KR20130093387A - Dna markers for ganoderma lucidum, primers for the markers, and method for discriminating ganoderma lucidum - Google Patents

Abstract

PURPOSE: A molecular marker of ganoderma lucidum is provided to distinguish ganoderma ucidum species by a molecular biological analyzing method, and to expect quality management of imported ganoderma lucidum. CONSTITUTION: A composition for marker of ganoderma lucidum comprises sequence number 1 of base sequence as an active ingredient. A pair of primers having sequence number 2 and 3 is provided. A composition for discriminating ganoderma lucidum comprises the pair of primers as an active ingredient. A distinguishing method of ganoderma lucidum comprises the steps of: separating genome DNA from the mushroom; amplifying a target sequence by performing an amplification using the oligonucleotide primer set, taking the separated genome DNA as a frame; and detecting the amplified product.

Description

DNA markers for Ganoderma lucidum, primers for the markers, and method for discriminating Ganoderma lucidum}

The present invention Ganoderma lucidum mushroom ( Ganoderma lucidum ) relates to the specific determination of the Ganoderma lucidum , and more particularly relates to a method of specifically identifying Ganoderma lucidum using a specific molecular marker, a primer specific to the marker in the genome of Ganoderma lucidum.

In general, Ganoderma lucidum is a yearly mushroom belonging to the genus Bolocho, and its shape varies according to the strain. The flesh is cork-like and the surface is polished like varnish. It is native to hardwood dead trees and stumps, and is widely distributed in the northern temperate hemisphere. Ganoderma lucidum has been used as a valuable medicine in China, Japan, etc. as well as Korea, and it has been called by many names such as ganoderma lucidum, bulchocho, and perennial mushroom.

Currently, a number of fiery fungi have been reported to several taxonomists, but there is some confusion because of disagreements among morphographers. In particular, problems of morphological classification have been pointed out, and in order to solve and supplement these problems, biochemical and molecular biological methods are used to explore new interpretation methods.

Currently, most of the Ganoderma lucidum is imported from China, North Korea, Japan, etc. in dried form, and Ganoderma lucidum is most commonly imported from China. Imported ganoderma lucidum mushroom has a limited morphological distinction, and in the case of powder, it is more difficult to discriminate varieties. In Korea, Ganoderma lucidum 1, Ganoderma lucidum 2, Kunyoung 1, and Jangsaeng Green Carp are registered and officially recognized as a Ganoderma lucidum variety, and they are not only selected and imported like Ganoderma lucidum, but also preserve the excellent Ganoderma lucidum variety. It is necessary to breed and develop.

As a related patent, Korean Patent Publication No. 1020110086265 relates to 'Specific primers for discriminating the Pleurotus eryngii strain and its use', and to identify the circular strain among oyster varieties consisting of oligonucleotide primer sets of SEQ ID NO: 1 and SEQ ID NO: 2 Primer set for the kit, a kit for determining a circular lineage among oyster varieties comprising the primer set, and a method for determining a circular lineage among oyster varieties using the primer set are described.

In another related patent, Korean Patent Publication No. 1020110086264 relates to 'Specific primers for determining the number of Pleurotus eryngii strains and their use', and to determine the number of strains of Pleurotus varieties consisting of oligonucleotide primer sets of SEQ ID NO: 1 and SEQ ID NO: 2 A primer set for a kit, a kit for determining a number of strains of a Pleurotus cultivar containing the primer set, and a method for determining the number of strains of a Pleurotus cultivar using the primer set are described.

Object of the present invention Ganoderma lucidum ( Ganoderma) comprising the nucleotide sequence of SEQ ID NO: 1 lucidum ) to provide molecular markers.

Another object of the present invention is to provide a pair of primers specific for the nucleotide sequence of SEQ ID NO: 1.

In addition, another object of the present invention Ganoderma lucidum ( Ganoderma) by PCR analysis using a pair of primers specific for the sequence of SEQ ID NO: 1 lucidum ) provides a way to determine

In order to achieve the above object, the present invention Ganoderma lucidum ( Ganoderma) comprising a nucleotide sequence of SEQ ID NO: 1 It provides a composition for molecular markers ( lucidum ).

