly Ile Glu Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu 290 295 300 AAC AGT ATA ACC ATC TAT ACG GAT GCT CAT AGA GGA GAA TAT TAT 945 Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr 305 310 315 TGG TCA GGG CAT CAA ATA ATG GCT TCT CCT GTA GGG TTT TCG GGG 990 Trp Ser Gly His Gln Ile Met Ala Ser Pro Val Gly Phe Ser Gly 320 325 330 CCA GAA TTC ACT TTT CCG CTA TAT GGA ACT ATG GGA AAT GCA GCT 1035 Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala 335 340 345 CCA CAA CAA CGT ATT GTT GCT CAA CTA GGT GAG GGC GTG TAT AGA 1080 Pro Gln Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg 350 355 360 ACA TTA TCG TCC ACT TTA TAT AGA AGA CCT TTT AAT ATA GGG ATA 1125 Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile 365 370 375 AAT AAT CAA CAA CTA TCT GTT CTT GAC GGG ACA GAA TTT GCT TAT 1170 Asn Asn Gln Gln Leu Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr 380 385 390 GGA ACC TCC TCA AAT TTG CCA TCC GCT GTA TAC AGA AAA AGC GGA 1215 Gly Thr Ser Ser Asn Leu Pro Ser Ala Val Tyr Arg Lys Ser Gly 395 400 405 ACG GTA GAT TCG CTG GAT GAA ATA CCG CCA CAG AAT AAC AAC GTG 1260 Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln As n Asn Asn Val 410 415 420 CCA CCT AGG CAA GGA TTT AGT CAT CGA TTA AGC CAT GTT TCA ATG 1305 Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His Val Ser Met 425 430 435 TTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGT ATA ATA AGA GCT 1350 Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile Arg Ala 440 445 450 CCT ATG TTC TCT TGG ATA CAT CGT AGT GCT GAA TTT AAT AAT ATA 1395 Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn Ile 455 460 465 ATT CCT TCA TCA CAA ATT ACA CAA ATA CCT TTA ACA AAA TCT ACT 1440 Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr 470 475 480 AAT CTT GGC TCT GGA ACT TCT GTC GTT AAA GGA CCA GGA TTT ACA 1485 Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr 485 490 495 GGA GGA GAT ATT CTT CGA AGA ACT TCA CCT GGC CAG ATT TCA ACC 1530 Gly Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr 500 505 510 TTA AGA GTA AAT ATT ACT GCA CCA TTA TCA CAA AGA TAT CGG GTA 1575 Leu Arg Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val 515 520 525 AGA ATT CGC TAC GCT TCT ACC ACA AAT TTA CAA TTC CAT ACA TCA 1620 Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser 530 535 540 ATT GAC GGA AGA CCT ATT AAT CAG GGG AAT TTT TCA GCA ACT ATG 1665 Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met 545 550 555 AGT AGT GGG AGT AAT TTA CAG TCC GGA AGC TTT AGG ACT GTA GGT 1710 Ser Ser Gly Ser Asn Leu Gln Ser Gly Ser Phe Arg Thr Val Gly 560 565 570 TTT ACT ACT CCG TTT AAC TTT TCA AAT GGA TCA AGT GTA TTT ACG 1755 Phe Thr Thr Pro Phe Asn Phe Ser Asn Gly Ser Ser Val Phe Thr 575 580 585 TTA AGT GCT CAT GTC TTC AAT TCA GGC AAT GAA GTT TAT ATA GAT 1800 Leu Ser Ala His Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp 590 595 600 CGA ATT GAA TTT GTT CCG GCA GAA GTA ACC TTT GAG GCA GAA TAT 1845 Arg Ile Glu Phe Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr 605 610 615 GAT TTA GAA AGA GCA CAA AAG GCG GTG AAT GAG CTG TTT ACT TCT 1890 Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Glu Leu Phe Thr Ser 620 625 630 TCC AAT CAA ATC GGG TTA AAA ACA GAT GTG ACG GAT TAT CAT ATT 1935 Ser Asn Gln Ile Gly Leu Lys Thr Asp Van Thr Asp Tyr His Ile 635 640 645 GAT CAA GTA TCC AAT TTA GTT GAG TGT TTA TCT GAT GAA TTT TGT 1980 Asp Gln Van Ser Asn Leu Val Glu Cys Leu Ser Asp Glu Phe Cys 650 655 660 CTG GAT GAA AAA AAA GAA TTG TCC GAG AAA GTC AAA CAT GCG AAG 2025 Leu Asp Glu Lys Lys Glu Leu Ser Glu Lys Val Lys His Ala Lys 665 670 675 CGA CTT AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC TTT AGA 2070 Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg 680 685 690 GGG ATC AAT AGA CAA CTA GAC CGT GGC TGG AGA GGA AGT ACG GAT 2115 Gly Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp 695 700 705 ATT ACC ATC CAA GGA GGC GAT GAC GTA TTC AAA GAG AAT TAC GTT 2160 Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyl Val 710 715 720 ACG CTA TTG GGT ACC TTT GAT GAG TGC TAT CCA ACG TAT TTA TAT 2205 Thr Leu Leu Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr 725 730 735 CAA AAA ATA GAT GAG TCG AAA TTA AAA GCC TAT ACC CGT TAT CAA 2250 Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Gln 740 745 750 TTA AGA GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA 2295 Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu 755 760 765 ATT CGC TAC AAT GCA AAA CAT GAA ACA GTA AAT GTG CCA GGT ACG 2340 Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr 770 775 780 GGT TCC TTA TGG CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT 2385 Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys 785 790 795 GGA GAG CCG AAT CGA TGC GCG CCA CAC CTT GAA TGG AAT CCT GAC 2430 Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp 800 805 810 TTA GAT TGT TCG TGT AGG GAT GGA GAA AAG TGT GCC CAT CAT TCG 2475 Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser 815 820 825 CAT CAT TTC TCC TTA GAC ATT GAT GTA GGA TGT ACA GAC TTA AAT 2520 His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn 830 835 840 GAG GAC CTA GGT GTA TGG GTG ATC TTT AAG ATT AAG ACG CAA GAT 2565 Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp 845 850 855 G GG CAC GCA AGA CTA GGG AAT CTA GAG TTT CTC GAA GAG AAA CCA 2610 Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro 860 865 870 TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA AGA GCG GAG AAA AAA 2655 Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys 875 880 885 TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA ACA AAT ATC GTT 2700 Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr Asn Ile Val 890 895 900 TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT TTA AAC TCT 2745 Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser 905 910 915 CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG ATT CAT 2790 Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His 920 925 930 GCG GCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG CCT 2835 Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu Pro 935 940 945 GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA 2880 Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu 950 955 960 TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CT A TAT GAT GCG AGA 2925 Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyl Asp Ala Arg 965 970 975 AAT GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG 2970 Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Asn Gly Leu Ser Cys Trp 980 985 990 AAC GTG AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT 3015 Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg 995 1000 1005 TCG GTC CTT GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA 3060 Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu 1010 1015 1020 GTT CGT GTC TGT CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG 3105 Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala 1025 1030 1035 TAC AAG GAG GGA TAT GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC 3150 Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile 1040 1045 1050 GAG AAC AAT ACA GAC GAA CTG AAG TTT AGC AAC AGC GTA GAA GAG 3195 Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu 1055 1060 1065 GAA ATC TAT CCA AAT AAC ACG GTA ACG TGT AAT GAT TAT ACT GTA 3240 Glu Ile Tyr Plo Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val 1070 1075 1080 AAT CAA GAA GAA TAC GGA GGT GCG TAC ACT TCT CGT AAT CGA GGA 3285 Asn Gln Gln Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly 1085 1090 1095 TAT AAC GAA GCT CCT TCC GTA CCA GCT GAT TAT GCG TCA GTC TAT 3330 Tyr Asn Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr 1100 1105 1110 GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA GAG AAT CCT TGT GAA 3375 Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu 1115 1120 1125 TTT AAC AGA GGG TAT AGG GAT TAC ACG CCA CTA CCA GTT GGT TAT 3420 Phe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr 1130 1135 1140 GTG ACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT AAG GTA TGG 3465 Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp 1145 1150 1155 ATT GAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC AGC GTG 3510 Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val 1160 1165 1170 GAA TTA CTC CTT ATG GAG GAA 1196 Glu Leu Leu Leu Met Glu Glu 1175

