JPH0870865A - Tropolone-resistant gene and new microorganism containing the same - Google Patents

Abstract

PURPOSE: To obtain a gene PP-151 which is a tropolone-resistant gene derived from Pseudomonas plantarii, having a prescribed base length and specific physical properties and useful for controlling bacterial damping-off of rice plant. CONSTITUTION: This gene PP-151 which is a tropolone-resistant gene, derived from Pseudomonas plantarii, having 1.9kb base length and represented by a restriction map of the formula or PP-161, PP-21-1, PP-21-2, PP-21-4, PP-21-5 or PP-30-1. Furthermore, Escherichia colt containing the gene is deposited as FERM P-14506, FERM P-14505, FERM P-14508, FERM P-14509, FERM P-14510 and FERM P-14511.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to Pseudomonas plantarii.
) -Producing tropolone-resistant Pseudomonas
Plantation-derived novel genes PP-151, PP-161, PP-2
The present invention relates to 1-1, PP-21-2, PP-21-4, PP-21-5 and PP-30-1, and a novel microorganism containing these genes. It is expected that the rice transformed with these genes will be endowed with resistance to tropolone produced by Pseudomonas plantaly and can contribute to the control of bacterial seedling bacterial disease.

[0002]

2. Description of the Related Art Rice seedling blight fungus has been regarded as an important disease at rice seedling raising all over the country since its occurrence was confirmed in Chiba Prefecture in 1982. This disease is a seed-borne bacterial disease, and it is urgently necessary to establish a control method because there are many unclear points regarding the ecology of occurrence and it is difficult to predict the occurrence of the disease and there are few control agents.

[0003] Tropolone produced by Pseudomonas plantaly is involved in the manifestation of disease symptoms of rice seedling bacterial killing on rice seedlings, and the rice seedlings treated with tropolone are similar to the rice seedling bacterial bacterial disease infected seedlings. Commitment of the aboveground part is observed due to inhibition of root elongation. Also, Pseudomonas
Tropolone is a non-host-specific toxin because tropolone treatment of plantless non-host cruciform and asteraceae plants shows root elongation inhibition, aerial part deprivation and yellowing. It is considered. Furthermore, tropolone has been shown to have antibacterial activity against bacteria and filamentous fungi other than Pseudomonas plantaly.

However, the mechanism of action of tropolone on plants and bacteria has not been clarified, and there has been no study of tropolone resistance gene. Also,
Neither rice cultivar showing resistance to bacterial seedling blight or tropolone has been found.

By the way, regarding the production of a host non-specific toxin resistance gene and a disease resistant plant using the same, P. syring (P. syring)
The inactive enzyme gene (ttr) of tabtoxin produced by P. ae pv. tabaci) was isolated from Pseudomonas syringae tabachi and an example of tobacco wildfire resistant tobacco transformed with ttr has been reported (Anzai, H. et al., Mol. Gen. Ge
net. 219, 492).

[0006] As shown in this report, when the producing bacterium itself is resistant to a substance which is a host non-specific toxin and has antibacterial activity against other bacteria and filamentous fungi, It itself has a resistance mechanism to this substance, and it is thought that the information is in the genomic DNA of the producing bacterium.

[0007]

An object of the present invention is to provide a tropolone resistance gene and a microorganism containing the gene.

[0008]

[Means for Solving the Problems] In order to solve the above problems, Pseudomonas plantaly which produces tropolone itself is resistant to tropolone (for example, Pseudomonas plantaly is present in the presence of 200 ppm of tropolone. However, as a result of the research, the tropolone resistance genes PP-151, PP-161, PP-21-1, PP-21-2, PP derived from Pseudomonas plantaly were obtained.
-21-4, PP-21-5 and PP-30-1 were successfully isolated, and the present invention was completed.

The present invention provides a gene PP-151, which is a tropolone resistance gene derived from Pseudomonas plantaly and has a base length of 1.9 kilobases and represented by the restriction enzyme map of FIG. Furthermore, the present invention provides a gene PP-161 derived from Pseudomonas plantaly, which has a tropolone resistance gene and has a base length of 1.2 kilobases and is represented by the restriction map of FIG.

Furthermore, the present invention provides a tropolone resistance gene derived from Pseudomonas plantaly which has a base length of 0.1 kilobase and is represented by the gene PP-21-1 represented by the restriction map of FIG. . Further, the present invention provides a gene PP-21-2, which is a tropolone resistance gene derived from Pseudomonas plantaly, has a base length of 1.3 kilobases and is represented by the restriction map of FIG. 4.

