CN110564885B - Specific molecular markers for identifying Ganoderma sinense and Ganoderma lucidum strains, and identification method and application thereof - Google Patents
Specific molecular markers for identifying Ganoderma sinense and Ganoderma lucidum strains, and identification method and application thereof Download PDFInfo
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Abstract
The invention discloses a group of molecular markers for identifying ganoderma sinense and ganoderma lucidum strains, wherein the nucleotide sequences of upstream primers GLMI001-F, GLMI002-F and GLMI003-F are respectively shown as SEQ ID No1, SEQ ID No3 and SEQ ID No5, and the nucleotide sequences of downstream primers GLMI001-R, GLMI002-R and GLMI003-R are respectively shown as SEQ ID No2, SEQ ID No4 and SEQ ID No 6. The specific molecular markers GLMI001, GLMI002 and GLMI003 are used for identifying and detecting Ganoderma sinense and Ganoderma lucidum strains, standardizing the Ganoderma lucidum strain market, detecting adulteration behaviors and ensuring the safety of Ganoderma lucidum medicinal materials; the method can also be used for detecting the ganoderma lucidum product, and has great application potential and better economic value for ensuring the ganoderma lucidum germplasm purity and improving the production efficiency.
Description
Technical Field
The invention belongs to the technical field of ganoderma lucidum strain molecular identification, and particularly relates to a group of specific molecular markers for identifying ganoderma sinense and ganoderma lucidum strains, an identification method and application.
Background
Ganoderma lucidum belongs to the kingdom of fungi, Basidiomycota, class of Aphyllophorales, order of Polyporales, family of Ganoderma lucidum, genus Ganoderma, and is widely distributed in tropical and subtropical areas. China has rich ganoderma germplasm resources, and is widely distributed in Jilin, Hebei, Shanxi, Henan, Zhejiang, Yunnan, Guizhou, Guangdong, Guangxi and other provinces. The ganoderma lucidum is a traditional Chinese medicinal material in China, is one of the beautiful names of the Jiuda sianchau, has mild nature, special smell and slightly bitter taste. Ganoderma lucidum is prepared from dried fruiting body of Ganoderma lucidum (Ganoderma lucidum) or Ganoderma sinense (Ganoderma sinense) in pharmacopoeia (2015) of the people's republic of China, has complicated chemical components, mainly contains triterpenes, polysaccharides, amino acid polypeptides, nucleosides, furans, alkaloids, oils and trace elements, and has various physiological activities and pharmacological activities, such as regulating immunity, preventing cardiovascular system diseases, resisting tumor, resisting virus, reducing blood sugar and resisting oxidation. But red glossy ganoderma is warm in nature and purple glossy ganoderma is neutral in nature; the red ganoderma lucidum is suitable for being used as a medicine, and the purple ganoderma lucidum is suitable for daily health care and conditioning. Moreover, purple Ganoderma lucidum is more expensive than red Ganoderma lucidum.
Along with the improvement of the living standard of people, the demand of high-quality lucid ganoderma is more and more, so that more and more lucid ganoderma are cultivated in a wild-like manner at present, and the culture, preservation and production of strains of good lucid ganoderma varieties are promoted. At present, some merchants do not produce and sell the acquired fulminant according to the edible fungus strain management method, and the classification of the ganoderma is disordered due to the long-term cultivation of the ganoderma, crossbreeding and the like, so that the strain names are not unified, and the healthy development of the ganoderma industry is hindered to a great extent. Two main species in the ganoderma lucidum industry, Ganoderma sinense and Ganoderma lucidum, are easily distinguished by appearance at the fruiting body stage, but in the strain stage, the two species are almost the same, so that the differentiation by molecular means is required to ensure the healthy and orderly development of the ganoderma lucidum industry.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a group of molecular markers for identifying ganoderma sinense and ganoderma lucidum strains, an identification method and application in order to standardize the cultivation, preservation and production of ganoderma lucidum strains.
