CN109666761B - Specific DNA fragment for identifying thelephora ganbajun zang, amplification primer, preparation method and application thereof - Google Patents
Specific DNA fragment for identifying thelephora ganbajun zang, amplification primer, preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a specific DNA fragment for identifying thelephora ganbajun zang, an amplification primer, a preparation method and application thereof, belonging to the technical field of biotechnology and enzyme engineering. The nucleotide sequence of the specific DNA fragment for identifying thelephora ganbajun is shown as SEQ ID No.1 in a sequence table. The primers for amplifying the specific DNA fragment identified by the Thelephora ganbajun comprise 158F primers and 437R primers. The application of the specific DNA fragment for identifying thelephora ganbajun zang, the amplification primer or the specific DNA fragment of thelephora ganbajun zang obtained by the preparation method in the specific identification of thelephora ganbajun zang has the characteristic of quickly and accurately completing the identification of thelephora ganbajun zang.
Description
Technical Field
The invention belongs to the technical field of strain molecular identification, and particularly relates to a specific DNA fragment and an amplification primer for identifying thelephora ganbajun, and a preparation method and application thereof.
Background
The wild edible fungi are important forest resources, play an important role in the industries of green food development, biological pharmacy, environmental protection and the like, and are particularly suitable for some wild edible fungi growing in forest regions. Thelephora (Thelephora) and Thelephora (Thelephora) belong to Thelephora Basidiomycota, are grown in Pinus sylvestris and Pinus sylvestris of Yunnan at an altitude of 600-2300 m and are ectomycorrhizal fungi capable of symbiotic with various Pinus sylvestris plants. The Thelephora ganbajun zang is a relatively distinctive wild edible fungus in Yunnan province, has unique taste, has the fragrance similar to dried beef, is rich in nutrition, and has high nutritional value and economic value. The novel compound separated from Thelephora ganbajun zang has been reported to have higher antioxidant and anticancer activity, and the compound is proved to have potential medicinal value.
At present, people mainly determine the classification of wild edible fungi and the identification of mycelium isolates thereof according to the morphological, anatomical and other characteristics of the fruiting bodies of the wild edible fungi. When the wild edible fungus does not produce fruit bodies or the fruit bodies are not found, the identification of the wild edible fungus strain can not be carried out. Even if pure strains are separated from fruit bodies of wild edible fungi, in order to avoid misleading caused by contamination of associated fungi and mixed fungi, a back grafting test should be carried out by following koch's law, and an identification method for producing the same fruit bodies is convincing. Unfortunately, many precious wild edible fungi cannot be artificially cultivated for a while, and in such a case, it is difficult to determine whether the isolated strain is true or false. Thelephora ganbajun can be identified by the appearance zone of the mature fruit body, but not by hyphae. DNA as genetic material can objectively and truly reflect the genetic relationship between species, and is a powerful basis and supplement for species identification. The DNA molecule based specific PCR technology is one efficient, fast and reliable method for identifying separately cultured strain and is widely used in the truth identification of fungus separating culture.
Disclosure of Invention
In view of the above, the present invention aims to provide a specific DNA fragment for identifying thelephora ganbajun, an amplification primer, a preparation method and an application thereof, wherein the specific DNA fragment is firstly provided and has a characteristic of high identification accuracy.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a specific DNA fragment for identifying thelephora ganbajun zang, wherein the nucleotide sequence of the specific DNA fragment for identifying thelephora ganbajun zang is shown as SEQ ID No.1 in a sequence table.
The invention provides a primer for amplifying a specific DNA fragment for identifying thelephora ganbajun, which comprises a 158F primer and a 437R primer; the nucleotide sequence of the 158F primer is shown as SEQ ID No.2 in a sequence table; the 437R primer has a nucleotide sequence shown as SEQ ID No.3 in the sequence table.
The invention provides a preparation method of a specific DNA fragment for identifying thelephora ganbajun zang, which comprises the following steps:
(1) extracting genome DNA of thelephora ganbajun;
(2) performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and using the primer to obtain an amplification product;
(3) and (3) cutting and recovering the amplification product in the step (2) to obtain a DNA fragment of the specific DNA fragment for identifying thelephora ganbajun.
