JPH06205682A - Transcription control sequence for expressing bt insecticidal protein - Google Patents

Abstract

PURPOSE:To obtain a new sequence capable of controlling the expression of a Bt insecticidal protein in Bacillus thuringiensis. CONSTITUTION:This transcription control sequence has a base sequence of the formula for expressing Bt insecticidal protein. The sequence is obtained by preparing a mutant deficient in a 5'-untranslatable region of an insecticidal ISRH4 gene of 134K daltons derived from Bacillus, thuringiensis var. israelensis and then constructing an expression plasmid of the insecticidal protein gene from the gene, transforming the Bacillus thuringiensis according to an electric pulse method, measuring the amount of expression and finding the sequence capable of controlling the expression of the Bt insecticidal protein.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a transcriptional regulatory sequence for the expression of Bt insecticidal protein derived from Bacillus thuringiensis, which contains the same and is capable of expressing the Bt insecticidal protein in Bacillus thuringiensis. The present invention relates to an expression plasmid, a microorganism carrying the plasmid, and a method for producing a Bt insecticidal protein, which comprises culturing the microorganism.

[0002]

BACKGROUND OF THE INVENTION Bacillus thuringiensis is a gram-negative soil bacterium consisting of an insecticidal protein during sporulation 1.
It produces crystals of ˜2 μm. It is known that some insects that eat this crystal will stop eating and eventually die due to dehydration. A BT agent is obtained by culturing Bacillus thuringiensis and preparing a mixture of crystals of insecticidal protein and spores, which is used as an insecticide. Hereinafter, this insecticidal protein derived from Bacillus thuringiensis is referred to as Bt insecticidal protein. Bacillus thuringiensis is classified into about 30 subspecies depending on the serotype and esterase type by the flagella antigen, and each strain produces an insecticidal protein that shows specific and different insecticidal activity. . This insecticidal protein gene
It exists on the plasmid or the chromosome of Chuuringensis, and many genes have already been cloned and its nucleotide sequence has been elucidated. Bacillus thuringiensis islaensis, which has a high insecticidal activity against Diptera larvae, has a characteristic not found in other strains, in which four different proteins mainly combine to form mature crystals. Have Therefore, it is not known at present what kind of control the individual insecticidal protein genes in Bacillus thuringiensis Islaensis are expressed under.

[0003]

[Problems to be Solved by the Invention] By incorporating an insecticidal protein gene, which has already been cloned using a genetic engineering technique, into an expression vector and introducing it into a host such as Escherichia coli, Bacillus subtilis, Pseudomonas, etc. Attempts have been made to produce in heterologous cells. However, in this case, industrially, a recombinant-specific manufacturing facility is required because the expression is carried out in a heterologous host. Further, when Bacillus thuringiensis is directly used, there are drawbacks that the activity is very specific and the insecticidal spectrum is low as described above. Conventionally, for the breeding of Bacillus thuringiensis, strains showing useful insecticidal activity were isolated from the natural world.
It has been promoted by selecting. However, in order to actively breed Bacillus thuringiensis, it is more effective to artificially improve the strain by expressing a highly active protein by a genetic engineering technique. However, regarding the expression of Bt insecticidal protein using a genetic engineering method using Bacillus thuringiensis as a host, a host vector system has been established because there is no efficient gene transfer method in Bacillus thuringiensis so far. The current situation is that it has not been done.

[0004]

Therefore, the present inventor has aimed to control the expression of the Bt insecticidal protein in Bacillus thuringiensis, and thus, to control the expression of Bt. Various deletion mutants of the 5'untranslated region of the insecticidal protein ISRH4 gene were constructed, expression plasmids for the gene and the insecticidal protein gene were constructed, and Bacillus thuringiensis was transformed using the electric pulse method. Then, the expression level was measured. As a result, they succeeded in finding a sequence controlling the expression of Bt insecticidal protein.

[0005]

EFFECTS OF THE INVENTION By using the Bt insecticidal protein expression control sequence of the present invention, it is possible to develop a host vector system in Bacillus thuringiensis for which an effective host vector system has not been developed so far. Become. By using this host vector system, it becomes possible to create a strain having a higher insecticidal activity by using a genetic engineering technique. When a Bt insecticidal protein is produced using Bacillus thuringiensis as a host, this transgenic strain is not a recombinant strain, and therefore can be distributed outdoors as a Bt insecticidal protein.

