JP3138727B2 - Three Insertion Sequence Factors from Rice Bacterial Blight - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a Psedomonas glumae
And a transposase encoded by the transposable element.

[0002]

BACKGROUND ART It is known that transposable elements not only inactivate or activate genes before and after transposition by moving around the genome, but also cause various genomic rearrangements (deletion, inversion, duplication, etc.). Have been. It is also known that these transposable elements generate genomic plasticity important for microbial evolution and environmental adaptation (Arber et al., FEMS Microb. Ecol., 15: 5-14 (199).
Four)).

[0003] Transposable elements can be roughly classified into transposons and insertion sequence elements (hereinafter, referred to as IS elements).

[0004] Transposons have phenotypic genes such as antibiotic resistance in addition to genes involved in metastasis. On the other hand, the IS element is defined as a gene having a size of generally 2 kb or less and having no phenotypic gene except for the gene transfer (Gay et al., J. Bacteriol., 164: 91).
8-921 (1985)).

[0005] The IS element was first discovered in the late 1960's as a factor causing mutation of E. coli. Subsequently, isolation and characterization of IS factors were advanced, mainly for the coliforms. 19
In the late 1980's, isolation of IS factors was reported in groups other than coliforms. Currently, about 300 IS factors have been isolated (Otsubo and Sekine (0htsubo and S.
ekine), Current topics in Microbiology and Immuno
logy, 204: l-26 (1996)).

[0006] The isolated IS elements are being compared with each other based on their base sequences and deduced amino acid sequences. As a result, the IS factors obtained to date are largely IS1,
It is being grouped into groups such as IS3 and IS4. In particular
The IS3 family is a large family, and IS factors belonging to this IS3 family have been isolated from many bacteria from gram-negative to gram-positive bacteria (Otsubo and Sekine, supra).

[0007] A characteristic feature of the structure of the IS element is that it has an inverted repeat sequence at the end and two open reading frames encoding a transposase. The terminal inverted repeat sequence is considered to play an important role in transposition since recombination occurs in this portion. Upon insertion, 2 to 13 base pairs of the inserted site are duplicated as isotropic repeats on both sides of the IS element (Galas and Chamdler, Mobile DNA, 109-162, ASM Press (Washin
gton, DC) 1989).

[0008] Transposase is an enzyme that catalyzes a gene insertion reaction. In the IS element, the transposase is encoded by two open reading frames and is expressed as a single protein during translation. For example, in the case of the IS3 family, the two open reading frames overlap. A frame shift occurs in the overlapping portion, and the transposase is expressed by continuous translation of the two open reading frames. (Otsubo and Sekine, supra).

[0009] IS factors are not only of academic interest,
It is also an industrially useful gene that can be used for identification diagnosis of bacteria and isolation of useful genes.

The mechanism by which the IS element causes metastasis is of scientific interest. These studies have been carried out using materials such as the IS factors IS1, IS3, IS150, and IS911 of the coliform group, which are easy to proceed with.
Details of the translocation mechanisms of the three families are becoming clearer (Otsubo and Sekine, supra).

[0011] Although the IS factor has a homology, it has a sequence unique to each microorganism, and is used for classification, identification and diagnosis of bacteria. The most practically used is Mycobacterium tuberculosi
s), which has contributed to epidemiological studies such as the diagnosis of M. tuberculosis and the elucidation of the transmission route (Otal et al., J. C.
lin. Microbiol., 29: 1252-1254 (1991)). Similar attempts have been made with many bacteria.

As a characteristic of the IS factor, by causing metastasis, genes before and after insertion can be activated or inactivated. Attempts have also been made to actively utilize this function to use IS factors as a tool for isolating useful genes (Haas et al., Molecular Biology of Pseudomon
as, pp238-249, ASM Press (Washington DC) (199
6)).

[0013] By the way, the isolation of the IS factor has mostly been obtained by accidental analysis of mutant strains, rather than being strategically advanced. This is because, as can be seen from the definition, since the IS element has no phenotypic traits other than those related to gene transfer, active isolation was difficult. Positive selection of transposable elements (a technique that utilizes the activation of a detectable marker by transposition of a transposable element to a part of a structural gene)
Was reported in 1985 (Gay et al., Supra), and a similar transposon trap vector has been developed.
Agrobacterium tumefaciens, Pseudomonas cepacia, Rh
This technique was used to isolate IS factors from bacteria such as izobium meliloti and Rhizobiumu leguminosarum. However, the number of isolated IS factors by this technique is more than ten, which is small compared to the total number of isolated elements. With the development of the positive selection method, the isolation of new IS factors from various bacteria is expected to accelerate in the future.

[0014]

At present, research on the isolation of IS factors has been little progressed for bacteria related to agriculturally important plants. An object of the present invention is to provide an IS factor derived from a bacterial wilt of rice, which is one of the important pathogens of rice that causes rice seedling rot and rice wilt in a southwestern warm region of Japan. Further, a transposase encoded by these IS elements is provided.

[0015]

Means for Solving the Problems In view of the above-mentioned object, the present inventors have attempted to isolate a new IS factor using a transposon trap vector pSHI1063 newly developed by Iida et al.

The present invention provides a terminal inverted repeat sequence comprising:
SEQ ID NO: 3 at the 5 'end and SEQ ID NO: 3 at the 3' end
And an IS element comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or a functional equivalent thereof as an open reading frame between the terminal inverted repeats. . Here, the open reading frames may be present in duplicate or independently.

Further, the present invention provides an IS factor ISmsp2.2 comprising the nucleotide sequence of SEQ ID NO: 1.

Further, the present invention provides a terminal inverted repeat sequence having a base sequence of SEQ ID NO: 7 at the 5 'end and a base sequence of SEQ ID NO: 8 at the 3' end, and As an open reading frame, an IS element comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10 or a functional equivalent thereof is provided. Here, the open reading frames may be present in duplicate or independently.

Further, the present invention provides an IS factor ISmsp8.1 consisting of the nucleotide sequence of SEQ ID NO: 6.

Further, an IS consisting of the nucleotide sequence of SEQ ID NO: 11
Provides the factor ISmsp1.1 or a functional equivalent thereof.

Furthermore, the present invention relates to the nucleotide sequence of
The present invention provides a transposase expressed from the base sequence of base 1296 and a functional equivalent.

Further, the present invention relates to the nucleotide sequence of SEQ ID NO: 6
A transposase expressed from the nucleotide sequence of base 856 or a functional equivalent thereof is provided.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION

(1) Definition In the present invention, the term “IS element” refers to two open reading frames encoding transposase and a terminal inverted gene having a size of 2 kb or less, having no expression trait gene other than a gene involved in gene transfer, and encoding a transposase. It is defined as a genetic unit containing orientation repeats.

ISmsp2.2 is defined as an IS element consisting of the nucleotide sequence of SEQ ID NO: 1.

ISmsp8.1 is defined as an IS element consisting of the nucleotide sequence of SEQ ID NO: 6.

ISmsp1.1 is defined as an IS element consisting of the nucleotide sequence of SEQ ID NO: 11.

ISmsp2.2 has 5 ′ as a terminal reverse sequence.
It has the base sequence of SEQ ID NO: 2 at the end and the base sequence of SEQ ID NO: 3 at the 3 ′ end.

ISmsp8.1 has 5 ′ as a terminal reverse sequence.
It has the nucleotide sequence of SEQ ID NO: 7 at the end and the nucleotide sequence of SEQ ID NO: 8 at the 3 'end.

In the present invention, "transposase" is defined as an enzyme that catalyzes a gene insertion reaction.