Preferably, the PCR amplification system is PCR amplification MIX liquid 22.5. mu.l, 158F 1. mu.l, 437R 1. mu.l and DNA 10. mu.l.
Preferably, the procedure of PCR amplification: pre-denaturation at 98 ℃ for 2 min; then denaturation at 98 ℃ for 20s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; finally, extension is carried out for 5min at 72 ℃.
The invention provides the specific DNA fragment for identifying thelephora ganbajun, the primer and the application of the specific DNA fragment for identifying thelephora ganbajun, which is prepared by the preparation method, in identifying thelephora ganbajun.
The specific DNA fragment for identifying the Thelephora ganbajun zang provided by the invention is firstly proposed as a molecular label for identification, and the identification accuracy rate of the Thelephora ganbajun zang is up to 100%. Meanwhile, the specific DNA fragment provided by the invention also has higher detection specificity.
Drawings
FIG. 1 is a pie chart of metagenomic analysis results of a soil sample from Thelephora ganbajun growing;
FIG. 2 is an electrophoretogram of PCR products of a soil sample of Thelephora ganbajun, wherein y is a negative control; a is a soil sample g1 for Thelephora ganbajun zang; b is a Thelephora ganbajun zang sample g 2; c is a Thelephora ganbajun zang sample g 3; m is 2000 marker.
Detailed Description
The invention provides a specific DNA fragment for identifying thelephora ganbajun zang, wherein the nucleotide sequence of the specific DNA fragment is shown as SEQ ID No.1 in a sequence table. The length of the specific DNA fragment is 280 bp. The gene is derived from Thelephora ganbajun zang.
The invention provides a primer for amplifying a specific DNA fragment for identifying thelephora ganbajun, which comprises a 158F primer and a 437R primer; the nucleotide sequence of the 158F primer is shown as SEQ ID No.2 in a sequence table; the 437R primer has a nucleotide sequence shown as SEQ ID No.3 in the sequence table. The source of the primer is not particularly limited in the present invention, and a primer synthesis company known to those skilled in the art may be used. In the embodiment of the present invention, the primer is synthesized by Kunming ShuoZhi Biotech, Inc. The method for designing the primer is not particularly limited, and a primer design method known in the art may be used.
The invention provides a preparation method of the specific DNA fragment, which comprises the following steps:
(1) extracting genome DNA of thelephora ganbajun;
(2) performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and using the primer to obtain an amplification product;
(3) and (3) cutting and recovering the amplification product in the step (2) to obtain a DNA fragment of the specific DNA fragment for identifying thelephora ganbajun.
The invention is toThe method of extraction is not particularly limited, and may be carried out by hand or using a kit known in the art. In the embodiment of the invention, the extraction method preferably adopts the method of Qiagen
DNA Isolation Kit (cat No.: 12888-50) DNA extraction was carried out according to the procedure of the specification.
And (4) carrying out nucleic acid quantitative detection on the extracted genome DNA. When the concentration of genomic DNA is high, the DNA is diluted with a buffer. The concentration of the genomic DNA is preferably 25 ng/. mu.l at the time of PCR amplification.
The method of PCR amplification according to the present invention is not particularly limited, and a PCR amplification method known to those skilled in the art may be used. The PCR amplification instrument is not particularly limited, and a PCR instrument of a type commonly used by those skilled in the art can be used. The PCR amplification system is preferably PCR amplification MIX liquid 22.5. mu.l, 158F 1. mu.l, 437R 1. mu.l and DNA 10. mu.l. The procedure for the PCR amplification is preferably as follows: pre-denaturation at 98 ℃ for 2 min; then denaturation at 98 ℃ for 20s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; finally, extension is carried out for 5min at 72 ℃.
The invention provides the specific DNA fragment for identifying thelephora ganbajun zang, the primer or the specific DNA fragment for identifying thelephora ganbajun zang prepared by the preparation method.
