JP2697783B2 - Primers for gene amplification of cordyceps and amplification method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a primer for amplifying 18S ribosomal RNA gene of Cordyceps sinensis and an amplification method.

[0002]

BACKGROUND OF THE INVENTION Cordyceps is a general term for fungi that parasitize insects and form fruiting bodies, and are reported to be classified into about 400 species according to morphological characteristics such as color and shape. Among them, there are bacterial species that are the raw materials of herbal medicines, and it is presumed that they still contain many unknown bacterial species that produce pharmacologically active substances. Cordyceps is rarely found in nature, and immature strains are often unclear in color or shape. Therefore, in order to select a bacterial species that produces a pharmacologically active substance from nature, it is necessary to specify the bacterial species by another approach. In recent years, the classification of fungi has changed from the conventional classification based on morphological characteristics, such as color and shape, which are susceptible to the environment and the like, to the classification based on ribosomal RNA genes, which are less susceptible to external factors.
There is a gene region called a fingerprint region on the ribosomal RNA gene that can distinguish individual species, and the nucleotide sequence of this gene is compared to identify the bacterial species. In this case, the base sequence of the gene cannot be analyzed and compared unless the gene is amplified to increase the amount. Therefore, in order to amplify the target region, a pair of synthetic DNA primers sandwiching the region is required. Since the primer is designed to be complementary to the gene, the amplification efficiency is significantly reduced when the strain is different. Primers have been developed to amplify the ribosomal RNA gene of yeast among fungi, but appropriate primers have not been developed for Cordyceps, so we attempted to amplify the gene using primers from other fungi (such as Matsutake). Was not successful. Thus, a primer that enables amplification of the ribosomal RNA gene of Cordyceps is desired.

[0003]

An object of the present invention is to provide a primer and an amplification method capable of efficiently amplifying a cordyceps 18S ribosomal RNA gene.

[0004]

Means for Solving the Problems In order to solve the above problems, the present inventors have repeatedly studied the design of a synthetic DNA primer capable of efficiently amplifying the 18S ribosomal RNA gene of Cordyceps sinensis, and found that a 25mer And a 20mer long pair of 18S ribosomes from Cordyceps
The inventors have found that the RNA gene can be efficiently amplified, and have completed the present invention.

That is, the present invention is a primer for amplifying Cordyceps sinensis gene comprising oligonucleotides having the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The present invention also provides a method for amplifying Cordyceps sinensis, which comprises using, as primers, oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 1 and 2. Hereinafter, the present invention will be described in detail.

[0006] The primer for amplifying a cordyceps gene of the present invention has the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2. Here, the primers are four different nucleotide triphosphates and DNA in an appropriate buffer solution.
An oligonucleotide that, when annealed to a DNA template at the appropriate temperature in the presence of a polymerase, acts as a starting point for DNA synthesis.

[0007] The nucleotide sequence having a length of 25mer shown in SEQ ID NO: 1 is an 18S ribosome R of Sclerotinia sclerotiorum, which belongs to the aspergillus fungus, Kinkakukinidae.
This sequence is homologous to positions 183 to 207 of the nucleotide sequence of the NA gene. On the other hand, the nucleotide sequence having a length of 20 mer shown in SEQ ID NO: 2 is composed of the ascomycetous fox swan (Sclerotinia scle).
rotiorum) 20 nucleotides of the 18S ribosomal RNA gene
This sequence is complementary to positions 88 to 2107. Cordyceps belongs to the ascomycetes fungus Bakakikinidae, but the genetic similarity between Bakakikinidae and Kakakikinidae is not clear.

(1) Selection of Primers Primers for amplifying a cordyceps cordyceps gene are Sclerotinia sclero belonging to the ascomycetes Kinukakukinidae.
tiorum) 18S ribosomal RNA gene nucleotide sequence (Syst
em. Appl. Microbiol., Vol. 16, 436-444 (1993)), and synthesized several types of oligonucleotides with a length of 20 to 25 mer to determine whether the gene of Cordyceps can actually be amplified. Select by confirming. Selection can be, for example, Cordyceps sinensi.
s) The DNA extracted from the cells was actually amplified by the PCR method using the above several types of oligonucleotides as primers, the amplified DNA was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and then transilluminated. And the intensity is selected as an index.

(2) Synthesis of primer A primer is synthesized based on the nucleotide sequence of the oligonucleotide selected in (1). The primer can be synthesized by, for example, an automatic synthesis in a DNA synthesizer using a phosphate triester method, a phosphodiester method, or a cyanoethylphosphoramidite method.