Nucleotide 95 to base 1296 of SEQ ID NO: 1
This is a region containing two open reading frames of 2.2, and nucleotides 54 to 856 of SEQ ID NO: 6 are regions containing two open reading frames of ISmsp8.1. These open reading frames do not have transposase activity when translated separately, but only become transposase when expressed as a single protein during translation (Otsubo and Sekine, supra).

[0031] As used herein, a "functional equivalent" is defined as having substantially the function or activity of the original IS element or transposase and optimally aligned with the original IS element or transposase. At least 90%, preferably 95%,
It is defined as an IS element or transposase having a homology of ≧%.

Such a functional equivalent of the IS element is obtained by substituting, adding, deleting, or deleting one or more nucleotides in addition to the original sequence in the terminal inverted repeat sequence or open reading frame which is a functional site. It may include insertion. Examples of such functional equivalents include those having a nucleotide substitution that causes a conservative substitution of an amino acid in the encoding transposase, those having an intervening nucleotide in the open reading frame, and the like.

[0033] Such functional equivalents of the transposase may also include, in addition to the original sequence, one or more amino acid substitutions (preferably conservative substitutions) or additional amino acid substitutions.
(Eg, a leader sequence, a secretory sequence, a sequence advantageous during purification, and the like). The creation of these functional equivalents is considered well within the skill of the art.

[0034] General molecular biology experimental techniques (electrophoresis of DNA, electrophoresed DNA) that can be used in each step of the present invention.
Methods for recovering NA from a gel, restriction enzyme digestion, PCR, DNA radiolabeling, hybridization, nucleotide sequencing, etc.) are described, for example, in Sambrook et al., A Laboratory Manual. Second Edition, Cold Spring Harbor Press, Cold Spring Harbo.
ur, New York (1989) as known to those skilled in the art.

(2) Method for Searching for Transposable Factors (a) Target Bacteria In the present invention, rice germ blight (Pseu) is used as a target bacterium.
domonas glumae). A preferred strain is MA
FF302744 strain. This strain is a rice germ blight fungus isolated in 1982 (Tsima et al., Nissho Disease Report, 52: 253-
259 (1986)).

(B) Principle As a means for isolating a transposable element, a transposon trap vector has been developed. The vector generally has a selectable marker gene, a repressor gene for transposable element trapping, and a detectable marker gene linked to a promoter under its control. A preferred transposon trap vector in the present invention is pSHI
1063 (FIG. 1). pSHI1063 has a kanamycin resistance gene linked to the P _R promoter in the under its control cI repressor gene λ phage for ampicillin resistance gene and spectinomycin gene and IS elements trap as a detection marker, as a selection marker It is a plasmid vector (see FIG. 1). pS
HI1063 is introduced into bacteria, transposable elements, such as IS elements possessed by the bacterium cI repressor when diving into cI repressor region of this plasmid is inactivated, resulting P _R
Since the promoter is activated and the kanamycin resistance gene starts to work, the bacteria become kanamycin resistant, which allows efficient selection of IS factors (Iida et al.
Abstracts of the 4th Annual Meeting of the Molecular Biology Society of Japan, pp216 (199
1)).

(C) Introduction of Plasmid DNA and Selection of Transformant The plasmid DNA was introduced into competent cells of P. glumae by standard methods (eg, electroporation), and the cells were transformed into kanamycin-resistant and spectinomycin. Select for resistance.

The introduction of a plasmid by general electroporation will be described. Competent cells of P. glumae suspended in a 10% glycerol solution are mixed with the pSHI1063 plasmid in a buffer, and an electric pulse is applied to the mixture. Ration. Next, the bacteria in the mixture are cultured, and the bacteria are collected by centrifugation and plated on a medium containing spectinomycin. This is cultured for an appropriate time, and a spectinomycin-resistant colony is selected. Next, the resulting cells derived from the spectinomycin-resistant colonies are cultured in a medium containing kanamycin and spectinomycin, whereby kanamycin- and spectinomycin-resistant colonies are selected.

(D) Extraction of Plasmid from Transformant The method of extracting a plasmid from selected cells having the desired resistance is well known to those skilled in the art. For example, the obtained kanamycin-resistant and spectinomycin-resistant colonies are further cultured, centrifuged, the supernatant is discarded, and the cells are dissolved in a SET solution (25% sucrose, 50 mM Tris-HCl (p
H8.0) Suspend in 20 mM EDTA-2Na). Next, a lysozyme solution is added and left at room temperature for an appropriate time. Then, TE buffer (1
0 mM Tris-HCl (pH 8.0) -1 mM EDTA) and add 1.
Mix 5% SDS solution (pH 12.85). 2M Tris
(PH 7.0) Add buffer and mix, add 2M KCl and leave for appropriate time. The mixture is centrifuged, the supernatant is removed, 0.125M KCl saturated phenol is added, and the mixture is further centrifuged. The supernatant is again taken out and chloroform phenol (chloroform: isoamyl alcohol: 0.125
MKCl saturated phenol = 24: 1: 25), mix and centrifuge. 3M sodium acetate and isopropanol are added to the obtained supernatant, and the mixture is left for an appropriate time. After centrifugation and discarding the supernatant, the precipitate is vacuum dried and sterile distilled water is added to isolate the plasmid.

(E) Amplification of recovered plasmid The obtained plasmid is subjected to agarose electrophoresis by a general method known to those skilled in the art (for example, Sambrook et al., Supra), and the size of the extracted plasmid is confirmed. I do.
Then, a plasmid larger than the size of the original plasmid is introduced into an appropriate host cell. The host cells are cultured on a medium containing ampicillin to amplify the desired plasmid.

(F) Amplification and analysis of plasmid DNA Plasmids are recovered from E. coli, for example by minirep, according to general methods known to those skilled in the art (eg, Sambrook et al., Supra). In order to clarify the insertion position and size of the transposable element in the recovered plasmid, the plasmid can be analyzed by the following method.

(A) Analysis of Recovered Plasmid with Restriction Enzyme Restriction enzyme analysis of transposable elements in the recovered plasmid can be performed by a general method known to those skilled in the art (for example, Sambrook et al., Supra). Restriction enzymes are mainly SalI, EcoRV, Pst
I, HindIII and the like may be used. After digestion with various restriction enzymes, the inserted transposable element can be classified by comparing the band pattern formed by subjecting the digestion product to electrophoresis.

(B) Southern Hybridization In order to identify the insertion position of the transposable element in the recovered plasmid, Southern hybridization can be carried out using a partial sequence of the pSHI1063 plasmid as a probe according to a conventional method (Sambrook et al., Supra). As the probe, for example, three types of fluorescently labeled partial fragments of pSHI1063, PstI / EcoRV fourth fragment (403 bp), HindIII / EcoRV third fragment (805 bp), HindIII
A third fragment (459 bp) may be used. Typically, a nylon membrane is pre-hybridized, and then hybridized by adding a probe solution. Thereafter, the nylon membrane is washed, a detection reagent is added, and the resultant is subjected to autoradiography using an X-ray film. By comparing the original plasmid with the plasmid having the transposable element of interest for the position of hybridization, the insertion position of the transposable element can be specified.

(C) Analysis of the recovered plasmid by PCR In order to specify the insertion position and size of the transposable element on pSHI1063 in more detail, a primer prepared based on the known nucleotide sequence of pSHI1063 was used in a conventional method. (Sambro
ok et al., supra) can perform PCR. After the PCR reaction, remove the reaction solution and confirm the DNA amplification by agarose electrophoresis. By comparing the primers used with the resulting amplified fragment, the approximate insertion position and size of the transposable element can be specified.

