The way to detect genes catabolism of the herbicide 2,4-d in the genomes of microorganisms

 

(57) Abstract:

The method can be used in biotechnology for the detection of genes for catabolism of the herbicide 2,4-D in the genomes of different organisms. DNA-hydrolyzing microorganisms by endonuclease Bam H 1, then hydrolyzing with radioactive DNA preparation plasmid RMK 16. The method allows to identify the strains, metaboliziruetsa the herbicide 2,4-D. table 1.

The present invention relates to the field of biotechnology and can be used to detect genes for catabolism of the herbicide 2,4-D in the genomes of different organisms.

Known methods of testing genetic structures in the cells of Pro - and eukaryotes, in particular the detection of mycoplasmal infections, a method for the diagnosis of virus Curling of leaves, testing the Y chromosome (1-3).

The closest in technical essence and the achieved positive effect is a method of diagnosis of the virus Curling of leaves, described in the author's certificate of the USSR N 1405314.

Detection methods in the genomes of prokaryotic genes for catabolism of 2,4-D were not previously offered.

The purpose of this invention is to develop a detection catabolism genes of herbicide 2,4-D in gepolabo the use of recombinant plasmid pMK 16 as a molecular probe is used to detect genes for catabolism of 2,4-D in the genomes of microorganisms.

The method is based on structural features of the recombinant plasmids pMK16, namely, that a plasmid is constructed from the vector pBR322 molecules and genes biodegradation of 2,4-dichlorophenoxyacetic acid Bacillus subtilis strain 16(6).

The method is carried out by performing the following operations:

1. Obtaining DNA preparations from cells of microorganisms.

2. Obtaining DNA preparation pMK 16.

3. Enzymatic hydrolysis of DNA preparations of microorganisms, fractionation and transfer to a membrane filter.

4. Getting drug radioactive DNA pMK 16.

5. Hybridization preparations of genomic DNA of the microorganisms on the filter with drug radioactive DNA pMK16. The exposure of membrane filters to x-ray film.

The essence of the method is illustrated by the following specific examples of execution.

Example 1. Obtaining DNA preparations from cells of microorganisms.

To obtain DNA preparations of increasing the biomass of the studied strains in 10 ml of liquid medium until it reaches a value of optical density of cell suspension (OD600), and 0.8 oE. Cells are harvested by centrifugation at 3000 rpm for 30 min, suspended in 0.1 ml of Buchi 1-10 mg/ml The mixture is incubated at -4oC for 10 minutes, the Cells are poorly suspendidas cultures homogenized in liquid nitrogen without treatment with lysozyme (4). Then hold the lysis of the cells in the presence of 0.2% SDS at 60oC for 10 minutes To the lysate add NaClO4to a final concentration of 1 M. the Mixture was incubated in ice for 20 minutes Then the lysate twice shaken with an equal volume mixture of chloroform-phenol (pH 8 (1:1) and once with a mixture of chloroform-isoamyl alcohol (24:1). After that, the supernatant add 2.5 volume of ethanol and allowed to sediment nucleic acids. The precipitate is collected by centrifugation at 5000 rpm for 15 min, dissolved in a buffer containing 10 mm Tris-HCl pH to 7.4; 1 mm EDTA and use in the future.

Example 2. Obtaining recombinant DNA pMK 16.

The recombinant plasmid pMK 16 is obtained from a strain of E. coli HB 101 (pMK 16) (5,6). The biomass of E. coli strain HB 101 (pMK 16) increase in 50 ml of LB broth with the addition of ampicillin to a final concentration of 30 μg/ml of the Culture incubated at 37oC to reach the value of optical density of cell suspension (OD600) of 0.8 oE. Cells are harvested by centrifugation at 3000 rpm for 20 min, suspension 1 m is ncentratio 5 mg/ml and incubated in ice for 7 minutes Then the lysate add 3 ml of 0.2 M NaOH and 1% SDS incubated until the lightening lysate. Next, make a 1.5 ml 5 M chilled potassium acetate. After 10-minute incubation in melting ice, the lysate centrifuged at 20000 rpm at -4oC 60 minutes To the supernatant add RNase A to a final concentration of 2 μg/ml and incubated for 40 min at room temperature. The mixture depotentiate 2 times with phenol pH 8 and 1 times with chloroform. Nucleic acids are precipitated with 2.5 volumes of ethanol, the precipitate is collected by centrifugation at 3000 rpm for 20 min, eavesdrop on the air and dissolved in a buffer of 10 mm Tris-HCl pH 8,1 mm EDTA, 10 mm NaCl. Then spend chromatography of the drug on sepharose CL-2B. Fractions containing DNA precipitated with 2.5 volumes of ethanol. The precipitate is collected by centrifugation at 5000 rpm for 15 min, dried, dissolved in a buffer containing 10 mm Tris-HCl pH 7,4; mm EDTA.

