KR102382106B1 - A set of specific molecular markers for differentiating between lychee and red lye species, and their methods and applications - Google Patents

Abstract

The present invention discloses a set of molecular marks for differentiating between rhododendron and erythrocyte species, and the nucleotide sequences of the upstream primers GLMI001-F, GLMI002-F and GLMI003-F are SEQ ID No. 1, SEQ ID No. 3, It is represented by SEQ ID No.5, and the downstream primers GLMI001-R, GLMI002-R and GLMI003-R nucleotide sequences are represented by SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, respectively. These specific molecular markers GLMI001, GLMI002, and GLMI003 are used for the identification and detection of Zinnia and Red Ginger strains, and can ensure the safety of Ganoderma medicinal products by standardizing the market for Ganoderma and mixing fakes. In addition, it can be used in the detection of reishi products to ensure the purity of reishi germplasm and improve production efficiency, so it has high application potential and economic value.

Description

A set of specific molecular markers for differentiating between lychee and red lye species, and their methods and applications

The present invention relates to the field of molecular identification of Ganoderma species, and more particularly, to a set of specific molecular markers for differentiating the species of Ganoderma and Red reishi, and the method and application of the differentiation.

Reishi belongs taxonomically to Fungi, Basidiomycetes, Agaricomycetes, Polyformoc, Gnosaceae, and Reishi, and is widely distributed in tropical and subtropical regions. China is rich in reishi germplasm resources, and is located in Jilin, Hebei, Shaanxi, Henan, Zhejiang, Yunnan, Guizhou, Guangdong and It is widely distributed in provinces such as Guangxi. Lingzhi is a traditional Chinese herbal medicine known as one of “Gudaeseoncho” (九大仙草). Ganoderma Ganoderma (Pharmaceuticals of the People's Republic of China (2015)) describes that the dried fruit of Ganoderma lucidum or Ganoderma sinense is used as a medicine, and its chemical composition is complex, mainly Contains triterpenoids and polysaccharides, amino acid polypeptides, nucleotides, furans, alkaloids, oils and fats and trace elements thereof, and includes immune regulation, cardiovascular disease prevention, It has various physiological and pharmacological activities such as antitumor, antiviral, hypoglycemic and antioxidant. However, Jeokyeongji has a mild temperament and Jayeongji has a soft nature. Red reishi is suitable for use as a medicine, and red reishi is suitable for daily health care and body care. In addition, private land is more expensive than red territory.

As people's living standards improve, the demand for high-quality manor is increasing. Accordingly, more and more manor is cultivated by mimicking the wild environment, which promotes the cultivation, storage and production of excellent varieties of manor. Currently, some companies do not produce and sell according to the <Edible fungus species management method> in order to take profit, and confusion in the classification of reishi due to long-term cultivation and hybrid breeding of reishi has caused confusion in the classification of reishi. impeding development. The two main species of reishi industry, jasmine and red reishi, are easily distinguished by appearance at the fruiting stage, but they are almost identical at the fungal stage, so it is necessary to distinguish them using molecular means to ensure the healthy and orderly development of the ganoderma industry.

In consideration of the problems of the prior art, it is an object of the present invention to standardize the culture, preservation and production of Ganoderma spp. .

The present invention is implemented through the following technical solutions.

In a set of specific molecular markers for differentiating between lyophilis and red ginseng species, primers of the specific molecular markers are GLMI001, GLMI002, and GLMI003, and the nucleotide sequences of each primer are as follows.

upstream primer GLMI001-F: 5'-ACCCGGTGAAACAACA-3', represented by SEQ.ID No.1;

Downstream primer GLMI001-R: 5'-GCAGTGAGTTTGGCTTAT-3', represented by SEQ.ID No.2;

upstream primer GLMI002-F: 5'-CGGACAGCGAACAGACG-3', represented by SEQ.ID No.3;

Downstream primer GLMI002-R: 5'-GGCAGCAATGAGGACACC-3', represented by SEQ.ID No.4;

Upstream primer GLMI003-F: 5'-AGCGACGCCGTCAACTC-3', represented by SEQ.ID No.5;

Downstream primer GLMI003-R: 5'-AAGCAGCCACAGCCAACT-3', represented by SEQ.ID No.6.