The present invention also provides a pair of primer sets of SEQ ID NO: 2 and SEQ ID NO: 3.

In addition, the present invention Ganoderma lucidum ( Ganoderma) comprising a pair of primer sets of SEQ ID NO: 2 and SEQ ID NO: 3 as an active ingredient lucidum ) provides a composition for determination.

The present invention also provides an oligonucleotide primer set according to the present invention; And it provides a ganoderma lucidum determination kit comprising a reagent for performing an amplification reaction.

In one embodiment of the present invention, the reagent for performing the amplification reaction preferably comprises a DNA polymerase, dNTPs and buffer, but is not limited thereto.

In another aspect, the present invention comprises the steps of: separating genomic DNA from a mushroom; amplifying a target sequence by performing the amplification reaction using the oligonucleotide primer set according to the isolated genomic DNA as a template; And it provides a method for determining the Ganoderma lucidum mushroom comprising the step of detecting the amplification product.

In one embodiment of the present invention, the detection of the amplification product is preferably carried out through capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto.

In one embodiment of the present invention, the amplification product preferably has a size of 559-bp, but is not limited thereto.

In another embodiment of the present invention, the amplification is preferably performed 25 times at 94 ℃ 15 seconds, 56 ℃ 15 seconds, 72 ℃ 30 seconds, but is not limited thereto.

Hereinafter, the present invention will be described.

The present inventors have used URP1 (Universal Rice) widely used for analyzing genetic diversity of various species and species for securing and preserving the genetic resources of selected Ganoderma lucidum varieties, breeding for excellent Ganoderma lucidum varieties, and identifying species of Ganoderma lucidum species. Primer) Performed random amplification of polymorphic DNA (RAPD) analysis using Primer (5'ATCCAAGGTCCGAGACAACC-3 '), Ganoderma Specific primers were prepared after cloning and sequencing of amplification products specific to lucidum . Based on this research, the present invention was completed by selecting a final molecular marker and developing specific primers for the determination of ganoderma lucidum by PCR analysis using the same.

Hereinafter, the present invention will be described in detail.

The present invention provides a molecular marker of Ganoderma lucidum each comprising the nucleotide sequence of SEQ ID NO: 1.

This molecular marker was selected based on the results of cloning and sequencing of the product specifically amplified for Ganoderma lucidum by RAPD (random amplification of polymorphic DNA) analysis using the URP1 primer (SEQ ID NO: 1). .

According to another aspect, the present invention provides a pair of primers specific for the nucleotide sequence of SEQ ID NO: 1.

Primers specific for the nucleotide sequence of SEQ ID NO: 1 are as follows.

KGSMF: 5'CCCTAAACCTCTCAAAGTCA-3 '(SEQ ID NO: 2)

KGSMR: 5'TATCGTACAGGTTCTCGTG -3 '(SEQ ID NO: 3)

The primer pairs of SEQ ID NOs: 2 and 3 allow amplification products of 559-bp to be obtained by PCR analysis using the nucleotide sequence of SEQ ID NO: 1 as a template.

Such primers can be obtained by synthetic techniques known in the art.

According to another aspect, the present invention is Ganoderma by PCR using primers specific for the sequence of SEQ ID NO: lucidum ) provides a method to specifically determine.

The method comprises the steps of i) preparing ganoderma lucidum mycelium sample and extracting genomic DNA, ii) conducting PCR in the sample using a pair of primers consisting of SEQ ID NOs: 2 and 3, and iii) by electrophoresis. Identifying the PCR product.

PCR can be performed by PCR techniques known in the art.

By checking the PCR product obtained by performing PCR according to the present method, when the PCR product of 559bp amplified by the primer pair used is confirmed, it is possible to determine the Ganoderma lucidum .