The invention is realized by the following technical scheme:
a group of specific molecular markers for identifying purple ganoderma and red ganoderma strains, primers of the specific molecular markers are GLMI001, GLMI002 and GLMI003, and nucleotide sequences of the primers are as follows:
upstream primer GLMI 001-F: 5'-ACCCGGTGAAACAACA-3', as shown in SEQ ID No. 1;
downstream primer GLMI 001-R: 5'-GCAGTGAGTTTGGCTTAT-3', as shown in SEQ ID No. 2;
upstream primer GLMI 002-F: 5'-CGGACAGCGAACAGACG-3', as shown in SEQ ID No. 3;
downstream primer GLMI 002-R: 5'-GGCAGCAATGAGGACACC-3', as shown in SEQ ID No. 4;
the upstream primer GLMI 003-F: 5'-AGCGACGCCGTCAACTC-3', as shown in SEQ ID No. 5;
downstream primer GLMI 003-R: 5'-AAGCAGCCACAGCCAACT-3', as shown in SEQ ID No. 6.
The three pairs of specific molecular marker primers (GLMI001-F/R, GLMI002-F/R and GLMI003-F/R) are used for carrying out common PCR amplification on the DNA of the samples of the ganoderma sinense and the ganoderma lucidum, carrying out electrophoresis on the amplified products, screening specific bands of the ganoderma sinense to carry out sequencing, then carrying out comparison analysis on the specific band sequences of the ganoderma sinense obtained by the sequencing, designing specific primers according to the specific primers, and verifying whether the specific bands of the ganoderma sinense can be obtained again. Therefore, the three pairs of primer pairs have extremely high specificity, and PCR amplification is carried out on the sample DNA of the ganoderma sinense and the ganoderma lucidum, only the ganoderma sinense can obtain DNA bands of 187bp, 365bp and 168bp, but the ganoderma lucidum does not; the nucleotide sequence of the specific fragment obtained by amplification of the ganoderma sinense strain is shown as SEQ.ID No7, SEQ.ID No8 and SEQ.ID No 9.
Another objective of the invention is to provide a method for rapidly identifying the ganoderma sinense and ganoderma lucidum strains by using the specific molecular marker primers (GLMI001-F/R, GLMI002-F/R and GLMI 003-F/R).
The invention adopts the specific molecular marker primers (GLMI001-F/R, GLMI002-F/R and GLMI003-F/R) as specific amplification primers, takes the genomic DNA of the ganoderma sinense and ganoderma lucidum strains as templates, performs PCR amplification, performs electrophoresis detection on the amplification products, and if a DNA band of 187bp, 365bp and 168bp appears in the electrophoresis result, the sample to be detected is the ganoderma sinense strain; and if the electrophoresis result does not show the DNA band, determining that the sample to be detected is the ganoderma lucidum strain.
In the above method, the selection of amplification primers, the extraction determination of genomic DNA, and the electrophoresis detection can be performed according to the conventional methods in the art.
It should be noted that the specific molecular marker primers and the detection method of the invention are only suitable for the identification of the ganoderma sinense and ganoderma lucidum strains, i.e. the samples to be detected are limited to the range of the samples of the ganoderma sinense and ganoderma lucidum strains.
The method comprises the following specific steps:
(1) taking 0.3-0.5g of ganoderma sinense and ganoderma lucidum strains, and extracting the genomic DNA of a sample to be detected by using a fungal genomic DNA extraction kit;
(2) performing common PCR amplification by taking the genomic DNA obtained in the step (1) as a template and the specific molecular marker primers (GLMI001-F/R, GLMI002-F/R and GLMI003-F/R) as amplification primers:
the PCR reaction system (total volume 30. mu.l) consisted of:
| components of the System | Volume (μ L) |
| 10 XPCR Buffer (containing Mg)2+) | 3 |
| 10mmol/L dNTPs | 0.9 |
| 20 mu mol/L upstream primer | 0.6 |
| 20 mu mol/L downstream primer | 0.6 |
| 5U/. mu.LTaq enzyme | 0.5 |
| DNA template (50 ng/. mu.L) | 1 |
| ddH2O | 23.4 |
The PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; 30 cycles (denaturation at 94 ℃ for 30 seconds, annealing temperature (GLMI001 at 54 ℃, GLMI002 at 60 ℃ and GLMI003 at 61 ℃) for 45 seconds, extension at 72 ℃ for 90 seconds); final extension at 72 ℃ for 10 min.
(3) Detecting the PCR amplification product obtained in the step (2) by using 1.5% agarose gel electrophoresis, and performing imaging photographing by using a gel imaging system, wherein if a DNA band of 187bp (GLMI001), 365bp (GLMI002) and 168bp (GLMI003) respectively appears in the electrophoresis result, the sample to be detected is the ganoderma sinense strain; and if the electrophoresis result does not show the DNA band, determining that the sample to be detected is the ganoderma lucidum strain.