Further, the present invention provides a tropolone resistance gene derived from Pseudomonas plantaly, which has a base length of 0.5 kilobase and is expressed by the restriction map of FIG. 5, PP-21-4. . Further, the present invention provides a gene PP-21-5, which is a tropolone resistance gene derived from Pseudomonas plantaly, has a base length of 0.3 kilobases and is represented by the restriction map of FIG. 6.

Further, the present invention provides a tropolone resistance gene derived from Pseudomonas plantaly, which has a base length of 0.2 kilobases and is represented by the restriction enzyme map of FIG. 7, PP-30-1. .

Furthermore, the present invention provides the above-mentioned genes PP-151 and PP.
-161, PP-21-1, PP-21-2, PP-21-4, PP-21-5 and
E. coli containing PP-30-1 is provided. Further, the present invention provides a novel Escherichia coli which is a tropolone resistance gene derived from Pseudomonas plantaly and which contains the gene PP-151 and the gene PP-161 in tandem.

The present invention will be described in detail below. Gene PP
-151, PP-161, PP-21-1, PP-21-2, PP-21-4, PP-21-
5 and PP-30-1 can be isolated from Pseudomonas plantaly genomic DNA. The method for separating these genes is as follows.

First, the genomic DNA prepared from Pseudomonas plantaly is cleaved with 22 kinds of restriction enzymes for 15 minutes to obtain 22 kinds of cleaved fragments.
22 kinds of restriction enzymes are Acc I, Bam HI, Bcl I, Bl
n I, Cla I, Eco RI, Hinc II, Hind III, Kpn I, N
ae I, Nhe I, Nru I, Nsp V, Pst I, Sac I, Sal
I, Sma I, Spe I, Sph I, Sse 8387I, Xba I and Xho I were used. Each of the obtained cut fragments is ligated to the plasmid pUC118 vector, and E. coli DH5-α strain is transformed.

After the transformation, each is cultured in the presence of ampicillin to obtain ampicillin-resistant colonies. Next, each of the obtained ampicillin-resistant colonies is cultured in the presence of 100 ppm of tropolone to obtain tropolone-resistant colonies. In addition, untransformed E. coli DH5-α
The strain does not grow even in the presence of 50 ppm tropolone. Each tropolone-resistant colony obtained was further cultured in the presence of tropolone at 150 ppm. In this way, tropolone-resistant clones 151, 161, 21-1, 21-2, 21-4, 21-5 and 30-1 which were stably grown could be isolated. In addition, tropolone-resistant clones 151 and 161 were 21-1, 21-2, 21-4 when the Pseudomonas plantaly genomic DNA was cleaved with Sal I.
And 21-5 are clones obtained from E. coli transformed with the plasmid pUC118 vector ligated with the cut fragment obtained when cut with Pst I, and 30-1 when cut with Nhe I.

Isolated tropolone resistant clones 151, 16
1, 21, 21-1, 21-2, 21-4, 21-5 and 30-1 are respectively LB
Propagate overnight in the medium and prepare the plasmid DNA by the alkali-SDS method. The plasmid DNA obtained from clones 151 and 161 was then Sal I, clone 21-
Plasmid DN obtained from 1, 21-2, 21-4 and 21-5
A is Pst I and is a plasmid D obtained from clone 30-1.
NA was cleaved with Eco RI and Sal I, and each DNA fragment was separated by agarose gel electrophoresis (agarose 1.5%). The electrophoresis pattern is shown in FIG.

Tropolone resistant clones 151, 161, 21-
The above-mentioned DNA fragments obtained from 1, 21-2, 21-4, 21-5 and 30-1 were respectively converted into PP-151, PP-161, PP-21-1,
They were named PP-21-2, PP-21-4, PP-21-5 and PP-30-1. The base length of each DNA fragment is 1.9 kilobases for PP-151, 1.2 kilobases for PP-161, 0.1 kilobase for PP-21-1, 1.3 kilobase for PP-21-2, PP-21- 4 for 0.5 kg base, PP-21-5 for 0.3 kg base and PP-30-1 for
It was 0.2 kilobase. Furthermore, PP-151, PP-161, PP
-21-1, PP-21-2, PP-21-4, PP-21-5 and PP-30-1
The restriction enzyme maps of are shown in FIGS. 1 to 7, respectively.

