KR970006163B1 - New strain having control activity of schizophrenia and gene fragments isolated therefrom - Google Patents

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Description

New strain having control activity of schizophrenia and gene fragments isolated therefrom

1 is a photograph showing that the strain Hafnia alvei W1 of the present invention and the bacterium mycelial growth inhibitor-coding gene clone C32 isolated therefrom inhibit the growth of the bacterium mycelium mycelia.

2 is a photograph of Southern blot development showing that gene clone C32 is derived from Hafnia alvei W1.

The present invention relates to a gene clone encoding a new strain Hafnia alvei having a control effect against rice blasts and a substance having an activity of inhibiting the growth of rice blast bacteria isolated therefrom.

Rice blast is the most damaging pathogen caused by rice cultivation. Therefore, the use of a lot of manpower and pesticides for the control of blast disease is inevitable. As a result, there are side effects such as residual toxicity of crops, poisoning to livestock and destruction of ecosystems. Many researches and investments are being made on the development of varieties by engineering techniques and the development of new pesticides by new materials.

Under these circumstances, the present inventors have made a thorough study to provide a method for controlling biological rice fever that does not have the above-mentioned problems. As a result, the present inventors have succeeded in separating the new strains that inhibit rice mycelial mycelial growth from the soil to complete the present invention. Reached.

That is, an object of the present invention is to provide a new strain Hernia alvei having the activity of inhibiting the mycelial growth of rice blast.

Another object of the present invention is to provide a gene clone encoding a substance that inhibits the growth of mycelia of rice blast bacteria isolated from the strain.

Another object of the present invention is a method for controlling rice blasts, characterized in that the microbial agent containing the strain Hafnia alvei is applied to rice plants with the risk of infection or plantation of infected plants or plants. To provide.

Hereinafter, the present invention will be described in more detail.

The strain of the present invention is a bacterium exhibiting antagonism against rice blast bacteria and is isolated from soil. The separation method from the soil is equivalent to that of conventional bacteria. Since the isolated strain exhibits control activity against rice blast, it can be made into a microbial agent by a conventional method and used as a control agent of rice blast. In the present invention, not only this, but also the gene of the strain was analyzed to purely separate the gene encoding a substance that inhibits the mycelial growth of rice blast bacteria.

Separation of a gene encoding a substance having an antagonistic activity against rice blast from the strain is carried out according to a conventional gene separation method. In other words, after separating the entire DNA from the strain to produce a gene bank, transforming these gene banks in Escherichia coli and replacing the transformed Escherichia coli with rice blast bacteria to isolate a gene clone having a mycelial inhibitory effect.

In this way, the isolated gene clone can be introduced into an appropriate plant transformation vector and then transformed into rice to create rice that is resistant to rice blast.

The present invention will be described in more detail with reference to the following examples.

Example 1 Isolation and Identification of Hafnia alvei W1

10 g of soil sample was added to 100 ml of acidic water and shaken for 30 minutes at room temperature, followed by dilution of the shaker with 101-105, followed by smearing onto LB medium (trypton 10g, yeast extract 5g, NaCl 10g bacto agar 15g). The bacteria were purely separated by an agar dilution plate method which forms. After inoculating heat bacillus on PDA medium (potato 200g, dextrose 20g, agar 20g) and incubating for 48 hours at 28 ℃, the selected bacteria were inoculated to check antagonism, and among them, the strain with the highest antagonism was selected. Selected and named W1.

As a result of analyzing the bacteriological properties for the identification of W1, the results shown in Table 1 were obtained.

The strain showed resistance to antibiotics such as ampicillin, tetracycline, kanamycin, and susceptibility to gentamicin.

Based on the results of Table 1, the strain of the present invention was identified as Hafnia alvei in comparison with Bergey's manual and API index.

The Hafnia Albay W1, first provided to the inventors, was deposited with the Korean spawn association on December 20, 1993 and was given an accession number of KFCC-10811.

Strain W1 provided by the present invention exhibits growth inhibitory activity against rice blast bacteria and can be used for the control of rice blast diseases. That is, the strain may be made into a microbial preparation in a conventional manner, and then the formulation may be sprayed on crops or applied to planting soil.

Example 2. Isolation of Chromosome DNA

In order to isolate the chromosomal DNA encoding a substance that inhibits the growth of mycelial bacteria from the strain, W1 strain and antibiotic ampicillin were inoculated in 5 ml of LB medium mixed at a concentration of 50 mg / ml, and then incubated at 30 ° C. After 16 hours, 1 ml of the culture solution was taken and inoculated again into 100 ml of LB (Amp) medium, followed by suspension culture for 16 hours. To collect the bacteria, the colonies were centrifuged at 4 ° C. for 10 minutes at 4000 rpm, and the colonies were washed again with TEN buffer (10 mM tris-HCl, pH 8.0, 6 mM NaCl; 1 mM EDTA), followed by 4 ml of bacterial colonies. In a TEN buffer solution, 500 μl of lysozyme (10 mg / ml), 200 μl of RNase (0.1 mg / ml), and 500 μl of EDTA (0.25M, pH 8.0) were added and iced for 20 minutes. 270 μl of 20% SDS and 500 μl of protease K (1 mg / ml) were added thereto, followed by incubation at 37 ° C. for 4 hours. After 4 hours, 5 ml of TEN buffer solution was added again.

To remove the protein, add 10 ml of phenol: chloroform (1: 1) solution, shake it slowly by hand for 5 minutes, and centrifuge (5000rpm, 5 minutes) to recover only the supernatant, and then mix with phenol: chloroform solution to remove protein. The procedure was repeated five times.