(3) Gene Amplification A DNA sample to be subjected to amplification is obtained by growing a target Cordyceps fungus body on an agar medium such as Sabouraud dextrose agar medium and then growing on a liquid medium such as Sabouraud dextrose broth. Take out and freeze-dry, mortar,
Alternatively, pulverize using a homogenizer, and
Can be obtained by extracting DNA extraction is performed by SDS (sodium dodecyl sulfate), Sarkosyl
Attached to DNA with an anionic surfactant such as (N-dodecylsarcosinate), a cationic surfactant such as cetyltrimethylammonium bromide (CTAB), or a nonionic surfactant such as Triton X-100 The micelles are formed by forming micelles with existing proteins and lipids, solubilizing the micelles, releasing the DNA, further denaturing the protein using phenol or chloroform as an organic solvent, and releasing the DNA from the DNA. Thereafter, ethanol precipitation or dialysis against Tris-HCl / EDTA is performed, and finally ribonuclease treatment is performed.

The obtained DNA sample is subjected to DNA amplification by PCR using the synthetic primers prepared in the above (2). DNA amplification by PCR is described, for example, in Science,
vol.239, 487 (1988), and the heat denaturation step of the DNA, the step of annealing the primer to the DNA to be amplified, and the step of extending the annealed primer with the DNA polymerase are performed. Including.

First, dNTPs and a heat-resistant DNA polymerase such as Taq DNA polymerase and Tth DNA polymerase are added to an Eppendorf tube containing a DNA sample, and the tube is heated at 94 ° C. for 5 minutes to thermally denature the DNA. Amplification is performed by repeating the following thermocycles after making the strands. The style of the thermocycle is
For example, 30 seconds at 94 ° C; slope within 1 ° C / 2 seconds to 55 ° C; 55
30 seconds at 72 ° C .; 1 ° C./1 second ramp to 72 ° C .; 90 seconds at 72 ° C. constitutes one cycle, and this cycle is repeated 30 cycles. afterwards,
For example, an extension reaction is performed at 72 ° C. for 10 minutes, and the mixture is cooled to 4 ° C. The above PCR amplification is performed by thermal cycler 2400 (PerkinElmer) which can automatically adjust the temperature change,
What is necessary is just to use commercially available apparatuses, such as a program balance control system PC-700 (Astec).

The obtained amplification product is analyzed by agarose gel electrophoresis and stained with ethidium bromide to estimate its molecular weight.

[0014]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[Example 1] Preparation of primer for amplifying Cordyceps sinensis gene (1) Selection of primer Since Cordyceps sinensis belongs to the ascomycetes Bakakukinidae, a region having high commonality to genes of other ascomycetes exists. It is expected that. In the present invention, based on the above findings, the base sequence of the 18S ribosomal RNA gene of Sclerotinia sclerotiorum belonging to the ascomycete Kinukakukinidae as a cordyceps primer (System. Appl. Microbiol., Vol. 16,
436-444 (1993)), several primers were synthesized based on the sequence, and primers capable of actually amplifying a cordyceps gene were selected. Specifically, 20-25
Cordyceps sinensis DNA using several primers of mer length
Was amplified by the PCR method. After amplification, 1% agarose gel electrophoresis and DN amplified by ethidium bromide staining
A was detected. The amount of amplified DNA was estimated based on the density of the detected DNA band, and a combination of primers having the largest amount of amplified DNA was selected.

(2) Synthesis of primers The synthesis of the primers selected in (1) was performed using an automatic DNA synthesizer (Applied Biosystems). Combine
The DNA sequence was programmed and synthesis was initiated by the cyanoamidite method. After completion of the synthesis, 24% ammonia water was added to cut out the synthesized DNA from the column. Next, the synthetic DNA was placed in a vial, sealed, and heated at 55 ° C. for about 8 hours. Thereafter, the temperature was returned to room temperature and dried by an evaporator. D to dryness
NA was dissolved by adding purified water, and ethanol was precipitated. An appropriate amount of purified water was added to dissolve the precipitate, and the concentration of the synthesized DNA was determined from the absorbance at 260 nm. 2 synthetic DNA primers
The concentration was adjusted to 000 pmol / μl.

Example 2 18S Ribosome of Cordyceps
Propagation of RNA Gene Using the primers prepared in Example 1, Cordyceps milialis, a Cordyceps militalis, an American Type Culture Collection (ATCC) -preserved strain, was prepared.
talis) 18S ribosomal RNA gene was amplified. After growing the cells on a Sabouraud agar medium, growing on a liquid medium, removing only the cells, freeze-drying, pulverizing in a mortar, extracting the DNA by the CTAB method,
I got 10ug. Next, DNA was amplified by the PCR method.
The above DNA was used in an amount of 0.2 ug, and the reaction solution was 25 ul (x10 T
aq buffer 2.5ul, 25mM MgCl ₂ 2.5ul, 2mM dNTPs, Taq
polymerase 0.05unit)
l was added. First, the PCR reaction conditions were 94 ° C, 5 min,
Next, 30 cycles of (94 ° C., 30 s; 55 ° C., 30 s; 70 ° C., 1.5 min) were carried out, and finally, the reaction was carried out at 72 ° C. for 10 min using a thermal cycler 2400 (Perkin Elmer).
After the reaction, 1 ul of the reaction solution was subjected to agarose gel electrophoresis,
DNA amplification was confirmed.