(G) Determination of base sequence of transposable element A target DNA fragment is prepared from a PCR reaction product containing the transposable element and subcloned into an appropriate vector. Plasmid DNA is prepared from the obtained positive clones and used as a template for nucleotide sequence determination. The nucleotide sequence of both strands of this plasmid DNA can be determined using, for example, the Primer Walking method (Sambrook et al., Supra).

(3) Characterization of Each Isolated Transposable Element Four different transposable elements were isolated from P. glumae by the above procedure. The nucleotide sequences of the obtained four types of transposable elements were determined, and commercially available databases (for example, GENE
Using TYX-MAC / CD1995), the homology analysis of the base sequence and amino acid sequence was advanced, and all of these transposable elements were IS elements. The properties of three of them are as follows.

(A) ISmsp2.2 This is an IS element consisting of the nucleotide sequence from base 78 to base 1412 in FIG. 8 (1335 bp in total length), and has an incomplete inverted repeat sequence (17 bp) at the end. The target overlapping sequence is 5 bp and the sequence is CAGGA. At the nucleotide sequence level, this IS element is IS2 (E.
coli) and ISRM1 (Rhizobium meliloti)
It has 61.3%, 59.5% with IS1312, 58.0% with IS136, and 57.5% with IS426 (Agrobacterium tumefaciens). Since these ISs belong to the IS3 family (Otsubo and Sekine, supra), ISmsp2.2 is considered to be a novel IS obtained from P. glumae belonging to the IS3 family.

(B) ISmsp8.1 Consists of the nucleotide sequence from base 93 to base 957 in FIG. 9 (total length: 865 bp)
It is an IS element and has an incomplete inverted repeat (15 bp) at the end. The target overlapping sequence is 3 bp and the sequence is TTA. At the nucleotide sequence level, this IS element is IS402 (Pse
udomonas cepacia), 59.6%, IS1237 (Clavibacter
xyli) is 57.7%, IS427 (Agrobacterium tumefacien)
s) is 48.8%, IS1030C is 47.4%, IS1031 (Acetobac
terium xylinum) with 47.1% homology. These ISs belong to IS4 groupB (Otsubo and Sekine, supra)
Therefore, ISmsp8.1 is considered to be a new IS obtained from P. glumae belonging to IS4 groupB.

(C) ISmsp1.1 ISmsp1.1 An IS element consisting of the nucleotide sequence from base 70 to base 1284 (1215 bp in total length) in FIG. 10, with an incomplete inverted repeat sequence (36 bp) at the end.
Having. The target duplication sequence is 7 bp and its sequence is GATCAA
G. Comparison of base sequence and amino acid sequence homology showed no homology at all with known IS and completely novel
It is considered an IS factor.

(4) Transposase encoded by each transposable element The nucleotide sequence analysis and the amino acid sequence homology analysis were performed, and it was confirmed that the transposable element of the present invention encodes a transposase like other transposable elements.

(A) Transposase encoded by ISmsp2.2 ISmsp2.2 has two overlapping open reading frames. The open reading frame (ORFA) present at bases 172 to 534 (FIG. 8) encodes a 121 amino acid protein. This protein is called ISmsp2.2 Orf
It was named A protein. The amino acid sequence is shown in SEQ ID NO: 4. ISmsp2.2 OrfA protein is 13.4kD of IS2
Has 70.5% amino acid sequence homology to protein.

The open reading frame (ORFB) present at bases 495 to 1373 (FIG. 8) encodes a 293 amino acid protein. This protein is called ISmsp2.2 Orf
It was named B protein. The amino acid sequence is shown in SEQ ID NO: 5. ISmsp2.2 OrfB protein is 33.4 kD of IS2
Has 59.3% amino acid sequence homology to protein.

Comparison with other members of the IS3 family shows that transposase is expressed from these two open reading frames.

(B) The transposase ISmsp8.1 encoded by ISmsp8.1 also has two open reading frames. The open reading frame (ORFC) present at bases 146 to 550 (FIG. 9) encodes a 135 amino acid protein having 49.0% amino acid sequence homology with the 16.2 kD protein of IS402. This protein is called ISmsp
8.1 Named OrfA protein. The amino acid sequence is shown in SEQ ID NO: 9.

The open reading frame (ORFD) present at bases 610 to 948 (FIG. 9) encodes a 113 amino acid protein having 51.0% amino acid sequence homology with the IS402 24 kD protein. This protein is
It was named msp8.1 OrfB protein. The amino acid sequence is shown in SEQ ID NO: 10. Comparison with other members of the IS4 family shows that transposase is expressed from these two open reading frames.

These transposases can be readily expressed by isolating the coding sequence from the transposable elements of the present invention and introducing them into host cells along with appropriate expression control sequences. Alternatively, it can be chemically synthesized. These procedures are known to those skilled in the art, and further, isolation and purification of the expressed protein can be performed using standard methods known to those skilled in the art.

(5) Functional Equivalents The IS factor or transposase identified as described above has a maximum of 70.5% as compared with a known IS factor or transposase at the nucleotide sequence or amino acid level.
With a homology of Thus, those with greater than 90% homology to these IS elements or transposases would clearly be distinguished from their equivalents in other known families.

[0058]

【Example】

(Example 1) 1) Used strain Pseudomonas glumae MAF was used as a target bacterium for IS factor search.
The F302744 strain was used.

For amplification of IS factor in E. coli, E. coli E.
coli DH5α strain was used.

2) Test plasmid As a transposon trap vector, the vector pSHI1063 developed by Iida et al. Was used. pSHI1063 is a full-length 11.5 Kb, a fusion plasmid of the broad host range plasmid pVS1 of Psudomonas aeruginosa and the plasmid pBR322 of Escherichia coli, and has a respective replication region. Is a plasmid capable of multiplying.
Further, it has an ampicillin resistance gene and a spectinomycin resistance gene as selection marker genes.

3) Medium P. glumae was added to a PTYG medium (bactopeptone in 1000 ml of distilled water).
0.25 g, Bacto tryptone 0.25 g, Bacto yeast extract 0.5
g, glucose _{0.5g, MgS0 4 · 7H 2 0} 30mg, CaCl 2 · 2H 2 03.5m
g, in the case of a solid medium, 15 g of agar powder) and cultured at 28 ° C. E. coli DH5α is LB (1000 g of distilled water, 10 g of bactotripton, 5 g of Bacto yeast extract, 10 g of NaCl, pH 7.4,
In the case of a solid medium, agar powder (15 g) was used and cultured at 37 ° C.

4) Isolation Procedure (l) Preparation of Competent Cells Competent cells were prepared in order to introduce a plasmid into P. glumae by electroporation.

P. gluma grown on PTYG agar plates
e. One colony was inoculated into 5 ml of PTYG medium, and
The cells were cultured with shaking for days. 4 ml of the culture was added to 200 ml of PTYG medium, and cultured at 28 ° C. with shaking until the OD ₆₀₀ reached approximately 0.2. After the culture, the culture was placed on ice for 20 minutes, and then centrifuged (8000 rpm / 10 min / 4 ° C.) to collect the cells. The cells were suspended in 10 ml of ice-cooled 1 mM HEPES.Na0H buffer (pH 7.0), centrifuged again to collect the cells, and the cells were collected in 10 ml of ice-cooled lmM HEPES.Na0H.
It was suspended in a buffer solution (pH 7.0). Centrifugation (8000rpm / 10min / 4 ℃)
The cells were collected, and the cells were suspended in 2 ml of an ice-cooled 10% glycerol solution. The obtained competent cells were dispensed into Eppendorf tubes in an amount of 40 μl each and stored at −80 ° C. until use.