The present invention will be described in more detail below. The present invention provides a transcriptional regulatory sequence for the expression of Bt insecticidal protein derived from Bacillus thuringiensis represented by the nucleotide sequence of Chemical formula 5,
Bt in Bacillus thuringiensis containing the sequence
The present invention relates to an expression plasmid capable of expressing an insecticidal protein, a microorganism carrying the plasmid, and a method for producing a Bt insecticidal protein, which comprises culturing the microorganism.

## STR00005 ## SEQ ID NO: 1 The present inventor first investigated four insecticidal protein genes (Agric.Bio) from Bacillus thuringiensis Islaensis strain.
L. Chem., 52, 873-878 (1988)) was deleted to create a strain that does not produce crystals of insecticidal protein. Then, a plasmid vector replicable in the strain was inserted with an insecticidal protein gene showing a strong insecticidal activity against Diptera and introduced into the same strain, whereby the gene was independently expressed in large amounts and expressed. It was found that the protein formed granules in sporulated cells and retained high insecticidal activity against larvae of Aedes mosquitoes. next,
Plasmids containing various deletion mutants of the 5'untranslated region of the insecticidal protein gene were constructed.

The constructed plasmid was introduced into a non-crystallizing strain of Bacillus thuringiensis Islaensis strain using the electric pulse method and the expression level of the insecticidal protein gene was measured to obtain By clarifying the region required for the expression of the insecticidal protein gene and further analyzing the transcript of the gene by the hybridization method or the primer extension method, the transcription start point of the gene in the strain into which the gene was introduced was determined. I succeeded in clarifying it.

[SEQ ID NO: 1] Since the sequence relating to the transcriptional regulation of the insecticidal protein gene in the Bacillus thuringiensis Islaensis strain was elucidated, Bt was linked by using the same sequence to the insecticidal protein gene. It became possible to control the expression of the insecticidal protein gene. Furthermore, it is possible to create a sequence having a higher expression activity by modifying this regulatory sequence using a genetic engineering technique.

[0008] In addition to Bacillus thuringiensis as a host for introducing the expression plasmid for insecticidal protein, which contains the sequence of the present invention, the transcriptional control sequence of the present invention functions as a promoter in the sporulation stage of Bacillus subtilis. Bacillus subtilis is also mentioned due to the fact that it is similar to the sequence. Regarding the electric pulse method for introducing the expression plasmid of the insecticidal protein of the present invention into a host, a known method can be used, and the method can be further modified for the strain. The transformed microorganism can be cultured using a known medium. The culturing temperature of the transformed microorganism may be 20 ° C. to 37 ° C., more preferably 30 ° C. Culture of transformed microorganisms
It is possible to culture by shaking culture which is usually used. Culturing is usually carried out for 1 to 4 days, preferably 2 days. After the transformed microorganisms cultured by the above method are collected by a method such as centrifugation, the cells are crushed by a method such as ultrasonic crushing, and crystals of insecticidal protein are collected by a method such as centrifugation. It is possible to recover insecticidal proteins. The insecticidal protein or the treated bacterial cell product thus recovered is used as a Bt insecticidal protein.

[0009]