The genes PP-151 and PP-16 obtained as described above
1, PP-21-1, PP-21-2, PP-21-4, PP-21-5 and PP-
Introduction of 30-1 into E. coli can be performed, for example, as follows.

A plasmid pUC1 treated with alkaline phosphatase using a ligation kit (Takara Shuzo)
After ligating the 18 vector and the above gene,
After mixing with Escherichia coli DH5-α strain competent cells and standing on ice for 2 minutes, SOC medium (tryptone 1 g, yeast extract
0.25g, NaCl 29mg, KCl 10mg, and glucose 180
After suspending mg in distilled water to prepare a suspension of 49.5 ml, 1M
MgSO ₄ / 1M MgCl ₂ solution 0.5ml added) 400μl
And shake culture at 37 ° C for 1 hour. LM agar medium containing 50 ppm ampicillin (NaCl 88 mg, yeast extract 0.75 g, tryptone 1.5 g and agar 2.25 g was dissolved in distilled water to prepare a 150 ml aqueous solution, which was autoclaved and cooled to 50-60 ° C. Then, the cells are cultured in 1 M MgSO ₄ (1.5 ml added) at 37 ° C. for 24 hours to obtain Escherichia coli transformed with the plasmid containing each gene.

Further, the plasmid pUC118 vector containing the genes PP-151 and PP-161 in tandem was used to transform Escherichia coli DH5-α.
When the strain was transformed, growth was observed in the presence of 200 ppm of tropolone, clone 151-161 and clone 161-151.
was gotten. This makes these clones clones
It can be seen that tropolone resistance is enhanced as compared with 151 and clone 161. The method for introducing the plasmid containing the genes PP-151 and PP-161 in tandem into Escherichia coli can be performed by the same method as the method for introducing a single gene such as the gene PP-151 described above into E. coli. it can.

[0022]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

(Example 1) (1) Preparation of genomic DNA of Pseudomonas plantaly Pseudomonas plantaley 301723 strain (a strain owned by the Institute for Biological Resources, Ministry of Agriculture, Forestry and Fisheries) was cultured in LB agar medium at 30 ° C for 2 days. I collected the bacteria. Next, the obtained strain was treated with TES (100 mM Na
The cells were suspended in 10 mM Tris buffer (pH 7.4) containing CL and 1 mM EDTA, and then centrifuged at 12,000 rpm for 2 minutes. The obtained precipitate fraction was suspended in TES again and centrifuged at 12,000 rpm. Suspend the obtained precipitate fraction in 500 μl of TES, and
90 μl of SDS was added. Do this for 10 minutes with Slow Rocking Sha
Shake at the lowest speed using a ker (hereinafter, for shaking and suspension, Slow Rocking Shake manufactured by Tokyo Glass Instrument Co., Ltd.
(using r) and 60 μl 3M sodium acetate and
450 μl of isopropanol was added and shaken well. After shaking, centrifuge at 10,000 rpm for 10 minutes, discard the supernatant and dry the remaining precipitate under reduced pressure for 2-3 minutes.
500 μl TE (10 mM Tris buffer containing 1 mM EDTA, pH 8.
0) was added and suspended. Further 10% SDS 60 μl and 20
12.5 μl of mg / mL proteinase K was added, and the mixture was shaken slowly at 60 ° C. until the precipitate was completely dissolved. 20 mg / mL proteinase K (12.5 μl) was added every 12 hours.
After the precipitate was completely dissolved, phenol extraction was performed twice, phenol / chloroform extraction was performed once, and chloroform extraction was performed once, followed by ethanol precipitation. The obtained ethanol solution was centrifuged at 10,000 rpm for 15 minutes, the obtained precipitation fraction was rinsed again with 80% ethanol, and then at 10,000 rpm.
Centrifugation was performed for 15 minutes. The obtained precipitate fraction was dried under reduced pressure, dissolved in 500 μl of RNase, and reacted at 37 ° C. for 2 hours. After completion of the reaction, phenol extraction was performed twice, phenol / chloroform extraction was performed once, and chloroform extraction was performed once, followed by ethanol precipitation. In this way, genomic DNA of Pseudomonas plantaly was obtained.