Ganoderma lucidum ( Ganoderma lucidum ) is commonly called Bullocho, a mushroom belonging to Aphyllophorales, Ganodermataceae, and Ganoderma. Since ganoderma lucidum is known to have many pharmacological effects other than anticancer activity, scientific research of pharmacologically effective ingredients on them is being actively conducted. Ganoderma lucidum mushrooms are divided into red, black, black, yellow, purple, blue, and white paper depending on the shape of fruiting body, color, and color of spores. Although it is classified into 6 species, it is known that there are more than 60 species in recent years. Also jannabi bulrocho belonging to the Ganoderma genus (genus) (Ganoderma applanatum ), Black-Blowed Candle ( Ganoderma) neo -japonicum , Ganoderma tsugae ) and the like are also known. Most of the Ganoderma lucidum mushrooms used to study the pharmacological effects to date have been studied without the accurate classification. This is because it is difficult to accurately classify the system based on morphological differences. The classification of Ganoderma lucidum should take into account not only the morphological differences such as the time of collection, location, and fruiting body, but also genetic differences. As a result, it is possible to refer to the comprehensive pharmacological effects on Ganoderma lucidum, but it is difficult to identify the characteristics or efficacy of Ganoderma species included in the Ganoderma genus.

Currently, the Ganoderma lucidum that is imported to Korea is mainly dried form about 1 billion won per year and is mainly imported from China. Ganoderma 1 No. 1 in Korea lucidum ), ganoderma 2 ( Ganoderma) lucidum), Kunyoung No.1 (Ganoderma species), Jang Saeng overgrown antler (Ganoderma species is registered as an official Ganoderma lucidum variety and its efficacy has also been tested.

By using molecular markers developed by the present invention, Ganoderma including domestic registered varieties such as Youngji No. 1, Youngji No. 2, Kun Young No. 1, Jangsaeng Greengap by molecular biological analysis method Accurate identification of lucidum species and quality control of imported ganoderma lucidum mushrooms are also possible. In addition, by using the molecular markers developed according to the present invention, it is considered that it is possible to breed, develop and preserve genetic resources of Korea's own excellent ganoderma lucidum species.

Figure 1 is a photograph of the RAPD (random amplification of polymorphic DNA) analysis using the URP1 primer of Ganoderma lucidum. M, marker; 1kb, 1kilo base, 631-bp, Ganoderma lucidum specific amplification band, Ganoderma lucidum , ganoderma lucidum ; Ganoderma species, Fireweed but species not known.
Figure 2 is the nucleotide sequence of the specific amplification product of Ganoderma lucidum isolated by RAPD analysis.
Figure 3 is a primer prepared from a specific DNA fragment site separated by RAPD analysis of Ganoderma lucidum.
Figure 4 is a picture of PCR analysis using the discrimination-specific primers prepared with 18 strains of Bulchochox and 59 strains and one strain ( laetiporus genus 1 strain). M, marker; 1kb, 1kilo base, 559-bp, Ganoderma lucidum specific amplification band, Ganoderma lucidum , ganoderma lucidum ; Ganoderma species, Fireweed but species not known.

The present invention may be understood in more detail by the following examples, which are intended to illustrate the present invention and are not intended to limit the protection scope of the present invention. On the other hand, the molecular biological experiments not specifically mentioned in the following examples, see Sambrook et al. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, second edition, 1989).

Example  1. Cultivation of Ganoderma lucidum mycelium

    The collected Ganoderma lucidum mycelium was inoculated in Potato Dextrose Broth (Difco, Becton Dickinson; 0.8% Potato starch, 4% Dextrose) liquid medium and incubated for 3 weeks in a 30 ° C. incubator. Strains used for PCR analysis are summarized in Table 1.