(2) Electroporation and Selection of Transformants A 40 μl competent cell was treated with TE buffer (10 mM Tr).
is.HCl (pH8.0) -1mMEDTA) about 0.36μg of pSH
Mix I1063 plasmid (1 μl solution), and add 4
Transferred to a cuvette chilled at <RTIgt; Electric pulses were applied to the cuvette using a high voltage pulse generator “Gene Pulser” manufactured by Bio-Rad. The conditions of the electric pulse were an electric resistance of 200Ω, an electric capacity of 25 μFD, and an electric field of 6.25 KV / cm. After applying the pulse, the cell / DNA mixture was quickly transferred to 2 ml of PTYG medium, cultured with shaking at 28 ° C. for 1 hour, centrifuged (8000 rpm / 10 min / 4 ° C.), and the cells were clarified with 1 ml of sterile distilled water. Suspended in water. The suspension was serially diluted 10-fold with sterile distilled water to obtain a cell dilution of up to 10 ^-4 . 100 μl thereof was plated on a PTYG agar plate containing 100 μg / ml of spectinomycin. In order to confirm the transformation efficiency, the same number of bacteria was seeded on a PTYG agar plate containing no antibiotic as a control. This was cultured at 28 ° C. for 2 days. The resulting spectinomycin-resistant transformant colonies were applied again to a PTYG plate containing spectinomycin using a platinum loop and cultured at 28 ° C. for 2 days to form single colonies.

(3) Culture of transformant and selection of mutant strain One spectinomycin-resistant colony that formed a single colony was picked up with a platinum loop and inoculated in 5 ml of PTYG medium containing 100 μg / ml spectinomycin. And shaking culture at 28 ° C. for 2 days. After incubation, the suspension was serially diluted 10-fold with sterile distilled water, bacterial dilutions 10 from suspension stock to 10 ^-6 dilution
0 μl was spread on PTYG agar plates containing 50 μg / ml kanamycin and 100 μg / ml spectinomycin. 100 as a control to confirm the emergence efficiency of the mutant strain
The same number of bacteria was inoculated on a PTYG agar plate containing μg / ml spectinomycin. This was cultured at 28 ° C. for 2 days, and the obtained kanamycin-resistant transformant colonies were again applied to a PTYG agar plate containing 50 μg / ml kanamycin and 100 μg / ml spectinomycin using a platinum loop, and then single-plated. Colonies were allowed to form.

(4) Extraction of plasmid from mutant strain and confirmation of size A single colony resistant to kanamycin and spectinomycin was inoculated in 5 ml of PTYG medium containing 50 μg / ml kanamycin and 100 μg / ml spectinomycin. And shaking culture at 28 ° C. for 2 days. Transfer 1.5 ml of the culture solution to an Eppendorf tube, centrifuge (8000 rpm / 1 min / 4 ° C),
1.5 ml of the culture was transferred to an Eppendorf tube and centrifuged (8000 rpm / 1 min / 4 ° C.). 50 μl of SET solution (25% sucrose, 50 mM Tris · HCl (pH 8.0), 20 mM EDTA-2Na)
The cells were suspended therein, 5 μl of a lysozyme solution (20 mg / ml) was added, and the mixture was left at room temperature for 1 hour. 100 μl of TE buffer (10 mM
Tris-HCl (pH 8.0)-1 mM EDTA) was added and mixed up and down quickly, and immediately 300 µl of 1.5% SDS solution (pH 12.85, 25
° C) and mixed by inverting. 50 μl of 2 MTris
(PH 7.0) Add buffer, mix up and down, add 2M KCl to 50
Add μl, mix up and down, leave on ice for 2 hours,
After centrifugation (15000 rpm / 15 min / 4 ° C.), the supernatant was transferred to a new Eppendorf tube. Add 560 μl of 0.125 M KCl saturated phenol, mix up and down, then centrifuge (15000 rpm / 1
(5 min / 4 ° C), transfer the supernatant to a new Eppendorf tube, add 560 μl of chloroform phenol (chloroform: isoamyl alcohol: 0.125M KCl saturated phenol = 24: 1: 25), mix up and down, and centrifuge (1500
0 rpm / 15min / 4 ° C). Transfer the supernatant to a new Eppendorf tube and add 50 μl of 3 M sodium acetate and 340 μl.
l of isopropanol was added, the mixture was allowed to stand at −20 ° C. for 15 minutes, and further centrifuged (15000 rpm / 15 min / 4 ° C.). The precipitate was dried under vacuum, and then 20 μl of sterile distilled water was added. Take 10 μl,
Agarose electrophoresis was performed by a conventional method (Sambrook et al., Supra) to confirm the size of the extracted plasmid. As a control, pSHI1063 was electrophoresed simultaneously.

(5) Transformation of the recovered plasmid into Escherichia coli E. coli E.coli prepared in advance according to the method of (1)
10 μl of the recovered plasmid was mixed with 40 μl of DH5α competent cells, and electroporation was carried out according to the procedure of (2).
Was seeded on an LB agar plate containing 100 μg / ml of ampicillin, and cultured at 37 ° C. for one day. Ampicillin using a platinum loop from the emerged ampicillin-resistant transformant colony
The cells were inoculated into 5 ml of LB medium containing 100 μg / ml, and cultured at 37 ° C. with shaking for one day.

(6) Amplification of Plasmid DNA Plasmids were recovered from Escherichia coli by miniprep by a conventional method (Sambrook et al., Supra). For some of the colonies, a large amount of plasmid was prepared.

(7) Analysis of Recovered Plasmid by Restriction Enzyme Restriction enzyme analysis was carried out by a conventional method (Sambrook et al., Supra) to clarify the insertion position and size of the transposable element in the recovered plasmid. Restriction enzymes are mainly SalI, EcoR
V, PstI, HindIII and the like were used.

(8) Analysis of Recovered Plasmid by Southern Hybridization In order to identify the insertion position of the transposable element in the recovered plasmid, a partial sequence of the pSHI1063 plasmid was used as a probe by a conventional method (Sambrook et al., Supra). Was done. After subjecting the recovered plasmid digested with the restriction enzyme to agarose electrophoresis, the DNA was converted to a nylon membrane (HybH) using a suction plotter (Pharmacia).
ond-N). The probe was fluorescently labeled using an ECL kit (Amersham). As partial probes of pSHI1063, three types of fragments, a PstI / EcoRV fourth fragment (403 bp), a HindIII / EcoRV third fragment (805 bp), and a HindIII third fragment (459 bp), were used as probes. After pre-hybridizing the nylon membrane at 42 ° C. for about 2 hours, a probe solution was added and hybridization was performed at 42 ° C. for about 16 hours. Then, Washing buffer (0.4% SDS, 0.1 × SSC)
After washing twice at 55 ° C. for 10 minutes, the plate was further washed twice with 2 × SSC, and an ECL detection reagent was added. The membrane was subjected to autoradiography using an X-ray film.

(9) Analysis of Recovered Plasmid by PCR In order to identify the insertion position of the transposable element on pSHI1063, a primer prepared based on the known nucleotide sequence of pSHI1063 was used in a conventional manner (Sambrook et al., Supra). ) Was used to perform PCR. The PCR reaction was performed 25 times at 94 ° C. for 1 min (DNA denaturation), 55 ° C. for 2 min (annealing), and 72 ° C. for 3 min (DNA extension) using a DNA amplifier (PC800 manufactured by ASTEC). After the PCR reaction, 10 μl was taken from the reaction solution, and DNA amplification was confirmed by agarose electrophoresis (1.5% agarose).