(2) Acquisition of tropolone-resistant Escherichia coli clone The obtained genomic DNA was digested with 22 kinds of restriction enzymes for 15 minutes to obtain 22 kinds of digested fragments.
22 kinds of restriction enzymes are Acc I, Bam HI, Bcl I, Bl
n I, Cla I, Eco RI, Hinc II, Hind III, Kpn I,
Nae I, Nhe I, Nru I, Nsp V, Pst I, Sac I, Sal
I, Sma I, Spe I, Sph I, Sse 8387I, Xba I and Xho I were used. Each of the obtained cut fragments was ligated to the plasmid pUC118 vector (Takara Shuzo). In addition, Acc I, BamHI, Eco RI, Hinc II
, Hind III, Kpn I, Pst I, Sac I, Sal I, Sma I
Ligation of the SphI, Sse8387I and XbaI digested fragments was performed after digesting the plasmid pCU118 vector with the same restriction enzymes. Also, Bcl I, Bln I, ClaI
, Nae I, Nhe I, Nru I, Nsp V, Spe I and Xho
The ligations of the I-cleavage fragments were Bam HI and X, respectively.
ba I, Acc I, Sma I, Xba I, Hinc II, Acc I, X
It was performed after cutting the plasmid pCU118 vector with baI and SalI. Escherichia coli Escherichia coli DH5-α strain was transformed with the thus obtained plasmid. After transformation, each is cultured in the presence of 50 ppm ampicillin to obtain ampicillin-resistant colonies. Next, each ampicillin-resistant colony obtained was treated with tropolone.
Cultivate in the presence of 100 ppm to obtain tropolone-resistant colonies. Furthermore, the obtained tropolone-resistant colonies were cultured again in the presence of 150 ppm of tropolone to obtain tropolone-resistant clones.

In this way, tropolone-resistant clones 151, 161, 21-1, 21-2, 21 which are stably grown are obtained.
-4, 21-5 and 30-1 could be isolated. The tropolone-resistant colonies 151 and 161 were 21- when the Pseudomonas plantaly genomic DNA was cleaved with Sal I.
1, 21-2, 21-4 and 21-5 are 30- when cut with Pst I
1 is a clone obtained from Escherichia coli transformed with the plasmid pU118 vector, which was ligated with the cut fragment obtained by cutting with Nhe I.

(3) Cloning of tropolone resistant gene Closing of tropolone resistant clones 151, 161, 21 isolated above
Plasmid DNAs are prepared from -1, 21-2, 21-4, 21-5 and 30-1 by the method described below. Then plasmid D obtained from colonies 151 and 161
NA is Sal I and colonies 21-1, 21-2, 21-4 and 21-5
The plasmid DNA obtained from was Pst I and colony 30
-1 is the plasmid DNA obtained from Eco RI and Sal
Cut with I and agarose gel electrophoresis of each DNA fragment
(Agarose 1.5%).

Method for preparing plasmid DNA The above-mentioned tropolone-resistant clone was treated at 37 ° C. in an LB medium containing 50 ppm ampicillin (consisting of 10 g of NaCl, 5 g of yeast extract, 10 g of tryptone and 1 L of distilled water).
The cells were shaken and cultured overnight. The resulting culture solution was centrifuged at 8,000 rpm for 10
Centrifuge for minutes, and precipitate the resulting precipitate with lysozyme buffer (50 m
After suspending in 3.2 ml of 50 mM Tris buffer, pH 8.0) containing M sucrose and 10 mM EDTA, 800 μl of 20 mg / ml lysozyme was added and suspended. The resulting suspension was allowed to stand at room temperature for 5 minutes, then SDS-alkaline solution (1M NaO
8 ml of H2, 1% SDS) was added and suspended, and the mixture was allowed to stand on ice for 10 minutes. Subsequently, ₆ ml of 3M CH ₃ C00H was added and suspended, and the mixture was allowed to stand on ice for 10 minutes and subsequently centrifuged at 8,000 rpm for 15 minutes. Phenol / chloroform extraction was performed on the obtained supernatant, 0.6 volume of isopropanol was added, the mixture was allowed to stand at room temperature for 15 minutes, centrifuged at 8,000 rpm for 15 minutes, and the obtained precipitate was extracted with 80% ethanol. After rinsing
It was again centrifuged at 8,000 rpm for 10 minutes, and the obtained precipitate was dried under reduced pressure. 1.6 ml TE (containing 1 mM EDTA) of the dried precipitate
After dissolving in 10 mM Tris buffer (pH 8.0), 1.74 g of CsCl was added and dissolved, and further 120 mg of 10 mg / ml EtBr was added and gently suspended. The obtained suspension was transferred to a centrifuge tube, centrifuged at 120,000 rpm for 180 minutes using an ultra-high speed centrifuge, and further centrifuged at 80,000 rpm for 60 minutes. The band of the plasmid DNA fraction is extracted with a tuberculin syringe, washed five times with TE-saturated isopropanol, and diluted with 4 volumes of TE. 1/10 the volume of diluent
Add CH ₃ C00Na and 2.5 volumes of ethanol and add 20 at -80 ℃.
Leave for a minute. After centrifugation at 15,000 rpm for 15 minutes, the obtained precipitate is rinsed with 80% ethanol and then centrifuged at 15,000 rpm for 10 minutes to obtain a plasmid DNA precipitate.