The protein completely removed was mixed with 1/10 portions of 3M sodium acetate solution and twice the amount of 100% ethanol cooled on ice, and shaken again to precipitate DNA. The DNA was recovered and washed twice with 70% ethanol, ethanol was removed with a vacuum dryer, dissolved in 3 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA) and stored in a 4 ° C. refrigerator. Used to prepare gene banks.

Example 3 Preparation of Gene Bank

To prepare a gene bank, first, 10 μg of a cosmid vector, supercos DNA (produced by Stratagene), was treated with 3 μl of BamH1 restriction enzyme (12 units / μ) at 37 ° C. for 3 hours, and CIAP (Calf Intestinal Alkaline) was used. Phosphatase: calf intestinal alkaline phosphatase) was treated with 0.5 unit to remove the 5'-phosphate group from the cleavage site of the vector supercos to prevent self-conjugation of the vector. 10 μg of the chromosomal DNA of the W1 strain was partially cleaved with 0.25 units of Sau3A1 enzyme, mixed with the vector DNA at a ratio of about 1: 1, and 1 μl of T4 DNA ligase (1 unit / μl) was incubated at 13 ° C. for 19 hours. The fragment and the vector DNA were conjugated. In order to package the conjugated DNA into lambda phage, after packaging using a commercially available in vitro packaging kit (manufactured by Gigapack gold Inc.), the resulting lambda phage was introduced into E. coli NM554 strain to obtain approximately 3,000 genes. Completed the bank.

Example 4 Isolation of DNA Fragments Encoding Substances That Inhibit Mycelial Growth of S. aureus

For the selection of Escherichia coli clones showing antagonistic ability to blast bacillus, 3000 Escherichia coli clones were alternately incubated with the blast bacterium from the above-described gene bank, and the mycelial growth inhibitory effect was confirmed. The effect was confirmed. In Figure 1, strain W1 of the present invention and Escherichia coli TG1 (clone C34) having the same DNA fragments as E. coli NM554 and C32 designated as clone C32 show the result of replacement culture that inhibits the growth of KJ 301, a rice blast bacterium. It was. In Figure 1 554 represents host E. coli NM554, which does not carry the DNA fragmentol of the present invention.

Alkaline lysis was used to separate DNA encoding a substance that inhibits the growth of rice blast bacteria from the C32 clone. That is, C32 clone E. coli NM554 was inoculated in 5 ml of LB and suspended in culture at 39 ° C. for 13 hours. After dispensing 1 ml of the culture solution into an Eppendorf tube, the supernatant was discarded after centrifugation and suspended in 200 μl of 10mN EDTA using the remaining colonies. Add 400 μL of 0.2 M NaOH, 0.1% SDS solution, add 2.5 M potassium acetate, 300 μL of 2.5 M acetic acid solution, shake the test tube, and centrifuge at 12,000 rpm for 5 minutes to separate the plasmid DNA. About 30 Kb of the W1 chromosome DNA was isolated.

The DNA fragment was named as pANTl, introduced into E. coli NM554, and deposited with the Korean spawn association on December 20, 1993 and was given accession number of KFCC-10810.

A portion of the Hind III fragment (1.2 kb) of the about 30 Kb gene inserted into the vector was used as a probe by labeling radiation (32P) using a Nick translation Kit (promega company). Meanwhile, the total chromosome DHA isolated from the W1 strain was cut at 37 ° C. for 5 hours using four restriction enzymes of EcoRI, HindIII, PstI and BamHI, and the fragments were subjected to electrophoresis (50V, 10 hours), the DNA contained in the gel was denatured into single strands, neutralized and transferred to a nitrocellulose filter for adsorption. Next, hybridization according to the homology of DNA was performed by mixing with the probe DNA labeled with the above-described radiation, and the results are shown in FIG.

In FIG. 2, lane 1 shows hybridization results of probe DNA with fragments obtained by EcoRI cleavage of total DNA of W1 strain, and lane 2 fragments and probe DNA cleaved by HindIII of total DNA of W1 strain. Shows the results of the hybridization with, lane 3 shows the results of hybridization with the pros DNA and fragments cut the total DNA of the W1 strain with Pstl, lane 4 shows the total DNA of the W1 strain with BamHI The results of hybridization of the fragments with the probe DNA are shown.

As can be seen from the results in FIG. 2, homology with the probe DNA was detected in all lanes, confirming that the pANT1 DNA inserted into the C32 clone was derived from the chromosomal gene of blast antagonist W1.

In addition, even when the pANT1 DNA is transformed into E. coli TG1 by a conventional method, it is observed that mycelial growth is inhibited (C34 clone of FIG. 1), which demonstrates that a gene that inhibits the growth of febrile mycelia is present in pANT1 DNA.

The pANT1 can be used for the transformation of plants by conventional plant transformation methods. In other words, by introducing into a plasmid, cosmid or phage and then introduced into the plant by an appropriate transformation method to transform the plant can create a plant resistant to rice fever.

Claims (3)

Hafnia alvei W1 strain (KFCC-10811) having the activity of inhibiting the mycelial growth of rice blast bacteria. Gene fragment pANT1 (KFCC-10810), derived from the Harnia alvei W1 strain (KFCC-10811), encoding a substance that inhibits the growth of rice blast bacteria. Microbial preparations, including the Hafnia alvei W1 strain (KFCC-10811), which have antimicrobial activity against rice blasts, are vulnerable to rice blasts or on plants or plants grown in rice blasts. A method for controlling rice blast, characterized by spraying.