(10) Determination of base sequence of transposable element A target DNA fragment was prepared from the PCR reaction product containing the transposable element, and subcloned into pT7Blue vector. Plasmid DNA was prepared from the obtained positive clone and used as a template for nucleotide sequence determination. The nucleotide sequence of both strands of this plasmid DNA was determined by the Primer Walking method using an auto sequencer (Model 137 manufactured by ABI).
7) (Sambrook et al., Supra).

(11) Search for homology of transposable element For the transposable element whose base sequence was determined, the homology between the base sequence and the amino acid sequence was searched using a commercially available database (GENETYX-MAC / CD1995), and a new gene was identified. Was characterized.

Results 1) Isolation and typing of mutant strain The transformation efficiency of pSHI1063 into Pseudomonas glumae MAFF302744 was 6 × 10 ^-4 . Furthermore, kanamycin-resistant colonies appeared at a frequency of 2 × 10 ⁻⁷ from the obtained transformants. Plasmids were extracted from 72 kanamycin-resistant strains and compared in size with pSHI1063.The plasmids recovered from 42 strains were larger than the 11.5 kb plasmid of pSHI1063, and the transposable element was inserted into these. Was suggested. On the other hand, the plasmids obtained from the remaining 30 strains were almost the same size as pSHI1063, and it was considered that kanamycin resistance was obtained in these plasmids by a point mutation in the kanamycin gene control region.

Of the 42 plasmids larger than pSHI1063, 19 were transformed into E. coli DH5α,
After amplification of the plasmid in E. coli, the plasmid was extracted by miniprep, digested with various restriction enzymes, and subjected to agarose electrophoresis to compare band patterns. As a result, 19 plasmids were classified into five types (Type 3.1, Typ
e2.2, Type1.1, Type8.1, Type5.1). FIG. 2 shows the results of the electrophoresis. In each photograph, lanes 1 and 8 are size markers (λ HindIII fragment), lane 2 is pSHI1063, lane 3 is Type 3.1 plasmid, lane 4 is Type 2.2 plasmid, lane 5 is Type 1.1 plasmid, and lane 6 is Type 8.1. Plasmid, lane 7 is Type5.1
It is a plasmid. FIG. 2A shows the results of SalI digestion. Ty
Only the pe1.1 plasmid was divided into two fragments, while the other plasmids were one fragment. FIG. 2B shows the results of double digestion with SalI and EcoRV. Type1.1 plasmid only 3
The fragments and the remaining plasmids were all two fragments, but the size of the second fragment was largest in Type 8.1, followed by Type 5.1.
Met. Type3.1 and Type2.2 were the same as pSHI1063. FIG. 2C shows the results of PstI digestion. Type2.2 and Type5.1
Was divided into four fragments, and the band patterns were the same. The remaining Type3.1, Type1.1 and Type8.1 were 3 fragments,
The size of each second fragment was different. FIG. 2D
The results of double digestion with PstI and EcoRV are shown. Type 5.1 had five distinct fragments. Type 8.1 was divided into four fragments. Some fragments overlapped in Type3.1, Type2.2 and Type1.1, but Type3.1 had 4 fragments, Type2.2 and Type1.
1 was divided into 5 fragments.

It should be noted that among the 19 plasmids examined, Ty
pe3.1 had the highest appearance rate of 9 pieces. Next, Type2.2
And Type8.1 and Type1.1, and Type1.1 and Type5.1 each had one.

2) Determination of Transposable Element Insertion Position Southern hybridization was carried out to clarify the insertion position of transposable elements in each type of plasmid (see FIG. 3). Activation of the kanamycin gene (nptII) gene on pSHI1063 requires either disruption of the cI repressor upstream of the nptII gene or insertion of a transposable element with promoter activity between the nptII gene and the cI repressor gene. Because of the possibility, Southern hybridization was performed using three types of probes. FIG. 3A shows nptI from the cI repressor region of pSHI1063.
The positional relationship of three types of probes in the SalI-PstI fragment (2587 bp) containing the I gene is shown. The position of each fragment is pS
When hybridized differently from that of HI1063, it is considered that a transposable element was inserted at that position. Probe A
(403 bp PstI-EcoRV fragment) (FIG. 3B)
Right), the hybridization position of Type8.1 and 5.1 was the same as that of pSHI1063, but the hybridization position was different in Type3.1, Type2.2 and Type1.1. This is Type3.1, Type2.2
And in Type 1.1, the transposable element was PstI-Ec
This indicates that it is on the oRV fragment. On the other hand, two types of probes covering the cI repressor (probe B; 805b
HindIII-EcoRV fragment of p (FIG. 3C left), probe C; 459b
Among the Southern hybridizations using the HindIII fragment of p (FIG. 3C right)), probe B was used for Type8.1 and Type5.1.
Is different from that of pSHI1063,
Type 8.1 and Type 5.1 transposable elements are cI repressor Hind
It turned out to be between III-EcoRV. In each of the photographs in FIG. 3, lanes 1 and 8 are size markers (λHIndIII fragment), lane 2 is pSHI1063, lane 3 is Type3.1 plasmid, lane 4 is Type2.2 plasmid, and lane 5 is Type
Lane 1.1 is a Type 8.1 plasmid, Lane 6 is a Type 8.1 plasmid, and Lane 7 is a Type 5.1 plasmid.

Further, PCR was performed to narrow down the insertion position and to grasp the size of the transposable element. Type3.1, Type2.2
And Type 1.1, primer set (1535S-161
0A and 329S-1610A) (see FIG. 4). Lane 1 is a size marker (λ HindIII fragment), lane 10
And 11 are also size markers (100 bp DNA ladder and 250 bp D
NA ladder), lanes 2-5 are primer set 329S-16
PCR by 10A, lanes 6 to 9 are primer set 1535S-
PCR by 1610A. The templates were pSHI1063 in lanes 2 and 6, Type 3.1 plasmid in lanes 3 and 7,
Lanes 4 and 8 are Type2.2 plasmid, lanes 5 and 9
Is a Type1.1 plasmid. As a result, an amplified fragment was found at a position different from pSHI1063 only with the 329S-1610A primer set, so that each of the Type 3.1, Type 2.2 and Type 1.1 transposable elements had an approximate position between EcoRV and PstI on pSHI1063. The insertion was found to be within the range of 70 bp. From comparison with the size marker, the approximate size of each transposable element is Type3.1 (1.3 kb), Type2.2 (1.3 kb) and Type1.
1 (1.1 kb). For Type8.1 and Type5.1, three primer sets (37650S-37864A, 3
7803S-37864A, 37803S-38040A).
As a result, in 37650S-37864A, an amplified fragment appeared at a position different from pSHI1063 in Type 5.1, and 37803S-38 in Type 8.1.
Since the amplified fragment appeared similarly in 040A (see FIG. 5), the insertion position was different between Type5.1 and Type8.1 although it was on the cI gene, and Type5.1 was more upstream than Type8.1. It turned out to be on the side. From comparison with the size marker, the approximate size of each transposable element was Type 8.1 (0.9 k
b), estimated to be Type5.1 (1.3 kb). Lane 1 in FIG. 5 is a size marker (λ HindIII fragment), lane 11
Also size marker (100bp DNA ladder), lanes 2-4
Is PCR with primer set 37650S-37864A, lane 5
7 to 7 are PCRs using the primer set 37803S-37864A, and lanes 8 to 10 are PCRs using the primer set 37803S-38040A. As for the template, lanes 2, 5, and 8 have pSHI1063,
Lanes 3, 6, and 9 are Type8.1 plasmids, Lane 4,
7 and 10 are Type 5.1 plasmids.