EXAMPLES Examples will be described below, but it goes without saying that the present invention is not limited thereto. Example 1. Transformation of non-crystallizing strain of Bacillus thuringiensis islaerensis. Bacillus thuringiensis Isla Ellensis ND52
2 strains [US Department of Agriculture (USDA) deposited strain Goldberg ONR
60] has all insecticidal protein genes present on the 70M Dalton plasmid pBTI-6 [Agric. Biol. Che.
m., 52, 873-878 (1988)]. By culturing the wild strain (HD522) at 42 ° C., a strain (HD522 C37-21) lacking the plasmid pBTI-6 was obtained. This strain was subjected to Southern hybridization [J. Mol. Biol., 98, 503-5 17 (197
5)], the deletion of ISRH3 and ISRH4 genes [Agric. Biol. Chem., 52, 873-878 (1988)] encoding insecticidal proteins was confirmed. Next, a method for introducing a gene was examined using the HD522 C37-21 strain as a host. 100 μl of overnight culture of HD522 C37-21 strain was added to 10 ml of LB medium [Bactotrypton (Difco) 10 g, yeast extract (Difco) 5 g, Na
5 g of Cl (Hanai Kagaku) was added to distilled water to make 1 liter], and the cells were shake-cultured at 30 ° C. until the OD ₆₀₀ reached about 0.5. Transfer the culture to a 15 ml polypropylene tube and place on ice for 10 minutes.
After leaving it for a minute, it was centrifuged (4 ° C., 3500 rpm, 10 minutes) to completely remove the supernatant. Add 1 ml of ice-cold SPB (400 mM sucrose,
Suspended in 1 mM MgCl ₂ , 7 mM phosphate buffer (pH 7.0),
Centrifugation (4 ° C., 3500 rpm, 10 minutes) again was performed to completely remove the supernatant. Suspend the cells in 400 μl of ice-cold SPB and add 200 μl.
Each was transferred to two Eppendorf tubes and placed on ice. After inputting the distance between the electrodes and the electric field strength into the gene transfer device GTE-10 (Shimadzu), HD522 C37-21 was added to the bacterial cell suspension.
0.01 ~ 0.1 μg (~
5 μl) was added and left on ice for 1 minute. A cell suspension (20 cm2) in which the DNA solution was added between the electrodes (0.2 cm) of the chamber (Shimadzu; FTC-03) that had been cooled on ice in advance.
0 μl) was immediately transferred and a pulse was applied. SOC medium (bactotryptone
20g, yeast extract 5g, NaCl 5g, 0.25M KCl 10ml, 5M
Add 0.2 ml of NaOH to distilled water to make 0.96 l, and after autoclaving, just before use, 10 ml of 1M MgSO ₄ and 1M MgCl ₂ 1
The bacterial cell suspension was rapidly transferred to 0 ml of a medium containing 20 ml of 1 M glucose), cultured at 37 ° C. for 90 minutes with shaking, and plated on an LB plate containing an antibiotic. After culturing at 30 ° C. for 30 to 40 hours, colonies were obtained. When the gene was transferred using the HD522 C37-21 strain as a host, the electric field strength was 5.0 kV / cm and the time constant was 1.
Under the condition of 0 msec., Plasmid pUB110 (Sigma)
6.8x10 ³ transformants / μg DNA, plasmid pHY300PLK
(Takara Shuzo) was introduced at an efficiency of 4.7 × 10 ³ transformants / μg DNA. The colonies generated on the drug plate were cultured, and the alkaline method [Nucl. Acid. Res., 7, 1513-1523 (19
79)] and performing Southern hybridization, it was confirmed that all of these plasmids could stably exist as plasmids in the HD522 C37-21 strain.

Embodiment 2. Bacillus thuringiensis
Expression of insecticidal protein gene in Islaelensis HD522 C37-21 strain pHY300PLK, a shuttle vector between E. coli and Bacillus subtilis
Then, the ISRH4 gene was cloned and introduced into the HD522 C37-21 strain. At this time, various deletions of the 5'- and 3'-untranslated regions of the gene were also attempted to determine the region required for expression. ISRH
Plasmid pBGH4 containing four genes [Agric. Biol. Che
m., 52, 873-878 (1988)] with restriction enzymes HindIII and PmaCI
, XmnI, XbaI, etc.
Five types of pIS4-series plasmids (Fig. 1) were constructed by inserting each into the multiple cloning site of 300PLK. These recombinant plasmids were prepared from E. coli and HD522 C37- was prepared by the method described in Example 1.
Introduced into 21 shares. Colonies formed on a plate containing tetracycline (Sigma) were cultured, the plasmid was extracted, and Southern hybridization was performed to confirm that the plasmid was introduced. In addition, a colony in which the introduction of the plasmid was confirmed was picked up, inoculated into an LB medium containing tetracycline, and cultured by shaking at 30 ° C. overnight. OD ₆₀₀
After confirming that the culture was in the almost stationary phase, transfer the culture solution to a 15 ml polypropylene tube and centrifuge (4
The supernatant was removed by heating at 3500 rpm for 15 minutes. 10 ml of sporulation medium [Nutrient Broth (Difco)]
Dissolve 3.2 g in distilled water to 0.4 l, and after autoclaving, dissolve 5 g of MgSO ₄ .7H ₂ O and 20 g of KCl in distilled water to 100 m.
1 ml Ca solution (4 ml), 1M Ca (NO ₃ ) ₂ 0.4 ml, 0.1M MnC
l ₂ was added to 0.04 ml and 0.01 M FeSO ₄ was added to 0.04 ml],
The culture was further incubated at 30 ° C. overnight to complete sporulation. The progress of sporulation was observed using a phase contrast microscope. After the sporulation was completed, proteins were extracted from the cells and analyzed for the expression of the introduced ISRH4 gene by sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (PAGE) [Nature, 227, 680-685 (1970)]. . That is, the culture medium 0.
5 to 1 ml was collected and the supernatant was completely removed. Bacterial cells to 1M Na
After washing with Cl, the cells were suspended in 100 to 200 μl of sterilized water. While cooling in ice water, the cells were sonicated 4 times for 30 seconds each to disrupt the cells. 3 μl of sample buffer (0.14M Tris-HCl pH6.8, 22.4% glycerol, 6% SDS, 0.2% bromphenol-blue, 10
% β-mercaptoethanol), heat in boiling water for 5 minutes, and then rapidly cool to 12 μm on SDS-polyacrylamide gel.
l was applied. After the migration, the protein was stained with Coomassie Brilliant Blue. As a result, the ISRH4 gene is HD522.
It was found that the C37-21 strain alone was highly expressed. The expression level of ISRH4 protein in the transformants carrying pIS402, pIS431, and pIS422 was approximately three times that of the wild type strain. It is considered that one of the reasons for high expression is that the gene is present in a high copy number. On the other hand, when the 5'-untranslated region was deleted up to the XmnI site (pIS412), the expression level was remarkably reduced, which was equal to or lower than that of the wild type strain.