The method for identifying thelephora ganbajun is not particularly limited in the present invention, and a PCR amplification method well known in the art may be used. If the target fragment appears in the sample to be detected, the sample contains thelephora ganbajun.
The specific DNA fragment for identifying Thelephora ganbajun zang, the amplification primer, the preparation method and the application thereof provided by the invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the invention.
Example 1
1. Material
Samples of soil for Thelephora ganbajun were collected in Wudingcounty, Yunnan province (see Table 1). The acquisition method comprises the following steps: in each Ganba strain growing place, soil with a radius of 5cm and a depth of 14cm with a fruiting body as a center is taken by a 5-point method, the soil is put into prepared sterile sampling bags and numbered, and soil samples are stored at-80 ℃ (the soil samples for growing the Ganba strains are mixed samples of the fungi containing the strain ponds).
TABLE 1 sample Collection information
2. Method of producing a composite material
2.1 DNA extraction
DNA extraction Using Qiagen
DNA isolation kit (cat No.: 12888-50) was used for DNA extraction. Taking a soil sample (about 300mg of a soil sample collected in the field) of thejordneria ganbajun growing listed in the table 1, transferring the soil sample into a PowerBead T mu bes rendered grinding tube (adding 60 mu l C1 solution before adding the soil sample), and gently mixing the soil sample and the solution; incubating at 70 ℃ for 5min, vortexing for 3-4 s, then incubating at 70 ℃ for 5min, vortexing for 3-4 s, and finally incubating at 70 ℃ for 5 min; grinding for 10min at a speed of 4 with a Tiss μ e PreP grinding instrument, and centrifuging for 30s at 1000 g; adding the supernatant into 250 μ l of C2 to 2ml centrifuge tube, mixing for 5s, incubating at 4 deg.C for 5min, and centrifuging at 1000g for 1 min; mixing 600 μ l of supernatant with 200 μ l C3 to 2ml of centrifuge tube, incubating at 4 deg.C for 5min, and centrifuging at 1000g for 1 min; adding 1200 mu l of C4 and 750 mu l of supernatant into a new 2ml centrifuge tube, mixing uniformly, taking 675 mu l of the mixed solution into MB Spin Col mu mn each time, centrifuging for 30s at 1000g, pouring out the liquid in the centrifuge tube, repeating for 2 times until all the mixed solution is transferred into the MB Spin Col mu mn, and pouring out all the liquid in the centrifuge tube; adding 500 μ l of C5, centrifuging for 30s at 1000g, pouring off the liquid in the centrifuge tube, and centrifuging for 1min at 1000 g; placing MB Spin Col μmn into a new 2ml centrifuge tube, adding 100 μ l C6 to a white filter membrane, centrifuging 1000g for 30s, collecting the liquid, and standing at-20 deg.CAnd (the obtained liquid is soil fungus DNA mixed liquid) for standby.
2.2 primer Synthesis
Thelephora genome and transcriptome sequences (https:// www.ncbi.nlm.nih.gov). Primers were designed for the specific gene fragment of Thelephora ganbajun by Primer3(http:// Primer3.ut. ee /), the designed primers are shown in Table 2, and the primers in Table 2 were synthesized by Kunming Shuoji Biotech Co., Ltd, to obtain primers for PCR amplification.
TABLE 2 primer information List
| Name of Gene | Primer name | Primer sequences |
| e_gw1.16.180.1 | 158f | 5’-gagatcagacacgccttcac-3’(SEQ ID No.2) |
| e_gw1.16.180.1 | 437r | 5’-tcgaccaacagcaggactag-3’(SEQ ID No.3) |
2.3 analysis results of metagenome of soil fungi
Soil samples with the same quantity as g1, g2 and g3 of the thelephora ganbajun growing soil samples are respectively taken and evenly mixed, and the mixture is sent to a company for library construction, sequencing and analysis according to the requirements of an Illumina Miseq sequencing platform. Soil samples were determined to be 190 fungal genera, with the most abundant of 190 genera being Russula (Russula), accounting for 18.39%, followed by Mortierella (Mortierella), accounting for 17.83%, and specifically shown in fig. 1 (genera less than 1% abundant are classified as others).
