RU2125263C1 - Method of detecting genes of catabolism of herbicide 2,4-d in genomes - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: radioactive preparation of RNA of plasmid pMK 16 on solid carrier is used. EFFECT: enabled revealing genes of catabolism of herbicide 2,4-D in RNA of genomes of eucaryotic cells. 1 dwg, 1 tbl, 7 ex

Description

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

 Known methods for testing genetic structures in eukaryotic cells, in particular a method for detecting globin genes, a method for testing Y chromosomes [1-2].

 The closest in technical essence and the achieved positive effect is a method for detecting globin genes of eukaryotes [1].

 Methods for detecting 2,4-D catabolism genes in eukaryotic genomes have not been previously proposed.

 The purpose of this invention is the development of a method for detecting genes for catabolism of the herbicide 2,4-D in the genomes of eukaryotes.

 This goal is achieved in that according to the proposed method, the use of the recombinant plasmid pMK16 as a molecular probe allows the detection of 2,4-D catabolism genes in eukaryotic genomes.

 The method is based on the structural features of the recombinant plasmid pMK16, which consists in the fact that the plasmid is constructed from the vector molecule pBR322 and the biodegradation genes of 2,4-dichlorophenoxyacetic acid.

The method is carried out by performing the following operations:
1. Obtaining DNA preparations from eukaryotic cells.

 2. Obtaining DNA preparation pMK16.

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

 4. Obtaining the preparation of radioactive DNA pMK16.

 5. Hybridization of eukaryotic genomic DNA preparations on the filter with pMK16 radioactive DNA preparation. Exposure of membrane filters with x-ray film.

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

 Example 1. Obtaining DNA preparations from eukaryotic cells.

To obtain preparations of plant DNA, seed is germinated for 3 days in an incubator at an optimum temperature (25 - 28 ^o C). 1-3 g of seedlings are homogenized with liquid nitrogen in a porcelain mortar [3]. 21 ml of buffer containing 100 mM Tris HCl pH 8, 15 mM EDTA, 1% SDS was added to the homogenate. The suspension is incubated for 10 minutes in a water bath at 60 ^° C. The homogenate is cooled and 5 M NaClO _{4 is} added to it to a final concentration of 1 M, incubated in ice for 30 minutes. Next, an equal volume of a phenol mixture of pH 8 - chloroform (1: 1) is added twice to the homogenate, shaken for 20 minutes in the cold. The supernatant was separated by centrifugation for 15 min at 3000 rpm. This procedure is repeated with chloroform. To obtain a DNA pellet, 2.5 volumes of ethanol are added to the supernatant. The DNA preparation was collected by centrifugation at 3000 rpm for 20 minutes, treated with RNase A, deproteinized and transferred to a buffer of 10 mM NaCl, 5 mM EDTA. In this form, they are used in the future.

 To obtain preparations of animal DNA, blood cells are used [4]. The homogenate of the cells in a buffer of 0.15 M NaCl - 0.015 M Na citrate is twice deproteinized in the cold with equal volumes of phenol pH 8 and once with a mixture of chloroform - isoamyl alcohol (24: 1). The supernatant was separated by centrifugation at 3000 rpm for 20 minutes After deproteinization, the DNA is treated with RNase A, deproteinized and precipitated with ethanol. Next, the preparations are transferred to a buffer containing 10 mm NaCl, 5 mm EDTA and in this form is used in the future.

To obtain preparations of fungal DNA, the biomass of the studied strain is increased on an agarized nutrient medium. The mass (1-3 g) of cells is removed from the agar with a spatula and homogenized in liquid nitrogen [5]. Next, cell lysis is performed in the presence of 0.2% SDS at 60 ^° C. for 10 minutes. NaClO _{4 was} added to the lysate to a final concentration of 1 M. The mixture was incubated in ice for 20 minutes. Then the lysate is shaken twice with an equal volume of a mixture of chloroform-phenol pH 8 (1: 1) and once with a mixture of chloroform-isoamyl alcohol (24: 1). After that, 2.5 volumes of ethanol are added to the supernatant and left to form a nucleic acid precipitate. The precipitate was collected by centrifugation at 5000 rpm for 15 min, dissolved in a buffer containing 10 mm Tris-HCl pH 7.4; 1 mm EDTA, and in this form is used in the future.

 Example 2. The preparation of recombinant DNA pMK16.