To test patterns selected 1 µg plasmid DNA pMK16 hydrolyzing the endonuclease BamH I in buffer containing 100 mm NaCl, 50 mm Tris-HCl pH 7.5, 10 mm MgCl2, 1 mm dithiothreitol for 2 hours at 37oC. After hydrolysis of recombinant DNA fractionary by electrophoresis in 1.0% agarose gel in Tris-acetate buffer. As a token of Espoo recombinant plasmid consists of three fragments with a length of 4.4; of 3.1 and 1.3, etc., N. This medication is used for further analysis.

Example 3. Enzymatic hydrolysis of DNA preparations of microorganisms, fractionation and transfer to a membrane filter.

10 μg of DNA preparation hydrolyzing the endonuclease BamH I in 35 μl of buffer containing 100 mm NaCl, 50 mm Tris-HCl pH 7.5, 10 mm MgCl2, 1 mm dithiothreitol for 2 hours at 37oC. the resulting BamH I DNA fragments fractionary 1.2% agarose gel by electrophoresis. For electrophoresis using a buffer system of the following composition: 20 mm sodium acetate, 40 mm Tris-HCl pH 8.2 mm EDTA pH 8. The fractionation is carried out in the gel size HH mm, at a current of 40 to 50 mA for 2 - 3 hours. As a marker for drug use DNA phage , hydrolyzed by endonuclease Pst I. after electrophoresis the gel is stained in a solution of Acidum bromide and viewed under ultraviolet light. After the painting fractionation marker DNA preparation of phage sure as traceconsole investigated DNA preparations. Next, the gel is placed in a solution containing 0.5 M NaOH, 1.5 M NaCl and incubated at room temperature for 60 minutes, the Gel was washed with distilled water and placed in a solution of 1.5 M NaCl, 0,7 M Tris pH 8 for 60 minutes Next geltona buffer, contains 10xSSC, 1 mm EDTA, for 18 to 20 hours. After this procedure, the filter is dried in air and kept at 80oC in vacuum at 0,09 MPa for 2 hours. The filter is incubated in a solution containing 5xSSC, 0,1% SDS, 5xdenhardt's solution, 100 μg/ml denaturating DNA salmon sperm. The filter is incubated at 65oC for 2 hours and then used for hybridization with a radioactive drug pMK 16.

Example 4. Getting drug radioactive DNA pMK 16.

Preparation of radioactive plasmid pMK16 produced by the method of substitution of nucleotides (5). For this 0.5-1 ág DNA plasmid pMK 16 make a 20 μl mixture containing 0.1 nm DSTF, -dTTP, -dCTP, PM [-32P]- dATP; 0.1 M MgSO4, 1 mm dithiothreitol, 500 μg/ml bovine serum albumina, 0.5 M Tris-HCl pH to 7.2. To the mixture was added 1 μl of DNA polymerase I (4 units) and 1 μl of Gnkazy I (2 of 10-3mg/ml). The mixture is incubated for 2 hours at -12oC. Check the specific activity of the probe. The specific activity of the probe should be 105-106pulse/ml to 1 µg DNA. Labeled plasmid DNA was separated from free [-32P]- dATP on microbalance with Sephadex G-50 (coarse). The drug plasmids are denatured at 90oC 3 min, cooled in ice and ispolzovaniem radioactive DNA pMK 16.

Preparation of radioactive plasmid DNA pMK 16 is mixed with a buffer for hybridization of the following composition: 5xSSC, 0.001 M EDTA, 0,1% SDS, 5xdenhardt's solution, 100 μg/ml denatured DNA salmon. This mixture is placed a membrane filter with immobilized thereon drugs total DNA of different organisms. The filter is incubated at 65oC for 16 - 18 hours. The filter is then treated twice in buffer containing 2xSSC and 0.1% SDS at 65oC for 2 hours and 0.5 hours in a buffer containing 0.2xSSC and 0.1% SDS at 65oC. Next, the filter is dried in air at room temperature and exhibiting a x-ray film PM-B and intensifying screens EC-B2 for 3 to 7 days. After developing, the film analyze hybridization (5). Positive evaluation of the hybridization indicates the presence of genes for the catabolism of 2,4-D in the sample.