The invention has the following beneficial effects: the specific molecular marker primers (GLMI001-F/R, GLMI002-F/R and GLMI003-F/R) can be used for carrying out molecular identification on ganoderma sinense strains and ganoderma lucidum strains, and the method is simple, accurate and high in sensitivity, and cannot be achieved by other existing identification methods.
Drawings
FIG. 1 is an electrophoresis chart of the specific molecular marker GLMI001-F/R of the invention after PCR amplification of Ganoderma sinense and Ganoderma lucidum strains;
FIG. 2 is an electrophoresis diagram of the specific molecular marker GLMI002-F/R of the present invention after PCR amplification of Ganoderma sinense and Ganoderma lucidum strains;
FIG. 3 is an electrophoresis diagram of the specific molecular marker GLMI003-F/R of the present invention after PCR amplification of Ganoderma sinense and Ganoderma lucidum strains;
wherein: the channel M is DNA standard molecular weight Marker DL 2000; 1-9 of a channel: (1) jilin red ganoderma lucidum 1, (2) Jilin red ganoderma lucidum 2, (3) Shandong red ganoderma lucidum 1, (4) Shandong red ganoderma lucidum 2, (5) Anhui red ganoderma lucidum 1, (6) Anhui red ganoderma lucidum 2, (7) Henan red ganoderma lucidum, (8) Zhejiang red ganoderma lucidum 1, and (9) Zhejiang red ganoderma lucidum 2 (Shouxianggu strain); passage 10: ganoderma sinense (longevity immortal cereal strain).
Detailed Description
The purpose and effect of the present invention will become more apparent from the following detailed description of the present invention when taken in conjunction with the accompanying drawings.
Example 1
1. Extraction of genomic DNA of Ganoderma sinense and Ganoderma lucidum strains to be detected
Taking 0.3-0.5g of hyphae of the purple lucid ganoderma and the erythroderma lucidum strain to be detected, extracting the genomic DNA of the sample of the purple lucid ganoderma and the erythroderma lucidum strain to be detected by using a fungal genomic DNA extraction kit of Shanghai bioengineering technology Limited, carrying out electrophoresis on the obtained DNA by using 1.5% agarose gel to detect the purity, detecting the concentration of the DNA by using a NanoDrop TM 1000 type ultramicro ultraviolet spectrophotometer of American Saimer Feishel, then diluting to 50 ng/mu L, and placing in a 4-degree refrigerator for later use.
2. Synthesis of specific molecular marker primers
Designing three pairs of molecular marker primers based on a specific sequence of ganoderma sinense, wherein an upstream primer GLMI 001-F: 5'-ACCCGGTGAAACAACA-3' and downstream primer GLMI 001-R: 5'-GCAGTGAGTTTGGCTTAT-3', respectively; upstream primer GLMI 002-F: 5'-CGGACAGCGAACAGACG-3' and downstream primer GLMI 002-R: 5'-GGCAGCAATGAGGACACC-3', respectively; the upstream primer GLMI 003-F: 5'-AGCGACGCCGTCAACTC-3' and downstream primer GLMI 003-R: 5'-AAGCAGCCACAGCCAACT-3', synthesized by Shanghai.
3. PCR amplification of specific molecular marker primers (GLMI001-F/R, GLMI002-F/R and GLMI003-F/R)
PCR amplification system (total volume 30. mu.L): 10 XPCR Buffer (containing Mg)2+) mu.L of 3. mu.L, 0.9. mu.L of 10mmol/L dNTPs, 0.6. mu.L of 20. mu.mol/L upstream primer, 0.6. mu.L of 20. mu.mol/L downstream primer, 0.5. mu.L of 5U/. mu.L Taq enzyme, 1. mu.L DNA template (50 ng/. mu.L), ddH2O 23.4μL。
The PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; 30 cycles (denaturation at 94 ℃ for 30 seconds, annealing temperature (GLMI001 at 54 ℃, GLMI002 at 60 ℃ and GLMI003 at 61 ℃) for 45 seconds, extension at 72 ℃ for 90 seconds); final extension at 72 ℃ for 10 min.
4. Electrophoretic detection
Taking 5-10 mu L of PCR amplification product in the step (3), and carrying out electrophoresis detection by using 1.5% agarose gel, wherein the electrophoresis result is shown in figures 1-3.