The preparation of the recombinant plasmid pMK16 is obtained from E. coli strain HB 101 (pMK16) [5,6,7]. For this, the biomass of E. coli strain HB 101 (pMK16) is expanded in 50 ml of LB broth with the addition of ampicillin to a final concentration of 30 μg / ml. The culture is incubated at 37 ^o C until the optical density of the cell suspension (OD ₆₀₀ ) of 0.8 oE is reached. Cells are harvested by centrifugation at 3000 rpm for 20 minutes, suspended in 1 ml of buffer containing 50 mM glucose, 25 mM Tris-HCl pH 8, 10 mM EDTA. Lysozyme is added to the cell suspension to a final concentration of 5 mg / ml and incubated in ice for 7 minutes. Then, 3 ml of 0.2 M NaOH and 1% SDS are added to the lysate, incubated until the lysate is clarified. Then make 1.5 ml of 5 M chilled potassium acetate. After a 10-minute incubation in melting ice, the lysate is centrifuged at 20,000 rpm at 4 ^° C. for 60 minutes. RNase A was added to the supernatant to a final concentration of 2 μg / ml and incubated for 40 min at room temperature. The mixture is deprotenized 2 times with phenol, pH 8, and 1 time with chloroform. Nucleic acids are precipitated with 2.5 volumes of ethanol, the precipitate is collected by centrifugation at 3000 rpm for 20 min, dried in air and dissolved in 10 mM Tris-HCl buffer, pH 8.1 mM EDTA, 10 mM NaCl. After that, chromatography of the drug on sepharose CL-2B is carried out. Fractions containing DNA precipitated with 2.5 volumes of ethanol. The precipitate was collected by centrifugation at 5000 rpm for 15 min, dried, dissolved in a buffer containing 10 mm Tris-HCl pH 7.4; 1 mM EDTA.

To verify the structure of the isolated plasmid, 1 μg of pMK16 DNA was hydrolyzed with Bam HI endonuclease in a buffer containing 100 mM NaCl, 50 mM Tris-HCl pH 7.5, 10 mM MgCl ₂ , 1 mM dithiothreitol for 2 hours at 37 ^° C. After hydrolysis the recombinant DNA preparation is fractionated by electrophoresis in a 1.0% agarose gel in Tris acetate buffer. As a marker, a phage λ DNA preparation hydrolyzed by Pst I endonuclease is used. As a result of electrophoresis, it is ascertained that the recombinant plasmid consists of three fragments 4.4 in length; 3.1 and 1.3 kb This drug is used for subsequent analysis.

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

10 μg of the DNA preparation is hydrolyzed with BamH I endonuclease in 35 μl of buffer containing 100 mM NaCl, 50 mM Tris-HCl pH 7.5, 10 mM MgCl ₂ , 1 mM dithiothreitol for 2 hours at 37 ^° C. After that, the obtained BamH I DNA fragments are fractionated in a 1.2% agarose gel by electrophoresis [7]. For electrophoresis, a buffer system of the following composition is used: 20 mM sodium acetate, 40 mM Tris-HCl pH 8.2 mM EDTA pH 8. Fractionation is carried out in a gel measuring 120x80x5 mm, at a current of 40-50 mA for 2-3 hours. Markers use the phage λ DNA preparation hydrolyzed by Pst I endonuclease. After electrophoresis, the gel is stained with ethidium bromide in a solution and viewed under ultraviolet light. From the picture of fractionation of marker preparation of DNA of phage λ, one can see the quality of fractionation of the studied DNA preparations. The gel is then placed in a solution containing 0.5 M NaOH, 1.5 M NaCl and incubated at room temperature for 60 minutes. The gel is washed with distilled water and placed in a solution of 1.5 M NaCl, 0.7 M Tris-HCl pH 8 for 60 minutes. Next, the gel is washed with distilled water and DNA fragments are transferred to a 0.2 μm membrane filter in a buffer stream containing 10 ^x SSC, 1 mM EDTA for 18-20 hours. After this procedure, the filter is dried in air and kept at 80 ^o C in vacuum at 0.09 MPa for 2 hours. The filter is incubated in a solution containing 5 ^x SSC, 0.1% SDS, 5 ^x Denhardt's solution, 100 μg / ml denatured salmon sperm DNA. The filter was kept at 65 ^° C for 2 hours and then used for hybridization with the radioactive preparation pMK16.

 Example 4. Obtaining a preparation of radioactive DNA pMK16.

The preparation of the radioactive plasmid pMK16 is obtained by nucleotide substitution [7]. For this, 0.5-1 μg of DNA of plasmid pMK16 is added to 20 μl of a mixture containing 0.1 nM dGTP, -dTTP, -dTCP, 100 pM [α- ³² P] -ATP; 0.1 M MgSO ₄ , 1 mM dithiothreitol, 500 μg / ml bovine serum albumin, 0.5 M Tris-HCl pH 7.2. 1 μl of DNA polymerase I (4'units) and 1 μl of DNase I (2 • 10 ^-3 mg / ml) are added to the mixture. The mixture was incubated for 2 hours at 12 ^° C. The specific activity of the probe was checked. The specific activity of the probe should be 10 ⁵ -10 ⁶ imp / min per 1 μg of DNA. Labeled plasmid DNA is separated from free [α- ³² P] -ATP on a micro column with Sephadex G-50 (coarse). The plasmid preparation was denatured at 90 ^° C. for 3 minutes, cooled in ice and used for hybridization.

 Example 5. Hybridization of eukaryotic genomic DNA preparations on a filter with pMK16 radioactive DNA preparation.