Since the sizes and restriction sites of Type 5.1 and Type 2.2 were similar, Southern hybridization was carried out with each plasmid using a Type 2.2 HindIII fragment (about 900 bp) as a probe. (See FIG. 7),
Hybridized at exactly the same position as Type 5.1. Therefore, the transposable elements in Type 2.2 and Type 5.1 are identical,
Due to the positional relationship between III and PstI restriction enzymes, Type 5.1
It was considered that the transposable element was inserted on the cI gene in the opposite direction to 2.2. Note that lane 1 in FIG. 7 is a size marker (λ
HindIII fragment), lane 2 is pSHI1063 (HindIII fragment),
Lane 3 is Type3.1 (HindIII fragment), Lane 4 is Type2.
2 (HindIII fragment), lane 5 is Type 1.1 (HindIII fragment), lane 6 is Type8.1 (HindIII fragment), lane 7 is
Type 5.1 (HindIII fragment), lane 8 is a size marker (100 bp DNA ladder).

FIG. 6 shows the results obtained by integrating the above experiments. The insertion position, restriction enzyme cleavage site, and size of each transposable element on pSHI1063 are disclosed. In the Type3.1 plasmid, the transposable element was 1.3 kb in total length and was inserted into the region between the cI and nptII genes. This transposable element had one PstI cleavage site. Nine of the 19 plasmids examined
It was a transposable element belonging to Type 3.1. In the Type2.2 plasmid, the transposable element was 1.3 kb in total length and was inserted into the region between the cI and nptII genes. This transposable element contains one PstI, Hind
III There were two cleavage sites. Five of the 19 examined plasmids were transposable elements belonging to Type 2.2. In Type 1.1 plasmid, the transposable element is 1.1 kb in full length, cI and
Insertion into the region between the nptII genes. This transposable element had one cleavage site each for SalI and PstI.
Of the 19 plasmids examined, only one was Type 1.
It was a transposable element belonging to 1. In the Type8.1 plasmid, the transposable element was 0.9 kb in total length and was inserted on cI. This transposable element had one HindIII cleavage site. Of the 19 plasmids examined, three were transposable elements belonging to Type 8.1. Transposable element is full length in Type5.1 plasmid
1.3 kb, insertion on cI. This transposable element includes PstI
There was one cleavage site and two HindIII cleavage sites. Also, out of the 19 investigated plasmids, only 1 was a transposable element belonging to Type 5.1.

(Example 2) A target DNA fragment was prepared from the PCR reaction product containing the transposable element obtained in Example 1, and pTBlue was prepared.
The vector was subcloned. Plasmid DNA was prepared from the obtained positive clone and used as a template for nucleotide sequence determination. About this plasmid DNA Primer Walkin
The nucleotide sequence of both strands was determined using the auto sequencer (ABI model 1377) using the g method. The results are shown in FIGS. 8, 9 and 10.

[0082]

[Effect of the Invention] "Moving genes" such as transposons
It has been pointed out that (transposable elements) may be greatly involved in the evolution and environmental adaptation of living organisms as a self-mechanism of self-modification of the living genome itself. The gene of the present invention has the property of moving around on the genome of a microorganism and activating or inactivating genes downstream of the jumped-in genome. By utilizing this property, it is possible to efficiently isolate industrially useful genes and the like. By using these genes, it may be possible to elucidate the transmission route of Psuedomonas glumae, identify bacteria, and diagnose rice. In addition, by promoting the transposable function of transposable elements using the transposase of the present invention, it is possible to increase the efficiency of isolation of industrially useful genes and the like, and to promote the reduction of pathogenicity by mutagenesis of Pseudomonas glumae. Expected to be a controlling agent effect.