2.4 verification of primer specificity
Respectively extracting 3 thelephora ganbajun growth soil samples of g1, g2 and g3 to obtain 3 parts of mixed fungus DNA samples as templates, and carrying out PCR reaction by using primers 158F and 437R, wherein the amplification reaction system is as follows: gold medal MIX (available from Kunming Shuichi Biotech Co., Ltd.) 22.5. mu.l, 158F 1. mu.l, 437R 1. mu.l, DNA 10. mu.l. The amplification conditions were: pre-denaturation at 98 ℃ for 2 min; then denaturation at 98 ℃ for 20s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; finally, extension is carried out for 5min at 72 ℃. Mu.l of the PCR product was run on a 2% agarose gel, GeneFinderTMDyeing and detecting by an ultraviolet transilluminator, wherein the result is shown in figure 2, and y is a negative control; a is thelephora ganbajun sample g 1; b is thelephora ganbajun sample g 2; c is Thelephora ganbajun sample g 3; m is 2000 marker. The 280bp fragment was amplified, and the amplified band was a single band. And (3) sending the PCR amplification product to a company (Kunming Shungzhi biotechnology limited) for sequencing, obtaining a good peak pattern without any miscellaneous peak after sequencing, and obtaining a specific DNA fragment sequence (SEQ ID No.1) for identifying thelephora ganbajun after splicing.
The mixed fungus DNA contains 190 fungi in the soil fungus metagenome, the primer 158F and 437R are used for amplifying a band as a single band, the pair of primers are inquired in https:// www.ncbi.nlm.nih.gov', and no primer with the same sequence as the pair of primers is available. The primer is explained above as a primer for specifically amplifying thelephora ganbajun zang.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university of Yunnan
<120> specific DNA fragment for identifying thelephora ganbajun zang, amplification primer, preparation method and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 280
<212> DNA
<213> Thelephora ganbajun Zang
<400> 1
tcgaccaaca gcaggactag gtcagcgacc ttcccaatgt caatcataga gttgaggtcg 60
ttgttgcact ctacgaacgt gagtctccgc cgcttcccgc tgacaacggt gataggcccc 120
tggacgtggt tcaaggtctg cttcgtggat cttcgaacga ggcttttgag gagtgttgtt 180
ttccccactc caggtgggcc gacaatggcc acgatgacag gaggagggtc ctcgtccggt 240
gtacgattga caagaggtac gtgaaggcgt gtctgatctc 280
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gagatcagac acgccttcac 20
<210> 3
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tcgaccaaca gcaggactag 20
Claims (4)
1. A specific DNA fragment for identifying thelephora ganbajun zang is characterized in that the nucleotide sequence of the specific DNA fragment is shown as SEQ ID No.1 in a sequence table.
2. A primer for amplifying the specific DNA fragment of claim 1, comprising a 158F primer and a 437R primer; the nucleotide sequence of the 158F primer is shown as SEQ ID No.2 in a sequence table; the 437R primer has a nucleotide sequence shown as SEQ ID No.3 in the sequence table.
3. A method for preparing a specific DNA fragment for Thelephora ganbajun identification according to claim 1, comprising the steps of:
(1) extracting genome DNA of thelephora ganbajun;
(2) performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and using the primer of claim 2 to obtain an amplification product; the PCR amplification system comprises 22.5 mul of PCR amplification MIX liquid, 1 mul of 158F, 1 mul of 437R and 10 mul of DNA; procedure for the PCR amplification: pre-denaturation at 98 ℃ for 2 min; then denaturation at 98 ℃ for 20s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; final extension at 72 deg.C for 5min
(3) And (3) cutting and recovering the amplification product in the step (2) to obtain a specific DNA fragment with the length of 280bp for identifying thelephora ganbajun.
4. Use of the specific DNA fragment for identifying Thelephora ganbajun according to claim 1, the primer according to claim 2, and the specific DNA fragment prepared by the preparation method according to claim 3 for identifying Thelephora ganbajun.
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