Thus, the gene PP-151 of the present invention,
PP-161, PP-21-1, PP-21-2, PP-21-4, PP-21-5 and PP-30-1 were obtained. The base length of each DNA fragment is 1.9 kilobases for PP-151, 1.2 kilobases for PP-161, 0.1 kilobase for PP-21-1, 1.3 kilobase for PP-21-2, PP-21- 4 was 0.5 kg base, PP-21-5 was 0.3 kg base and PP-30-1 was 0.2 kg base.

(4) Construction of restriction enzyme map Furthermore, PP-151, PP-161, PP-21-1, PP-21-2, PP-21-
The restriction enzyme maps of 4, PP-21-5 and PP-30-1 are shown in FIGS. 1 to 7, respectively.

(5) Introduction of tropolone resistance gene into Escherichia coli Genes PP-151, PP-161, PP-21-1, PP-2 obtained above
1-2, PP-21-4, PP-21-5 and PP-30-1 were introduced into E. coli respectively as follows. Using the ligation kit (Takara Shuzo), the plasmid pUC118 vector treated with alkaline phosphatase and the above gene were ligated, mixed with Escherichia coli DH5-α strain competent cells, and allowed to stand on ice for 2 minutes, then SOC medium ( Tryptone 1g, yeast extract 0.25g, NaCl 29mg, KCl
Suspend 10 mg and 180 mg glucose in distilled water for 49.5
After preparing 1 ml suspension, add 1M MgSO ₄ / 1M MgCl ₂ solution.
Add 0.5 μl) (400 μl) and incubate at 37 ° C for 1 hour with shaking. LM agar medium containing 50 ppm ampicillin (NaCl 88 mg, yeast extract 0.75 g, tryptone)
Dissolve 1.5 g and agar 2.25 g in distilled water to prepare 150 ml aqueous solution, autoclave and cool to 50-60 ° C.
Escherichia coli transformed with the plasmid containing each gene can be obtained by culturing at 37 ° C. for 24 hours in 1 M MgSO ₄ (1.5 ml). Gene PP-151, PP-161, PP-21-1
Escherichia coli into which PP-21-2, PP-21-4, PP-21-5 and PP-30-1 had been introduced was deposited at the Institute of Biotechnology, Institute of Biotechnology, as indicated in Table 1. Table 1 shows each consignment number
As shown in.

[0031]

[Table 1]

Example 2 The clone obtained in Example 1
The plasmid DNA prepared from 151 was digested with Hind III and treated with alkaline phosphatase, and the plasmid DNA prepared from clone 161 obtained in Example 1 was digested with Sma I and ligated with a Hind III linker. When PP-161 was introduced and the E. coli DH5-α strain was transformed, colonies formed faster than clones 151 and 161 in the presence of tropolone at 150 ppm, and growth was observed in the presence of tropolone at 200 ppm. I got -161.

The plasmid DNA prepared from the clone 151 obtained in Example 1 was digested with HindIII and treated with alkaline phosphatase to obtain the plasmid DNA prepared from the clone 151 obtained in Example 1. Was digested with Sma I and PP-151 ligated with a Hind III linker was introduced to transform E. coli DH5-α strain. As a result, clone 161-151 showing similar results was obtained. That is, it was shown that tropolone resistance was enhanced by ligating PP-151 and PP-161 in tandem.

[0034]

INDUSTRIAL APPLICABILITY The gene of the present invention exhibits tropolone resistance, and the rice transformed with the gene of the present invention is endowed with the tropolone resistance produced by Pseudomonas plantaly. Therefore, it is expected that the gene of the present invention can contribute to the control of rice seedling bacterial wilt disease.

[Brief description of drawings]

FIG. 1 shows a restriction map of gene PP-151 of the present invention.