[0083]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1335 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: genomic DNA Origin Organism: Rice blight bacterium (Pseudomonas glumae) Strain name: MAFF302744 Characteristic of the sequence Symbol indicating the characteristic: CDS Location: 95..457 Method for determining the characteristic: S Symbol indicating the characteristic: CDS Location: 418.1296 Sequence TGGAGCAGCC CCCTGAAACA CCGGACACAG TCACCCACTT ACAATAACGG GTAGGCATAA 60 GACCGTGTTT TTGACTCACA GCAGGCAGGATGGGATGG 120 TCGGCGGCGC TGGACGGCAG AGCAAAAGCT GGCGATGGTT CGCGAGAGTT TCGAGCCGGG 180 GAAATCGGTT TCGATGGTAG CGCGCCAGCA CGGCGTGAAT CCGAACTAGC TCTTTCACTG 240 GCGCAAGCTT TACCAGGATG GAAGCCTGTC GGCGGTCAAG GCTGGCGAGG AAGTGGTTCC 300 GGCCTCGGAG CTGGCCGACG CGCTCAAACA GATTCGCGAG CTGCAGCGCA TGCTCGGCAA 360 GAAGACGATG GAGAATGAGA TTCTTCGCGA GGCTGTCGAA TACGGCCGGG CAAAAAAATG 420 GATAGCGCAC TCGCCATCGT TGCCGGAGGA CAGCCAGTGA AACTGGTCTG CGAAGTCCTC 480 GGCGTGTCGC GCTCGAACGT ATCGGCACGC AGGTCACGCG AGGCGACTTG GCGCGACGCA 540 CGGCAGTCCA GAGCGACCCA CGATGCACCA GTCGTCGAAG CCATTCAGCG GGTGATCAGC 600 GAGCTGCCCA GCTACGGCTA TCGACGCGTA TGGGGAGCGT TGCGCCGCGA GCGCATTGCA 660 GCGGGACAGG CACCGCTGAA CGCCAAGCGC ATTTACCGCG TAATGCGCAT GCATGGCCTG 720 CTGATGCAAC GCAGAGCGAC ACCGGTTCGG CCGCAGCGCC GTCACGATGG CAAGGTTGCG 780 GTGGAGCGCA GCAATCAACG GTGGTGTTCC GACGGCTTCG AGTTTCGCTG CGACAACGGC 840 GAGCCGTTGC GCGTGACGTT CGCACTCGAC TGCTGCGACC GCGAAGCGAT GAGCTGGGCG 900 GCCACGACGG CCGGCCATAG CGGCGACATC GTGCGCGACG TGATGCTGGC TGCGGTGGAA 960 AGCCGGTTTG GGGATGTGCT GCATACCGAG TCCGAAATCG AGTGGCTGAG CGACAACGGC 1020 TCGGGCTATA CGGCCGAGGA GACGCGTCAG TTTGCGGCGC TGCTCGGCCT GAAGCCGTTG 1080 ACCACGCCGG TGTGCAGCCC GCAGAGCAAC GGCATGGCGG AAAGCTTCGT GAAGACCATG 1140 AAGCGCGATT ACGTCGCTAT CATGCCGAAG CCGGACGCAG CGACCGCGGC CAGGAATCTG 1200 GCCATCGCAT TCGAGCACTA CAACGAAAAG CATCCCCATA GCGCGTTGAA GTACCGCTCG 1260 CCTCGCGAGT TTCGACGTTC GACGGATTCA GCAACCTAAG TGGGTGCCGT GTCCTGAGTT 1320 ACGGGGGCAA CTCCA 1335 SEQ ID NO: : 2 array Length: 17 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA sequence TGGAGCAGCC CCCTGAA 17 SEQ ID NO: 3 Sequence length: 17 Sequence type: Number of nucleic acid strand : Double-stranded topology: linear sequence type: genomic DNA sequence TTACGGGGGC AACTCCA 17 SEQ ID NO: 4 Sequence length: 121 sequence type: amino acid Number of chains: double-stranded topology: linear sequence type: Protein HYPOTHETICAL: YES sequence Met Asp Val Leu Thr Gly Pro Glu Arg Arg Arg Arg Trp Thr Ala Glu 1 5 10 15 Gln Lys Leu Ala Met Val Arg Glu Ser Phe Glu Pro Gly Lys Ser Val 20 25 30 Ser Met Val Ala Arg Gln His Gly Val Asn Pro Asn-Leu Phe His 35 40 45 Trp Arg Lys Leu Tyr Gln Asp Gly Ser Leu Ser Ala Val Lys Ala Gly 50 55 60 Glu Glu Val Val Pro Ala Ser Glu Leu Ala Asp Ala Leu Lys Gln Ile 65 70 75 80 Arg Glu Leu Gln Arg Met Leu Gly Lys Lys Thr Met Glu Asn Glu Ile 85 90 95 Leu Arg Glu Ala Val Glu Tyr Gly Arg Ala Lys Lys Trp Ile Ala H is 100 105 110 Ser Pro Ser Leu Pro Glu Asp Ser Gln 115 120 SEQ ID NO: 5 Sequence length: 293 Sequence type: Amino acid Topology: Linear Sequence type: Protein HYPOTHETICAL: YES sequence Met Asp Ser Ala Leu Ala Ile Val Ala Gly Gly Gln Pro Val Lys Leu 1 5 10 15 Val Cys Glu Val Leu Gly Val Ser Arg Ser Asn Val Ser Ala Arg Arg 20 25 30 Ser Arg Glu Ala Thr Trp Arg Asp Ala Arg Gln Ser Arg Ala Thr His 35 40 45 Asp Ala Pro Val Val Glu Ala Ile Gln Arg Val Ile Ser Glu Leu Pro 50 55 60 Ser Tyr Gly Tyr Arg Arg Val Trp Gly Ala Leu Arg Arg Glu Arg Ile 65 70 75 80 Ala Ala Gla Gly Gln Ala Pro Leu Asn Ala Lys Arg Ile Tyr Arg Val Met 85 90 95 Arg Met His Gly Leu Leu Met Gln Arg Arg Ala Thr Pro Val Arg Pro 100 105 110 Gln Arg Arg His Asp Gly Lys Val Ala Val Glu Arg Ser Asn Gln Arg 115 120 125 Trp Cys Ser Asp Gly Phe Glu Phe Arg Cys Asp Asn Gly Glu Pro Leu 130 135 140 Arg Val Thr Phe Ala Leu Asp Cys Cys Asp Arg Glu Ala Met Ser Trp 145 150 155 160 Ala Ala Thr Thr Ala Gly His Ser Gly A sp Ile Val Arg Asp Val Met 165 170 175 Leu Ala Ala Val Glu Ser Arg Phe Gly Asp Val Leu His Thr Glu Ser 180 185 190 Glu Ile Glu Trp Leu Ser Asp Asn Gly Ser Gly Tyr Thr Ala Glu Glu 195 200 205 Thr Arg Gln Phe Ala Ala Leu Leu Gly Leu Lys Pro Leu Thr Thr Pro 210 215 220 Val Cys Ser Pro Gln Ser Asn Gly Met Ala Glu Ser Phe Val Lys Thr 225 230 235 240 Met Lys Arg Asp Tyr Val Ala Ile Met Pro Lys Pro Asp Ala Ala Thr 245 250 255 Ala Ala Arg Asn Leu Ala Ile Ala Phe Glu His Tyr Asn Glu Lys His 260 265 270 Pro His Ser Ala Leu Lys Tyr Arg Ser Pro Arg Glu Phe Arg Arg Ser 275 280 285 285 Thr Asp Ser Ala Thr 290 SEQ ID NO: 6 Sequence length: 865 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: genomic DNA Origin Organism: Rice blight bacterial bacterium (Pseudomonas)
Strain name: MAFF302744 Characteristic of sequence Symbol of characteristic: CDS Location: 54..458 Sequence determination method: S Symbol of characteristic: CDS Location: 518..856 Sequence GAGCCGCTAA CATAACCTAG ACATGACGTA GCAGAAATCG TATCGTCTGC GATATGGCAA 60 GACGCAAAAT CAGCAATGAA TTGTGGGTGG CACTGGAACC TCTGATTCCG GAATTCACCC 120 CATCGCCCAG GGGCGGACGG CGGCGCACGG TCGATGACCG AGCTGCGCTC AACGGCATCC 180 TGTATGTGCT GCAAACCGGC ATCCCGTGGG AAGACCTCCC GCAAGAACTG GGCTTCGGCA 240 GTGGCATGAC GTGTTGGCGA CGTCTGCGGG ACTGGCAAGC GGCCGGTGTA TGGCATCGGC 300 TGCATCTGGC GATGCTGCGT CGGCTGCGTG AGCACGACCA GATCGATTGG GAGCGCGCCA 360 GCCTCGATGG AGCCAGCGTC TCCAGCCCCC GGGGGGCCAG GAAACCGGCC CCAACCCGAC 420 CGACCGCGGC AAGCTCGGAT CGAAGCGACA CATCGTCGTA GATGCACGCG GCATTCCTCT 480 GGCCGTCACG ATCACGGGAG CCAACCGCCA CGATTCGATG GCATTCGAGT CCACGCTCGA 540 TGCCATTCCT GCGGTACCCG GGCTGAACGG TCCGCCACGC AAGCGCCCGA GCAAACTGCA 600 CGCTGACAAA GGGTATGACT TCGGGCGTTG CCGACGTTAT CTGAGACAGC GCGGCATGTGCGT CGCG GTATCGAAAG CAGCGAACGG CTGGGCAGGC ATCGTTGGGT 720 CGTCGAGCGT ACGCATGCCT GGTTCGCCGG ATTCGGCAAG CTTCGAATTC GCTTCGAACG 780 ACGCCTCGAT ATTCATACCG CTCTGCTTGT CCTGGCTGCC GCCGTCATCT GCTCCAGATT TGCTGTGT CGTGCTGGTGC No. Type Sequence type: genomic DNA sequence GAGCCGCTAA CATAA 15 Sequence number: 8 Sequence length: 15 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: Genomic DNA sequence TTGTGTTAGC GACTC 15 Sequence number : 9 Sequence length: 135 Sequence type: Amino acid Topology: Linear Sequence type: Protein HYPOTHETICAL: YES Sequence Met Ala Arg Arg Lys Ile Ser Asn Glu Leu Trp Val Ala Leu Glu Pro 1 5 10 15 Leu Ile Pro Glu Phe Thr Pro Ser Pro Arg Gly Gly Arg Arg Arg Thr 20 25 30 Val Asp Asp Arg Ala Ala Leu Asn Gly Ile Leu Tyr Val Leu Gln Thr 35 40 45 Gly Ile Pro Trp Glu Asp Leu Pro Gln Glu Leu Gly Phe Gly Ser Gly 50 55 60 Met Thr Cys Trp Arg Arg Leu Arg Asp Trp Gln Ala Ala Gly Val Trp 65 70 75 80 His Arg Leu His Leu Ala Met Leu Arg Arg Leu Arg Glu His Asp Gln 85 90 95 Ile Asp Trp Glu Arg Ala Ser Leu Asp Gly Ala Ser Val Ser Ser Pro 100 105 110 Arg Gly Ala Arg Lys Pro Ala Pro Thr Arg Pro Thr Ala Ala Ser Ser 115 120 125 Asp Arg Ser Asp Thr Ser Ser 130 135 SEQ ID NO: 10 Sequence length: 113 Sequence type: amino acid Topology: linear Sequence type: protein HYPOTHETICAL: YES sequence Met Ala Phe Glu Ser Thr Leu Asp Ala Ile Pro Ala Val Pro Gly Leu 1 5 10 15 Asn Gly Pro Pro Arg Lys Arg Pro Ser Lys Leu His Ala Asp Lys Gly 20 25 30 Tyr Asp Phe Gly Arg Cys Arg Arg Tyr Leu Arg Gln Arg Gly Ile Lys 35 40 45 Ala Arg Ile Ala Arg Arg Gly Ile Glu Ser Ser Glu Arg Leu Gly Arg 50 55 60 His Arg Trp Val Val Glu Arg Thr His Ala Trp Phe Ala Gly Phe Gly 65 70 75 80 Lys Leu Arg Ile Arg Phe Glu Arg Arg Leu Asp Ile His Thr Ala Leu 85 90 95 Leu Val Leu Ala Ala Ala Val Ile Cys Ser Arg Phe Val Asp Asp Leu 100 105 110 Cys SEQ ID NO: 11 Sequence length: 1215 Sequence type: Nucleic acid Sequence type: Double-stranded Topology: Linear Sequence type: Genomic DNA Origin Organism: Rice blight bacterial bacterium ( Pseudomonas glumae) strain name: MAFF302744 sequence TGTAGAAGTT CAGACTTGAT CTGACAGTAA GGCCTTGCCA AGAGGCGGGG TTACCCGTTT 60 TGATAGAGGT GCGAATCTTC ATCAAAACAG CGCGCAAGCG CCAAGGAGTA ACCCCGTGAG 120 TAGTTTCAAC CAAAATGTCA TCCGCCACAA GATCGGTCTG CTGAATCTGG CCGCCGAGCT 180 GGGCAACGTG TCGAAAGCCT GCAAGGTGAT GGGGCTGTCG CGCGATACGT TCTACCGCTA 240 TCAGAACGCC GTGGCCGAGG GCGGCGTCGA TGCCCTGTTC GACAGCAATC GGCGCAAGCC 300 GAATCCCAAG AATCGAGTCG ATGAAGCGAC GGAAATCGCC GTGCTGGCCT ATGCCATCGA 360 GCAGCCCGCC CACGGGCAGG TTCGGGTCAG CAACGAATTA CGCCGGCGCG GCATTTTCGT 420 GTCTGCATCC GGCGTTCGTT CGATCTGGCT GCGTCACGCG TTGTCGTCCT TCAAGCTGAG 480 GCTCGTGGCGCTGGAGAAGC AGGTCGTCGCAGGCGCGCAGAGGCGAGCAGGCGAGCAGGCGAGCAGGCAGGCAGGCGAGCAGGCAGGCGAGCAGGCAGGCGAGCAGGCAGGCAGGCAG T CTACGTGGGC ACGATCAAGG GCGTAGGCCG 660 GATTTACCAG CAGACCTTCG TCGACACCTA CAGCAAAGTG GCGATGGCCA AGCTGTACAC 720 GACCAAGACA CCGATCACGG CGGCCGATCT GCTCAATGAC CGGGTGTTGC CGTTCTTCGA 780 GGAGCACGGC ATGGGTGTGA TCCGCATGCT GACCGATCGA GGCACGGAGT ATTGCGGCAA 840 GCCGGAATCG CACGATTATC AGCTGTACCT GGCGCTGAAC GACATCGAGC ACACCAAAAC 900 CAAGGCGCGA CATCCGCAGA CCAATGGCAT CTGCGAGCGG TTCCATAAAA CCATCCTGCA 960 GGAGTTTTAT CAGGTCGCGT TCCGCCACAA GCTCTATCTG ACGCTGGCGG AACTGCAGGT 1020 CGATCTCGAT ACTTGGCTGA TGTACTACAA CGGCGAGCGA ACGCATCAAG GTAAGATGTG 1080 TTGTGGTCGC ACGCCTTTGC AAACGCTCAT CGCGGGCAAG GAGGTGTGGA AGGAGAAAGT 1140 GAGCCACCTG AATCTGATCT GACAGTCACG CACCGTCGGA ACGGGTAACT GTCAGATCGG 1200 GTCGCGACTT CTACA 1215