Embodiment 3. Bacillus thuringiensis
Insecticidal activity of ISRH4 protein produced by Isla-elensis HD522 C37-21 strain A part of the culture solution was collected for the wild-type strain (HD522) having completed sporulation and the HD522 C37-21 strain having pIS431. Ultrasonication was performed according to the method described. The disrupted cell suspension was centrifuged, and the supernatant and the precipitate were subjected to SDS-PAGE. As a result, a crystalline protein band was observed on the precipitate side in the wild strain. Similarly, a band of ISRH4 was also found on the precipitate side in the transformant. As a result of microscopic observation, granules could be observed in the mother cells of the transformant in which sporulation was completed. While the crystals of the wild strain were oval, the granules of the transformant had a cubic structure and were large in size, as expected from the expression level by SDS-PAGE. The insecticidal activity was measured using this granule. HD522 shares and
Cells of HD522 C37-21 strain that had completed sporulation were collected by aligning OD units, ultrasonically disrupted, and the resulting precipitate fractions (spores and crystals) were given to Culex pipiens larvae at the 3rd stage.
The number of dead insects after the lapse of time was measured. As a result, 20 of 20 cows died when the precipitate fraction of HD522 strain was given, while HD52
2 When the precipitate fraction of C37-21 strain was given, 7 out of 20 died. Furthermore, a precipitate fraction was prepared from HD522 strain and HD522 C37-21 strain harboring plasmid pIS431 in the same manner and solubilized with an alkaline solution [0.2 M glycine-NaOH (pH 10.5)], and the amount of protein was adjusted to latex. Beads (Sigma,
0.8 μm) and then given to Culex pipiens larvae of the third instar, and the number of dead larvae was measured 48 hours later. In the case of the sample of HD522 strain (0.1 μg / ml), 19 out of 20 died, while the sample of HD522 C37-21 strain (0.1 μg / m) carrying pIS431.
In l), 18 out of 20 died. From the above, pIS
In the HD 522 C37-21 strain holding 431, Culex pipiens
It was revealed that a protein showing insecticidal activity was produced in larvae.