The three pairs of specific molecular marker primers (GLMI001-F/R, GLMI002-F/R, and GLMI003-F/R) used SCoT random primers to perform general PCR amplification on the sample DNA of the S. After performing sequencing by performing electrophoresis on the amplified product, screening for a specific band of Myobacteriaceae, a comparative analysis is performed on the sequence of the specific band of S. G. spp. obtained by sequencing, and based on this After designing a specific primer with . Therefore, these three pairs of primers have very high specificity, and PCR amplification is performed on the sample DNA of the Yingji and Red Ginger strains using this, and only the Y. Yingji strains can obtain DNA bands of 187bp, 365bp, and 168bp. There is, and the species of Red Youngji can not be obtained. Nucleotide sequences of the specific fragments obtained by amplification of S. Gypsy strains are represented by SEQ.ID No.7, SEQ.ID No.8, and SEQ.ID No.9.

Another object of the present invention is to provide a method for rapidly identifying Xyrrhosis and Red Reishi strains using the above-described specific molecular marker primers (GLMI001-F/R, GLMI002-F/R and GLMI003-F/R). is in

In the present invention, the specific molecular marker primers (GLMI001-F/R, GLMI002-F/R, and GLMI003-F/R) are used as specific amplification primers, and the genomic DNA of the S. PCR amplification is performed, and electrophoretic detection is performed on the amplification product. If one DNA band of 187 bp, 365 bp, and 168 bp appears in the electrophoresis result, the test target sample is a yeast strain, and if the DNA band does not appear in the electrophoresis result, the test target sample is a red yeast strain.

In the above method, the steps of selection of amplification primers, extraction and determination of genomic DNA, and electrophoretic detection may all be performed according to conventional methods in the art.

The specific molecular marker primers and detection method of the present invention are suitable only for the differentiation of S. Youngji and Red Youngji strains, and the sample to be tested is limited within the range of Yingzhi and Red Ginger strains. By adopting the method of , it is possible to quickly identify the fungal species of jasmine and red lye, and since this method is simple, accurate, and quick, it cannot be implemented by other conventional identification methods.

The specific method is as follows.

(1) Take 0.3 to 0.5 g of Zinnia and Red Ginger strains, and extract the genomic DNA of the sample to be tested using a fungal genomic DNA extraction kit.

(2) General PCR amplification using the genomic DNA obtained in step (1) as a template and using specific molecular marker primers (GLMI001-F/R, GLMI002-F/R and GLMI003-F/R) as amplification primers carry out

PCR reaction system (total volume 30 μl) configuration

The PCR reaction procedure is as follows. i.e., pre-denaturing at 94° C. for 5 minutes; 30 cycles (denaturation at 94°C for 30 seconds, annealing temperature (54°C for GLMI001, 60°C for GLMI002, and 61°C for GLMI003) for 45 seconds, stretched at 72°C for 90 seconds); Finally, it is stretched at 72°C for 10 minutes.

(3) The PCR amplification product obtained in step (2) is detected using 1.5% agarose gel electrophoresis, and imaging is performed using a gel imaging system. If DNA bands of 187 bp (GLMI001), 365 bp (GLMI002), and 168 bp (GLMI003) appear in the electrophoresis result, the sample to be tested is S. If the DNA band does not appear in the electrophoresis result, the sample to be tested is a strain of Red Gyneungji.

The advantageous effects of the present invention are mainly realized as follows. That is, the specific molecular marker primers (GLMI001-F/R, GLMI002-F/R, and GLMI003-F/R) of the present invention can perform molecular analysis of the S. The method is simple, accurate, and highly flexible, and thus cannot be implemented by other conventional differentiation methods.

1 is an electrophoresis diagram after PCR amplification was performed for the strains of jayongji and jeokyeongji by adopting the specific molecular marker GLMI001-F/R of the present invention.
FIG. 2 is an electrophoresis diagram after PCR amplification was carried out on the strains of jayongji and jeokyeongji by adopting the specific molecular marker GLMI002-F/R of the present invention.
3 is an electrophoresis diagram after PCR amplification was performed for the strains of jayongji and jeokyeongji by adopting the specific molecular marker GLMI003-F/R of the present invention.
Here, channel M is DNA standard molecular weight Marker DL2000, and channels 1 to 9 are (1) Jilin Jeokyeongji 1, (2) Jilin Jeokyeongji 2, (3) Shandong Jeokyeongji 1, (4) Shandong Jeokyeongji 2, (5) Anhui Red Ying Zhuo 1, (6) Anhui Red Ying Zhuo 2, (7) Henan Red Ying Zhuo, (8) Storage Red Ying Zone 1, (9) Storage Red Ying Zone 2 (Xu Xian District) Channel 10 is jayongji (Shouxiangu fungus).