[Brief description of the drawings]

FIG. 1 shows the structure of the transposon trap vector pSHI1063.

FIG. 2 is a photograph of agarose electrophoresis after restriction enzyme digestion of a plasmid obtained from a mutant strain.

FIG. 3 is an electrophoresis photograph showing the results of Southern hybridization for determining the insertion position of a transposable element on pSHI1063.

FIG. 4 is a set of PCR primers used for narrowing down the insertion position of each transposable element on pSHI1063 and grasping the size of the transposable element, and an electrophoresis photograph analyzed using the primers. A: Mutual positional relationship of primers used for PCR and their respective base sequences. B: Electrophoresis after PCR.

FIG. 5 is a set of PCR primers used for narrowing down the insertion position of each transposable element on pSHI1063 and grasping the size of the transposable element, and an electrophoresis photograph analyzed using the primers. A: Mutual positional relationship of primers used for PCR and their respective base sequences. B: Electrophoresis after PCR.

FIG. 6 is a diagram showing the insertion position of each transposable element on pSHI1063, restriction enzyme cleavage site, and size of transposable element.

FIG. 7 is an electrophoretic photograph showing the results of Southern blot hybridization using a HindIII fragment (about 900 bp) in a transposable element of Type 2.2 plasmid as a probe.
A: agarose gel electrophoresis photograph, B: Southern blot hybridization.

FIG. 8 shows the nucleotide sequence of ISmsp2.2 obtained from the Type2.2 plasmid and its both side regions. The underlined arrow indicates a terminal incomplete inverted repeat (17 bp). The boxed region indicates the target overlapping sequence (5 bp).

FIG. 9 shows the nucleotide sequence of ISmsp8.l obtained from the Type8.1 plasmid and the regions on both sides thereof. The underlined arrow indicates a terminal incomplete inverted repeat sequence (15 bp). The boxed region indicates the target overlapping sequence (3 bp).

FIG. 10: ISmsp1.1 obtained from Type1.1 plasmid
And the nucleotide sequences of both sides thereof. The underlined arrow indicates an incomplete terminal inverted repeat (36 bp). The boxed region indicates the target overlapping sequence (7 bp).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ identification code FI C12R 1:38) (58) Investigated field (Int.Cl. ⁷ , DB name) C12N 15/31 C12N 9/10 BIOSIS (DIALOG) ) GenBank / EMBL / DDBJ / PIR / SwissProt / Geneseq JICST file (JOIS) MEDLINE (STN) WPI (DIALOG)

Claims (7)

(57) [Claims] 1. A terminal inverted repeat sequence having a base sequence of SEQ ID NO: 2 at the 5 ′ end and a base sequence of SEQ ID NO: 3 at the 3 ′ end, as an open reading frame between the terminal inverted repeat sequences. , An IS element or a functional equivalent thereof comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5. 2. An IS factor IS consisting of the nucleotide sequence of SEQ ID NO: 1.
msp2.2. 3. The terminal inverted repeat sequence has a base sequence of SEQ ID NO: 7 at the 5 ′ end and a base sequence of SEQ ID NO: 8 at the 3 ′ end, and as an open reading frame between the terminal inverted repeat sequences. , An IS element or a functional equivalent thereof comprising a base sequence encoding the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. 4. An IS factor IS comprising the nucleotide sequence of SEQ ID NO: 6.
msp8.1. 5. An IS factor IS consisting of the nucleotide sequence of SEQ ID NO: 11.
msp1.1 or its functional equivalent. 6. A transposase expressed from the nucleotide sequence from base 95 to base 1296 of SEQ ID NO: 1 or a functional equivalent thereof. 7. A transposase expressed from the base sequence from base 54 to base 856 of SEQ ID NO: 6, or a functional equivalent thereof.