Example 4. Elucidation of transcriptional regulatory region of ISRH4 gene (Step 1) Examination of transcriptional regulatory region by CAT assay It was shown that the insecticidal protein ISRH4 gene was expressed by its own promoter in pBTI-6 deletion strain. ,
In order to determine the promoter region of ISRH4 gene and quantitatively show its activity, an assay system for promoter activity in Bacillus thuringiensis Islaelensis was established. First, promoter search vector pKK232
-8 (Pharmacia) was cut with Eco47III, and at the cut site,
A fragment obtained by cutting pUB110 with PvuII was inserted (Fig. 2). This was designated as pKKUBIV and introduced into the HD522 C37-21 strain by the gene introduction method described in Example 1. Contains kanamycin
By culturing the resulting colonies on the LB plate, extracting the plasmid, and performing Southern hybridization, it was confirmed that this vector could replicate in the HD522 C37-21 strain. This shuttle vector pKKUBIV
SmaI and HindII present at the multi-cloning site
It was cleaved at the I site, and an approximately 2.15 kb PvuII-HindIII fragment containing a sufficient 5'-untranslated region believed to contain the promoter of the ISRH4 gene and the first half of the reading frame was inserted at this cleavage site. In the obtained recombinant plasmid pKKUBPv, the ISRH4 gene and the cat gene of the vector are fused in the form of an operon, and it is expected that the cat gene will be expressed by read through from the promoter of the ISRH4 gene. We introduced this pKKUBPv into the HD522 C37-21 strain,
Cell proteins were prepared for each transformant at each growth stage, and chloramphenicol acetyltransferase (CAT) activity was measured for each protein sample as follows. Transfer 10 ml of culture solution in the sporulation period to a 15 ml polypropylene tube and centrifuge (4 ° C, 3500 rpm, 15 ° C).
Then, the supernatant was completely removed. Suspend the cells in 1 ml of washing buffer (1M NaCl, 50mM Tris-HCl pH8.0) and
Transferred to l eppendorf tube. Centrifuge (4 ℃, 1
After completely removing the supernatant by centrifugation at 0,000 rpm for 3 minutes, add glass beads (Sigma, 0.1-0.11 mm) in an equivalent volume to 2 times that of the cells, and add 400 μl of extraction buffer (50 mM Tris). -Hydrochloric acid
pH 8.0, 0.1 M phenylmethylsulfonyl fluoride, 50 μM dithiothreitol) was added. The cells were crushed with a bead beater 3 times each for 1 minute while cooling on ice. After centrifugation (4 ° C., 10,000 rpm, 3 minutes), the supernatant was transferred to a new Eppendorf tube, and the protein concentration of the bacterial cell extract solution was measured using a Protein assay kit (Bio-Rad). DTNB solution (5,5'-dithiobis-2-nitrobenzoic acid
Dissolve 4mg in 0.1M Tris-HCl pH7.8 10ml) 3ml
20 μl of 5 mM acetyl-CoA and 20 μl of cell extract solution were added, and the well mixed mixture was transferred to 1 ml of the cell for absorbance measurement. As a control, the absorbance at 412 nm was set to 0 using DTNB solution, and then 60 μl was added to the cell containing the sample.
l of 5 mM chloramphenicol was added, and the resulting mixture was immediately mixed, and the time-dependent change in absorbance at 412 nm was recorded on the chart. The reaction rate v (ΔABS ₄₁₂ / min) was calculated from the curve of the chart, and the nmol number of acetyl groups transferred per minute per 1 mg of protein was calculated according to the following formula, and this was defined as the CAT activity. CAT activity = vx 220.6 x 1/20 μl of protein contained in
mg number (nmol / min.mg) CAT activity increased with the progress of sporulation, and a sharp increase was observed about 10 hours after entering the stationary phase.
On the other hand, in the HD522 C37-21 strain into which the promoter-free plasmid (pKKUBIV) had been introduced, no increase in CAT activity was observed at any growth stage. This suggests that transcriptional level regulation is involved in the temporal regulation of ISRH4 gene expression. Then, the transcriptional activity of the ISRH4 gene gradually increases during the sporulation stage, and the mid-sporulation stage, (stage III to stage IV according to the definition of Bacillus subtilis sporulation), that is, sporulation It was found to increase sharply about 10 hours after the start. Since the CAT protein was found to be stable in Bacillus thuringiensis sporulating cells, the first half of the ISRH4 gene with various deletions in the 5'-untranslated region was constructed in the same manner as pKKUBPv was constructed. PKKU part
It was inserted into BIV (Fig. 3). Then, the CAT activity was measured at the time when the HD522 C37-21 strain into which these recombinant plasmids had been introduced completed sporulation (FIG. 4). In this case, it is unclear how long the plasmid once introduced will remain stable in the HD522C37-21 strain.Therefore, for all constructions to be assayed, the colonies generated on the kanamycin plate were carried out simultaneously from the gene introduction. Two of each were picked up and cultured. In addition, the progress of sporulation was always observed using a phase contrast microscope, and cells were collected from a strain in which sporulation was almost completely completed. As a result, a plasmid without promoter inserted (pKKUBIV), ISRH4 gene and cat
Those constructed so that the transcription direction of the gene is opposite (pKK
UBH ') introduced strains had very low CAT activity. On the other hand, it exists upstream of the ISRH4 gene.
About 30 times the activity obtained from pKKUBIV, pKKUBH'-introduced strains containing pKKUBH and pKKUBPv containing the upstream of the stop codon of the reading frame (IS240) transcribed in the same direction as the ISRH4 gene. High activity was obtained. Then, if the entire region upstream from the PmaCI site existing at a position of about 630 bp upstream of the initiation codon of the ISRH4 gene is deleted (p
KKUBPm) and CAT activity decreased to about 20%. Furthermore,
When the XmnI site located approximately 260 bp upstream of the start codon was deleted (pKKUBX), CAT activity was completely reduced to the background level. From these experiments, it was found that at least the region upstream from XmnI is required for the expression of the ISRH4 gene, which is consistent with the result of the analysis of the fluctuation of the protein level expression in Example 2.
(pIS412; see Figure 1). In addition, it was found that a region further upstream of PmaCI is required for maximum expression of ISRH4 gene. From pKKUBPv, 0.37 kb Pm
When the aCI-XmnI fragment was deleted, (pKKUBdelPm-X) CAT activity was about 15% of the maximum level, but did not decrease to background. From the above experiment, PmaCI-Xm
There are two possibilities in the 0.37 kb region of nI: whether there is a promoter that works by itself, or whether there is a sequence that exists downstream of XmnI and that performs an enhancer-like action on a promoter that does not function by itself. Was thought. And 0.24 kb PvuII-Pm existing upstream
A similar possibility was considered for the function of the aCI fragment.