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings, and the object and effect of the present invention will be further clarified.

Example 1

1. Extraction of genomic DNA of the test target Jayongji and Redyoungji strains

Take 0.3 to 0.5 g of the mycelium of the Xyrrhosis and Red Youngji mycelium to be tested, and use the fungal genomic DNA extraction kit of Shanghai Biotechnology Co., Ltd. DNA was extracted, and the purity was detected by electrophoresis of the DNA obtained with 1.5% agarose gel, and the DNA concentration was detected using a NanoDropTM 1000 ultra-fine ultraviolet spectrophotometer from Thermo Fisher, USA, and then 50ng/μL Dilute and store in the refrigerator at 4 degrees.

2. Synthesis of specific molecular marker primers

Design a three-pair molecular marker primer based on a locust specific sequence, upstream primer GLMI001-F: 5'-ACCCGGTGAAACAACA -3', downstream primer GLMI001-R: 5'-GCAGTGAGTTTGGCTTAT-3', upstream primer GLMI002-F : 5'-CGGACAGCGAACAGACG-3' and downstream primer GLMI002-R: 5'-GGCAGCAATGAGGACACC-3', upstream primer GLMI003-F: 5'-AGCGACGCCGTCAACTC-3' and downstream primer GLMI003-R: 5'-AAGCAGCCACAGCCCAACT- 3' is synthesized by Shanghai Biotechnology Co., Ltd.

3. PCR amplification of specific molecular marker primers (GLMI001-F/R, GLMI002-F/R and GLMI003-F/R)

PCR amplification system (total volume 30 μL): 3 μL of 10×PCR Buffer (with Mg ²⁺ ), 0.9 μL of 10 mmol/L dNTPs, 0.6 μL of 20 μmol/L upstream primer, 0.6 μL of 20 μmol/L downstream primer, 0.6 μL of 5U/μLTaq enzyme µL, 1 µL of DNA template (50 ng/µL), 23.4 µL of ddH ₂ O.

PCR reaction procedure: pre-denaturing at 94°C for 5 minutes; 30 cycles (denaturation at 94°C for 30 seconds, annealing temperature (54°C for GLMI001, 60°C for GLMI002, and 61°C for GLMI003) for 45 seconds, stretched at 72°C for 90 seconds); Finally, it is stretched at 72°C for 10 minutes.

4. Electrophoretic detection

Step (3) 5 to 10 μL of the PCR amplification product was taken, and electrophoretic detection was performed using a 1.5% agarose gel, and the electrophoresis results are shown in FIGS. 1 to 3 .

According to the above method, Jayeongji and Redyoungji strains were detected using the specific molecular marker GLMI001, and the electrophoresis diagram is as shown in FIG. amplified; The rest of the numbers were jeokyeongji strains, and all 187bp DNA bands were not amplified.

According to the above method, Jayeongji and Redyoungji strains were detected using the specific molecular marker GLMI002, and the electrophoresis diagram is as shown in FIG. amplified; The rest of the numbers were of the species of Red Youngji, and the 365 bp DNA band was not amplified.

According to the above method, Jayeongji and Redyoungji strains were detected using the specific molecular marker GLMI003, and the electrophoresis diagram is as shown in FIG. amplified; The rest of the numbers were jeokyeongji strains, and all 168bp DNA bands were not amplified.

This means that the specific molecular markers GLMI001, GLMI002 and GLMI003 of the present invention have a fairly high specificity, and thus can be used for rapid identification of hyacinth and erythrae species.