NO . Species Collection sites Collection ID Origin One Ganoderma annulare Korean Collection for Type Culture KCTC16803 Brazil 2 Ganoderma applanatum The American Type Culture Collection ATCC44053 Japan 3 Ganoderma applanatum incheon University IUM-3985 Netherlands 4 Ganoderma carnosum The Centraalbureau voor Schimmelcultures CBS 516.96 Netherlands 5 Ganoderma curtisii The Centraalbureau voor Schimmelcultures CBS 100132 Netherlands 6 Ganoderma lobatum The American Type Culture Collection ATCC 42985 Canada 7 Ganoderma lobatum Mushroom Division at RDA ASI-7061 U.S.A 8 Ganoderma lucidum (Governor 1) Mushroom Division at RDA ASI-7004 Korea 9 Ganoderma lucidum Mushroom Division at RDA ASI-7013 Korea 10 Ganoderma lucidum (Manor 2) Mushroom Division at RDA ASI-7071 Korea 11 Ganoderma lucidum Mushroom Division at RDA ASI-7074 Korea 12 Ganoderma lucidum Mushroom Division at RDA ASI-7091 Korea 13 Ganoderma lucidum Mushroom Division at RDA ASI-7094 Korea 14 Ganoderma lucidum Mushroom Division at RDA ASI-7135 Korea 15 Ganoderma lucidum incheon University IUM-0047 Korea 16 Ganoderma lucidum incheon University IUM-0757 Korea 17 Ganoderma lucidum incheon University IUM-0938 Korea 18 Ganoderma lucidum incheon University IUM-3986 Korea 19 Ganoderma lucidum incheon University IUM-4002 Korea 20 Ganoderma lucidum incheon University IUM-4100 Korea 21 Ganoderma lucidum Mushroom Division at RDA ASI-7117 unknown 22 Ganoderma lucidum incheon University IUM-4303 Bangladesh 23 Ganoderma lucidum incheon University IUM-4304 Bangladesh 24 Ganoderma lucidum incheon University IUM-4310 Bangladesh 25 Ganoderma lucidum The American Type Culture Collection ATCC46755 Canada 26 Ganoderma lucidum incheon University IUM-4242 China 27 Ganoderma lucidum Mushroom Division at RDA ASI-7037 Papuanewguinea 28 Ganoderma lucidum The American Type Culture Collection ATCC 64251 Taiwan 29 Ganoderma lucidum Korean Collection for Type Culture KCTC 16802 Thailand 30 Ganoderma meredithae The American Type Culture Collection ATCC 64492 U.S.A 31 Ganoderma meredithae Mushroom Division at RDA ASI-7140 unknown 32 Ganoderma mirabile The Centraalbureau voor Schimmelcultures CBS 218.36 Philippines 33 Ganoderma neo - japonicum Mushroom Division at RDA ASI-7032 unknown 34 Ganoderma oregonense Mushroom Division at RDA ASI-7049 U.S.A 35 Ganoderma oregonense Mushroom Division at RDA ASI-7062 U.S.A 36 Ganoderma oregonense Mushroom Division at RDA ASI-7063 U.S.A 37 Ganoderma oregonense Mushroom Division at RDA ASI-7067 U.S.A 38 Ganoderma pfeifferi The Centraalbureau voor Schimmelcultures CBS 747.84 Netherlands 39 Ganoderma resinaceum The American Type Culture Collection ATCC 52416 Argentina 40 Ganoderma resinaceum The Centraalbureau voor Schimmelcultures CBS 152.27 UK 41 Ganoderma resinaceum The Centraalbureau voor Schimmelcultures CBS 220.36 U.S.A 42 Ganoderma resinaceum Mushroom Division at RDA ASI-7142 unknown 43 Ganoderma resinaceum Mushroom Division at RDA ASI-7143 unknown 44 Ganoderma resinaceum incheon Univercity IUM-3651 Czech 45 Ganoderma subamboinense there . laecisporum The American Type Culture Collection ATCC52420 Argentina 46 Ganoderma Species Konkuk University KU-4033 Korea 47 Ganoderma Species Konkuk University KU-4035 Korea 48 Ganoderma Species Mushroom Division at RDA ASI-7033 Papuanewguinea 49 Ganoderma Species Mushroom Division at RDA ASI-7034 Papuanewguinea 50 Ganoderma Species Mushroom Division at RDA ASI-7038 Papuanewguinea 51 Ganoderma Species Jangsaeng Green Carving Mushroom Division at RDA ASI-7146 unknown 52 Ganoderma Species Mushroom Division at RDA ASI-7150 unknown 53 Ganoderma Species Mushroom Division at RDA ASI-7151 unknown 54 Ganoderma Species (Keun Young) Mushroom Division at RDA ASI-7152 unknown 55 Ganoderma tsugae The American Type Culture Collection ATCC 64795 Canada 56 Ganoderma tsugae Mushroom Division at RDA ASI-7064 U.S.A 57 Ganoderma tornatum The Centraalbureau voor Schimmelcultures CBS 109679 Netherlands 58 Ganoderma valesiacum The Centraalbureau voor Schimmelcultures CBS 428.84 U.S.A 59 Ganoderma webeianum The Centraalbureau voor Schimmelcultures CBS 219.36 Philippines 60 laetiporus persicinus The Centraalbureau voor Schimmelcultures CBS 274.92 Netherlands