Example 3 18S Ribosome of Cordyceps
Proliferation of RNA Gene Using the primers prepared in Example 1, 18S ribosomal RNA genes of three kinds of cordyceps A, B, and C collected from nature (Ibaraki Prefecture) were propagated. The amount of cordyceps used for DNA extraction was A, 10.1 mg; B, 6 mg; C, 8.8 mg. Perform DNA extraction using CTAB method, A, 0.5ug (20ul);
B, 0.2ug (20ul); C, 0.1ug (20ul) DNA was obtained. Next, DNA was amplified by the PCR method. The above DNA used was 2 ul, and the reaction solution was 25 ul (x10 Taq buffer).
2.5ul, 25mM MgCl ₂ 2.5ul, 2mM dNTPs, Taqpolymerase
The above primers were added in an amount of 200 pmol as 0.05 unit). The PCR reaction conditions were as follows: 94 ° C, 5 min, then (94
℃, 30s; 55 ℃, 30s; 72 ℃, 1.5min) for 30 cycles,
Lastly, set to 72 ° C for 10 minutes, thermal cycler 2400
(Perkin Elmer). After the reaction,
1 ul of the reaction solution was subjected to agarose gel electrophoresis to confirm DNA amplification in all samples (FIG. 1).

Example 4 18S Ribosome of Cordyceps
Propagation of RNA gene Using the primers prepared in Example 1,
The 18S ribosomal RNA gene of the culture A 'obtained by spore culture of A was amplified. DNA extraction was performed by the CTAB method.
From 0 mg to 10 ug (50 ul) of DNA was obtained. Next, DNA was amplified by the PCR method. Using 1 ul of the above DNA, the reaction mixture was 25 ul in total (2.5 ul of x10 Taq buffer, 25 mM MgCl ₂ 2.
5ul, 2mM dNTPs, Taq polymerase 0.05unit), and added 200pmol of each primer. The PCR reaction conditions were as follows: 94 ° C, 5 min, then (94 ° C, 30s; 55 ° C, 30s; 72 ° C,
(1.5 min) for 30 cycles, and finally at 72 ° C. for 10 min, and the reaction was carried out using a thermal cycler 2400 (Perkin Elmer). After the reaction, 1 ul of the reaction solution was subjected to agarose gel electrophoresis to confirm DNA amplification (FIG. 1).

Example 5 18S Ribosome of Cordyceps
Propagation of RNA gene Using the primers prepared in Example 1, 18S ribosome of Cordyceps sinensis, Cordyceps sinensis from Sichuan, China, which is commercially available as a Chinese medicine
Propagation of the RNA gene was performed. DNA extraction was performed by the CTAB method, and 780 ug of DNA was obtained from 200 mg of the sample. By PCR method
DNA amplification was performed. Using 0.2 ug of the above DNA, the reaction solution was 25 ul in total (2.5 ul of 10 Taq buffer, 2.5 uM of 25 mM MgCl ₂
l, 2 mM dNTPs, and Taq polymerase 0.05 unit) were added at 200 pmol each. The PCR reaction conditions were as follows: 94 ° C, 5 min, then (94 ° C, 30s; 55 ° C, 30s; 72
℃, 1.5min) for 30 cycles and finally 72 ℃, 10min.
The reaction was performed using Thermal Cyclic 2400 (PerkinElmer). After the reaction, 1 ul of the reaction solution was subjected to agarose gel electrophoresis to confirm DNA amplification.

[0021]

Industrial Applicability According to the present invention, the 18S ribosomal RNA gene of Cordyceps can be amplified, so that the nucleotide sequence of the gene can be quickly determined and analyzed. As a result, the species of Cordyceps fungi that produces the pharmacologically active substance can be identified from the genetic characteristics, and the search for bacteria that produce a new physiologically active substance can be made much more efficient.

[0022]

[Sequence list] SEQ ID NO: 1 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CGACTTCGGA AGGGGTGTAT TTAAT

SEQ ID NO: 2 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TAATGATCCT TCCGCAGGTT

[Brief description of the drawings]

FIG. 1 shows a photograph of agarose gel electrophoresis of a 1.6-1.9 kb cordyceps 18S ribosomal RNA gene amplified by the method of the present invention. M: molecular weight marker Lane 1: Cordyceps A spore culture A ′ Lane 2: Cordyceps A Lane 3: Cordyceps B Lane 4: Cordyceps C

Claims (3)

(57) [Claims] 1. A primer for amplifying Cordyceps sinensis comprising oligonucleotides having the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. 2. A method for amplifying Cordyceps sinensis, which comprises using, as primers, oligonucleotides having the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. 3. A Cordyceps 18S ribosomal RNA gene having a molecular weight of 1.6 to 1.9 kb, amplified by the method of claim 2.