SEQUENCE LISTING <110> HANGZHOU NORMAL UNIVERSITY ZHEJIANG SHOUXIANGU BOTANICAL DRUG INSTITUTE CO., LTD <120> SPECIFIC MOLECULAR MARKERS OF ONE SET AND DIFFERENTIATION METHODS AND APPLICATIONS FOR DISCRIMINATING BACTERIA SPECIES BETWEEN GANODERMA SINENSE AND GANODERMA LUCIDUM <160> 9 <170> SIPOSequenceListing 1.0 <210> 1 <211> 16 <212> DNA <213> primer <400> 1 acccggtgaa acaaca 16 <210> 2 <211> 18 <212> DNA <213> primer <400> 2 gcagtgagtt tggcttat 18 <210> 3 <211> 17 <212> DNA <213> primer <400> 3 cggacagcga acagacg 17 <210> 4 <211> 18 <212> DNA <213> primer <400> 4 ggcagcaatg aggacacc 18 <210> 5 <211> 17 <212> DNA <213> primer <400> 5 agcgacgccg tcaactc 17 <210> 6 <211> 18 <212> DNA <213> primer <400> 6 aagcagccac agccaact 18 <210> 7 <211> 187 <212> DNA <213> Ganoderma sinense <400> 7 gcagtgagtt tggcttatgc gcatgttgaa ctgaaattca cctacgccag ccgccttctt 60 cgaggacttc tcgttgacga atttttctct tcgtcggacg aggtgctttt ttccggcgac 120 aggttttgaa ggggatgggg gaggagggat tgacggcgta ccaactggca ttgttgtttc 180 accgggt 187 <210> 8 <211> 365 <212> DNA <213> Ganoderma sinense <400> 8 ggcagcaatg aggacaccct tggagaacga gtgccgggag acgatctccg actgcgggct 60 ctcgcccgtt aggtagcgga cgtcggcgcg gagctggggc acggagatgt tgctcacaat 120 agacgcaacg acgggatcaa acttgaggcc atgaaggatg tccttcacgc gctggacggc 180 ctcggatggc acggggacaa gcggaacggg agcgggaggg aggggcgatg ccttccagaa 240 gcggggcagg tggaggtcga gcgtgagtgc ggtagaagag tccacggaga tgagggcgga 300 ggtgggggtg cggaggagaa cctttgctgc gttttggttg ctgaacaccg tctgttcgct 360 gtccg 365 <210> 9 <211> 168 <212> DNA <213> Ganoderma sinense <400> 9 aagcagccac agccaactgc aacgcctaag aaggacgcgc agtctgatgc gaagacgact 60 gtaagtggaa ccactgccgt tgcatcctcc accgctacag caacctcgag cccgaagaag 120 agcaaacaac gaaaggccgg gaagaagtag agagttgacg gcgtcgct 168

Claims (5)

In a set of specific molecular marker compositions for differentiating between lychee and red ginseng species,
The primers of the specific molecular marker are GLMI001, GLMI002 and GLMI003, and the nucleotide sequence of each primer is,
The upstream primer GLMI001-F is represented by SEQ.ID No.1;
The downstream primer GLMI001-R is represented by SEQ.ID No.2;
The upstream primer GLMI002-F is represented by SEQ.ID No.3;
The downstream primer GLMI002-R is represented by SEQ.ID No.4;
The upstream primer GLMI003-F is represented by SEQ.ID No.5;
Downstream primer GLMI003-R is a set of specific molecular marker composition for differentiating the strains of rhinoceros and erythrasma, characterized in that it is represented by SEQ.ID No.6. In the differentiation method using the specific molecular marker composition for differentiating the fungal species of Jagyeongji and Red Ginger according to claim 1,
The identification method is
Step 1) of extracting the genomic DNA of the specimen of the test target Jagyeongji and Red Ginger species as a template, and performing PCR amplification using specific molecular markers GLMI001, GLMI002, and GLMI003 to obtain an amplification product; and
Electrophoretic detection is performed on the amplification product obtained in step 1), and if DNA bands of 187 bp, 365 bp, and 168 bp appear in the electrophoresis result, the sample to be tested is S. If the 187bp, 365bp, and 168bp DNA bands do not appear in the electrophoresis result, the test target sample is a red lye spp. Step 2); Differentiation method comprising: 3. The method of claim 2,
PCR amplification system, based on a total volume of 30 μL, 3 μL of 10×PCR Buffer containing Mg ²⁺ , 0.9 μL of 10 mmol/L dNTPs, 0.6 μL of 20 μmol/L upstream primer, 0.6 μL of 20 μmol/L downstream primer, 5U/μLTaq A method of differentiation comprising 0.5 μL of enzyme, 1 μL of 50 ng/μL of DNA template, and 23.4 μL of ddH ₂ O. 3. The method of claim 2,
The PCR reaction procedure was performed pre-denaturation at 94°C for 5 minutes; 30 cycles, performing 30 second denaturation at 94°C, GLMI001 annealing temperature is 54°C, GLMI002 annealing temperature is 60°C, GLMI003 annealing temperature is 61°C, recovery for 45 seconds, 72°C for 90 seconds stretched during; Finally, the differentiation method, characterized in that the stretching for 10 minutes at 72 ℃. delete

Images