Example  2. From Ganoderma lucidum mycelium Genomic DNA  extraction

Liquid nitrogen was added to Ganoderma lucidum mycelium, ground, and then taken in an appropriate amount. 500μl of extraction buffer (200mM Tris-HCl (PH8.0), 200mM NaCl, 25mM EDTA, 0.5% SDS) and 10μl of Proteinase K (20mg / ml) were added and 50 ℃. Reaction was carried out for 1 hour. 250μl of 2X CTAB (2% CTAB, 100mM Tris-HCl (PH8.0), 5% SDS) was added and 500μl of Phenol: Chloroform: isoamylalcohol (25: 24: 1) was added and centrifuged at 12,000rpm for 10 minutes. Separated. 550μl of the supernatant was taken and transferred to a new 1.5ml tube. After adding 0.6 volume of isopropanol and 1/30 volume of 3M Sodium acetate (PH 5.2), the mixture was allowed to react sufficiently at room temperature and centrifuged at 12,000 rpm for 15 minutes. . The precipitated DNA pallet was washed with 70% ethanol, dissolved in 60 μl TE buffer (Tris-HCl 10 mM, EDTA 1 mM; PH 8.0), and 6 μl RNase (25 mg / ml) was added thereto, followed by reaction at 37 ° C. for 30 minutes. Genomic DNA was carried out PCR by adjusting the concentration to 100ng / μl.

Example  3. Specific for the detection and determination of Ganoderma lucidum primer  making

In order to prepare a specific primer for PCR analysis for determination of Ganoderma lucidum, RAPD (random amplification of polymorphic DNA) analysis using URP1 primer was performed (FIG. 1). PCR amplification reactions were carried out using a TaKaRa thermalcycler (TaKaRa, Japan), and the reaction was carried out in a Pro-Hot Start PCR Master Mix (TNT research, Korea). μl URP1 primer (500ng / μl) and 1μl of each genomic DNA (100ng) were added and the total amount was 20μl. The PCR reaction was carried out 30 times at 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes. After the reaction, the PCR amplification product was electrophoresed on an agarose gel at 1.2% concentration and stained with ethidium bromide to be identified on UV. It was. As a result, used in the present invention Ganoderma Among the lucidum (Table 1), G. lucidum from Korea, Bangladesh, Papua New Guinea, and Thailand showed the same or similar amplification pattern. In addition, two Ganoderma species (genus 47, 50; Table 1), which are genus Ganodrma , but whose species are not defined, are currently registered as Ganoderma lucidum species in Korea, but the species is not clearly defined. Jangsaeng-tung green and dry young (No. 51, 54; Table 1) were the same or similar to those of Ganoderma lucidum. However, Ganoderma lucidum in Canada, China, and Taiwan origin Ganoderma lucidum (No. 25, 26, 28; Table 1) shows an amplification pattern completely different from that of Ganoderma lucidum, which may be misclassified. In addition, G is also a clone-specific amplified fragment (631-bp) for G. lucidum based on the results of the first to obtain the nucleotide sequence of G. lucidum specific fragment by nucleotide sequence analysis (Fig. 2) Based on this . to prepare a specific primer to determine the 1 pair for PCR analysis for lucidum (Fig. 3).