(Step 2) Determination of ISRH4 Gene Transcription Start Point In order to determine the ISRH4 gene transcription start point, first, ISRH
Total RNA was prepared from cells expressing 4 genes, slot blot analysis was performed, and 0.34 Kb of XmnI containing the translation region was analyzed.
-EcoRV fragment and the upstream PmaCI-XmnI fragment of 0.37 kb were used as probes for hybridization. Preparation of total RNA was performed as follows. 20 ml of the culture solution of the HD522 C37-21 strain into which the plasmid pIS431 was introduced during the sporulation period was transferred to a 50 ml polypropylene tube in which ice blocks had been previously placed, and the cells were rapidly cooled. Centrifuge this (4 ° C, 3500 rpm, 3-5 minutes) to remove the supernatant and ice cubes, and add 40 μl of Vanagyl ribonucleoside Complex to the cells.
700 μl RNA buffer (0.05M Tris-HCl pH8.0, 0.
1M NaCl, 10 mM EDTA) was added and suspended. 600 in advance
2m containing μl glass beads (0.1 to 0.11mm)
It was transferred to an l eppendorf tube (1.5 ml) and treated with phenol / chloroform. Vanagyl Ribonucleosi
The phenol / chloroform treatment was repeated 4 to 5 times until the dark green color of de Complex was almost completely removed, followed by chloroform / isoamyl alcohol treatment and ethanol precipitation. This precipitate was added to 400 μl of DNase buffer (50 mM CH ₃ COON
apH6.5, 10 mM MgCl ₂ , 2 mM CaCl ₂ )
μl of DNase I (Vesesta Research Laboratory, RN
ase-free) was added, and the mixture was reacted at 37 ° C for 25 minutes. This was immediately followed by phenol / chloroform treatment, phenol / isoamyl alcohol treatment, and ethanol precipitation. The resulting RNA precipitate was 400-500 μl of solution IV (0.02
M CH ₃ COONa pH5.5, 0.5% SDS, 1 mM EDTA),
It was used as an RNA standard. The slot blot of RNA was performed as follows. Regarding the RNA solution prepared by the above method,
The concentration was measured with an absorptiometer. 7 μg of RNA sample to be adsorbed on the membrane was placed in an Eppendorf tube and sterilized water was added to make the total volume 60 μl. To this, 120 μl of a formamide-formaldehyde mixed solution (a mixture of 750 μl formamide and 250 μl formaldehyde) was added, and the mixture was kept at 50 ° C. for 1 hour to denature RNA. After this,
The solution was transferred onto ice and rapidly cooled, and 520 μl of sterilized water was added. On the other hand, nylon membrane (Gene Screen Plus, NEN Rese
arch products) were cut according to a slot blot apparatus (Bio-Dot SF Microfiltration apparatus), and immersed in distilled water for 15 minutes. This was superposed on three filter papers which were also soaked in distilled water, fixed on a slot blot apparatus, and the denatured RNA sample was poured into the wells. After confirming that all the RNA solution had flowed down, the membrane was removed from the device and air-dried on filter paper. Furthermore, after being left under vacuum at 80 ° C. for 2 hours, RNA was immobilized on the membrane, and then Southern hybridization was carried out with a labeled DNA probe. The hybridization temperature and the filter washing temperature were 60 ° C. As a result, the same level of signal was obtained regardless of which probe was used. From this, it was revealed that at least one promoter was present at a position upstream from XmnI. Then, upstream of the start codon 274-29
Oligomeric DNA complementary to the position of 6 bases (base numbers 601 to 579 of the base sequence 1) was synthesized and used for primer extension. Synthesized primer DN
A 10 pmol to 5xkination buffer (0.25M Tris-HCl p
H7.6, 50 mM MgCl ₂ , 25 mM dithiothreitol, 0.5 mM
Spermidine hydrochloric acid, 0.5 mM EDTA pH8.0) 4 μl, [γ-
5 μl of ³² P] ATP (Amersham, 5000 Ci / mmol) and sterilized water were added to make the total volume 19 μl. Add this to T4 polynuc