[Brief description of drawings]

FIG. 1 shows a method for constructing a gene encoding a fusion crystal toxin protein. In the restriction enzyme action site in the figure, A is A
haIII and E represent EcoRI and K represents KpnI. Also, the thin line indicates E. coli plasmid pUC9, and the open and dotted boxes indicate the cry-1-1 and cry-1-2 crystal toxin protein genes, respectively.

FIG. 3 shows the structure of a fusion crystal toxin protein gene-integrated expression vector. In the restriction enzyme action site in the figure, E represents EcoRI, K represents KpnI, and S represents SalI. The thin line is the E. coli plasmid pCU19, and the white line is CAT for C.
m resistance gene, the diagonal line is the DNA fragment upstream of the spoVG gene promoter, the thick black line is the spoVG promoter, and the white and dotted boxes are cry-1-1 and cry-1-
2 represents the crystal toxin gene.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] January 5, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Figure 2

[Correction method] Change

[Correction content]

FIG. 2 shows the structure of a fusion crystal toxin protein gene-integrated expression vector. In the restriction enzyme action site in the figure, E represents EcoRI, K represents KpnI, and S represents SalI. The thin line is the E. coli plasmid pCU19, and the white line is CAT for C.
m resistance gene, the diagonal line is the DNA fragment upstream of the spoVG gene promoter, the thick black line is the spoVG promoter, and the white and dotted boxes are cry-1-1 and cry-1-
2 represents the crystal toxin gene.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display area A01N 63/02 E 9159-4H C12P 21/02 C 8214-4B (C12N 15/32 C12R 1:07 ) (C12P 21/02 C12R 1: 125)

Claims (2)

[Claims] 1. A crystalline toxin protein of Bacillus thuringiensis var. Kristaki HD-1 having C-terminal side of CRY-1-2 is Bacillus thuringiensis var. Kristaki HD.
1. A fused crystalline toxin protein substituted on the C-terminal side of CRY-1-1 which is a crystalline toxin protein of -1. 2. A gene encoding the fusion crystal toxin protein according to claim 1.