JP2600090B2 - Host vector system - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a plasmid pBR32
2. It relates to a host vector system using pBR325 or pBR328 as a vector and Pseudomonas avenae as a host, and is transformed with a plasmid pBR322, pBR325 or pBR328 and is a proline-requiring mutant, Pseudomonas avenae. ). The host vector system and the novel bacterium of the present invention are expected to be used in the field of microbial industry such as production of useful substance-producing bacteria using Pseudomonas bacteria, and in the field of agriculture such as production of biocontrol microorganisms.

[0002]

2. Description of the Related Art As a means for clarifying the gene expression mechanism of Pseudomonas genus bacteria, a gene is isolated from the bacterium by genetic recombination technology. As the method, the following two means are generally adopted. Cloning is performed using an E. coli host vector system. Cloning is performed using a Pseudomonas host vector system. However, the method (1) has a drawback that expression of a gene derived from Pseudomonas bacteria in E. coli is generally low in efficiency. In the method (1), the transformation efficiency is low and the expression is often insufficient depending on the bacterial species.

On the other hand, a pBR plasmid (pBR32) which has been developed as a host vector vector for Escherichia coli.
2, pBR325, pBR328, etc.) have extremely excellent advantages as cloning vectors. However, these plasmids could not replicate in Pseudomonas sp. And could not be used as vectors.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have proposed a rice brown streak fungus (Pseudomonasa) isolated from rice.
venae) can be transformed and replicated with a pBR-based plasmid and transformed as efficiently as the Escherichia coli system. Furthermore, a proline-requiring strain selected from the bacterium can be more efficiently The present inventors have found that transformation can be performed using a pBR plasmid, and have completed the present invention. According to the present invention, the cloning of the gene of Pseudomonas bacteria, particularly Pseudomonas avenae, can be performed extremely easily.

[0005]

That is, the present invention relates to a plasmid pBR322, pBR325 or pBR.
The present invention relates to a host vector system using 328 as a vector and Pseudomonas avenae as a host, which is transformed with a plasmid pBR322, pBR325 or pBR328, and is a proline-requiring mutant, a rice brown streak (Pseudomonas avenae). It is.

[0006] The rice brown streak, which is a proline-requiring mutant transformed with pBR322, pBR325 or pBR328 of the present invention, is produced as follows. The production of a proline-requiring mutant from the brown streak fungus strain K1 is performed as follows. Bacteria in logarithmic growth phase [K1 (MA
FF03-01752)] is treated with nitrosoguanidine at a concentration of 100 μg / ml at 37 ° C. for 30 minutes, and then cultured overnight. The bacteria are plated to form colonies. Individual colonies are cultured in a minimal medium and a medium in which casamino acid and tryptophan are added to the minimal medium, and amino acid mutants that grow only in the latter are selected. Proline-requiring mutants can be obtained by further culturing these strains in a minimal medium and a medium in which proline is added to the minimal medium, and selecting strains that grow only in the latter. The proline-requiring strain obtained in the present invention is Pr
The strain was named 47 strains.

The K1 strain used in the present invention was obtained from Fermentation Research Institute (2-17-17, Jusanhoncho, Yodogawa-ku, Osaka-shi).
No. TEL: 06-302-7281) and the accession number is “IFO15113”. Also, P
The r47 strain has also been deposited with the above-mentioned Fermentation Research Institute, and the accession number is “IFO15114”. Any fungus can be furnished for the practice of the present invention.

[0008] The bacteriological properties of the K1 (NR8201) strain have been investigated in detail, and from those results,
Rice brown streak fungus (Pseudomonas avena)
e) (Tsuchiya et al., Agricultural and Environmental Research Institute 3)
No. 8, 1983). The Pr47 strain has the same bacteriological properties as the K1 strain, but differs only in proline requirement. Furthermore, both the K1 strain and the Pr47 strain can grow well on a general growth medium such as an LB medium or a potato semi-synthetic medium which is usually used for culturing bacteria.