1 μl of leotido kinase (Takara Shuzo, 10 units / μl) was added and reacted at 37 ° C for 45 minutes. 0.5 μl of kinase was newly added and further reacted at 37 ° C. for 30 minutes to label the 5 ′ end of the primer. After heating at 68 ° C for 10 minutes to inactivate the kinase, ethanol precipitation was performed. The end-labeled primer was collected and then suspended in 50 μl of sterilized water. 10 ⁴ to 10 ⁶ cpm of primer was added to 50 to 100 μg of RNA sample, ethanol precipitation was performed, and the sample was naturally dried at room temperature. 30 to this
μl hybridization buffer (40mM PIPES, 1m
M EDTA pH8.0, 0.4 M NaCl, 80% formamide, prepared immediately before use) was added, and the precipitate was well dissolved by pipetting. After heating at 85 ° C for 10 minutes to denature the RNA and primer DNA, an annealing reaction was performed at 30 ° C overnight.
170 μl of sterilized water was added to the reaction solution for ethanol precipitation. At that time, leave it at -20 ℃ for 1 hour, then at 14,000 rpm for 15
Centrifugation centrifugation was performed, washing with 70% ethanol, and natural drying. Precipitate with 20 μl Reverse transcriptase buffer (50
mM Tris-HCl pH 7.6, 60 mM KCl, 10 mM MgCl ₂ , 2 mM d
NTP, 1 mM dithiothreitol, 50 μg / ml actinomycin D, prepared immediately before use) and dissolved in M-MLV Reverse
0.5 μl of transcriptase (RT, Vestista Research Laboratory, 200 units / μl) was added. CDNA was synthesized by reacting at 37 ° C for 2 hours. 0.5M EDTA
Add 1 μl of (pH8.0) to stop the RT reaction, and add RNase A (20
1 μl of (μg / ml) was added and incubated at 37 ° C. for 30 minutes to decompose RNA. TE containing 10 mM Tris (10 mM Tris)
-Hydrochloric acid pH8.0, 1 mM EDTA) (150 μl) was added, phenol / chloroform treatment and ethanol precipitation were performed. At this time, the mixture was left at -20 ° C for 1 hour and then centrifuged at 14000 rpm for 15 minutes. After vacuum drying, dissolve the precipitate in 9 μl of formamide dye solution (80% formamide, 1 mM EDTA, 0.1% bromphenol blue, 0.1% xylene cyanol) and
After heating at ℃ for 3 minutes and quenching, apply 5 μl to 8M urea-5% polyacrylamide gel (low resolution gel),
Electrophoresis was performed. After confirming the bands by autoradiography, the remaining 4 μl was electrophoresed on a high-resolution gel with a sequence ladder. The RNA sample at this time had a transformant in the sporulation stage (having pIS431).
HD522 C37-21) or those prepared from transformants in the vegetative growth phase were used. As a result, a band with a length of about 90 bp, which was considered to be elongated by Reverse transcriptase, was confirmed specifically for the RNA sample obtained from cells in the sporulation phase. A single band was obtained when run on a high resolution gel with a sequence ladder. As a result of the primer extension shown above, it was found that the ISRH4 gene was transcribed from the position of 364 bp upstream of the start codon (base sequence 1, base number 511). Then, upstream of the transcription initiation point, there was a sequence highly similar to the -10, -35 consensus sequence which is considered to be recognized by Bacillus subtilis E σ ^H (Fig. 5). Comparing with the promoter region of the Bacillus subtilis gene under the control of E σ ^H that has been known to date, the promoter region of the ISRH4 gene that was determined this time was completely -10, -35 consensus sequences and the region between them. The spacing regions had the same length. In addition, about 70 bp upstream of the transcription initiation point, the AbrB box, which is often found in the upstream region of the promoter of a gene specifically expressed in Bacillus subtilis at the sporulation stage, is also found.
(CAAAA) was present.