FIG. 2 shows a restriction map of gene PP-161 of the present invention.

FIG. 3 shows a restriction map of the gene PP-21-1 of the present invention.

FIG. 4 shows a restriction map of the gene PP-21-2 of the present invention.

FIG. 5 shows a restriction map of the gene PP-21-4 of the present invention.

FIG. 6 shows a restriction map of the gene PP-21-5 of the present invention.

FIG. 7 shows a restriction map of the gene PP-30-1 of the present invention.

FIG. 8: PP-151, PP-161, PP-21- which are genes of the present invention
1 is an electrophoretic photograph showing the result of separating 1, PP-21-2, PP-21-4, PP-21-5, and PP-30-1 by agarose electrophoresis (1.5% agarol).

[Explanation of symbols]

 A: It contains the gene PP-151. B: Contains the gene PP-161. C: contains the gene PP-21-1. D: It contains the gene PP-21-2. E: It contains the gene PP-21-4. F: Contains the gene PP-21-5. G: Contains the gene PP-30-1. M: λ / Hind III 118: plasmid pUC118 vector / Sal I

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location // (C12N 15/09 C12R 1:38) (C12N 1/21 C12R 1:19) C12R 1: 38) (72) Inventor Mitsuru Saito 1-32-14, Nishi-Odori, Hanamaki, Iwate Prefecture Park Heights Hakozaki I-106 (72) Inventor, Ako Okumura 15-11 Murasakino, Kitakami City, Iwate Sankopo Numata B-106

Claims (15)

[Claims] 1. A tropolone resistance gene derived from Pseudomonas plantaly, which has a base length of 1.9 kilobases and is represented by the restriction map of FIG. 1, PP-15.
1. 2. A tropolone resistance gene derived from Pseudomonas plantaly, which has a base length of 1.9 kilobases and is represented by the restriction enzyme map of FIG. 1, PP-151.
A novel E. coli containing. 3. A tropolone resistance gene derived from Pseudomonas plantaly, which has a base length of 1.2 kilobases and is represented by the restriction enzyme map of FIG. 2 PP-16.
1. 4. A tropolone resistance gene derived from Pseudomonas plantaly, which has a base length of 1.2 kilobases and is expressed by the restriction map of FIG.
A novel E. coli containing. 5. A tropolone resistance gene derived from Pseudomonas plantaly, which has a base length of 0.1 kilobase and is expressed by the restriction map of FIG.
1. 6. A tropolone resistance gene derived from Pseudomonas plantaly, which has a base length of 0.1 kilobase and is expressed by the restriction map of FIG.
A novel E. coli containing 1. 7. A tropolone resistance gene derived from Pseudomonas plantaly, which has a base length of 1.3 kilobases and is expressed by the restriction map of FIG.
2. 8. A tropolone resistance gene derived from Pseudomonas plantaly, which has a base length of 1.3 kilobases and is expressed by the restriction map of FIG.
A novel E. coli containing 2. 9. A tropolone resistance gene derived from Pseudomonas plantaly, which has a base length of 0.5 kilobase and is expressed by the restriction map of FIG.
Four . 10. A tropolone resistance gene derived from Pseudomonas plantaly, which has a base length of 0.5 kilobase and is represented by the restriction enzyme map of FIG. 5, PP-2.
Novel E. coli containing 1-4. 11. A tropolone resistance gene derived from Pseudomonas plantaly, which has a base length of 0.3 kilobase and is expressed by the restriction enzyme map of FIG. 6, PP-2.
1-5. 12. A tropolone resistance gene derived from Pseudomonas plantaly, which has a base length of 0.3 kilobases and is represented by the restriction enzyme map of FIG. 6 PP-2.
A new E. coli containing 1-5. 13. A tropolone resistance gene derived from Pseudomonas plantaly, which has a base length of 0.2 kilobases and is expressed by the restriction enzyme map of FIG. 7, PP-3.
0-1. 14. A tropolone resistance gene derived from Pseudomonas plantaly, which has a base length of 0.2 kilobase and is expressed by the restriction enzyme map of FIG. 7, PP-3.
New E. coli containing 0-1. 15. A tropolone resistance gene derived from Pseudomonas plantaly, which has a base length of 1.9 kilobases and is represented by the restriction enzyme map of FIG. 1, PP-1.
A novel E. coli containing 51 and the base length of 1.2 kilobases in tandem with the gene PP-161 expressed in the restriction map of FIG.