The pMK16 radioactive plasmid DNA preparation was mixed with a hybridization buffer of the following composition: 5 ^x SSC, 0.001 M EDTA, 0.1% SDS, 5 ^x Denhardt's solution, 100 μg / ml denatured salmon DNA. A membrane filter with genomic DNA preparations immobilized on it is placed in this mixture. The filter is incubated at 65 ^° C. for 16-18 hours. The filter is then treated twice in a buffer containing 2 ^x SSC and 0.1% SDS at 65 ^° C. for 2 hours and 0.5 hours in a buffer containing 0.2 ^x SSC and 0.1% SDS at 65 ^o C. Next, the filter is dried in air at room temperature and exposed with an X-ray film PM-B and intensifying screens EU-B2 for 3-7 days. After the development of the film, a hybridization analysis is carried out [7]. A positive assessment of hybridization means the presence of 2,4-D catabolism genes in the test sample.

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

 Hybridization of genomic DNA preparations with pBR322 radioactive DNA preparations is carried out in order to exclude non-specific hybridization of genomic DNA with the pBR322 vector molecule, which is part of plasmid pMK16.

For hybridization of genomic DNA preparations with pBR radioactive DNA preparations, the in situ hybridization method is used [7]. To do this, 2 μg of each preparation of the studied DNA is heated for 3 min in a water bath at 100 ^o C in a buffer of the following composition: 5 mm Tris pH 7.5; 0.1 mM EDTA. Then the drug is quickly cooled in ice and applied to a membrane filter. The drug is placed on a small area in the form of a dot. After drying the filter in air, it is kept for 2 hours in a vacuum oven at 0.09 MPa and 80 ^° C. Next, the filter is used for DNA hybridization, as described in Example 5. Hybridization is carried out with the radioactive preparation pBR322, which is obtained as described in Example 4.

 Example 7. Experimental laboratory verification of the method for detecting genes for catabolism of the herbicide 2,4-D in the genomes of eukaryotes.

 Laboratory verification of the proposed method was carried out using DNA samples of various eukaryotes. Genomic DNA preparations of maize Zea mays (Vir 44) (see drawing, item 4), wheat Triticum aestivum (Moscow variety 35) (item 2), and pea Pisum sativum (variety Chishminsky) (item 3 were included in the study). ), Aspergillus niger van Zeghem fungus (item 5), Galliformes birds (domestic chicken breed) (item 1). The research results are presented in the drawing and in the table.

 From the drawing and table it can be seen that positive hybridization with the radioactive plasmid pMK16, indicating the presence of 2,4-D catabolism genes, has all the studied eukaryotic genomic DNA preparations. Hybridization bands are observed in the region of Bam HI fragments 4.4-4.5 kbp long. Less intense hybridization bands are found with Bam HI fragments whose length exceeds 10 kb. In addition, a hybridizing Bam HI fragment of less than 4.4 kbp was found in Aspergillus niger. The table also shows the results of control experiments on the hybridization of the radioactive plasmid pBR322 with the genomic DNA of eukaryotes. The table shows that the molecules of the vector pBR322 do not hybridize with the studied eukaryotic preparations and, therefore, cannot affect the resolving capabilities of the proposed method.

 Thus, it is shown that the inventive method allows to detect genes for catabolism in DNA preparations of eukaryotic genomes.

 This method is proposed for the first time.

 The method is applicable for analysis of the genomes of various representatives of eukaryotes.

 The method is applicable for the simultaneous analysis of a significant number of DNA samples.

 The method is applicable for molecular mapping of eukaryotic genomes.

 Thus, the proposed method is new, has no significant restrictions on the application, expands the possibilities of molecular mapping of eukaryotic genomes.

Sources of information
1. Annual review of genetics, v. 14, 1980, p. 145-178.

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

 3. Genetic engineering of plants. Ed. J. Dreiper, R. Scott, F. Armitage, R. Walden, Moscow, Mir, 1991, pp. 232-252.

 4. Chemistry and biochemistry of nucleic acids. Ed. I. B. Zbarsky, S. S. Debov, Leningrad, Medicine, 1968, pp. 107-115.

 5. Methods of general bacteriology. Ed. F. Gerhard, Moscow, Mir, 1984, pp. 112-118.

 6. RF patent 2064501, 02.08.91. T.V. Markusheva, V. S. Nikitina, I. V. Kusova, M. N. Sultanbekova, R. N. Churaev, E. Yu. Zhurenko, E. G. Katkova, R. S. Shakirova "Bacterial strain Escherichia coli biodegradation of 2,4-dichlorophenoxyacetic acid, recombinant plasmid DNA pMK16 containing the biodegradation genes of 2,4-dichlorophenoxyacetic acid, and a method for constructing recombinant plasmid DNA pMK16 containing 2,4-dichloroacetic acid biodegradation genes.

 7. Molecular cloning. T. Maniatis, E. Fritsch, J. Sambrook. Moscow, Mir, 1984, pp. 332-337, 344-350.

Claims (1)

 A method for detecting 2,4-D herbicide catabolism genes in eukaryotic genomes, which consists in hybridizing the DNA of test samples with a radioactive DNA preparation of plasmid pMK16 on a solid support.

Images