Example 6. Hybridization preparations of genomic DNA of microorganisms with drugs radioactive DNA pBR322.

Hybridization preparations of genomic DNA mikroorganizmov with drugs radioactive DNA pBR322 performed to exclude non-specific hybridization of the DNA of the microorganisms with the vector molecular pBR322, which is part of the plasmid pMK16.oC in a buffer of the following composition: 5 mm Tris pH 7.5; 0.1 mm EDTA. Then the drug is rapidly cooled in ice and put on a membrane filter. The drug is placed on a small square in the form of a point. After drying the filter in the air it stand 2 hours in a vacuum Cabinet at 0,09 MPa and 80oC. Further, the filter used for DNA hybridization as described in example 5. Hybridization is carried out with a radioactive drug pBR322, which is obtained as described in example 4.

Example 7. The experimental verification of the method for detecting genes catabolism of the herbicide 2,4-D in the genomes of microorganisms.

Laboratory validation of the proposed method performed using 17 DNA samples of different strains of microorganisms. Among the investigated strains were included strains, capable and not capable of metabolismrelated the herbicide 2,4-D. as a control we used a strain of Bacillus subtilis 16 containing the genes for the catabolism of 2,4-D that are included with the composition of the plasmid pMK 16 and strains of Escherichia coli HB101 (pMK 16) and Escherichia coli HB101 (pBR322). The table shows the results of hybridization preparations of genomic DNA of different strains microorganis the samples DNA positive assessment of hybridization with a radioactive drug pMK 16 have drugs 9 strains. This control DNA preparations of strains of Bacillus subtilis 16 and Escherichia coli (pMK 16), and the DNA preparations 7 the following strains: Agrobacterium rhizogenes, Agrobacterium tumefaciens, Artrobacter globiformis, Asopergillus niger, Pseudomonas fluorescens, Pseudomonas putida, Serratia marcescens. These data indicate that the above strains have genes for catabolism of 2,4-D. This conclusion is supported by previously established facts of the use of 2,4-D indicated strains as a source of carbon and energy (6 - 10). From the table it is seen that the molecules of the vector pBR322 not hybridize with the investigated DNA preparations. Positive hybridization was observed only with the control DNA preparation Escherichia coli (pBR322). These data suggest that the presence of molecules of the vector pBR322 in plasmide pMK 16 does not affect allow the possibility of the proposed method. Thus it is shown that the inventive method can effectively identify genes for catabolism of 2,4-D in the genomes of different strains of microorganisms.

This method is proposed for the first time.

Method of application for analysis of the genomes of microorganisms, regardless of their taxonomic position.

The method does not involve time-consuming and laborious methods using varying parameters to the ptx2">

The method is applicable for the simultaneous analysis of a large number of strains.

Thus, the proposed method is new, does not have significant limitations when applied to different objects of microorganisms, reduces the time and resources searching for strains, metaboliziruemah the herbicide 2,4-D.

Information taken into consideration

1. USSR author's certificate N 1374783, 24.12.85.

2. USSR author's certificate N 1405314, 30.12.86.

3. The Y Chromosome. Part A: basic Characteristics of the Y Chromosome. Jau Yun-Fai. 1985, 177 c.

4. Methods of General bacteriology/Ed. by F. Gerhardt. - M.: Mir, 1984, pp. 112-118.

5. Mandates T. , Fritsch E., Sambrook J., Molecular cloning. - M.: Mir, 1984, S. 332 - 337, 344 - 350.

6. Patent of the Russian Federation on the application N 5035856/13, 02.08.91.

7. RF patent N 1742226, 22.05.89.

8. Zhurenko E. Yu, Kusova I. C., Sultanbekov M. N., Katkov E. G, Savenko O. S. , Markushev Century So the Search for and study of microorganisms for the degradation of the herbicide 2,4-D. Coll. : The main directions of biotechnology in solving economic problems. - Ufa: 1991, S. 81-92.

9. Nikitin, C. S., Markushev T. C., Zhurenko E. Y., Kusova I. C., Churaev R. N. A comparative study of the dynamics of degradation of the herbicide 2,4-D by bacteria razlichayutsya microorganisms in organic synthesis. - M.: Nauka, 1976, S. 300 - 305.

The way to detect genes catabolism of the herbicide 2,4-D in the genomes of microorganisms, consisting of DNA hybridization studied strains with radioactive DNA preparation plasmid RMK 16 on solid media.

 

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