The host vector system of the present invention is produced as follows. The method of Hanahan [J. Mol. Bio
l. , Vol. 166, pp. 557-580, (19
83)] to prepare susceptible bacteria for transformation. 2xTY medium (16 g of bactotryptone, 10 g of yeast extract, 5 g of sodium chloride, 1 g of distilled water)
1), the bacteria reaching 0.3-0.4 OD (660 nm) are collected by a centrifuge, replaced with RF1 solution, and replaced with 4 ° C.
For 1.5-2.0 hours.

The composition of the RF1 solution is as follows. Further, the bacteria placed in the RF2 solution for 15 to 30 minutes, dispensed in 50 to 100 μl aliquots into microtubes are rapidly frozen in dry ice / ethanol and stored at −80 ° C. The composition of the RF2 solution is as follows.

[0011] The transformation can be carried out according to the above-mentioned method of Hanahan. That is, 50 μl of the susceptible bacteria
Add 0 ng of plasmid and let stand for 1 hour on ice.
The sample heated at 42 ° C. for 90 seconds and returned to ice for several minutes is incubated at 35 ° C. for 3 hours. These samples are cultured in a selective medium to which an antibiotic has been added, and the transformation efficiency can be measured by the number of colonies formed.

[0012]

[Action] with the plasmid pBR322, pBR325 or pBR328, the strain K1 or Pr47 strain, it is possible to make the transformed recombinant at ^{10 4-5} high rate per plasmid 1 [mu] g.

[0013]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the present invention is limited thereto. [Example 1] Transformation with plasmid 20 ng to susceptible strain (50 µl) of K1 strain and Pr47 strain
Of PBR322 strain and several μl of distilled water as a control were mixed well, and allowed to stand on ice for 1 hour. After leaving still at 42 ° C. for 1 minute and again placing on ice for several minutes, the SOC medium (20 g of bactotryptone, yeast extract 5) was added.
g, sodium chloride 0.5 g, 20 mM glucose, 20 g
After culturing for 3 hours at 35 ° C. in 1 l of mM magnesium sulfate and distilled water, the cells are cultured overnight at 35 ° C. in an LB plate medium containing tetracycline (25 μg / ml). The number of formed colonies was measured, and the conversion efficiency was calculated by converting the number of colonies per 1 μg of plasmid. Table 1 shows the average value of 5 repetitions. In the case of pBR325 and pBR328, the cells were cultured in an LB medium containing chloramphenicol (30 μg / ml). From these results, the K1 strain was pB
R322, pBR325 and pBR328 were transformed at high rates. Furthermore, the efficiency of the Pr47 strain was about 10 to 50 times higher than that of the K1 strain, and was 1.3 to 3.0 × 10 ⁶ (Table 1).

Example 2 Confirmation of Transformation by Electrophoresis To confirm that the plasmid was transformed into the Pr47 strain, the formed colonies were subjected to liquid culture, and the plasmid was extracted. Twelve colonies obtained by transforming the Pr47 strain using pBR325 were picked and cultured in a 1.5 ml liquid medium containing chloramphenicol (30 μg / ml) with shaking at 35 ° C. overnight. The plasmid was extracted by the alkaline method and subjected to 1% agarose electrophoresis.
It showed the same mobility as R325, and it was confirmed that all 12 cells were transformed. The results are shown in FIG. In FIG. 1, lane A shows marker DNA, lanes show plasmid pBR325, and lanes 1 to 6 show plasmids extracted from transformants.

[Brief description of the drawings]

FIG. 1 is a view showing an electrophoresis pattern of agarose gel electrophoresis of a plasmid extracted from a transformant.

Claims (2)

(57) [Claims] 1. The plasmids pBR322 and pBR32
5 or pBR328 as a vector, and Pseudomonas avenae as a host. 2. The plasmids pBR322, pBR325
Alternatively, PBR328, which is a proline-requiring mutant, is a rice brown streak (Pseudomonas).
avenae).