Detecting Ganoderma sinense and Ganoderma lucidum strain with specific molecular marker GLMI001 according to the above method, wherein the electrophoresis chart is shown in FIG. 1, the number 10 is Ganoderma sinense strain individual, and a 187bp DNA band is amplified; no other DNA band of 187bp is amplified in the other strains with the numbers of the Ganoderma lucidum.
Detecting Ganoderma sinense and Ganoderma lucidum strain with specific molecular marker GLMI002 according to the above method, and amplifying to obtain 365bp DNA band with an electrophoretogram shown in FIG. 2, wherein the number of 10 is Ganoderma sinense strain individual; no 365bp DNA band is amplified in other Ganoderma lucidum strains.
Detecting Ganoderma sinense and Ganoderma lucidum strain with specific molecular marker GLMI003 according to the above method, and amplifying a 168bp DNA band with an electrophoretogram shown in FIG. 3, wherein the number 10 is Ganoderma sinense strain individual; no 168bp DNA band is amplified in other Ganoderma lucidum strains.
This shows that the specific molecular markers GLMI001, GLMI002 and GLMI003 of the present invention have very high specificity, and thus, can be used for the rapid identification of Ganoderma sinense and Ganoderma lucidum strains.
Sequence listing
<110> university of teachers in Hangzhou
Zhejiang shouxian grain botanical drug research institute Co., Ltd
<120> a group of specific molecular markers for identifying Ganoderma sinense and Ganoderma lucidum, and identification method and application thereof
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Claims (5)
1. A group of specific molecular markers for identifying purple ganoderma and red ganoderma strains is characterized in that primers of the specific molecular markers are GLMI001-F/R, GLMI002-F/R and GLMI003-F/R, and nucleotide sequences of the primers are as follows:
the upstream primer GLMI001-F is shown as SEQ ID No. 1;
the downstream primer GLMI001-R is shown as SEQ ID No. 2;
the upstream primer GLMI002-F is shown as SEQ ID No. 3;
the downstream primer GLMI002-R is shown as SEQ ID No. 4;
the upstream primer GLMI003-F is shown as SEQ ID No. 5;
the downstream primer GLMI003-R is shown as SEQ ID No. 6.
2. An identification method using the specific molecular marker for identifying Ganoderma sinense and Ganoderma lucidum strains as claimed in claim 1, characterized in that the identification method comprises the steps of:
1) firstly, extracting genome DNA of a sample of a purple lucid ganoderma and a red lucid ganoderma strain to be detected as a template, and carrying out PCR amplification by utilizing specific molecular markers GLMI001-F/R, GLMI002-F/R and GLMI003-F/R to obtain an amplification product;
2) carrying out electrophoresis detection on the amplification product obtained in the step 1), wherein if DNA bands of 187bp, 365bp and 168bp appear in the electrophoresis result, the sample to be detected is a ganoderma sinense strain; if the electrophoresis result does not have DNA bands of 187bp, 365bp and 168bp, the sample to be detected is the ganoderma lucidum strain.
3. The method for identifying specific molecular markers of Ganoderma sinense and Ganoderma lucidum as claimed in claim 2, wherein the PCR amplification system is used in a total volume of 30 μ L: containing Mg2+10 XPCR Buffer 3. mu.L, 10mmol/L dNTPs 0.9. mu.L, 20. mu. mol/L upstream primer 0.6. mu.L, 20. mu. mol/L downstream primer 0.6. mu.L, 5U/. mu.L of LTaq enzyme 0.5. mu.L, DNA template 1. mu.L at a concentration of 50 ng/. mu.L, ddH2O 23.4μL。
4. The method for identifying specific molecular markers of Ganoderma sinense and Ganoderma lucidum as claimed in claim 2, wherein the PCR reaction process is as follows: pre-denaturation at 94 ℃ for 5 min; 30 cycles: denaturation at 94 ℃ for 30 seconds, annealing at GLMI001 at 54 ℃, GLMI002 at 60 ℃, annealing at GLMI003 at 61 ℃, renaturation at 45 seconds, and extension at 72 ℃ for 90 seconds; final extension at 72 ℃ for 10 min.
5. The use of the specific molecular markers GLMI001, GLMI002 and GLMI003 of claim 1 in the identification of Ganoderma sinense and Ganoderma lucidum strains.
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