Example  4. Specific for the determination of Ganoderma lucidum Primer  Used PCR

PCR was carried out using Ganoderma genus 18 strains 59 strains and the control strain ( laetiporus genus 1 strain 1) cultured in Example 1 using a specific primer prepared in Example 3. PCR amplification reaction was carried out using a TaKaRa thermalcycler (TaKaRa, Japan), and the reaction was performed with 2 μl (200 ng) of genomic DNA and 0.5 μl and 2.5 dNTP of KGSM1-F and KGSM1-R primers respectively. 0.5μl, 25mM MgCl ₂ 2μl, 5x Buffer (10mM Tris-Cl (PH8.3), 50mM KCl, 0.25mM MgCl ₂ ) 5μl, Taq polymerase (Promega, USA) 0.25μl and then sterile distilled water (DDW) PCR reaction was performed by correcting the total amount to 25 μl. PCR reaction was carried out 25 times at 94 ℃ 15 seconds, 56 ℃ 15 seconds, 72 ℃ 30 seconds. After the reaction, the PCR amplification product was electrophoresed in agarose gel of 1.2% concentration, and then stained with ethidium bromide, and confirmed on UV. As a result of performing PCR analysis on 18 strains of Ganoderma genus 59 and control strains (one strain of laetiporus genus 1) using Ganoderma lucidum specific DNA marker-derived primers developed by the present invention, it was registered as a Ganoderma lucidum variety in Korea. No. 1 on territory, No.2 manor, Kunyoung No. 1, and Jang Saeng overgrown antler; including (8, 10, 54 and 51 on Table 1) Ganoderma Only lucidum species were able to identify 559-bp amplification products. However, Ganoderma from Canada, China, and Taiwan as described above in Example 3 Amplification products of 559-bp could not be identified in lucidum (FIG. 4).

<110> Konkuk University Industrial Cooperation Corp. <120> DNA markers for Ganoderma lucidum, primers for the markers, and          method for discriminating Ganoderma lucidum <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 631 <212> DNA <213> Ganoderma <400> 1 atccaaggtc cgagacaacc ctgatgcccc taaacctctc aaagtcacga tatccaagaa 60 agctcgagca gtcgaagcca taccagacta cgagcatgat tgggaacgat gggcaaaggt 120 ttacgatcga tagcagaaac ggcgacgaca acaaggactc gacccgctcc cggatcctat 180 cactccacca ctccttattt ttgcacaata ctccacataa acccctttaa tagccgttga 240 agggatgccc gatggccaga gacctgattc tacggaatcc ccggttaatg acccctgacg 300 gatccatgta ctacctcccc cgactctacc gtgtgggaaa agctaggcag gaaagcgatt 360 tcgcccttga caggaatgga attccgtccc catcacactc ctttaagttt acgataccag 420 gggtagtttt aaagatcacc ttatttatcc aacggccaac cctataccca ctacgtactt 480 ggtttggaac cccaagtctg aaaggggggt aatgtcacga tacgattttc gcttttagta 540 gttttctctc acgattacag cattagtcac gagaacctgt acgatacgat atctccgaac 600 gttctcggtg tggttgtctc ggaccttgga t 631 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ccctaaacct ctcaaagtca 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tatcgtacag gttctcgtg 19

Claims (9)

Composition for molecular markers of Ganoderma lucidum containing a nucleotide sequence of SEQ ID NO: 1 as an active ingredient. A pair of primer sets of SEQ ID NO: 2 and SEQ ID NO: 3. Ganoderma lucidum ( Ganoderma) comprising a pair of primer sets of SEQ ID NO: 2 and SEQ ID NO: 3 as an active ingredient lucidum ) Discrimination composition. An oligonucleotide primer set according to claim 2; And a ganoderma lucidum mushroom kit comprising a reagent for performing an amplification reaction. The kit of claim 4, wherein the reagent for carrying out the amplification reaction comprises DNA polymerase, dNTPs and a buffer. Separating genomic DNA from mushrooms;
Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the oligonucleotide primer set according to claim 2; And
Detecting the amplification product. The method of claim 6, wherein the detection of the amplification product is carried out through capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement. The method of claim 6, wherein the amplification product has a size of 559-bp. The method of claim 6, wherein the amplification is performed 25 times at 94 ° C. 15 seconds, 56 ° C. 15 seconds, and 72 ° C. 30 seconds.

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