[0014]

[Sequence list]

SEQ ID NO: 1 Sequence length: 874 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA
． Origin organism name: Bacillus thuringiensis Islaensis
(Bacillus thuringiensis var. Israelensis) Strain name: H
D522 CAGCTGTAAA AATGATTGCT GGAATAGAAG CCATGCATAT GGTCAAAAAA GGTCAACTCA 60 AATTAAGGGC ACAATCTGCC CAAAATCAGA ATAGATGTAT TCATCAGTTA TTTGGATTAA 120 CTGCTTAGGA GTGGATTCCA GAAGGAATCT ATGCCTTTTT TTGCGTTTGT CCATTATTTG 180 CACCAGAACC TTTGATGTTA TTAAGGCGTA GAATATCCAG ACATTGCAAA CCAGGTGGAA 240 TGCCACGTGA GTAATTTGTA TAATGAGTTT CAAATGCCTA AAATAATAAA TTTACGCGAA 300 ATAATAACCT GTACGGTAGC AGTACCATAG CTTAAAAAAC ATCATCAGTT CCTATACTAT 360 TTGATGTAAA AGGCATATAA AGGGATATTG AAGGGGATTA GATAGCTTTT TTAAAAATTA 420 ACAGGCTGGT TGATTGTCAA AAAATGACGC GGTTATTTGT TGAAAGAATG TAACAGGAAT 480 AGATTATTTA AATTACGAAT ACTTTAAAAT GCATAAGGGA AATTTTGATA GCTTGGAGTG 540 TAAAAGAAAG AGAGATTACT ACATAGGAGA GGATAAATGC ATAAGGGTCA AAGATACCAA 600 GAATTCTTGT AAAGAAGGTT TTCTTATTTT TGTGTGAGGT TTTGTAACAA ACCATATGAA 660 ATACAAAGTT TTAAAAATTT AACGCAAAAA TTTTTAAAAT GTATAAATTA CGAATAATTA 720 TATTGGTACA TTAATATGTA TATAGGATTT GTAAAAGTAA TCAATAATTT TCAATTTTGT 780 ACTTTTTAAA CCATATCAAT TTAAAAATGA TTTAAAATAA TCTTTGTTGC TACAGAAAAA 840 GAGTGTAT CT AAATTGAGTA TGGGAGGAAC AAAT 874

[Brief description of drawings]

FIG. 1 shows the structure of the plasmid pBGH4 containing the ISRH4 gene and the structure of the insert fragment carried by the pIS4-series plasmid. * Indicates that the tetracycline resistance gene of the vector and the ISRH4 gene have opposite transcription directions. White arrows indicate the coding region for the insecticidal protein ISRH4. H
Indicates HindIII, Pm indicates PmaCI, Xm indicates XmnI, and Xb indicates XbaI.

FIG. 2 shows a method for constructing the vector pKKUBIV for CAT assay. CIP stands for alkaline phosphatase. CA
T represents a chloramphenicol acetyltransferase gene, Amp ^r represents an ampicillin resistance gene, Km ^r represents a kanamycin resistance gene, and rrnBT ₂ T ₁ represents ribosomal RNA T ₂ and T ₁ terminators, respectively.

FIG. 3 shows the structure of a plasmid used for examination of the transcriptional regulatory region of the ISRH4 gene. CAT is the chloramphenicol acetyltransferase gene, ISRH4 'is the carboxy-terminal deletion of the ISRH4 gene, and IS240' is the amino-terminal deletion of the insertion sequence IS240.

FIG. 4 shows the results of a CAT assay carried out to examine the transcriptional regulatory region of the ISRH4 gene. The assay results are all the average value of duplicates.

FIG. 5 shows the promoter sequence recognized by E σ ^{H of} Bacillus subtilis and the sequence upstream of the transcription start site of the ISRH4 gene, which are aligned. Boxes indicate consensus sequences for the -10 and -35 regions.

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

Claims (7)

[Claims] 1. A transcriptional regulatory sequence for the expression of Bt insecticidal protein represented by the nucleotide sequence of Chemical formula 1 [SEQ ID NO: 1] 2. An expression plasmid capable of expressing a Bt insecticidal protein by cloning the insecticidal protein gene downstream of the transcriptional control sequence for expressing the Bt insecticidal protein represented by the nucleotide sequence of Chemical formula 2. 2] SEQ ID NO: 1 3. An expression plasmid capable of expressing a Bt insecticidal protein is cloned in which the insecticidal protein gene is cloned downstream of the transcriptional control sequence for expressing the Bt insecticidal protein represented by the nucleotide sequence of Chemical formula 3. Microorganisms that act [Sequence No. 1] 4. The microorganism according to claim 3, which is Bacillus thuringiensis. 5. The microorganism according to claim 4, which is Bacillus thuringiensis Islaensis. 6. The microorganism according to claim 3, which is Bacillus subtilis. 7. An expression plasmid capable of expressing a Bt insecticidal protein is obtained by cloning the insecticidal protein gene downstream of a transcriptional control sequence for expressing the Bt insecticidal protein represented by the nucleotide sequence of Chemical formula 4. A method for producing a Bt insecticidal protein, which comprises culturing a microorganism that reacts with SEQ ID NO: 1